transforming-growth-factor-beta and Pancreatic-Neoplasms

Cachexia is a metabolic syndrome consisting of massive loss of muscle mass and function that has a severe impact on the quality of life and survival of cancer patients. Up to 20% of lung cancer patients and up to 80% of pancreatic cancer patients are diagnosed with cachexia, leading to death in 20% of them. The main drivers of cachexia are cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), macrophage inhibitory cytokine 1 (MIC-1/GDF15) and transforming growth factor-beta (TGF-β). Besides its double-edged role as a tumor suppressor and activator, TGF-β causes muscle loss through myostatin-based signaling, involved in the reduction in protein synthesis and enhanced protein degradation. Additionally, TGF-β induces inhibin and activin, causing weight loss and muscle depletion, while MIC-1/GDF15, a member of the TGF-β superfamily, leads to anorexia and so, indirectly, to muscle wasting, acting on the hypothalamus center. Against this background, the blockade of TGF-β is tested as a potential mechanism to revert cachexia, and antibodies against TGF-β reduced weight and muscle loss in murine models of pancreatic cancer. This article reviews the role of the TGF-β pathway and to a minor extent of other molecules including microRNA in cancer onset and progression with a special focus on their involvement in cachexia, to enlighten whether TGF-β and such other players could be potential targets for therapy.

Topics: Animals; Cachexia; Humans; Mice; Pancreatic Neoplasms; Quality of Life; Transforming Growth Factor beta; Transforming Growth Factors

Transforming growth factor β (TGFβ) signalling pathway switches between anti-tumorigenic function at early stages of cancer formation and pro-tumorigenic effects at later stages promoting cancer metastasis. A similar contrasting role has been uncovered for reactive oxygen species (ROS) in pancreatic tumorigenesis. Down-regulation of ROS favours premalignant tumour development, while increasing ROS level in pancreatic ductal adenocarcinoma (PDAC) enhances metastasis. Given the functional resemblance, we propose that ROS-mediated processes converge with the spatial and temporal activation of TGFβ signalling and thereby differentially impact early tumour growth versus metastatic dissemination. TGFβ signalling and ROS could extensively orchestrate cellular processes and this concerted function can be utilized by cancer cells to facilitate their malignancy. In this article, we revisit the interplay of canonical and non-canonical TGFβ signalling with ROS throughout pancreatic tumorigenesis and metastasis. We also discuss recent insight that helps to understand their conflicting effects on different stages of tumour development. These considerations open new strategies in cancer therapeutics.

Topics: Humans; Neoplasm Metastasis; Pancreatic Neoplasms; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta

Pancreatic cancer is a common malignant digestive disease. Epidemiological and clinical studies have demonstrated that pancreatic cancer is closely related to diabetes mellitus. Diabetic patients are more likely to develop pancreatic cancer, which is linked with poor outcomes. Pancreatic cancer is complicated with abnormal blood sugar and insulin resistance and promotes the development of diabetes mellitus. Understanding the molecular mechanisms linking diabetes mellitus and pancreatic cancer is essential for the treatment of diabetes cancer patients. The transforming growth factor-β (TGF-β) signaling pathway is deregulated in cancer and has a dual role in different stages of cancer as a suppressor or a promoter. More important, The TGF-β signaling pathway is also another important reason for diabetic complications. This review summarizes the relationship between diabetes and pancreatic cancer, in particular, focusing on the role of the TGF-β signaling pathway. It is possible to find drugs like metformin that can prevent and treat pancreatic cancer by targeting the TGF-β signaling pathway.

Topics: Diabetes Mellitus; Humans; Insulin Resistance; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death worldwide. PDAC is an aggressive disease with an 11-month median overall survival and a five-year survival of less than 5%. Incidence of PDAC is constantly increasing and is predicted to become the second leading cause of cancer in Western countries within a decade. Despite research and therapeutic development, current knowledge about PDAC molecular mechanisms still needs improvements and it seems crucial to identify novel therapeutic targets. Genomic analyses of PDAC revealed that transforming growth factor β (TGFβ) signaling pathways are modified and the

Topics: Animals; Carcinoma, Pancreatic Ductal; Epithelial-Mesenchymal Transition; Humans; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancerrelated mortality in the USA, and the overall incidence of the disease is increasing such that it is expected to be the third leading cause of cancer-related deaths in the next decade. Minimal improvements in therapy have not changed the overall mortality rate over the past decade for patients with PDAC. The purpose of this review is to identify new data regardign the role of Transforming growth factor beta (TGF-β) based therapeuics in patients with PDAC.. The literature was searched for peer reviewed manuscripts regarding the use of TGF-β inhibitors in PDAC therapy and the mechanism in which TGF-β intracellular signaling effects patient survival.. TGF-β plays a vital, context-dependent role as both a tumor suppressor and promoter of PDAC. The downstream effects of this duality play a significant role in the immunologic response of the tumor microenvironment (TME), epithelial-mesenchymal transformation (EMT), and the development of metastatic disease. Immunologic pathways have been shown to be successful targets in the treatment of other diseases, though they have not been shown efficacious in PDAC. TGF-β-mediated EMT does play a critical role in PDAC progression in the development of metastases. The use of anti-TGF-β-based therapies in phase I and II clinical trials for metastatic PDAC demonstrate the importance of understanding the role of TGF-β in PDAC progression.. This review clarifies the recent literature investigating the role of anti-TGF-β-based therapy in PDAC and areas ripe for targeted investigations and therapies.

Topics: Antineoplastic Agents; beta Catenin; Carcinoma, Pancreatic Ductal; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic cancer management remains a major challenge to society. Poor prognosis results in dismal patient survival rates and quality of life post chemo/radiation therapies. Although progress has been made in drug development for targeting pancreatic cancer, accompanying issues of drug resistance renders it futile. While intake of fruits and vegetables in routine diets has been linked to reduced risk of pancreatic cancer, a wide variety of natural agents are being evaluated as adjuvant therapies in combination with frontline chemotherapeutics in pancreatic cancer clinical trials.. This review highlights the emerging area of cancer chemoprevention with natural/ dietary compounds serving as novel agents possessing strong anticancer properties; these are pleiotropic agents targeting multiple pathways with minimal toxicity in normal tissue.. We employed extensive literature search and considered all the relevant information presented in a concise, well-balanced and structured format.. Completed and ongoing human studies with natural agents have shown surprisingly successful rates for regulating pancreatic carcinogenesis. Combinatorial therapies with synthetic, approved drugs and natural agents not only improved the patient response rates, but also helped in overcoming drug resistance and inducing chemosensitivity to the resistant tumors, as opposed to monotherapies for pancreatic cancer chemoprevention.. The current review focuses on the available chemotherapeutic drugs and their limitations, and moves on to discuss the wide realm of chemopreventive efficacy that the natural agents have to offer. It discusses the underlying mechanisms of action and available information, from extensive literature analysis, to highlight the novelty of these agents for their antitumor effects against pancreatic cancer.

Topics: Antineoplastic Agents; Chemoprevention; Hedgehog Proteins; Humans; Insulin-Like Growth Factor I; Pancreatic Neoplasms; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Transforming growth factor (TGF)-β regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-β have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-β as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-β, in relation to crosstalk with some other signaling pathways, and the roles of TGF-β in lung and pancreatic cancers, in which TGF-β has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-β signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-β plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-β is produced as latent high molecular weight complexes, and the latent TGF-β complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-β activities through regulation of the latent TGF-β complex activation will be discussed.

Topics: Drug Discovery; Humans; Lung Neoplasms; Membrane Proteins; Pancreatic Neoplasms; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

This article focusses on the role of TGF-β and its signaling crosstalk with the RHO family GTPases RAC1 and RAC1b in the progression of breast and pancreatic carcinoma. The aggressive nature of these tumor types is mainly due to metastatic dissemination. Metastasis is facilitated by desmoplasia, a peculiar tumor microenvironment and the ability of the tumor cells to undergo epithelial-mesenchymal transition (EMT) and to adopt a motile and invasive phenotype. These processes are controlled entirely or in part by TGF-β and the small RHO GTPase RAC1 with both proteins acting as tumor promoters in late-stage cancers. Data from our and other studies point to signaling crosstalk between TGF-β and RAC1 and the related isoform, RAC1b, in pancreatic and mammary carcinoma cells. Based on the exciting observation that RAC1b functions as an endogenous inhibitor of RAC1, we propose a model on how the relative abundance or activity of RAC1 and RAC1b in the tumor cells may determine their responses to TGF-β and, ultimately, the metastatic capacity of the tumor.

Topics: Animals; Breast Neoplasms; Humans; Pancreatic Neoplasms; rac1 GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) regulates cell functions and has key roles in pancreatic cancer development. SMAD4, as one of the Smads family of signal transducer from TGF-β, mediates pancreatic cell proliferation and apoptosis and is specifically inactivated in half of advanced pancreatic cancers. In recent years, many advances concerning SMAD4 had tried to unravel the complex signaling mechanisms of TGF-β and its dual role of tumor-suppressive and tumor-promoting efforts in pancreatic cancer initiation and progression through SMAD4-dependent TGF-β signaling and SMAD4-independent TGF-β signaling pathways. Meanwhile, its potential prognostic value based on immunohistochemical expression in surgical sample was variably reported by several studies and short of a systematic analysis. This review aimed to discuss the structure, functions, and regulation of this principal protein and its effects in determining the progression and prognosis of pancreatic cancer.

Topics: Animals; Epithelial-Mesenchymal Transition; Humans; Pancreatic Neoplasms; Prognosis; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Advanced pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC) are non-curable diseases with a particularly poor prognosis. Over the last decade, research has increasingly focused on the microenvironment surrounding cancer cells, and its role in tumour development and progression. PDAC and HCC differ markedly regarding their pathological features: PDAC are typically stromal-predominant, desmoplastic, poorly vascularized tumours, whereas HCC are cellular and highly vascularized. Despite these very different settings, PDAC and HCC share transforming growth factor-β (TGF-β) as a common key-signalling mediator, involved in epithelial-to-mesenchymal transition, invasion, and stroma-tumour dialogue. Recently, novel drugs blocking the TGF-β pathway have entered clinical evaluation demonstrating activity in patients with advanced PDAC and HCC. TGF-β signalling is complex and mediates both pro- and anti-tumoural activities in cancer cells depending on their context, in space and time, and their microenvironment. In this review we provide a comprehensive overview of the role of the TGF-β pathway and its deregulation in PDAC and HCC development and progression at the cellular and microenvironment levels. We also summarize key preclinical and clinical data on the role of TGF-β as a target for therapeutic intervention in PDAC and HCC, and explore perspectives to optimize TGF-β inhibition therapy.

Topics: Animals; Carcinoma, Hepatocellular; Carcinoma, Pancreatic Ductal; Humans; Liver Neoplasms; Molecular Targeted Therapy; Pancreatic Neoplasms; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) plays a crucial role and takes part in many processes in the human body both in physiology and pathology. This cytokine is involved in angiogenesis, regulates apoptosis and stimulates divisions of cells, such as hepatocytes, lymphocytes or hematopoietic cells. SMAD proteins family is a unique group of particles responsible for transducting the signal induced by TGF-β into the nucleus. This molecules, after receiving a signal from activated TGF-β, act on transcription factors in the nucleus, leading directly to the expression of the corresponding genes. According to current knowledge, disturbances in the functioning of SMAD proteins are present in a number of diseases. The reduced expression was observed, for example in cardiovascular diseases such as primary pulmonary hypertension or myocardial infarction, autoimmune diseases for instance systemic lupus erythematosus and multiple sclerosis, Alzheimer's disease or osteoporosis. The latest clinical data showed the presence of mutations in SMAD proteins in cancerogenesis. Mutation of SMAD-4 protein can be detected in half of the patients with pancreatic cancer, 20% of patients with colorectal cancer and 10% of patients with lung cancer. However, mutation in SMAD-2 protein was observed in 7% of both patients with colorectal cancer and lung cancer. On the basis of numerous works, SMAD protein expression would be valuable prognostic factor in some of neoplastic diseases.

Topics: Apoptosis; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mutation; Neoplasms; Neovascularization, Pathologic; Pancreatic Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Pancreatic cancer constitutes one of the most aggressive tumours with a 5-year survival rate of less than 5%. It is characterized by a high degree of resistance to apoptosis which is associated by high expression levels of multiple prosurvival proteins of the extrinsic and intrinsic apoptosis signalling cascades. This review focuses on current knowledge of apoptotic pathways involved in pancreatic cancer and mechanisms of resistance to apoptosis, including alterations in the death receptor and mitochondrial pathways, as well as anti-apoptotic effects of NF-kB and Akt signalling and the impact of histone-modifying enzymes such as histondeacetylases (HDAC). Furthermore, the therapeutic implications of modulating pro-survival pathways by specific inhibitors investigated in preclinical and clinical trials will be discussed.

Topics: Animals; Apoptosis; Humans; Mitochondria; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Receptors, Death Domain; Signal Transduction; Transforming Growth Factor beta

Pancreatic adenocarcinoma remains among the most lethal of human malignancies. Overall 5-y survival is less than 5%, and only 20% of patients presenting with localized disease amenable to surgical resection. Even in patients who undergo resection, long-term survival remains extremely poor. A major contributor to the aggressiveness of multiple cancers, and pancreatic cancer in particular, is the process of epithelial-to-mesenchymal transition (EMT). This review highlights the growing evidence of EMT in pancreatic cancer progression, focusing on the contribution of EMT to the development of cancer stem cells and on interaction of EMT with other pathways central to cancer progression, such as Hedgehog signaling, the K-ras oncogene, and transforming growth factor-beta (TGF-β). We will also discuss EMT-targeting agents currently in development and in clinical trials that may help to reduce the morbidity and mortality associated with pancreatic cancer.

Topics: Adenocarcinoma; Disease Progression; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; Neoplastic Stem Cells; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

PDAC (pancreatic ductal adenocarcinoma) is among the most deadly of human malignances. A hallmark of the disease is a pronounced collagen-rich fibrotic extracellular matrix known as the desmoplastic reaction. Intriguingly, it is precisely these areas of fibrosis in which human PDAC tumours demonstrate increased expression of a key collagenase, MT1-MMP [membrane-type 1 MMP (matrix metalloproteinase); also known as MMP-14]. Furthermore, a cytokine known to mediate fibrosis in vivo, TGF-β1 (transforming growth factor-β1), is up-regulated in human PDAC tumours and can promote MT1-MMP expression. In the present review, we examine the regulation of PDAC progression through the interplay between type I collagen (the most common extracellular matrix present in human PDAC tumours), MT1-MMP and TGF-β1. Specifically, we examine the way in which signalling events through these pathways mediates invasion, regulates microRNAs and contributes to chemoresistance.

Topics: Carcinoma, Pancreatic Ductal; Collagen Type I; Disease Progression; Drug Resistance, Neoplasm; Fibrosis; Humans; Matrix Metalloproteinase 14; MicroRNAs; Myofibroblasts; Pancreatic Neoplasms; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

TGFβ signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies.

Topics: Animals; Carcinoma, Squamous Cell; Cholangiocarcinoma; Colonic Neoplasms; Disease Progression; Fanconi Anemia; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Intestinal Polyposis; Male; Mice; Models, Biological; Neoplastic Syndromes, Hereditary; Pancreatic Neoplasms; Prostatic Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Pancreatic adenocarcinoma is one of the most aggressive human cancers. It displays many different chromosomal abnormalities and mutations. To design new therapeutic strategies, it is important to identify the signaling pathways and gene networks within this apparent complexity that are predominantly altered. The TGFβ signaling pathway and associated transcription network emerges as a central actor of pancreatic oncogenesis. Its tumor suppressor function in this tissue can be affected by several alterations.

Topics: DNA-Binding Proteins; GATA6 Transcription Factor; Humans; Models, Biological; Nuclear Proteins; Pancreatic Neoplasms; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Pancreatic cancer is an aggressive malignancy with poor prognosis. Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths in the United States. Due to the aggressive nature of this malignancy, there is a serious concern for identifying effective targets, and adopting novel strategies for therapy. Members of the Specificity Protein (Sp) family of transcription factors, Sp1, Sp3, and Sp4 regulate the expression of a number of genes associated with cancer cell proliferation, differentiation, and metastasis. Sp1 levels are upregulated in pancreatic cancer cell lines, and surgically resected human pancreatic adenocarcinoma. Sp1 overexpression in tumor tissues is associated with aggressive disease, poor prognosis and inversely correlated with survival. Sp1 is also known to affect angiogenesis by regulating the expression of vascular endothelial growth factor and its receptors. Results from clinical studies suggest Sp1 as new biomarker to identify aggressive pancreatic ductal adenocarcinoma. The pharmacological inhibition of Sp1 using agents such as celecoxib, mithramycin, curcumin, and tolfenamic acid has showed promising results in pre-clinical studies and demonstrated Sp transcription factors as potential targets for pancreatic cancer therapy. This review summarizes studies showing the association of Sp proteins with this malignancy, with a special emphasis on pre-clinical studies that tested strategies to target Sp transcription factors for inhibiting human pancreatic cancer cell proliferation and tumor growth in laboratory animals. The results showed remarkable efficacy and suggest that such approaches have the potential for high success in developing clinically relevant strategies for treating pancreatic cancer.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Endoplasmic Reticulum Stress; Epithelial-Mesenchymal Transition; Humans; Keratin-19; Mucins; Neovascularization, Pathologic; Pancreatic Neoplasms; Prognosis; Sp Transcription Factors; Transforming Growth Factor beta

Pancreatic cancer has high incidence and mortality rates, and effective treatment remains a clinical challenge. As deregulation of the cytokine transforming growth factor beta (TGF-β) contributes to the progression of pancreatic carcinoma, the TGF-β pathway has been targeted using various strategies, including small molecule inhibitors of TGF-βRI, TGF-β-specific neutralizing antibodies and antisense compounds. As increased TGF-β2 levels in serum or tumor tissue of patients with pancreatic cancer correlated with poor prognosis, inhibition of TGF-β2 synthesis via the antisense oligonucleotide trabedersen (AP 12009) is a promising approach.

Topics: Animals; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms; Transforming Growth Factor beta

Pancreatic cancer treatment remains a challenge for clinicians and researchers. Despite undisputable advances in the comprehension of the molecular mechanisms underlying cancer development and progression, early disease detection and clinical management of patients has made little, if any, progress in the past 20 years. Clinical development of targeted agents directed against validated pathways, such as the EGF/EGF receptor axis, the mutant KRAS protein, MMPs, and VEGF-mediated angiogenesis, alone or in combination with gemcitabine-based standard chemotherapy, has been disappointing.. This review explores the preclinical rationale for clinical approaches aimed at targeting the TGF-β, IGF, Hedgehog, Notch and NF-κB signaling pathways, to develop innovative therapeutic strategies for pancreatic cancer.. Although some of the already clinically explored approaches (particularly EGFR and KRAS targeting) deserve further clinical consideration, by employing more innovative and creative clinical trial designs than the gemcitabine-targeted agent paradigm that has thus far invariably failed, the targeting of emerging and relatively unexplored signaling pathways holds great promise to increase our understanding of the complex molecular biology and to advance the clinical management of pancreatic cancer.

Topics: Hedgehog Proteins; Humans; NF-kappa B; Pancreatic Neoplasms; Receptors, Notch; Signal Transduction; Somatomedins; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by a plethora of molecular alterations that include major and minor driving mutations, the presence of intense desmoplasia exhibiting numerous proliferating pancreatic stellate cells (PSC) and cancer-associated fibroblasts that produce fibronectin and collagens, and foci of inflammatory cells that produce mitogenic cytokines. This review will focus on signaling by tyrosine kinase receptors, and the role of transforming growth factor beta in this malignancy is described briefly. Potential for therapeutic interventions will be discussed in relation to specific pathways.

Topics: Animals; Carcinoma, Pancreatic Ductal; Disease Models, Animal; ErbB Receptors; Humans; Pancreatic Neoplasms; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; Receptors, Somatomedin; Signal Transduction; Transforming Growth Factor beta

Inflammation is a primary defense process against various extracellular stimuli, such as viruses, pathogens, foods, and environmental pollutants. When cells respond to stimuli for short periods of time, it results in acute or physiological inflammation. However, if the stimulation is sustained for longer time or a pathological state occurs, it is known as chronic or pathological inflammation. Several studies have shown that tumorigenesis in the gastrointestinal (GI) tract is closely associated with chronic inflammation, for which abnormal cellular alterations that accompany chronic inflammation such as oxidative stresses, gene mutations, epigenetic changes, and inflammatory cytokines, are shared with carcinogenic processes, which forms a critical cross-link between chronic inflammation and carcinogenesis. Transforming growth factor (TGF)-beta is a multi-potent cytokine that plays an important role in regulation of cell growth, apoptosis and differentiation. Most importantly, TGF-beta is a strong anti-inflammatory cytokine that regulates the development of effector cells. TGF-beta has a suppressive effect on carcinogenesis under normal conditions by inhibiting abnormal cell growth, but on the other hand, many GI cancers originate from uncontrolled cell growth and differentiation by genetic loss of TGF-beta signaling molecules or perturbation of TGF-beta adaptors. Once a tumor has developed, TGF-beta exerts a promoting effect on the tumor itself and stromal cells to enhance cell growth, alter the responsiveness of tumor cells to stimulate invasion and metastasis, and inhibited immune surveillance. Therefore, novel development of therapeutic agents to inhibit TGF-beta-induced progression of tumor and to retain its growth inhibitory activities, in addition to anti-inflammatory actions, could be useful in oncology. In this review, we discuss the role of TGF-beta in inflammation and carcinogenesis of the GI tract related to abnormal TGF-beta signaling.

Topics: Animals; Colorectal Neoplasms; Esophageal Neoplasms; Esophagitis, Peptic; Gastritis, Atrophic; Gastroenteritis; Gastrointestinal Neoplasms; Homeostasis; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Ligands; Pancreatic Neoplasms; Pancreatitis, Chronic; Signal Transduction; Stomach Neoplasms; Transforming Growth Factor beta

Epithelial to mesenchymal transition (EMT) is a physiologic process that allows morphological and genetic changes of carcinoma cells from an epithelial to a mesenchymal phenotype, which is the basis of the high metastatic potential of pancreatic cancer cells. EMT is triggered by various tumor microenvironmental factors, including cytokines, growth factors, and chemotherapeutic agents. This review summarizes the state-of-the-art knowledge on the molecular mechanisms that support pancreatic cancer EMT and the evidences that support its involvement in invasiveness/ aggressiveness, and the drug resistance of pancreatic cancer cells.

Topics: Adenocarcinoma; Animals; Epithelial Cells; Humans; Mesoderm; MicroRNAs; Models, Biological; Pancreatic Neoplasms; Smad Proteins; Transforming Growth Factor beta

The fact that pancreatic ductal adenocarcinoma (PDAC) is still an exceptionally lethal disease with an annual mortality almost equivalent to its annual incidence has stimulated intense research efforts directed at understanding the underlying molecular mechanisms. By enabling simultaneous expression analysis of thousands of genes, microarray technology has significantly contributed to illuminating the pathophysiology of PDAC. Gene expression profiling studies have been performed for molecular classification of clinically relevant tumor subtypes and have shed light on various signaling pathways associated with tumor progression. Altered expression levels of several genes have been identified as correlating with functional in vitro data as well as patient survival, indicating the potential clinical value of transcriptional profiling. However, broad clinical use of array techniques for patient characterization has been hampered by their cost intensity and by limited inter-study comparability. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), as the most sensitive technique for mRNA detection and quantification, will complement arrays for the confirmation of individual transcripts in larger sample cohorts. This review highlights recent studies that addressed gene expression analysis with both methodologies and that identified components of the TGF-beta signaling pathway, BNIP3, or periostin to be differentially expressed in PDAC. These studies demonstrated that the combination of microarray and RT-PCR technologies is a highly efficient and reliable approach for the identification of clinically important diagnostic and prognostic biomarkers, as well as for the discovery of novel therapeutic target candidates.

Topics: Carcinoma, Pancreatic Ductal; Cell Adhesion Molecules; Drug Resistance, Neoplasm; Gene Expression Profiling; Humans; Membrane Proteins; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Topics: Extracellular Matrix; Fibrosis; Humans; Models, Biological; Pancreas; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wound Healing

Pancreatic cancer is a lethal disease with a 5-year survival rate of 4%. The only opportunity for improved survival continues to be complete surgical resection for those with localized disease. Although chemotherapeutic options are limited for the few patients with resectable disease, this problem is even more magnified in the majority (85%) of patients with unresectable or metastastic disease. Therefore, there is an urgent need for improved therapeutic options. The recent success of inhibitors of signal transduction for the treatment of other cancers supports the need to identify and validate aberrant signaling pathways important for pancreatic tumor growth. This review focuses on the validation of specific signaling networks and the present status of inhibitors of these pathways as therapeutic approaches for pancreatic cancer treatment.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Disease Progression; Drug Delivery Systems; Drug Design; ErbB Receptors; Forecasting; Genes, ras; Hedgehog Proteins; Humans; Immunotherapy; MAP Kinase Signaling System; Neoplasm Proteins; NF-kappa B; Oncogene Protein p21(ras); Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-vav; Proto-Oncogene Proteins p21(ras); Receptors, Notch; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Pancreatic cancer is the 4th leading cause of cancer-related death in the United States. The number of diagnoses per year equals the number of deaths per year, making it the deadliest of all malignancies. Modern advances and breakthroughs in molecular oncology have allowed researchers to gain a better understanding of the mechanisms responsible for the pathogenesis of this disease. The transforming growth factor-beta (TGF-beta) pathway is one of the signaling systems that has been identified as a major contributor. TGF-beta plays a paradoxical role as both a tumor suppressor and a tumor promoter in pancreatic cancer. The purpose of this review is to provide the practicing clinician a thorough review of this molecule and its associated signaling partners in the context of its duplicitous role and behavior in patients with pancreatic cancer.

Topics: Animals; Carcinogens; Cell Cycle; Humans; Latent TGF-beta Binding Proteins; Pancreas; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Suppressor Proteins

Transforming growth factor beta (TGF-beta) is a dominant mediator of pancreatic fibrosis. The objective of this study was to identify cellular sources of TGF-beta mRNA and compare the results with previous immunohistochemical/in situ hybridization studies.. In situ hybridization of TGF-beta was conducted for 9 human tissues of chronic obstructive pancreatitis (COP) and 2 control specimens. By classifying these 9 COP tissues into 3 fibrosis phases by the amount of fibrotic space, histopathologic changes were examined for each fibrosis phase. Whether or not TGF-beta-positive cells were closely distributed to fibrosis was also investigated in control and COP cases.. Three cases were categorized in early, intermediate, and advanced stages of fibrosis. Transforming growth factor beta mRNA was identified for a part of small duct epithelia, that is, intercalated ductule cells, centroacinar cells, and/or metaplastic ductal structures adjacent to acinar cells. The number of TGF-beta-positive cells was greater in COP cases than in controls. In controls and in the early stage of fibrosis, no fibrosis was seen near TGF-beta-positive cells.. Small duct epithelia are the main cellular sources of TGF-beta in COP, and many of them may be working for COP fibrosis either directly or indirectly.

Topics: Aged; Aged, 80 and over; Disease Progression; Epithelial Cells; Female; Fibrosis; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Pancreas, Exocrine; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis, Chronic; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta (TGFbeta) functions as a growth inhibitor for many cell types by inhibiting cell cycle progression. Loss of TGFbeta responsiveness can lead to deregulated cell proliferation and ultimately tumor progression. For example, the TGFbeta signaling pathway is a frequent target for inactivation in pancreatic cancer. Functional connection between the potent growth inhibitory activity of TGFbeta and the tumor suppressor activity of Smads has been well documented. Smads directly modulate transcription of the genes involved in cell cycle progression in response to TGFbeta, and that abrogation of this regulation leads to tumor progression. In this review, we summarize recent research progress on TGFbeta signaling and pancreatic cancer.

Topics: Cell Proliferation; DNA-Binding Proteins; Humans; Pancreatic Neoplasms; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Regulatory T-cells (Treg) protect the host from autoimmune disease by suppressing self-reactive immune cells. As such, Treg may also block antitumor immune responses. Recent observations by us and others showed that the prevalence of Treg is increased in cancer patients, particularly in the tumor environment. Our studies in a mouse pancreas cancer model suggest that the tumor actively promotes the accrual of Treg through several mechanisms involving activation of naturally occurring Treg as well as conversion of non-Treg into Treg. Our studies focus on further defining these mechanisms with the ultimate goal of designing strategies that block Treg-mediated suppression in cancer patients.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cancer Vaccines; Carcinoma, Ductal, Breast; CD4 Antigens; Dendritic Cells; Female; Forkhead Transcription Factors; Humans; Immune Tolerance; Lymphocyte Depletion; Mice; Mice, Knockout; Neoplasms; Pancreatic Neoplasms; Phenotype; Receptors, Interleukin-2; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Pancreatic ductal adenocarcinomas continue to have the worst prognosis of any adult malignancy with a five-year survival rate of less than 4%. One approach to improve patient survival from pancreatic cancer is to identify new biological targets that contribute to the aggressive pathogenecity of this disease and to develop reagents that will interfere with the function of these targets. Apart from the identification of the genetic profile of pancreatic cancer, a number of studies have focused on aberrant cell signaling pathways and their role in pancreatic cancer biology and response to therapy. This review, although not comprehensive, will discuss the salient features of several of these pathways. These include the roles of TGF beta signaling in both tumor suppression and tumor promotion and the effects of deregulation of phosphotyrosine kinase receptor signaling pathways in pancreatic cancer.

Topics: Adult; Carcinoma, Pancreatic Ductal; Humans; Pancreatic Neoplasms; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

TGF-beta is a multifunctional cytokine which regulates cell growth, extracellular matrix deposition, cell differentiation, and immunosuppression etc. The signal is mainly mediated through Smad pathway to inhibit epithelial cell growth. In 50% of pancreatic cancer, Smad4/DPC4 gene is deleted or mutated, which might cause pancreatic carcinogenesis and be associated with highly invasive and metastatic character of the disease. On the other hand, TGF-beta signal itself has recently been shown to act in favor of cancer cells, especially in the late phase of tumor progression. In Smad4-inactivated pancreatic cancer cells, TGF-beta signal regulates a number of genes involved in tumor suppression and progression. The regulated genes and signaling pathways of TGF-beta signal should be investigated to obtain an effective therapeutic target molecule for pancreatic cancer.

Topics: Animals; Mice; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

In recent years, numerous studies have provided novel insights into the pathomechanisms of pancreatic fibrogenesis. This includes in particular the identification and characterization of the pancreatic stellate cells (PSCs) and their role in the synthesis of extracellular matrix (ECM) proteins. It has become clear that pancreatic stellate cell activation is regulated by a complex network of growth factors and cytokines and results in increased expression and release of collagens I and II, fibronectin and other components of ECM. Among the cytokines involved in PSC activation and other fundamental mechanisms of pancreatic fibrosis, transforming growth factor beta (TGFbeta) is of particular relevance. TGFbeta stimulates PSC activation and induces transcription of ECM proteins mainly via activation of the Smad proteins which regulate gene expression through functional interaction with co-operating partner proteins such as the zinc finger transcription factor Sp1. Recent progress in understanding of the biochemical and molecular mechanisms of pancreatic fibrosis, is reviewed here.

Topics: DNA-Binding Proteins; Extracellular Matrix Proteins; Fibrosis; Humans; Neoplasm Proteins; Pancreas; Pancreatic Diseases; Pancreatic Neoplasms; Pancreatitis; Repressor Proteins; Smad Proteins; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Pancreatic cancer ranks fifth as a cause of cancer-related death in the world with an overall 5-year survival rate of less than 1% and a median survival of less than a year after tumour detection. Most of these patients have already metastases at the time of diagnosis. The oncologic strategies such as chemotherapy, radiotherapy, antihormonal modalities or the systemic use of specific monoclonal antibodies have not achieved a significant improvement in the survival of pancreatic cancer patients. Recent studies suggest that alterations in molecular pathways, particularly in growth factor mediated mechanisms, that regulate cell proliferation and differentiation play a pivotal role in the pathogenesis of this cancer. The molecular knowledge regarding changes in the expression of growth factors in pancreatic cancer has the potential to improve diagnostic and therapeutic treatment strategies in the near future.

Topics: Animals; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Nerve Growth Factor; Pancreatic Neoplasms; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The biological cause of fibrosis is the accumulation of excessive amounts of extracellular matrix (ECM) which leads to tissue dysfunction and organ failure. A strong correlation can be found between pancreatic diseases and fibrotic processes, in particular chronic pancreatitis and pancreatic cancer. There is growing evidence that pancreatic fibrosis represents a dysregulation of the normal repair processes after injury. This concept is based on the findings that fibrosis and tissue repair involve similar biological reactions regulated by the same group of molecules. The best characterized example for these regulatory molecules are the members of the transforming growth factor beta family (TGFbeta). TGFbeta1 represents the prototype of this family of highly similar growth factors, with the unique ability to stimulate the expression and deposition of extracellular matrix and to inhibit its degradation. Growth factor-induced fibrotic events are targeted by a myofibroblast-like cell called pancreatic stellate cell (PSC). These cells show enhanced expression of all-important ECM proteins after TGFbeta stimulation including collagen, fibronectin and proteoglycans. At the same time TGFbeta inhibits the degradation of ECM by blocking the secretion of proteases and stimulating the production of naturally occurring protease inhibitors.

Topics: Collagen; Endopeptidases; Extracellular Matrix; Fibrosis; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state.. To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another.. Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-beta, a cell-transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic anti-leukemic drugs such as STI571 (Gleevec). Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high-efficacy nongenetic novel targets for cancer therapies.

Topics: Apoptosis; Carbon Radioisotopes; Cell Cycle; Cell Differentiation; Cell Division; Citric Acid Cycle; Deuterium; Enzyme Inhibitors; Genistein; Glucose; Glycolysis; Humans; Isotopes; Mass Spectrometry; Pancreatic Neoplasms; Phenotype; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Endocrine tumors are characteristically hypervascularized. This property recalls that of normal endocrine tissues, which possess a dense and specialized capillary network. The cellular and molecular mechanisms of the angiogenesis process associated with endocrine tumorigenesis are poorly known. Most normal endocrine cells constituvely express high levels of angiogenic factors, such as VEGF, which likely play an important role in the development of the characteristic vascular architecture of normal endocrine tissues. Clinical and experimental data suggest that a surexpression of such angiogenic factors is unlikely to be involved in the induction of the angiogenic process associated with endocrine tumorigenesis. In contrast, according to some experimental observations, the loss of endocrine-specific anti-angiogenic factors may be required for the initiation of the angiogenic process and the transition from endocrine hyperplasia to endocrine neoplasia. Such inhibitory factors remain to be identified and characterized. A better understanding of the mechanisms of angiogenesis in endocrine tumors is important for the delineation of novel therapeutic strategies.

Topics: Animals; Capillaries; Cell Division; Endocrine Gland Neoplasms; Endocrine System; Endothelial Growth Factors; Endothelium, Vascular; Humans; Lymphokines; Mice; Mice, Transgenic; Neovascularization, Pathologic; Pancreatic Neoplasms; Transforming Growth Factor alpha; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Pancreatic cancer has one of the worst prognoses of all human malignancies and the molecular mechanisms underlying this aggressive disease have been extensively investigated in the past years. Tyrosine kinase growth factor receptors and their ligands act to influence tumor cell growth, differentiation, invasion, metastasis, and angiogenesis. In pancreatic cancer a variety of these growth factor receptors and ligands are expressed at increased levels and this overexpression influences the clinical course of the disease. For example, the concomitant presence of the EGF receptor and its ligands EGF, TGF-alpha, and/or amphiregulin is associated with enhanced tumor aggressiveness and shorter survival periods following tumor resection. Furthermore, the growth inhibitory effects of the TGF-beta superfamily of serine-threonine kinase receptors and their ligands are often blocked in pancreatic cancer cells. In addition to these alterations, mutations of the p53 tumor-suppressor gene, the K-ras proto-oncogene, and the Smad4 gene are frequently present in these tumors. Taken together, the abundance of growth-promoting factors, the disturbance of growth inhibitory pathways, and the presence of gene mutations combine to give pancreatic cancer cells a distinct growth advantage which clinically results in rapid tumor progression and poor survival.

Topics: ErbB Receptors; Genetic Therapy; Humans; Mutation; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; ras Proteins; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Human pancreatic cancers overexpress a number of important tyrosine growth factor receptors and their ligands. These include the epidermal growth factor (EGF) receptor (EGFR) and related receptors, multiple ligands that bind to EGFR, certain fibroblast growth factors (FGF) receptors (FGFR) and ligands, and insulin-like growth factor I (IGF-I) and its receptor. The excessive activation of mitogenic signaling cascades that are modulated by these overexpressed ligands and receptors is compounded by the presence of mutations in the K-ras oncogene. Pancreatic cancers also overexpress transforming growth factor betas (TGF-betas) that usually inhibit the growth of epithelial cells. Pancreatic cancers, however, underexpress the type I TGF-beta receptor and harbor mutations in the smad4 gene, alterations that prevent TGF-betas from inhibiting cancer cell growth but that do not confer onto pancreatic actions that promote cancer growth in vivo. Together, these perturbations confer onto pancreatic cancer cells a tremendous growth advantage.

Topics: Animals; ErbB Receptors; Fibroblast Growth Factors; Growth Substances; Humans; Pancreatic Neoplasms; Receptors, Growth Factor; Somatomedins; Transforming Growth Factor beta

Circulating transforming growth factor-β (TGF-β)-specific T cells that recognize TGF-β-expressing immune regulatory cells have been described in patients with cancer. TGF-β-derived peptide vaccination modulates the tumor microenvironment and has shown clinical effects in animal models of pancreatic cancer (PC). TGF-β-expressing regulatory cells are especially elevated in PC and may prevent the clinical response to immune checkpoint inhibitors (ICIs). Thus, in the present study we investigated the significance of TGF-β-specific T-cell immunity in patients with PC treated with ICI combined with radiotherapy in a randomized phase 2 study (CheckPAC).. Immune responses to a TGF-β-derived epitope entitled TGF-β-15 as well as epitopes from. PBMCs from 32 patients were analyzed for immune responses to the TGF-β-derived epitope entitled TGF-β-15. Patients with a strong TGF-β-specific immune response at treatment initiation had longer progression-free and overall survival, compared with patients with a weak or no TGF-β-specific immune response. This remained significant in multivariate analysis. Patients with weak and strong TGF-β-specific responses displayed similar responses towards viral antigens. Furthermore, we show that TGF-β-specific T cells from a clinical responder specifically reacted to and lysed autologous, regulatory immune cells. Finally, mimicking a TGF-β-15 vaccination, we showed that repeated stimulations with the TGF-β-15 epitope

Topics: Cancer Vaccines; Epitopes; Humans; Immune Checkpoint Inhibitors; Immunity, Cellular; Leukocytes, Mononuclear; Pancreatic Neoplasms; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment; Vaccines, Subunit

Galunisertib is the first-in-class, first-in-human, oral small-molecule type I transforming growth factor-beta receptor (ALK5) serine/threonine kinase inhibitor to enter clinical development. The effect of galunisertib vs. placebo in patients with unresectable pancreatic cancer was determined.. This was a two-part, multinational study: phase 1b was a non-randomised, open-label, multicentre, and dose-escalation study; phase 2 was a randomised, placebo- and Bayesian-augmented controlled, double-blind study in patients with locally advanced or metastatic pancreatic adenocarcinoma considered candidates for first-line chemotherapy with gemcitabine. Patients were randomised 2:1 to galunisertib-gemcitabine (N = 104) or placebo-gemcitabine (N = 52). Gemcitabine dose was 1000 mg/m. Dose-escalation suggested a 300-mg/day dose. Primary objective was met: median survival times were 8.9 and 7.1 months for galunisertib and placebo, respectively (hazard ratio [HR] = 0.79 [95% credible interval: 0.59-1.09] and posterior probability HR < 1 = 0.93). Lower baseline biomarkers macrophage inflammatory protein-1-alpha and interferon-gamma-induced protein 10 were associated with galunisertib benefit.. Galunisertib-gemcitabine combination improved overall survival vs. gemcitabine in patients with unresectable pancreatic cancer, with minimal added toxicity. Future exploration of galunisertib in pancreatic cancer is ongoing in combination with durvalumab.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Deoxycytidine; Dose-Response Relationship, Drug; Female; Gemcitabine; Humans; Male; Middle Aged; Pancreatic Neoplasms; Placebos; Pyrazoles; Quinolines; Signal Transduction; Transforming Growth Factor beta

Epithelium-specific ETS transcription factor 1 (ESE1) has been implicated in epithelial homeostasis, inflammation, as well as tumorigenesis, and cancer progression. However, numerous studies have reported contradictory roles-as an oncogene or a tumor suppressor of ESE1 in different cancers, and its function in the development and progression of pancreatic ductal adenocarcinoma (PDAC) has remained largely unexplored. Herein, we report that ESE1 was found upregulated in primary PDAC compared to normal pancreatic tissue, but high expression of ESE1 correlated to better relapse-free survival in patients with PDAC. Interestingly, ESE1 was found to exhibit dual roles in regulation of malignant properties of PDAC cells in that its overexpression promoted cell proliferation, whereas its downregulation enhanced epithelial-mesenchymal transition (EMT) phenotype. In the context of TGF-β-induced EMT, ESE1 is markedly downregulated at post-transcriptional level, and reconstituted ESE1 expression partially reversed TGF-β-induced EMT marker expression. Furthermore, we identify AGR2 as a novel transcriptional target of ESE1 that participates in TGF-β-induced EMT in PDAC. Collectively, our findings reveal an ESE1/AGR2 axis that interacts with TGF-β signaling to modulate EMT phenotype in PDAC.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mucoproteins; Neoplasm Recurrence, Local; Oncogene Proteins; Pancreatic Neoplasms; Transforming Growth Factor beta

Pancreatic cancer (PaCa) is one of the most fatal malignancies of the digestive system, and most patients are diagnosed at advanced stages due to the lack of specific and effective tumor-related biomarkers for the early detection of PaCa. miR-492 has been found to be upregulated in PaCa tumor tissue and may serve as a potential therapeutic target. However, the molecular mechanisms by which miR-492 promotes PaCa tumor growth and progression are unclear. In this study, we first found that miR-492 in enhancer loci activated neighboring genes (NR2C1/NDUFA12/TMCC3) and promoted PaCa cell proliferation, migration, and invasion in vitro. We also observed that miR-492-activating genes significantly enriched the TGF-β/Smad3 signaling pathway in PaCa to promote epithelial-mesenchymal transition (EMT) during tumorigenesis and development. Using CRISPR-Cas9 and ChIP assays, we further observed that miR-492 acted as an enhancer trigger, and that antagomiR-492 repressed PaCa tumorigenesis in vivo, decreased the expression levels of serum TGF-β, and suppressed the EMT process by downregulating the expression of NR2C1. Our results demonstrate that miR-492, as an enhancer trigger, facilitates PaCa progression via the NR2C1-TGF-β/Smad3 pathway.

Topics: Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; NADPH Dehydrogenase; Pancreatic Neoplasms; Smad3 Protein; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with high mortality and recurrence rate. In this study, we generated a human immune system mouse model by transplanting human peripheral blood mononuclear cells into NSG-B2m mice followed by xenografting AsPC-1 cells, after which we assessed the role of transforming growth factor-β2 (TGF-β2) in T-cell-mediated anti-tumor immunity. We observed that inhibiting the TGF-β2 production by TGF-β2 antisense oligonucleotide (TASO) combined with IL-2 delays pancreatic cancer growth. Co-treatment of TASO and IL-2 had little effect on the SMAD-dependent pathway, but significantly inhibited the Akt phosphorylation and sequentially activated GSK-3β. Activation of GSK-3β by TASO subsequently suppressed β-catenin and α-SMA expression and resulted in attenuated fibrotic reactions, facilitating the infiltration of CD8 + cytotoxic T lymphocytes (CTLs) into the tumor. TGF-β2 inhibition suppressed the Foxp3 + regulatory T-cells in peripheral blood and tumors, thereby enhancing the tumoricidal effects of CTLs associated with increased granzyme B and cleaved caspase-3. Moreover, changes in the T-cell composition in peripheral blood and at the tumor site by TASO and IL-2 induced the increase of pro-inflammatory cytokines such as IFN-γ and TNF-α and the decrease of anti-inflammatory cytokines such as TGF-βs. These results indicate that the TGF-β2 inhibition by TASO combined with IL-2 enhances the T-cell mediated anti-tumor immunity against SMAD4-mutated PDAC by modulating the tumor-associated fibrosis, suggesting that TASO in combination with IL-2 may be a promising immunotherapeutic intervention for PDAC.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cytokines; Glycogen Synthase Kinase 3 beta; Humans; Interleukin-2; Leukocytes, Mononuclear; Mice; Oligonucleotides, Antisense; Pancreatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta2

The absence of T cells in the tumor microenvironment of solid tumors is a major barrier to cancer immunotherapy efficacy. Oncolytic viruses, including reovirus type 3 Dearing (Reo), can recruit CD8. Blockade of the pleiotropic molecule TGF-β can both improve and impair the efficacy of viro-immunotherapy, depending on the tumor model. While TGF-β blockade antagonized Reo&CD3-bsAb combination therapy in the KPC3 model for pancreatic cancer, it resulted in 100% complete responses in the MC38 colon model. Understanding factors underlying this contrast is required to guide therapeutic application.

Topics: Animals; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Immunotherapy; Mice; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) exhibits a pronounced extracellular matrix (ECM)-rich response, which is produced by an excessive amount of transforming growth factor β (TGF-β), resulting in tumor progression and metastasis. In addition, TGF-β signaling contributes to rapidly acquired resistance and incomplete response to gemcitabine. Recently, selective inhibitors of the TGF-β signaling pathway have shown promise in PDAC treatment, particularly as an option for augmenting responses to chemotherapy. Here, we investigated the synergistic anticancer effects of a small-molecule TGF-β receptor I kinase inhibitor (vactosertib/EW-7197) in the presence of gemcitabine, and its mechanism of action in pancreatic cancer. Vactosertib sensitized pancreatic cancer cells to gemcitabine by synergistically inhibiting their viability. Importantly, the combination of vactosertib and gemcitabine significantly attenuated the expression of major ECM components, including collagens, fibronectin, and α-SMA, in pancreatic cancer compared with gemcitabine alone. This resulted in potent induction of mitochondrial-mediated apoptosis, gemcitabine-mediated cytotoxicity, and inhibition of tumor ECM by vactosertib. Additionally, the combination decreased metastasis through inhibition of migration and invasion, and exhibited synergistic anti-cancer activity by inhibiting the TGF-β/Smad2 pathway in pancreatic cancer cells. Furthermore, co-treatment significantly suppressed tumor growth in orthotopic models. Therefore, our findings demonstrate that vactosertib synergistically increased the antitumor activity of gemcitabine via inhibition of ECM component production by inhibiting the TGF-β/Smad2 signaling pathway. This suggests that the combination of vactosertib and gemcitabine may be a potential treatment option for patients with pancreatic cancer.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Deoxycytidine; Gemcitabine; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-β to the activin A pathway. We found that TGF-β upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

Topics: Activins; Animals; Cell Line, Tumor; Cell Plasticity; Humans; Mice; MicroRNAs; Pancreatic Neoplasms; Transforming Growth Factor beta

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Ethanol; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Intrinsic drug resistance mechanisms of tumor cells often reduce intracellular drug concentration to suboptimal levels. Epithelial-to-mesenchymal transition (EMT) is a pivotal process in tumor progression and metastasis that confers an aggressive phenotype as well as resistance to chemotherapeutics. Therefore, it is imperative to develop novel strategies and identify new targets to improve the overall efficacy of cancer treatment. We developed SN38 (active metabolite of irinotecan)-assembled glycol chitosan nanoparticles (cSN38) for the treatment of pancreatic ductal adenocarcinoma (PDAC). Furthermore, cSN38 and the TGF-β1 inhibitor LY364947 formed composite nanoparticles upon self-assembly (cSN38 + LY), which obviated the poor aqueous solubility of LY364947 and enhanced drug sensitivity. The therapeutic efficacy of cSN38 + LY nanotherapeutics was studied in vitro and in vivo using suitable models. The cSN38 nanoparticles exhibited an antitumor effect that was significantly attenuated by TGF-β-induced EMT. The cellular uptake of SN38 was impeded during EMT, which affected the therapeutic efficacy. The combination of LY364947 and cSN38 markedly enhanced the cellular uptake of SN38, increased cytotoxic effects, and inhibited EMT in PDAC cells in vitro. Furthermore, cSN38 + LY significantly inhibited PDAC xenograft growth in vivo. The cSN38 + LY nanoparticles increased the therapeutic efficacy of cSN38 via repressing the EMT of PDAC cells. Our findings provide a rationale for designing nanoscale therapeutics to combat PDAC.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Transforming growth factor-b (TGFb) plays pleiotropic roles in pancreatic cancer, including promoting metastasis, attenuating CD8 T-cell activation, and enhancing myofibroblast differentiation and deposition of extracellular matrix. However, single-agent TGFb inhibition has shown limited efficacy against pancreatic cancer in mice or humans.. We evaluated the TGFβ-blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)-paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment.. Blockade of TGFβ with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFβ blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2. TGFβ regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFβ blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens.

Topics: Albumins; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Gemcitabine; Humans; Mice; Paclitaxel; Pancreatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Microenvironment

Pancreatic cancer (PC) ranked fourth among cancer-related death worldwide with a survival rate less than 5%. The abnormal proliferation and distant metastasis are major obstacles for the diagnosis and treatment of pancreatic cancer, therefore, it is urgent for researchers to uncover the molecular mechanisms underlying the PC proliferation and metastasis. In current study, we found that USP33, a member of deubiquitinating enzyme family, was upregulated among PC samples and cells, meanwhile, the high expression of USP33 correlated with poor prognosis of patients. Function experiments revealed that USP33 overexpression promoted the proliferation, migration and invasion of PC cells while the inhibition of USP33 expression in PC cells exhibited the opposite effect. The mass spectrum and luciferase complementation assay screened TGFBR2 as the potential binding protein of USP33. Mechanistically, USP33 triggered the deubiquitination of TGFBR2 and prevented its degradation by lysosome, therefore promoted TGFBR2 accumulation in cell membrane and eventually contributed to the sustained activation of TGF-β signaling. Moreover, our results revealed that the activation of TGF-β targeted gene ZEB1 promoted the transcription of USP33. In conclusion, our study found that USP33 contributed to the proliferation and metastasis of pancreatic cancer through a positive feedback loop with TGF-β signaling pathway. Moreover, this study suggested that USP33 may serve as a potential prognostic and therapeutic target in PC.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Phenotype; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Transforming Growth Factor beta; Ubiquitin Thiolesterase

Topics: Animals; Cachexia; Early Growth Response Transcription Factors; Humans; Kruppel-Like Transcription Factors; Mice; Muscle, Skeletal; Muscular Atrophy; Pancreatic Neoplasms; Transforming Growth Factor beta

Among digestive tract tumours, pancreatic ductal adenocarcinoma (PDAC) shows the highest mortality trend. Moreover, although PDAC metastasis remains a leading cause of cancer-related deaths, the biological mechanism is poorly understood. Recent evidence demonstrates that circular RNAs (circRNAs) play important roles in PDAC progression.. Differentially expressed circRNAs in normal and PDAC tissues were screened via bioinformatics analysis. Sanger sequencing, RNase R and actinomycin D assays were performed to confirm the loop structure of circEIF3I. In vitro and in vivo functional experiments were conducted to assess the role of circEIF3I in PDAC. MS2-tagged RNA affinity purification, mass spectrometry, RNA immunoprecipitation, RNA pull-down assay, fluorescence in situ hybridization, immunofluorescence and RNA-protein interaction simulation and analysis were performed to identify circEIF3I-interacting proteins. The effects of circEIF3I on the interactions of SMAD3 with TGFβRI or AP2A1 were measured through co-immunoprecipitation and western blotting.. A microarray data analysis showed that circEIF3I was highly expressed in PDAC cells and correlated with TNM stage and poor prognosis. Functional experiments in vitro and in vivo revealed that circEIF3I accelerated PDAC cells migration, invasion and metastasis by increasing MMPs expression and activity. Mechanistic research indicated that circEIF3I binds to the MH2 domain of SMAD3 and increases SMAD3 phosphorylation by strengthening the interactions between SMAD3 and TGFβRI on early endosomes. Moreover, AP2A1 binds with circEIF3I directly and promotes circEIF3I-bound SMAD3 recruitment to TGFβRI on early endosomes. Finally, we found that circEif3i exerts biological functions in mice similar to those of circEIF3I in humans PDAC.. Our study reveals that circEIF3I promotes pancreatic cancer progression. circEIF3I is a molecular scaffold that interacts with SMAD3 and AP2A1 to form a ternary complex, that facilitates the recruitment of SMAD3 to early endosomes and then activates the TGF-β signalling pathway. Hence, circEIF3I is a potential prognostic biomarker and therapeutic target in PDAC.

Topics: Animals; Carcinoma, Pancreatic Ductal; Endosomes; Humans; In Situ Hybridization, Fluorescence; Mice; Pancreatic Neoplasms; RNA, Circular; Smad3 Protein; Transforming Growth Factor beta

Tumor-associated macrophages (TAMs), as a major and essential component of tumor microenvironment (TME), play a critical role in orchestrating pancreatic cancer (PaC) tumorigenesis from initiation to angiogenesis, growth, and systemic dissemination, as well as immunosuppression and resistance to chemotherapy and immunotherapy; however, the critical intrinsic factors responsible for TAMs reprograming and function remain to be identified. By performing single-cell RNA sequencing, transforming growth factor-beta-induced protein (TGFBI) was identified as TAM-producing factor in murine PaC tumors. TAMs express TGFBI in human PaC and TGFBI expression is positively related with human PaC growth. By inducing TGFBI loss-of-function in macrophage (MΦs) in vitro with siRNA and in vivo with Cre-Lox strategy in our developed TGFBI-floxed mice, we demonstrated disruption of TGFBI not only inhibited MΦ polarization to M2 phenotype and MΦ-mediated stimulation on PaC growth, but also significantly improved anti-tumor immunity, sensitizing PaC to chemotherapy in association with regulation of fibronectin 1, Cxcl10, and Ccl5. Our studies suggest that targeting TGFBI in MΦ can develop an effective therapeutic intervention for highly lethal PaC.

Topics: Animals; Drug Resistance, Neoplasm; Humans; Macrophages; Mice; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic cancer (PC), a commonly recognized malignancy, arises within the digestive tract. Somatostatin (SOM) is a regulatory peptide that acts on secretion. First, the effects of SOM and transforming growth factor-β (TGF-β) on the proliferation of PC cells was determined by Cell Counting Kit-8 (CCK-8) assay. Next, we assessed the impact of SOM and TGF-β on the metastasis and apoptosis of PC cells using transwell assays and flow cytometry. Finally, we evaluated the effects of SOM and TGF-β on the expression of EMT-related proteins, apoptosis-related proteins, and proteins related to the TGF-β/Smad signaling pathway in PC cells using western blot analysis.. SOM suppressed the growth and metastasis of PC cells, and facilitated their apoptosis (. SOM suppressed the EMT progression in PC cells through its regulation of the TGF-β/Smad signaling pathway.

Topics: Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Pancreatic Neoplasms; Signal Transduction; Somatostatin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-β-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

Topics: Animals; Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Cells, Cultured; Embryo, Nonmammalian; Fibrosis; Fluvastatin; Humans; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; NIH 3T3 Cells; Pancreatic Neoplasms; Radiation Tolerance; Transforming Growth Factor beta; Zebrafish

Pancreatic cancer is a deadly disease with little response to standard therapies. Irreversible electroporation (IRE) has emerged as a novel ablative technique for the clinical treatment of pancreatic cancer. Combinations of IRE and immunotherapies, including anti-programmed death 1 (αPD1) immune checkpoint blockade, have shown promising efficacy in both preclinical and clinical studies. However, tumor recurrence remains an obstacle that needs to be overcome. It herein is shown that IRE induces a substantial infiltration of neutrophils into pancreatic tumors. These neutrophils are then polarized into a protumor phenotype by immunosuppressive cues, in particular transforming growth factor β (TGF-β). Using glutathione-responsive degradable mesoporous silica nanoparticles loaded with SB525334, an inhibitor of TGF-β1 receptor, it is demonstrated that local inhibition of TGF-β within the tumor microenvironment promotes neutrophil polarization into an antitumor phenotype, enhances pancreatic cancer response to combined IRE and αPD1 therapy, and induces long-term antitumor memory. The therapeutic efficacy is also attributed to tumor infiltration by CD8

Topics: CD8-Positive T-Lymphocytes; Electroporation; Humans; Immunotherapy; Neutrophils; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-β (TGF-β) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-β and the role of glycosylation therein is not well known. Here, we investigated the TGF-β-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-β signaling. PaTu-S cells responded to TGF-β by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-β induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-β stimulation. TGF-β treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-β-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-β treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-β stimulation, and its depletion blocked TGF-β-induced N-glycomic changes. Thus, TGF-β-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4-dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-β signaling in SMAD4-inactivated PDAC.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Glycosphingolipids; Humans; Pancreatic Neoplasms; Polysaccharides; Transforming Growth Factor beta

Pancreatic cancer has the highest prevalence of cancer-associated cachexia among all cancers. ZIP4 promotes pancreatic cancer progression by regulating oncogenic miR-373, and perturbation of circular RNAs (circRNAs) is associated with cancer aggressiveness. This study aimed to identify circRNAs involved in ZIP4/miR-373-driven cancer growth and cachexia and decipher the underlying mechanism.. Differentially expressed circRNAs and potential targets of microRNA were identified through in silico analysis. The RNA interactions were determined by means of biotinylated microRNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. The function of circRNA in ZIP4-miR-373 signaling axis were examined in human pancreatic cancer cells, 3-dimensional spheroids and organoids, mouse models, and clinical specimens. Mouse skeletal muscles were analyzed by means of histology.. We identified circANAPC7 as a sponge for miR-373, which inhibited tumor growth and muscle wasting in vitro and in vivo. Mechanistic studies showed that PHLPP2 is a downstream target of ZIP4/miR-373. CircANAPC7 functions through PHLPP2-mediated dephosphorylation of AKT, thus suppressing cancer cell proliferation by down-regulating cyclin D1 and inhibiting muscle wasting via decreasing the secretion of transforming growth factor-β through STAT5. We further demonstrated that PHLPP2 induced dephosphorylation of CREB, a zinc-dependent transcription factor activated by ZIP4, thereby forming a CREB-miR-373-PHLPP2 feed-forward loop to regulate tumor progression and cancer cachexia.. This study identified circANAPC7 as a novel tumor suppressor, which functions through the CREB-miR-373-PHLPP2 axis, leading to AKT dephosphorylation, and cyclin D1 and transforming growth factor-β down-regulation to suppress tumor growth and muscle wasting in pancreatic cancer.

Topics: Animals; Cachexia; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Mice; MicroRNAs; Muscles; Pancreatic Neoplasms; Phosphoprotein Phosphatases; Proto-Oncogene Proteins c-akt; RNA, Circular; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDA) tumors have a highly immunosuppressive desmoplastic tumor microenvironment (TME) where immune checkpoint inhibition (ICI) therapy has been exceptionally ineffective. Transforming growth factor-β (TGF-β) receptor activation leads to cancer and immune cell proliferation and phenotype, and cytokine production leading to tumor progression and worse overall survival in PDA patients. We hypothesized that TGF-β receptor inhibition may alter PDA progression and antitumor immunity in the TME. Here, we used a syngeneic preclinical murine model of PDA to explore the impact of TGF-β pathway inhibitor galunisertib (GAL), dual checkpoint immunotherapy (anti-PD-L1 and CTLA-4), the chemotherapy gemcitabine (GEM), and their combinations on antitumor immune responses. Blockade of TGF-β and ICI in immune-competent mice bearing orthotopically injected murine PDA cells significantly inhibited tumor growth and was accompanied by antitumor M1 macrophage infiltration. In contrast, GEM treatment resulted in increased PDA tumor growth, decreased antitumor M1 macrophages, and decreased cytotoxic CD8+ T cell subpopulation compared to control mice. Together, these findings demonstrate the ability of TGF-β inhibition with GAL to prime antitumor immunity in the TME and the curative potential of combining GAL with dual ICI. These preclinical results indicate that targeted inhibition of TGF-β may enhance the efficacy of dual immunotherapy in PDA. Optimal manipulation of the immune TME with non-ICI therapy may enhance therapeutic efficacy.

Topics: Animals; Carcinoma, Pancreatic Ductal; Deoxycytidine; Gemcitabine; Humans; Immunotherapy; Mice; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Microenvironment

The current standard therapy for metastatic pancreatic cancer is ineffective, necessitating a new treatment approach for prognosis improvement. The urokinase-plasmin activator (uPA) is a critical factor in epithelial-mesenchymal transition (EMT) and cancer metastasis, but its underlying mechanisms in pancreatic cancer remains elusive.. We investigated uPA expression in our pancreatic cancer cohort. A bioinformatics approach was used to further determine the role of uPA in pancreatic cancer. We employed MiaPaCa-2 and PANC-1 cell lines to investigate how uPA regulates EMT and metastasis in pancreatic cancer and present a novel approach aimed at inhibiting uPA in pancreatic cancer.. We observed that higher uPA mRNA expression was significantly associated with overall-poor survival and progression-free survival in pancreatic cancer. uPA was highly expressed in tumor tissue. Gene set enrichment analysis revealed a positive association between uPA mRNA expression and EMT and transforming growth factor β (TGF-β) signaling pathways. Moreover, shRNA-mediated uPA gene knockdown reduced plasmin, MMP14, and TGF-β activation, leading to the inhibition of PANC-1 cells' EMT marker expression, migration, invasion, and cell viability. Notably, 4-acetyl-antroquinonol B (4-AAQB) treatment suppressed MiaPaCa-2 and PANC-1 cell migratory and invasive abilities by inhibiting the uPA/MMP14/TGF-β axis through upregulation of miR-181d-5p. In the xenograft mouse model of orthotropic pancreatic cancer, 4-AAQB treatment has reduced tumor growth and metastasis rate by deactivating uPA and improving the survival of the mice model.. Accordingly, to extent of our knowledge and previous studies, we demonstrated that 4-AAQB is an anti Pan-Cancer drug, and may inhibit pancreatic cancer EMT and metastasis and serve as a new therapeutic approach for patients with late-stage pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Fibrinolysin; Humans; Matrix Metalloproteinase 14; Mice; Pancreatic Neoplasms; RNA, Messenger; Transforming Growth Factor beta; Ubiquinone; Urokinase-Type Plasminogen Activator

Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4

Topics: Cancer-Associated Fibroblasts; Fibroblasts; Humans; Pancreatic Neoplasms; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Pancreatic cancer (PC) is one of the malignant tumors with the worst prognosis worldwide because of a lack of early diagnostic markers and efficient therapies. Integrin, beta-like 1 (ITGBL1) is a β-integrin-related extracellular matrix protein and is reported to promote progression of some types of cancer. Nevertheless, the function of ITGBL1 in PC is still not clear. Herein, we found that ITGBL1 was highly expressed in PC tissues compared to normal tissues (P<0.05) and PC patients with higher TGBL1 expression showed worse prognosis. PANC-1 and AsPC-1 cells were used for gain/loss-of-function experiments. We found that ITGBL1-silenced cells exhibited decreased proliferation, migration, and invasion abilities and delayed cell cycle, whereas ITGBL1 overexpression reversed these malignant behaviors. ITGBL1 was also demonstrated to activate the TGF-β/Smad pathway, a key signaling pathway in PC progression. Additionally, ITGBL1 expression was found to be suppressed by a suppressor of PC progression, c-Jun dimerization protein 2 (JDP2). Results of dual-luciferase assay indicated that transcription factor JDP2 could inhibit TGBL1 promoter activity. ITGBL1 overexpression inversed the effects of JDP2 up-regulation on cell function. Collectively, we concluded that ITGBL1 may be transcriptionally suppressed by JDP2 and promote PC progression through the TGF-β/Smad pathway, indicating that ITGBL1 may have therapeutic potential for the treatment of PC.

Topics: Cell Line, Tumor; Humans; Integrin beta1; Pancreatic Neoplasms; Repressor Proteins; Transforming Growth Factor beta

Although HOXB9 induces tumor proliferation and chemoresistance in several cancer cells, little is known in pancreatic ductal adenocarcinoma (PDAC). In the present study, increased expression of HOXB9 in PDAC was associated with the induction of angiogenic factors and poor overall survival through the TGFβ pathway. Taken together, these results suggested that HOXB9 expression in PDAC could be a surrogate marker in clinical treatment.. HOXB9 expression regulated the expression of TGFβ1 signature, angiogenic factors, and EMT markers. In conclusion, HOXB9 expression could mediate angiogenesis, EMT, and cancer stemness through the TGFβ pathway, thereby resulting in chemoresistance and poor overall outcomes in patients with pancreatic cancer. Our results suggested that HOXB9 may clinically serve as a novel surrogate biomarker.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) has a very low survival rate due to the late detection and poor response to chemotherapy. Epithelial-to-mesenchymal transition (EMT) is considered an important step in tumor progression with regard to invasion and metastasis, and Transforming Growth Factor-beta (TGF-β) signaling has been shown to play an important role in EMT. Therefore, we aimed to investigate whether indomethacin, an anti-inflammatory and analgesic drug, has any effect on TGF-β-induced EMT in pancreatic cancer cell line and analyze the changes in their molecular structures by Raman spectroscopy and other molecular techniques. Indomethacin treated Panc-1 cells were analyzed with Raman spectroscopy, quantitative polymerase chain reaction and immunofluorescence techniques after the induction of EMT with TGF-β. The exposure of Panc-1 cells to TGF-β resulted in characteristic morphological alterations of EMT, and indomethacin inhibits TGF-β-induced EMT through up-regulation of E-cadherin and down-regulation of N-cadherin and Snail expressions. Raman spectroscopy supported by principal component analysis (PCA) confirmed the effects of both TGF-β and indomethacin. Raman spectra were further analyzed using the PCA-assisted vector machine algorithm and it was seen that the data could be classified with 97.6% accuracy. Our results suggest that indomethacin may have a significant effect on PDAC metastasis, and Raman spectroscopy was able to probe EMT-related changes and the efficacy of indomethacin in a short time and without the need for specific reagents compared to other molecular techniques.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Indomethacin; Pancreatic Neoplasms; Spectrum Analysis, Raman; Transforming Growth Factor beta

Serotonin producing pancreatic neuroendocrine tumors (SP-PanNET) account for 0.58-1.4% of all pancreatic neuroendocrine tumors (PanNET). They may present with atypical symptoms, such as acute pancreatitis and are often radiologically characterized by main pancreatic duct dilatation. SP-PanNET are well differentiated neuroendocrine tumors (NET) distinct from classical PanNET by atypical serotonin secretion and abundant dense stroma deposition, like serotonin producing ileal NET leading in some cases to difficulties to reliably distinguish SP-PanNET from ileal NET metastases. The biology and molecular profile of SP-PanNET remain poorly characterized and the cell of origin within the pancreas is unclear. To address these questions, we analyzed a large cohort of SP-PanNET by immunohistochemistry (n = 29; ATRX, DAXX, MENIN, Islet1, PAX6, PDX1, ARX, CDX2), whole genome copy number array (Oncoscan™) and a large NGS panel (NovoPM™) (n = 10), FISH (n = 13) and RNA sequencing (n = 24) together with 21 ileal NET and 29 nonfunctioning PanNET (NF-PanNET). These analyses revealed a unique genomic profile with frequent isolated loss of chromosome 1 (14 cases-61%) and few pathogenic mutations (KMT2C in 2 cases, ARID1A in 1 case). Unsupervised RNAseq-based clustering showed that SP-PanNET were closer to NF-PanNET than ileal NET with an exclusive beta cell-like signature. SP-PanNET showed TGF-β pathway activation signatures associated with extracellular matrix remodeling and similar signature were reproduced in vitro when pancreatic stellate cells were exposed to serotonin. SP-PanNET immunohistochemical profile resemble that of ileal NET except for PDX1 and PAX6 expression to a lesser extend suggesting that these two markers may be useful to diagnose SP-PanNET. Taken together, this suggests that SP-PanNET are a very specific PanNET entity with a peculiar biology leading to the characteristic fibrotic aspect.

Topics: Acute Disease; Humans; Neuroendocrine Tumors; Pancreatic Neoplasms; Pancreatitis; Serotonin; Transforming Growth Factor beta

The epithelial-to-mesenchymal transition (EMT) describes a biological process in which polarized epithelial cells are converted into highly motile mesenchymal cells. It promotes cancer cell dissemination, allowing them to form distal metastases, and also involves drug resistance in metastatic cancers. Transforming growth factor β (TGFβ) is a multifunctional cytokine that plays essential roles in development and carcinogenesis. It is a major inducer of the EMT. The MIR31 host gene (

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; RNA, Long Noncoding; Transforming Growth Factor beta

Transforming Growth Factor β (TGFβ) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFβ-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFβ-induced EMT is largely unkonwn.. Real-time qPCR analyses were performed to determine the effect of TGFβ1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFβ1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFβ1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFβ1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFβ1 on E-cadherin and mesenchymal genes in the cells.. Data provided suggest that TGFβ1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFβ1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFβ1-associated EMT in tumor cells.

Topics: A549 Cells; Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dual-Specificity Phosphatases; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Pancreatic Neoplasms; RNA, Small Interfering; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Pancreatic ductal adenocarcinoma (PDAC) is a hard-to-treat cancer due to the collagen-rich (fibrotic) and immune-suppressed microenvironment. A major driver of this phenomenon is transforming growth factor beta (TGF-β). TGF-β is produced in an inactive complex with a latency-associated protein (LAP) that can be cleaved by plasma kallikrein (PLK), hereby releasing active TGF-β. The aim of this study was to evaluate LAP cleaved by PLK as a non-invasive biomarker for PDAC and tumor fibrosis. An ELISA was developed for the quantification of PLK-cleaved LAP-TGF-β in the serum of 34 patients with PDAC (stage 1−4) and 20 healthy individuals. Biomarker levels were correlated with overall survival (OS) and compared to serum type III collagen (PRO-C3) and type VI collagen (PRO-C6) pro-peptides. PLK-cleaved LAP-TGF-β was higher in patients with PDAC compared to healthy individuals (p < 0.0001). High levels (>median) of PLK-cleaved LAP-TGF-β were associated with poor OS in patients with PDAC independent of age and stage (HR 2.57, 95% CI: 1.22−5.44, p = 0.0135). High levels of PLK-cleaved LAP-TGF-β were associated with high PRO-C3 and PRO-C6, indicating a relationship between the PLK-cleaved LAP-TGF-β fragment, TGF-β activity, and tumor fibrosis. If these preliminary results are validated, circulating PLK-cleaved LAP-TGF-β may be a biomarker for future clinical trials.

Topics: Biomarkers; Carcinoma, Pancreatic Ductal; Collagen Type III; Collagen Type VI; Complement C3; Fibrosis; Humans; Pancreatic Neoplasms; Plasma Kallikrein; Prognosis; Transforming Growth Factor beta; Tumor Microenvironment

Cancer cells and their stromal microenvironment are mutually supportive. Either destroying cancer cells or damaging stromal components cannot guarantee a satisfactory outcome in the long-term treatment. Herein, we showed that the tumor-stroma crosstalk was disturbed by nanoparticle-based photodynamic therapy (PDT) in pancreatic tumor models, leading to the persistent inhibition of extracellular matrix (ECM) secretion and the enhanced therapeutic effect. By employing a conditioned medium method, we found that the nanoparticulate PDT at a sub-lethal dosage down-regulated TGFβ signaling pathways, leading to the decrease in drug resistance, proliferation, and migration of the cancer cells. Meanwhile, pancreatic stellate cells (PSCs) were inactivated by PDT, hindering the secretion of ECM. Combining the results that PDT indiscriminately killed PSCs and cancer cells, we showed that the mutual support between the cancer cells and the stroma was interrupted. We further presented the inhibition of the crosstalk persistently enhanced tumor penetration in stroma-rich pancreatic tumor models. The loosened stroma not only facilitated tumor eradication by subsequent therapy but also improved the efficiency of gemcitabine treatment on monthly later recurrent tumors. Therefore, our work may boost the potential of PDT to be a valuable individual or adjuvant treatment for desmoplastic cancers.

Topics: Culture Media, Conditioned; Humans; Nanoparticles; Neoplasm Recurrence, Local; Pancreatic Neoplasms; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)

Topics: Animals; B7-H1 Antigen; Cancer-Associated Fibroblasts; CD8-Positive T-Lymphocytes; Humans; Membrane Proteins; Mice; Myofibroblasts; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

For pancreatic ductal adenocarcinoma (PDAC) treatment, the deactivation of pancreatic stellate cells (PSCs) by blocking the transforming growth factor β (TGF-β) pathway is a promising strategy to inhibit stroma, enhance drug penetration, and greatly amplify chemotherapeutic efficacy. It is known that photothermal therapy (PTT) locally depletes stroma and enhances permeability but whether and how PTT reacts in the molecular pathway to induce PSC deactivation in PDAC has rarely been investigated so far. Herein, C-G NPs are synthesized by loading both acid-responsive photothermal molecules and gemcitabine for investigating both the combinatory chemophotothermal therapy and the interaction between the PTT and TGF-β pathway in PDAC. Notably, C-G NPs exhibit tumoral acidic pH-activated PTT and succeeded in deactivating PSCs and suppressing the expression level for both TGF-β and collagen fiber. Furthermore, hyperthermia remodels the tumoral extracellular matrix, significantly improves NP penetration, and boosts the ultimate synergistic chemophotothermal therapeutic efficacy. Importantly, the molecular biology study reveals that hyperthermia leads to the decrease in the mRNA expression of

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Collagen; Humans; Hydrogen-Ion Concentration; Pancreatic Neoplasms; Photothermal Therapy; Transforming Growth Factor beta

TGF-β signaling is involved in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis, representing one of the four major pathways genetically altered in 100% of PDAC cases. TGF-β exerts complex and pleiotropic effects in cancers, notably via the activation of SMAD pathways, predominantly SMAD2/3/4. Though SMAD2 and 3 are rarely mutated in cancers, SMAD4 is lost in about 50% of PDAC, and the role of SMAD2/3 in a SMAD4-null context remains understudied. We herein provide evidence of a SMAD2/3 oncogenic effect in response to TGF-β1 in SMAD4-null human PDAC cancer cells. We report that inactivation of SMAD2/3 in SMAD4-negative PDAC cells compromises TGF-β-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-β gene signature related to aggressiveness mediated by SMAD2/3 in the absence of SMAD4. Using a PDAC patient cohort, we reveal that SMAD4-negative tumors with high levels of phospho-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC leads to an oncogenic gain-of-function of SMAD2/3, and to the onset of associated deleterious effects.

Topics: Carcinogenesis; Carcinoma, Pancreatic Ductal; Humans; Pancreatic Neoplasms; RNA; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Tumor-initiating cells (TIC), also known as cancer stem cells, are considered a specific subpopulation of cells necessary for cancer initiation and metastasis; however, the mechanisms by which they acquire metastatic traits are not well understood.. LAMC2 transcriptional levels were evaluated using publicly available transcriptome data sets, and LAMC2 immunohistochemistry was performed using a tissue microarray composed of PDAC and normal pancreas tissues. Silencing and tracing of LAMC2 was performed using lentiviral shRNA constructs and CRISPR/Cas9-mediated homologous recombination, respectively. The contribution of LAMC2 to PDAC tumorigenicity was explored in vitro by tumor cell invasion, migration, sphere-forming and organoids assays, and in vivo by tumor growth and metastatic assays. mRNA sequencing was performed to identify key cellular pathways upregulated in LAMC2 expressing cells. Metastatic spreading induced by LAMC2- expressing cells was blocked by pharmacological inhibition of transforming growth factor beta (TGF-β) signaling.. We report a LAMC2-expressing cell population, which is endowed with enhanced self-renewal capacity, and is sufficient for tumor initiation and differentiation, and drives metastasis. mRNA profiling of these cells indicates a prominent squamous signature, and differentially activated pathways critical for tumor growth and metastasis, including deregulation of the TGF-β signaling pathway. Treatment with Vactosertib, a new small molecule inhibitor of the TGF-β type I receptor (activin receptor-like kinase-5, ALK5), completely abrogated lung metastasis, primarily originating from LAMC2-expressing cells.. We have identified a highly metastatic subpopulation of TICs marked by LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in PDAC patients. Our results prompt further study of this TIC population in pancreatic cancer and exploration as a potential therapeutic target and/or biomarker.

Topics: Activin Receptors; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Humans; Laminin; Neoplastic Stem Cells; Pancreatic Neoplasms; Receptor, Transforming Growth Factor-beta Type I; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta

Less than 11% of pancreatic cancer patients survive 5-years post-diagnosis. The unique biology of pancreatic cancer includes a significant expansion of its desmoplastic tumor microenvironment, wherein cancer-associated fibroblasts (CAFs) and their self-produced extracellular matrix are key components. CAF functions are both tumor-supportive and tumor-suppressive, while normal fibroblastic cells are solely tumor-suppressive. Knowing that CAF-eliminating drugs are ineffective and can accelerate cancer progression, therapies that "normalize" CAF function are highly pursued. Eribulin is a well-tolerated anti-microtubule drug used to treat a plethora of neoplasias, including advanced/metastatic cancers. Importantly, eribulin can inhibit epithelial to mesenchymal transition via a mechanism akin to blocking pathways induced by transforming growth factor-beta (TGFβ). Notably, canonical TGFβ signaling also plays a pivotal role in CAF activation, which is necessary for the development and maintenance of desmoplasia. Hence, we hypothesized that eribulin could modulate, and perhaps "normalize" CAF function.. To test this premise, we used a well-established in vivo-mimetic fibroblastic cell-derived extracellular matrix (CDM) system and gauged the effects of eribulin on human pancreatic CAFs and cancer cells. This pathophysiologic fibroblast/matrix functional unit was also used to query eribulin effects on CDM-regulated pancreatic cancer cell survival and invasive spread.. Demonstrated that intact CAF CDMs modestly restricted eribulin from obstructing pancreatic cancer cell growth. Nonetheless, eribulin-treated CAFs generated CDMs that limited nutrient-deprived pancreatic cancer cell survival, similar to reported tumor-suppressive CDMs generated by TGFβ-deficient CAFs.. Data from this study support the central proposed premise suggesting that eribulin could be used as a CAF/matrix-normalizing drug.

Topics: Calmodulin-Binding Proteins; Cancer-Associated Fibroblasts; Epithelial-Mesenchymal Transition; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic cancer is one of the most lethal cancers, owing to its late diagnosis and resistance to chemotherapy. The tumor suppressor WW domain-containing oxidoreductase (WWOX), one of the most active fragile sites in the human genome (FRA16D), is commonly altered in pancreatic cancer. However, the direct contribution of WWOX loss to pancreatic cancer development and progression remains largely unknown. Here, we report that combined conditional deletion of Wwox and activation of KRasG12D in Ptf1a-CreER-expressing mice results in accelerated formation of precursor lesions and pancreatic carcinoma. At the molecular level, we found that WWOX physically interacts with SMAD3 and BMP2, which are known activators of the TGF-β signaling pathway. In the absence of WWOX, TGFβ/BMPs signaling was enhanced, leading to increased macrophage infiltration and enhanced cancer stemness. Finally, overexpression of WWOX in patient-derived xenografts led to diminished aggressiveness both in vitro and in vivo. Overall, our findings reveal an essential role of WWOX in pancreatic cancer development and progression and underscore its role as a bona fide tumor suppressor.

Topics: Animals; Bone Morphogenetic Protein 2; Genes, Tumor Suppressor; Humans; Mice; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase

Pancreatic ductal adenocarcinoma (PDAC) is associated with very poor survival, making it the third and fourth leading cause of all cancer-related deaths in the USA and European Union, respectively. The tumor microenvironment (TME) in PDAC is highly immunosuppressive and desmoplastic, which could explain the limited therapeutic effect of immunotherapy in PDAC. One of the key molecules that contributes to immunosuppression and fibrosis is transforming growth factor-β (TGFβ). The aim of this study was to target the immunosuppressive and fibrotic TME in PDAC using a novel immune modulatory vaccine with TGFβ-derived peptides in a murine model of pancreatic cancer.. C57BL/6 mice were subcutaneously inoculated with Pan02 PDAC cells. Mice were treated with TGFβ1-derived peptides (major histocompatibility complex (MHC)-I and MHC-II-restricted) adjuvanted with Montanide ISA 51VG. The presence of treatment-induced TGFβ-specific T cells was assessed by ELISpot (enzyme-linked immunospot). Changes in the immune infiltration and gene expression profile in tumor samples were characterized by flow cytometry, reverse transcription-quantitative PCR (RT-qPCR), and bulk RNA sequencing.. This study demonstrates the antitumor activity of TGFβ-derived multipeptide vaccination in a murine tumor model of PDAC. The data suggest that the vaccine targets immunosuppression and fibrosis in the TME by polarizing the cellular composition towards a more pro-inflammatory phenotype. Our findings support the feasibility and potential of TGFβ-derived peptide vaccination as a novel immunotherapeutic approach to target immunosuppression in the TME.

Topics: Animals; Cancer Vaccines; Carcinoma, Pancreatic Ductal; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Fibrosis; Immunity; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment; Vaccines, Subunit

Immunotherapy, such as checkpoint inhibitors against anti-programmed death-ligand 1 (PD-L1), has not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and the TGF-β cytokine are critical in anti-cancer immunity. We hypothesized that TGF-β enhances the immunosuppressive effects of TAM, MDSC, and DC presence in tumors.. Using a murine PDAC cell line derived from a genetically engineered mouse model, we orthotopically implanted treated cells plus drug embedded in Matrigel into immunocompetent mice. Treatments included saline control, TGF-β1, or a TGF-β receptor 1 small molecule inhibitor, galunisertib. We investigated TAM, MDSC, DC, and TAM PD-L1 expression with flow cytometry in tumors. Separately, we used the TIMER2.0 database to analyze TAM and PD-L1 gene expression in human PDAC tumors in TCGA database.. TGF-β did not alter MDSC or DC frequencies in the primary tumors. However, in PDAC metastases to the liver, TGF-β decreased the proportion of MDSCs (P=0.022) and DCs (P=0.005). TGF-β significantly increased the percent of high PD-L1 expressing TAMs (32 ± 6 % vs. 12 ± 5%, P=0.013) but not the proportion of TAMs in primary and metastatic tumors. TAM PD-L1 gene expression in TCGA PDAC database was significantly correlated with tgb1 and tgfbr1 gene expression (P<0.01).. TGF-β is important in PDAC anti-tumor immunity, demonstrating context-dependent impact on immune cells. TGF-β has an overall immunosuppressive effect mediated by TAM PD-L1 expression and decreased presence of DCs. Future investigations will focus on enhancing anti-cancer immune effects of TGF-β receptor inhibition.

Topics: Animals; B7-H1 Antigen; Carcinoma, Pancreatic Ductal; Dendritic Cells; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Macrophages; Mice; Pancreatic Neoplasms; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) cells are surrounded by a dense extracellular matrix (ECM), which greatly restricts the access of therapeutic agents, resulting in poor clinical response to chemotherapy. Transforming growth factor-β1 (TGF-β1) signaling plays a crucial role in construction of the desmoplastic stroma and provides potential targets for PDAC therapy. To surmount the pathological obstacle, we developed a size switchable nanosystem based on PEG-PLGA nanospheres encapsulated within liposomes for the combined delivery of vactosertib (VAC), a TGF-β1 receptor kinase inhibitor, and the cytotoxic drug paclitaxel (TAX). By surface modification of the liposomes with a peptide, APT

Topics: Adenocarcinoma; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Topics: 4-1BB Ligand; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Lymphatic Metastasis; Macrophage Activation; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Snail Family Transcription Factors; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

A novel, selective, next-generation transforming growth factor beta (TGFβ) receptor type-1 small molecule inhibitor, LY3200882, demonstrated promising preclinical data. This first-in-human trial evaluated safety, tolerability, recommended phase II dose (RP2D), pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of LY3200882 as monotherapy or with other anticancer agents in patients with advanced cancer.. This phase I multicenter study of oral LY3200882 (NCT02937272) comprised dose escalation, monotherapy expansion in grade 4 glioma, and combination therapy in solid tumors (LY3200882 and PD-L1 inhibitor LY3300054), pancreatic cancer (LY3200882, gemcitabine, and nab-paclitaxel), and head and neck squamous cell cancer (LY3200882, cisplatin, and radiation).. Overall, 139 patients with advanced cancer were treated. The majority (93.5%) of patients experienced ≥1 treatment-emergent adverse events (TEAE), with 39.6% LY3200882-related. Grade 3 LY3200882-related toxicities were only observed in combination therapy arms. One patient in the pancreatic cancer arm experienced cardiovascular toxicity. The LY3200882 monotherapy RP2Ds were established in two schedules: 50 mg twice a day 2-weeks-on/2-weeks-off and 35 mg twice a day 3-weeks-on/1-week-off. Four patients with grade 4 glioma had durable Revised Assessment in Neuro Oncology (RANO) partial responses (PR) with LY3200882 monotherapy (. LY3200882 as monotherapy and combination therapy was safe and well tolerated with preliminary antitumor activity observed in pancreatic cancer. Further studies to evaluate the efficacy of LY3200882 with gemcitabine and nab-paclitaxel in advanced pancreatic cancer are warranted.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Head and Neck Neoplasms; Humans; Maximum Tolerated Dose; Paclitaxel; Pancreatic Neoplasms; Transforming Growth Factor beta

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Culture Media, Conditioned; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Humans; Liver Neoplasms; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Pancreatic Neoplasms; Prognosis; Smad2 Protein; Smad3 Protein; Smad4 Protein; Snail Family Transcription Factors; THP-1 Cells; Transforming Growth Factor beta; Tumor-Associated Macrophages

Topics: Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

Interleukin (IL)-33 cytokine plays a critical role in allergic diseases and cancer. IL-33 also has a nuclear localization signal. However, the nuclear function of IL-33 and its impact on cancer is unknown. Here, we demonstrate that nuclear IL-33-mediated activation of SMAD signaling pathway in epithelial cells is essential for cancer development in chronic inflammation. Using RNA and ChIP sequencing, we found that nuclear IL-33 repressed the expression of an inhibitory SMAD, Smad6, by interacting with its transcription factor, RUNX2. IL-33 was highly expressed in the skin and pancreatic epithelial cells in chronic inflammation, leading to a markedly repressed Smad6 expression as well as dramatically upregulated p-SMAD2/3 and p-SMAD1/5 in the epithelial cells. Blocking TGF-β/SMAD signaling attenuated the IL-33-induced cell proliferation in vitro and inhibited IL-33-dependent epidermal hyperplasia and skin cancer development in vivo. IL-33 and SMAD signaling were upregulated in human skin cancer, pancreatitis, and pancreatitis-associated pancreatic cancer. Collectively, our findings reveal that nuclear IL-33/SMAD signaling is a cell-autonomous tumor-promoting axis in chronic inflammation, which can be targeted by small-molecule inhibitors for cancer treatment and prevention.

Topics: Animals; Carcinogenesis; Cell Line; Cell Line, Tumor; Cell Nucleus; Core Binding Factor Alpha 1 Subunit; Epithelial Cells; Female; Inflammation; Interleukin-33; Male; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Signal Transduction; Skin Neoplasms; Smad6 Protein; Transforming Growth Factor beta

Topics: Cell Line, Tumor; DNA-Binding Proteins; Humans; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; RNA-Binding Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-β) pathway plays a paradoxical, context-dependent role in pancreatic ductal adenocarcinoma (PDAC): a tumor-suppressive role in non-metastatic PDAC and a tumor-promotive role in metastatic PDAC. We hypothesize that non-SMAD-TGF-β signaling induces PDAC progression.. We investigated the expression of non-SMAD-TGF-β signaling proteins (pMAPK14, PD-L1, pAkt and c-Myc) in patient-derived tissues, cell lines and an immunocompetent mouse model. Experimental models were complemented by comparing the signaling proteins in PDAC specimens from patients with various survival intervals. We manipulated models with TGF-β, gemcitabine (DNA synthesis inhibitor), galunisertib (TGF-β receptor inhibitor) and MK-2206 (Akt inhibitor) to investigate their effects on NF-κB, β-catenin, c-Myc and PD-L1 expression. PD-L1 expression was also investigated in cancer cells and tumor associated macrophages (TAMs) in a mouse model.. We found that tumors from patients with aggressive PDAC had higher levels of the non-SMAD-TGF-β signaling proteins pMAPK14, PD-L1, pAkt and c-Myc. In PDAC cells with high baseline β-catenin expression, TGF-β increased β-catenin expression while gemcitabine increased PD-L1 expression. Gemcitabine plus galunisertib decreased c-Myc and NF-κB expression, but induced PD-L1 expression in some cancer models. In mice, gemcitabine plus galunisertib treatment decreased metastases (p = 0.018), whereas galunisertib increased PD-L1 expression (p < 0.0001). In the mice, liver metastases contained more TAMs compared to the primary pancreatic tumors (p = 0.001), and TGF-β increased TAM PD-L1 expression (p < 0.05).. In PDAC, the non-SMAD-TGF-β signaling pathway leads to more aggressive phenotypes, TAM-induced immunosuppression and PD-L1 expression. The divergent effects of TGF-β ligand versus receptor inhibition in tumor cells versus TAMs may explain the TGF-β paradox. Further evaluation of each mechanism is expected to lead to the development of targeted therapies.

Topics: Animals; B7-H1 Antigen; Carcinoma, Pancreatic Ductal; Disease Progression; Humans; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor-Associated Macrophages

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Intestinal Neoplasms; Neoplasm Invasiveness; Neuroendocrine Tumors; Oncostatin M; Pancreatic Neoplasms; RNA, Long Noncoding; Signal Transduction; Stomach Neoplasms; Transcription, Genetic; Transforming Growth Factor beta

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-β, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Coculture Techniques; Disease Models, Animal; Female; Interferon-gamma; Interleukin-2; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucin-1; Myeloid-Derived Suppressor Cells; Pancreatic Neoplasms; Proto-Oncogene Proteins c-myc; Signal Transduction; Spleen; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

The purpose of this study was to identify ferroptosis-related genes (FRGs) associated with the prognosis of pancreatic cancer and to construct a prognostic model based on FRGs.. Based on pancreatic cancer data obtained from The Cancer Genome Atlas database, we established a prognostic model from 232 FRGs. A nomogram was constructed by combining the prognostic model and clinicopathological features. Gene Expression Omnibus datasets and tissue samples obtained from our center were utilized to validate the model. The relationship between risk score and immune cell infiltration was explored by CIBERSORT and TIMER.. The prognostic model was established based on four FRGs (ENPP2, ATG4D, SLC2A1 and MAP3K5), and the risk score was demonstrated to be an independent risk factor in pancreatic cancer (HR 1.648, 95% CI 1.335-2.035, p < 0.001). Based on the median risk score, patients were divided into a high-risk group and a low-risk group. The low-risk group had a better prognosis than the high-risk group. In the high-risk group, patients treated with chemotherapy had a better prognosis. The nomogram showed that the model was the most important element. Gene set enrichment analysis identified three key pathways, namely, TGFβ signaling, HIF signaling pathway and the adherens junction. The prognostic model may be associated with infiltration of immune cells such as M0 macrophages, M1 macrophages, CD4 + T cells and CD8 + T cells.. The ferroptosis-related prognostic model can be employed to predict the prognosis of pancreatic cancer. Ferroptosis is an important marker, and immunotherapy may be a potential therapeutic target for pancreatic cancer.

Topics: Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Ferroptosis; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Nomograms; Pancreatic Neoplasms; Prognosis; Risk Factors; Transforming Growth Factor beta

TGFβ is an important tumor suppressor in pancreatic ductal adenocarcinoma (PDA), yet inactivation of TGFβ pathway components occurs in only half of PDA cases. TGFβ cooperates with oncogenic RAS signaling to trigger epithelial-to-mesenchymal transition (EMT) in premalignant pancreatic epithelial progenitors, which is coupled to apoptosis owing to an imbalance of SOX4 and KLF5 transcription factors. We report that PDAs that develop with the TGFβ pathway intact avert this apoptotic effect via ID1.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Inhibitor of Differentiation Protein 1; Mice; Mice, Nude; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

To explore novel biomarkers for patients with pancreatic ductal adenocarcinoma (PDAC), from the perspective of tumor hypoxia.. We screened 29 differentially expressed and hypoxia-upregulated genes from the Oncomine database. A total of 12 secretory proteins that interact with hypoxia-inducible factor 1 (HIF-1A) were selected by STRING (protein-protein interaction networks). After excluding enzymes and collagens, insulin-like growth factor-binding protein 3 (IGFBP3), glycoprotein NBM (GPNMB), transforming growth factor-β-induced (TGFBI), and biglycan (BGN) were detected by sandwich enzyme-linked immunosorbent assay (ELISA) in patients with cancer and healthy control individuals.. The serum level of TGFBI was significantly elevated in patients with PDAC, compared with healthy controls; the assay could discriminate among cases of PDAC in different clinical stages. The amount of TGFBI was significantly decreased after treatment. The combination of TGFBI and cancer antigen (CA) 19-9 was more accurate than TGFBI or CA 19-9 alone as diagnostic markers. Also, TGFBI might be used as a prognostic marker according to the PROGgeneV2 Pan Cancer Prognostics Database.. Serum TGFBI, combined with CA 19-9, offers higher diagnostic value than other methods for patients with PDAC. Also, TGFBI might be used as a prognostic marker.

Topics: Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Extracellular Matrix Proteins; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Hypoxia

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15

Topics: Animals; B7-H1 Antigen; Cancer-Associated Fibroblasts; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Lineage; Clinical Trials as Topic; Computational Biology; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Immune Checkpoint Inhibitors; Membrane Proteins; Mice; Myofibroblasts; Pancreatic Neoplasms; RNA-Seq; Single-Cell Analysis; Transforming Growth Factor beta; Treatment Outcome; Tumor Microenvironment

Transforming growth factor-beta (TGF-β) is a multifunctional regulatory factor. Here we measured serum soluble TGF-β (sTGF-β) levels and evaluated its dynamics and prognostic capabilities during chemotherapy in unresectable pancreatic cancer patients.. We prospectively enrolled 60 patients treated with FOLFIRINOX as the first-line palliative chemotherapy. We collected blood samples at the time of diagnosis, first response assessment, and disease progression and measured serum sTGF-β using an enzyme-linked immunosorbent assay.. The patients' median overall survival (OS) and progression-free survival (PFS) were 10.3 (95% confidence interval [CI], 8.5-12.1) and 6.5 (95% CI, 4.9-8.1) months, respectively. Patients with low sTGF-β at diagnosis (<31.2 ng/mL) had better OS and PFS than patients with high sTGF-β, respectively, (OS, 13.7 vs 9.2 months; hazard ratio [HR], 2.602; P = .004; PFS, 9.0 vs 5.8 months; HR, 2.010; P = .034). At the time of disease progression, sTGF-β was increased compared with that of diagnosis (mean, 26.4 vs 23.9 ng/mL). In particular, sTGF-β was significantly increased at disease progression in patients with a partial response (mean, 25.7 vs 31.0 ng/mL; P = .049).. Pretreatment sTGF-β levels can serve as a prognostic indicator in unresectable pancreatic cancer patients treated with FOLFIRINOX chemotherapy. Likewise, the dynamics of sTGF-β during chemotherapy have prognostic value.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Disease Progression; Female; Fluorouracil; Humans; Irinotecan; Kaplan-Meier Estimate; Leucovorin; Male; Middle Aged; Oxaliplatin; Palliative Care; Pancreatic Neoplasms; Prognosis; Progression-Free Survival; Prospective Studies; Transforming Growth Factor beta

Pancreatic cancer is one of the most lethal malignant tumors due to a late diagnosis and highly invasion and metastasis. Transforming growth factor-β (TGF-β) signaling plays a vital role in the progression of pancreatic cancer. The delicate activity of TGF-β signaling is particular important for the development of aggression and metastasis which must be fine-tuned. Here, we investigated the role of super-enhancers in regulating the expression of TGF-β signaling pathway in pancreatic cancer. TGFBR2 owns the modification of H3K27Ac around the gene in pancreatic cancer cells. Inhibition of BRD4 by JQ1 robustly blocked the expression of TGFBR2 in a dose dependent manner. We successfully mapped a super-enhancer in TGFBR2 by sgRNA. Deletion of the super-enhancer in TGFBR2 (sgTGFBR2-SEΔ) significantly reduced the expression of TGFBR2 in pancreatic cancer cells. TGF-β-induced p-SMAD2/3 was greatly impaired in TGFBR2 super-enhancer deleted cells. Both migration and EMT induced by TGF-β in pancreatic cancer cells were impaired after deleting the super-enhancer of TGFBR2. Our data suggest a novel molecular mechanism by which a super-enhancer regulates TGFBR2, affecting the activity of TGF-β as well as its function in pancreatic cancer progression.

Topics: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Receptor, Transforming Growth Factor-beta Type II; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is intractable due to its strong invasiveness and metastatic ability. Epithelial-mesenchymal transition (EMT) is the pivotal driver of tumor invasion and metastasis. The four-and-a-half LIM domain (FHL) family is involved in regulating transforming growth factor (TGF)-β and Ras signaling, which might control the EMT process. In this study, we found that higher expression of four-and-a-half LIM domains 3 (FHL3) predicted poor prognosis in PDAC. The decreasing of FHL3 changed the EMT phenotype by blocking the TGFβ/Atk/GSK3β/ubiquitin pathways. Interestingly, the GSK3β inhibitor could abrogate the role of FHL3 in the regulation of snail1 and twist1 expression, which implied that GSK3β plays a pivotal role in the FHL3-mediated EMT process. Furthermore, we found that FHL3 can directly bind to GSK3β, which weakened the interaction between GSK3β and snail1/twist1. We also found that the LIM-3 domain of FHL3 was required for the binding of FHL3 to GSK3β. Collectively, our study implied that FHL3, as a binding partner of GSK3β, promoted tumor metastasis in PDAC through inhibiting the ubiquitin-degradation of snail1 and twist1.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Models, Biological; Neoplasm Metastasis; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Ubiquitination

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies. TGF-β is strongly expressed in both the epithelial and stromal compartments of PDAC, and dysregulation of TGF-β signalling is a frequent molecular disturbance in PDAC progression and metastasis. In this study, we investigated whether blockade of TGF-β signalling synergizes with nal-IRI/5-FU/LV, a chemotherapy regimen for malignant pancreatic cancer, in an orthotopic pancreatic tumour mouse model. Compared to nal-IRI/5-FU/LV treatment, combining nal-IRI/5-FU/LV with vactosertib, a TGF-β signalling inhibitor, significantly improved long-term survival rates and effectively suppressed invasion to surrounding tissues. Through RNA-sequencing analysis, we identified that the combination treatment results in robust abrogation of tumour-promoting gene signatures and positive enrichment of tumour-suppressing and apoptotic gene signatures. Particularly, the expression of tumour-suppressing gene Ccdc80 was induced by vactosertib and further induced by vactosertib in combination with nal-IRI/5-FU/LV. Ectopic expression of CCDC80 suppressed migration and colony formation concomitant with decreased expression of epithelial-to-mesenchymal transition (EMT) markers in pancreatic cancer cells. Collectively, these results indicate that combination treatment of vactosertib with nal-IRI/5-FU/LV improves overall survival rates in a mouse model of pancreatic cancer by suppressing invasion through CCDC80. Therefore, combination therapy of nal-IRI/5-FU/LV with vactosertib could provide clinical benefits to pancreatic cancer patients.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinogenesis; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Drug Synergism; Epithelial-Mesenchymal Transition; Fluorouracil; Gene Expression Regulation, Neoplastic; Irinotecan; Leucovorin; Liposomes; Mice, Inbred C57BL; Nanoparticles; Neoplasm Invasiveness; Pancreatic Neoplasms; Signal Transduction; Survival Analysis; Transcriptome; Transforming Growth Factor beta; Triazoles; Tumor Stem Cell Assay; Up-Regulation

Calycosin is a bioactive isoflavonoid of the medicinal plant Astragalus membranaceus that exhibits a wide range of pharmacological properties. In the present study, we have attempted to explore the anti-tumorigenic potential of calycosin in pancreatic cancer.. MTT assay was used to determine cancer cell viability. Cell cycle analysis and detection of apoptosis were performed using flow cytometry. A wound healing assay was employed to study the migratory activity of cancer cells. Western blotting and RT-PCR were used to explore the mechanism by assessing the target proteins and genes. An orthotopic tumor xenograft mouse model was also used to study the drug effects in vivo.. Findings of this study provide innovative insights about the impact of calycosin in pancreatic cancer progression through induction of cell cycle arrest and apoptosis while possessing certain tumor-promoting property by modulation of the tumor microenvironment.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Survival; Epithelial-Mesenchymal Transition; Humans; Isoflavones; Male; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; RAW 264.7 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment; Xenograft Model Antitumor Assays

Transforming growth factor β (TGF-β) signaling pathway is one of the core pathways in pancreatic ductal adenocarcinoma (PDAC). Prognostic value of TGF-β pathway genes as a functionally related group in PDAC is rarely studied.. Seventy-two PDAC patients who underwent surgery between November 30, 2015, and September 13, 2017, in West China Hospital, Sichuan University, were identified and included in this study. Whole-exome sequencing or targeted next-generation sequencing was performed with tumor tissue. Clinicopathologic characteristics and survival data were retrospectively collected and analyzed.. Genetic alterations were detected in 71 patients (98.6%). Although 1 patient (1.4%) had one genetic alteration, 33 patients (45.8%) had 2 to 4 alterations and 37 patients (51.4%) had 5 or more alterations. Twenty-five patients with TGF-β pathway alteration were identified as TGF-βm+ group. Other 47 patients were TGF-βm- group. Mutation of TGF-β pathway was independently associated with inferior survival (hazard ratio, 2.22, 95% confidence interval, 1.05-4.70, P = 0.04), especially in patients accepting radical surgery (hazard ratio, 3.25, 95% confidence interval, 1.01-10.49, P = 0.04).. Inferior prognosis was observed in PDACs with mutations of TGF-β pathway. Genomic information could help screen out patients at risk after surgery, and adjuvant therapy might benefit this subgroup of PDACs.

Topics: Adult; Aged; Asian People; Brachytherapy; Carcinoma, Pancreatic Ductal; DNA, Neoplasm; Exome Sequencing; Female; Genes, Neoplasm; High-Throughput Nucleotide Sequencing; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Mutation; Neoplasm Proteins; Palliative Care; Pancreatic Neoplasms; Prognosis; Proportional Hazards Models; Radiotherapy, Adjuvant; Receptors, Transforming Growth Factor beta; Retrospective Studies; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer of the digestive tract that has a high potential for metastasis and a poor prognosis. Girdin was first reported in 2005 as an actin‑binding protein and was designated as Akt‑phosphorylation enhancer (APE); thus, Girdin has been revealed to have an important role in regulating cancer development. There is additional evidence indicating that Girdin is associated with cell proliferation, migration, invasion and survival in certain cancers. However, the potential mechanisms involving Girdin and mobility in pancreatic cancer have not been elucidated. In the present study, it was revealed that Girdin was highly expressed in pancreatic cancer tissue and was associated with tumor grade. The present study, to the best of our knowledge, is the first aimed at investigating the unknown role of Girdin in PDAC metastasis. A short hairpin RNA for Girdin (sh‑Girdin) was successfully constructed with recombinant adenoviral vectors to suppress the expression of Girdin in pancreatic cancer cell lines (PANC‑1 and BXPC‑3). The silencing efficiency of the Girdin shRNA was determined by RT‑qPCR and western blot analysis, and decreased Girdin expression in the cytoplasm was revealed by immunofluorescence detection. Then, sulforhodamine B (SRB) and colony formation assays were used to confirm that the knockdown of Girdin inhibited proliferation in vitro, and Transwell assays were used to examine the influence of Girdin knockdown on cellular mobility. Animal experiments also confirmed that silencing the expression of Girdin in pancreatic cancer cells inhibited the growth and metastasis of pancreatic cancer in vivo. Transforming growth factor‑β (TGF‑β) is a common inducer of epithelial‑mesenchymal transition (EMT) and can effectively induce EMT in PDAC. Notably, TGF‑β‑treated cells exhibited changes in the classic biological markers of EMT. The expression of E‑cadherin, a marker of the epithelial phenotype, increased, and the expression of N‑cadherin and vimentin, markers of the interstitial phenotype, decreased in response to sh‑Girdin. According to these experiments, Girdin may affect pancreatic cancer progression and development by interacting with vimentin. Therefore, there is evidence indicating that Girdin could be designated as a prognostic biological indicator and a candidate therapeutic target for pancreatic cancer.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Male; Microfilament Proteins; Neoplasm Grading; Neoplasm Metastasis; Pancreatic Neoplasms; Prognosis; Transforming Growth Factor beta; Up-Regulation; Vesicular Transport Proteins; Vimentin

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-β-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

Topics: Amino Acids, Branched-Chain; Cancer-Associated Fibroblasts; Carcinoma, Pancreatic Ductal; Computational Biology; Energy Metabolism; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Keto Acids; Oxidation-Reduction; Pancreatic Neoplasms; Smad5 Protein; Stromal Cells; Transaminases; Transforming Growth Factor beta; Tumor Stem Cell Assay

Pancreatic ductal adenocarcinoma (PDAC) has a dense extracellular matrix (ECM) surrounding tumor cells to sequester CD8+ T cell infiltration and prevent drug penetration. Concomitant inhibition of both the TGF-β pathway and the PD-1/PD-L1 checkpoint is a viable strategy to increase T cell infiltration and cytotoxicity. Here, we used an acidic tumor extracellular pH (pHe) responsive clustered nanoparticle (LYiClustersiPD-L1) to deliver TGF-β receptor inhibitors (LY2157299) and siRNA targeting PD-L1 (siPD-L1) for PDAC stroma microenvironment regulation and antitumor immunotherapy. LY2157299 encapsulated in the hydrophobic core of the nanoparticle can effectively inhibit the activation of pancreatic stellate cells (PSCs) and result in a reduction in type I collagen. siPD-L1 adsorbed on the surface of the nanoparticle was released with small size poly(amidoamine) (PAMAM) at the surface of LYiClustersiPD-L1 under pHe and penetrated into the tumors to silence PD-L1 gene expression in tumor cells. Compared to monotherapy, LYiClustersiPD-L1 significantly increased tumor infiltrating CD8+ T cells and provoked antitumor immunity to synergistically suppress tumor growth in both a subcutaneous Panc02 xenograft model and an orthotopic tumor model.

Topics: Animals; B7-H1 Antigen; Hydrogen-Ion Concentration; Immunotherapy; Nanoparticles; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and the therapeutic outcomes remain undesirable. Increasing evidence shows that aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) plays crucial roles in tumorigenesis of multiple tumors. However, the expression status and functions of ARNTL2 in PDAC remain elusive. Here we showed that ARNTL2 expression was markedly upregulated in PDAC tissues and cell lines. elevated expression of ARNTL2 was positively related to unfavorable prognosis. Knockdown of ARNTL2 could suppress motility and invasive ability of PDAC cells in vitro, as well as tumor development in vivo. In addition, microRNA-26a-5p (miR-26a-5p) was identified as the crucial specific arbitrator for ARNTL2 expression and the expression of miR-26a-5p was inversely correlated with ARNTL2 expression in PDAC tissues. Functionally, elevated expression of miR-26a-5p was found to inhibit the proliferation, migration, and invasion of PDAC cells in vitro, while ARNTL2 increased expression could partially abolish the suppressive effect of miR-26a-5p. Mechanism study indicated that elevated expression of miR-26a-5p suppressed TGF/BETA signaling pathway by targeting ARNTL2 in PDAC cells. In conclusion, our data suggested that ARNTL2 acted as an oncogene to regulate PDAC growth. MiR-26a-5p/ARNTL2 axis may be a novel therapeutic candidate target in PDAC treatment.

Topics: Aged; Aged, 80 and over; Apoptosis; ARNTL Transcription Factors; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; Pancreatic Neoplasms; Prognosis; Transforming Growth Factor beta

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by Kras

Topics: 3-Hydroxysteroid Dehydrogenases; Animals; Atorvastatin; Biosynthetic Pathways; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Line, Tumor; Cholesterol, LDL; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kaplan-Meier Estimate; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cellular plasticity contributes to intratumoral heterogeneity, metastatic spread, and treatment resistance of cancers. In this issue of Cancer Cell, Gabitova-Cornell et al. identify the potential to inadvertently develop an undifferentiated and more aggressive pancreas cancer with agents commonly prescribed to manage heart disease risk.

Topics: Cell Differentiation; Cell Plasticity; Cholesterol; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta

We investigated how pancreatic cancer developed resistance to focal adhesion kinase (FAK) inhibition over time.. Pancreatic ductal adenocarcinoma (PDAC) tumours from KPC mice (p48-CRE; LSL-KRas. Stromal depletion by FAK inhibitor therapy leads to eventual treatment resistance through the activation of STAT3 signalling. These data suggest that, similar to tumour-targeted therapies, resistance mechanisms to therapies targeting stromal desmoplasia may be critical to treatment durability.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Collagen; Down-Regulation; Drug Resistance, Neoplasm; Female; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Humans; Mice, Inbred Strains; Pancreatic Neoplasms; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Stromal Cells; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

B7-1 proteins are routinely expressed on the surface of antigen presenting cells (APC) and within the innate immune system. They function to establish a biologically optimal and dynamic balance between immune activation and inhibition or self-tolerance. Interactions between B7-1 and its receptors, which include CD28, CTLA4 and PD-L1, contribute to both stimulatory as well as inhibitory or homeostatic regulation. In the current study, we investigated whether the tumor-promoting actions of transforming growth factor beta (TGF-β) disrupted this equilibrium in pancreatic cancer to promote malignant progression and an enhanced means to evade immune detection. The data show that B7-1 is (i) upregulated following treatment of pancreatic carcinoma cells with TGF-β; (ii) induced by TGF-β via both Smad2/3-dependent and independent pathways; (iii) required for pancreatic tumor cell in vitro migration/invasion; and (iv) necessary for TGF-β regulated epithelial-mesenchymal transition (EMT) through induction of Snail family members. Results from the proposed studies provide valuable insights into mechanisms whereby TGF-β regulates both the innate immune response and intrinsic properties of pancreatic tumor growth.

Topics: B7-1 Antigen; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Immunomodulation; Neoplasm Invasiveness; Pancreatic Neoplasms; Smad Proteins; Transforming Growth Factor beta

TGFβ is important during pancreatic ductal adenocarcinoma (PDA) progression. Canonical TGFβ signaling suppresses epithelial pancreatic cancer cell proliferation; as a result, inhibiting TGFβ has not been successful in PDA. In contrast, we demonstrate that inhibition of stromal TGFβR2 reduces IL-6 production from cancer-associated fibroblasts, resulting in a reduction of STAT3 activation in tumor cells and reversion of the immunosuppressive landscape. Up to 7% of human PDA have tumor cell-specific deficiency in canonical TGFβ signaling via loss of TGFβR2. We demonstrate that in PDA that harbors epithelial loss of TGFβR2, inhibition of TGFβ signaling is selective for stromal cells and results in a therapeutic benefit. Our study highlights the potential benefit of TGFβ blockade in PDA and the importance of stratifying PDA patients who might benefit from such therapy.

Topics: Carcinoma, Pancreatic Ductal; Cardiomegaly; Humans; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

Cancer can be conceptualized as arising from somatic mutations resulting in a single renegade cell escaping from the constraints of multicellularity. Thus, the era of precision medicine has led to intense focus on the cancer cell to target these mutations that result in oncogenic signaling and sustain malignancy. However, in pancreatic ductal adenocarcinoma (PDAC) there are only four abundantly common driver mutations (KRAS, CDKN2A, TP53, and SMAD4), which are not currently actionable. Thus, precision therapy for PDAC must look beyond the cancer cell. In fact, PDAC is more than a collection of renegade cells, instead representing an extensive, supportive ecosystem, having developed over several years, and consisting of numerous interactions between the cancer cells, normal mesenchymal cells, immune cells, and the dense extracellular matrix. In this issue, Huang and colleagues demonstrate how elucidation of these complex relationships within the tumor microenvironment (TME) can be exploited for therapeutic intervention in PDAC. They identify in a subset of PDAC with mutations in TGFβ signaling, that a paracrine signaling axis can be abrogated to modulate the TME and improve outcomes.

Topics: Carcinoma, Pancreatic Ductal; Ecosystem; Humans; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is poorly responsive to therapies and histologically contains a paucity of neoplastic cells embedded within a dense desmoplastic stroma. Within the stroma, cancer-associated fibroblasts (CAF) secrete tropic factors and extracellular matrix components, and have been implicated in PDAC progression and chemotherapy resistance. We recently identified two distinct CAF subtypes characterized by either myofibroblastic or inflammatory phenotypes; however, the mechanisms underlying their diversity and their roles in PDAC remain unknown. Here, we use organoid and mouse models to identify TGFβ and IL1 as tumor-secreted ligands that promote CAF heterogeneity. We show that IL1 induces LIF expression and downstream JAK/STAT activation to generate inflammatory CAFs and demonstrate that TGFβ antagonizes this process by downregulating IL1R1 expression and promoting differentiation into myofibroblasts. Our results provide a mechanism through which distinct fibroblast niches are established in the PDAC microenvironment and illuminate strategies to selectively target CAFs that support tumor growth. SIGNIFICANCE: Understanding the mechanisms that determine CAF heterogeneity in PDAC is a prerequisite for the rational development of approaches that selectively target tumor-promoting CAFs. Here, we identify an IL1-induced signaling cascade that leads to JAK/STAT activation and promotes an inflammatory CAF state, suggesting multiple strategies to target these cells

Topics: Animals; Apoptosis; Cancer-Associated Fibroblasts; Carcinoma, Pancreatic Ductal; Cell Proliferation; Female; Humans; Inflammation; Interleukin-1; Janus Kinase 1; Mice; Mice, Nude; Mutation; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Signal Transduction; STAT1 Transcription Factor; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

The metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), plays multifaceted roles in inhibiting oncogenic signaling and can suppress the epithelial mesenchymal transition (EMT), a key step in metastasis. In this investigation, NDRG1 inhibited the oncogenic effects of transforming growth factor-β (TGF-β) in PANC-1 pancreatic cancer cells, promoting expression and co-localization of E-cadherin and β-catenin at the cell membrane. A similar effect of NDRG1 at supporting E-cadherin and β-catenin co-localization at the cell membrane was also demonstrated for HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG and ZEB1. To dissect the mechanisms how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the nuclear factor-κB (NF-κB) pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signaling at multiple sites in this pathway, suppressing NEMO, Iĸĸα and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/β and IĸBα. NDRG1 also reduced the levels, nuclear co-localization and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-κB and TGF-β signaling to upregulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering this, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anticancer agents, namely di-2-pyridylketone thiosemicarbazones, which upregulate NDRG1. These agents downregulated SNAIL, SLUG and ZEB1 in vitro and in vivo using a PANC-1 tumor xenograft model, demonstrating their marked potential.

Topics: Antigens, CD; Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Humans; Intracellular Signaling Peptides and Proteins; Neoplasm Metastasis; NF-kappa B; Pancreatic Neoplasms; RNA, Messenger; Signal Transduction; Thiosemicarbazones; Transforming Growth Factor beta

State-of-the-art genomic analyses of pancreatic adenocarcinoma (PDAC) have yielded insight into signaling pathways underlying carcinogenesis. PDAC is characterized by substantial genomic heterogeneity. We aimed to determine whether early-onset PDAC (EOPC; ≤55 years) displays a distinctive molecular landscape from average-age onset PDAC (AOPC; ≥70 years).. Three distinct datasets for PDAC were analyzed. In the first, patients undergoing treatment at Memorial Sloan Kettering (MSK) were consented for MSK-IMPACT next-generation sequencing. The second cohort analyzed was The Cancer Genome Atlas (TCGA) dataset for differences in somatic mutations, gene expression, and protein expression. The third dataset was an Australian cohort of PDAC. Clinical data were correlated with genomic analyses.. A total of 293 samples were analyzed, yielding 90 patients aged ≤55 years and 203 patients aged ≥70 years. Among the genes known to be associated with carcinogenesis,. These exploratory analyses suggest that there may be somatic gene alterations within the population of patients with early-onset PDAC that involve unique cellular pathways compared with average-onset PDAC. Former studies imply these cellular pathways may play a role in smoking-related PDAC carcinogenesis. Larger genomic datasets are warranted for future evaluation to extend these observations.

Topics: Adenocarcinoma; Adult; Age Factors; Aged; Aged, 80 and over; Biomarkers; Cohort Studies; Female; Genetic Predisposition to Disease; Genome-Wide Association Study; Genomics; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; SEER Program; Signal Transduction; Transforming Growth Factor beta

IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to immunosuppression by driving differentiation of myeloid suppressor cells together with TGF-β. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40 stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mechanisms of LOAd713, an oncolytic adenovirus designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected pancreatic cancer cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-β-1 and collagen type I. Virus infection prevented IL-6/GM-CSF-mediated differentiation of myeloid suppressors, but not CD163 macrophages, whereas infection of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-γ. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer.

Topics: Adenoviridae; Animals; B7-H1 Antigen; CD40 Ligand; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Collagen Type I; Dendritic Cells; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Interleukin-6; Leucine Zippers; Lymphocyte Activation; Melanoma, Experimental; Mice; Oncolytic Virotherapy; Pancreatic Neoplasms; Pancreatic Stellate Cells; Programmed Cell Death 1 Receptor; Receptors, Interleukin-6; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

We develop a mathematical model of pancreatic cancer that includes pancreatic cancer cells, pancreatic stellate cells, effector cells and tumor-promoting and tumor-suppressing cytokines to investigate the effects of immunotherapies on patient survival. The model is first validated using the survival data of two clinical trials. Local sensitivity analysis of the parameters indicates there exists a critical activation rate of pro-tumor cytokines beyond which the cancer can be eradicated if four adoptive transfers of immune cells are applied. Optimal control theory is explored as a potential tool for searching the best adoptive cellular immunotherapies. Combined immunotherapies between adoptive ex vivo expanded immune cells and TGF-[Formula: see text] inhibition by siRNA treatments are investigated. This study concludes that mono-immunotherapy is unlikely to control the pancreatic cancer and combined immunotherapies between anti-TGF-[Formula: see text] and adoptive transfers of immune cells can prolong patient survival. We show through numerical explorations that how these two types of immunotherapies are scheduled is important to survival. Applying TGF-[Formula: see text] inhibition first followed by adoptive immune cell transfers can yield better survival outcomes.

Topics: CD8-Positive T-Lymphocytes; Combined Modality Therapy; Computer Simulation; Cytokines; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Mathematical Concepts; Models, Immunological; Nonlinear Dynamics; Pancreatic Neoplasms; RNA, Small Interfering; Transforming Growth Factor beta

LASP2 (LIM and SH3 protein 2), a member of the LIM-protein subfamily of the nebulin group, was first identified as a splice variant of the nebulin gene. In the past, investigators mainly focused on the impact of LASP2 on cardiac diseases because of its identification in the myocardium. Recently, several studies have reported that LASP2 is associated with the progression of various cancers. However, there have been no investigations on the expression and function of LASP2 in pancreatic cancer (PC). In this study, we performed the quantitative real-time polymerase chain reaction and Western blot analysis to detect the expression of LASP2 in PC tissues and cell lines. PC cells were transfected with LASP2 overexpression plasmid or the negative control in the presence or absence of tumor growth factor-β (TGF-β). The transwell assays were used to measure the effects of LASP2 on PC cell migration and invasion. The protein expression of epithelial-mesenchymal transition (EMT) markers was detected using Western blot assay. Our results demonstrated that LASP2 was downregulated in PC tissues and cell lines. In addition, upregulation of LASP2 inhibited the PC cell migration and invasion. We also found that LASP2 upregulation reversed TGF-β-induced EMT in PC cells. Taken together, we provided novel evidence supporting the tumor-suppressor role of LASP2 in PC and suggested it as a potential therapeutic target in PC treatment.

Topics: Carrier Proteins; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; LIM Domain Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Transforming Growth Factor beta; Up-Regulation

Pancreatic cancer is a highly aggressive malignancy that strongly resists extant treatments. The failure of existing therapies is majorly attributed to the tough tumor microenvironment (TME) limiting drug access and the undruggable targets of tumor cells. The formation of suppressive TME is regulated by transforming growth factor beta (TGF-β) signaling, while the poor response and short survival of almost 90% of pancreatic cancer patients results from the oncogenic KRAS mutation. Hence, simultaneously targeting both the TGF-β and KRAS pathways might dismantle the obstacles of pancreatic cancer therapy. Here, a novel sequential-targeting strategy is developed, in which antifibrotic fraxinellone-loaded CGKRK-modified nanoparticles (Frax-NP-CGKRK) are constructed to regulate TGF-β signaling and siRNA-loaded lipid-coated calcium phosphate (LCP) biomimetic nanoparticles (siKras-LCP-ApoE3) are applied to interfere with the oncogenic KRAS. Frax-NP-CGKRK successfully targets the tumor sites through the recognition of overexpressed heparan sulfate proteoglycan, reverses the activated cancer-associated fibroblasts (CAFs), attenuates the dense stroma barrier, and enhances tumor blood perfusion. Afterward, siKras-LCP-ApoE3 is efficiently internalized by the tumor cells through macropinocytosis and specifically silencing KRAS mutation. Compared with gemcitabine, this sequential-targeting strategy significantly elongates the lifespans of pancreatic tumor-bearing animals, hence providing a promising approach for pancreatic cancer therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apolipoprotein E3; Benzofurans; Drug Administration Schedule; Drug Carriers; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Molecular Targeted Therapy; Mutation; Nanoparticles; NIH 3T3 Cells; Pancreatic Neoplasms; Peptide Fragments; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Herein, we report that the TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.

Topics: Activin Receptors, Type I; Activins; Animals; Apoptosis; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Transformation, Neoplastic; Female; Heterografts; Homeostasis; Humans; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Mice, SCID; Neoplasm Metastasis; Neoplasms; Pancreatic Neoplasms; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Tumor Microenvironment

BAG3 is constitutively expressed in multiple types of cancer cells and its high expression is associated with tumour progression and poor prognosis of PDAC. However, little is known about the role of BAG3 in the regulation of stromal microenvironment of PDAC. The current study demonstrated that beside PDAC tumour cells, BAG3 was also expressed in some activated stroma cells in PDAC tissue, as well as in activated PSCs. In addition, the current study demonstrated that BAG3 expression in PSCs was involved in maintenance of PSCs activation and promotion of PDACs invasion via releasing multiple cytokines. The current study demonstrated that BAG3-positive PSCs promoted invasion of PDACs via IL-8, MCP1, TGF-β2 and IGFBP2 in a paracrine manner. Furthermore, BAG3 sustained PSCs activation through IL-6, TGF-β2 and IGFBP2 in an autocrine manner. Thereby, the current study provides a new insight into the involvement of BAG3 in remodelling of stromal microenvironment favourable for malignant progression of PDAC, indicating that BAG3 might serve as a potential target for anti-fibrosis of PDAC.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL2; Cytokines; Humans; Immunohistochemistry; Insulin-Like Growth Factor Binding Proteins; Interleukin-6; Interleukin-8; Pancreatic Neoplasms; Pancreatic Stellate Cells; Transforming Growth Factor beta; Tumor Microenvironment

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Topics: Adenocarcinoma; Animals; CD8-Positive T-Lymphocytes; Cell Line; Cyclooxygenase 2; Ephrin-A2; Female; Gene Deletion; Humans; Immunosuppression Therapy; Immunotherapy; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Neoplasms; Receptor, EphA2; Transforming Growth Factor beta

Given its aggressive tumor biology and its exceptional therapy resistance, pancreatic ductal adenocarcinoma (PDAC) remains a major challenge in cancer medicine and is characterized by a 5-year survival rate of <8%. At the cellular level, PDAC is largely driven by the activation of signaling pathways that eventually converge in altered, tumor-promoting transcription programs. In this study, we sought to determine the interplay between transforming growth factor β (TGFβ) signaling and activation of the inflammatory transcription factor nuclear factor of activated T cells (NFATc1) in the regulation of transcriptional programs throughout PDAC progression. Genome-wide transcriptome analysis and functional studies performed in primary PDAC cells and transgenic mice linked nuclear NFATc1 expression with pro-proliferative and anti-apoptotic gene signatures. Consistently, NFATc1 depletion resulted in downregulation of target genes associated with poor PDAC outcome and delayed pancreatic carcinogenesis in vivo. In contrast to previous reports and consistent with a concept of retained tumor suppressive TGFβ activity, even in established PDAC, TGFβ treatment reduced PDAC cell proliferation and promoted apoptosis even in the presence of oncogenic NFATc1. However, combined TGFβ treatment and NFATc1 depletion resulted in a tremendous abrogation of tumor-promoting gene signatures and functions. Chromatin studies implied that TGFβ-dependent regulators compete with NFATc1 for the transcriptional control of jointly regulated target genes associated with an unfavorable PDAC prognosis. Together, our findings suggest opposing consequences of TGFβ and NFATc1 activity in the regulation of pro-tumorigenic transcription programs in PDAC and emphasize the strong context-dependency of key transcription programs in the progression of this devastating disease.

Topics: Animals; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chromatin; Chromatin Immunoprecipitation; Disease Progression; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Ontology; Humans; Mice; Mice, Transgenic; NFATC Transcription Factors; Pancreatic Neoplasms; Prognosis; RNA-Seq; Signal Transduction; Transcriptome; Transforming Growth Factor beta

The aim of this study was to investigate the effects of retinoid X receptor α (RXRα) on the proliferation and apoptosis of pancreatic cancer cells through the transforming growth factor-β (TGF-β)/Smad signaling pathway.. The expression of RXRα in pancreatic cancer tissues and para-carcinoma tissues was detected via immunohistochemistry. Human pancreatic cancer PANC-1 cells were cultured and treated with RXRα in vitro. The apoptosis rate of cells was detected via flow cytometry. Furthermore, changes in the protein expression level of TGF-β/Smad signaling pathway were detected via Western blotting.. The protein expression level of RXRα in pancreatic cancer tissues was significantly higher than that of para-carcinoma tissues. RXRα significantly promoted the proliferation and inhibited the apoptosis of pancreatic cancer cells. Moreover, RXRα could also activate the TGF-β/Smad signaling pathway.. RXRα promotes the proliferation and inhibits the apoptosis of pancreatic cancer cells through the TGF-β/Smad signaling pathway.

Topics: Apoptosis; Cell Proliferation; Cell Survival; Gene Expression Profiling; Humans; Pancreatic Neoplasms; Retinoid X Receptor alpha; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Exosomes from cancer cells or immune cells, carrying bio-macromolecules or microRNAs (miRNAs), participate in tumor pathogenesis and progression by modulating microenvironment. Our study aims to investigate the role of these microRNA-501-3p (miR-501-3p) containing exosomes derived from tumor-associated macrophage (TAM) in the progression of pancreatic ductal adenocarcinoma (PDAC).. Firstly, the function of TAM recruitment in PDAC tissues was assessed, followed by identification of the effects of M2 macrophage-derived exosomes on PDAC cell activities and tumor formation and metastasis in mice. In silico analysis was conducted to predict differentially expressed genes and regulatory miRNAs related to PDAC treated with macrophages, which determined miR-501-3p and TGFBR3 for subsequent experiments. Next, gain- and loss-of-function experiments were performed to examine their role in PDAC progression with the involvement of the TGF-β signaling pathway.. TAM recruitment in PDAC tissues was associated with metastasis. Highly expressed miR-501-3p was observed in PDAC tissues and TAM-derived exosomes. Both M2 macrophage-derived exosomes and miR-501-3p promoted PDAC cell migration and invasion, as well as tumor formation and metastasis in nude mice. MiR-501-3p was verified to target TGFBR3. PDAC cells presented with down-regulated TGFBR3, which was further decreased in response to M2 macrophage treatment. TGF-β signaling pathway activation was implicated in the promotion of miR-501-3p in PDAC development. The suppression of macrophage-derived exosomal miR-501-3p resulted in the inhibition of tumor formation and metastasis in vivo.. M2 macrophage-derived exosomal miR-501-3p inhibits tumor suppressor TGFBR3 gene and facilitates the development of PDAC by activating the TGF-β signaling pathway, which provides novel targets for the molecular treatment of PDAC.

Topics: Adult; Aged; Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Exosomes; Female; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; Neoplasm Transplantation; Pancreatic Neoplasms; Proteoglycans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Pancreatic cancer, composed of heterogeneous cancer cells, alters epithelial to mesenchymal features during growth and metastasis. In this study, we aimed to characterize pancreatic ductal adenocarcinoma (PDAC) cells showing epithelial or mesenchymal features in 3D culture. In 3D culture, PK-1 cells had high E-cadherin and low vimentin expression and exhibited a round-like appearance encircled by flat cells. PANC-1 cells had high vimentin and low E-cadherin expression and formed grape-like spheres. PK-1 cells had secretary granules and many microvilli, desmosomes, and adherens junctions, while PANC-1 cells had few microvilli, adherens junction, and no desmosomes. Cytokeratin 7, trypsin, CA19-9, and E-cadherin were highly expressed in PK-1 cells but not in PANC-1 cells. Ki-67 was diffusely expressed in PANC-1 spheres but was restricted to the peripheral flat cells of PK-1 spheres. PANC-1 and PK-1 cells were positive for transforming growth factor (TGF) β receptor II and phosphorylated smad2/3, but PK-1 cells were smad4 negative. Taken together, 3D culture enhanced morphofunctional differences of PDAC cells showing epithelial or mesenchymal characteristics, and epithelial phenotype maintenance may be due to the ineffectiveness of the TGF- β pathway. Clarification of heterogeneity using 3D culture may be useful for development of individualized diagnostic and therapeutic methods in patients with PDAC.

Topics: Antigens, Tumor-Associated, Carbohydrate; Cadherins; Carcinoma, Pancreatic Ductal; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Shape; Cell Transformation, Neoplastic; Desmosomes; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Keratin-7; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Vimentin

Phenotypic heterogeneity in cancer cells is widely observed and is often linked to drug resistance. In several cases, such heterogeneity in drug sensitivity of tumors is driven by stochastic and reversible acquisition of a drug tolerant phenotype by individual cells even in an isogenic population. Accumulating evidence further suggests that cell-fate transitions such as the epithelial to mesenchymal transition (EMT) are associated with drug resistance. In this study, we analyze stochastic models of phenotypic switching to provide a framework for analyzing cell-fate transitions such as EMT as a source of phenotypic variability in drug sensitivity. Motivated by our cell-culture based experimental observations connecting phenotypic switching in EMT and drug resistance, we analyze a coarse-grained model of phenotypic switching between two states in the presence of cytotoxic stress from chemotherapy. We derive analytical results for time-dependent probability distributions that provide insights into the rates of phenotypic switching and characterize initial phenotypic heterogeneity of cancer cells. The results obtained can also shed light on fundamental questions relating to adaptation and selection scenarios in tumor response to cytotoxic therapy.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Models, Genetic; Oxaliplatin; Pancreatic Neoplasms; Phenotype; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma is one of the most aggressive cancers, with a 5-year survival rate of less than 8%. The complicated tumor microenvironment, particularly TGF-β, provides possible convenience for the progression of PC cells. TGF-β regulates critical cellular processes, including autophagy. However, the mechanism and effects of TGF-β-mediated autophagy are still poorly understood.. Bioinformatics analysis, western blot, transmission electron microscopy and confocal microscopy were used to identify that TFEB is the key factors in TGF-β-induced autophagy. The biological effects of TFEB-driven autophagy were investigated in vitro using transwell and wound healing assays and in vivo using liver metastasis and LSL-KrasG12D/Pdx1-Cre mice models. Luciferase assays and motif analysis were used to assess regulation of RAB5A gene promoter activity by TGF-β-induced TFEB. TFEB levels were measured by real-time PCR, western blot and immunohistochemical staining in clinical pancreatic ductal adenocarcinoma tissues.. We demonstrated that TGF-β induces TFEB expression via the canonical smad pathway in Smad4-positive PC cells and facilitates TFEB-mediated autophagic activation. TFEB-driven autophagy caused by TGF-β regulates RAB5A-dependent endocytosis of Itgα5 and promotes progression of PC cells. We further showed that enhanced TFEB expression and its direct target RAB5A both predict poor prognosis in PC patients.. Our findings reveal TFEB-driven autophagy is required for TGF-β induced migration and metastasis of PC cells by promoting endocytosis of Itgα5β1 and focal adhesion disassembly through the TGF-β-TFEB-RAB5A axis. Our results highlight the potential utility of suppressing TFEB-driven autophagy to block PC metastasis.

Topics: Animals; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Endocytosis; Female; Gene Expression; Humans; Integrins; Mice; Mice, Transgenic; Pancreatic Neoplasms; RNA Interference; Smad Proteins; Transforming Growth Factor beta

Resident fibroblasts that contact tumor epithelial cells (TEC) can become irreversibly activated as cancer-associated-fibroblasts (CAF) that stimulate oncogenic signaling in TEC. In this study, we evaluated the cross-talk between CAF and TEC isolated from tumors generated in a mouse model of KRAS/mut p53-induced pancreatic cancer (KPC mice). Transcriptomic profiling conducted after treatment with the anticancer compound Minnelide revealed deregulation of the TGFβ signaling pathway in CAF, resulting in an apparent reversal of their activated state to a quiescent, nonproliferative state. TEC exposed to media conditioned by drug-treated CAFs exhibited a decrease in oncogenic signaling, as manifested by downregulation of the transcription factor Sp1. This inhibition was rescued by treating TEC with TGFβ. Given promising early clinical studies with Minnelide, our findings suggest that approaches to inactivate CAF and prevent tumor-stroma cross-talk may offer a viable strategy to treat pancreatic cancer.

Topics: Animals; Apoptosis; Cancer-Associated Fibroblasts; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Proliferation; Disease Models, Animal; Diterpenes; Epithelial Cells; Epoxy Compounds; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred C57BL; Mutation; Organophosphates; Pancreatic Neoplasms; Phenanthrenes; Proto-Oncogene Proteins p21(ras); Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

In this study, we investigated the ability of pancreatic cancer cell lines to form spheroids with the aim of identifying factors involved in cell invasiveness, a property that leads to a poor prognosis in pancreatic cancer. For this purpose, 8 cell lines derived from human pancreatic cancer tissues were cultured in non-adherent culture conditions to form spheroids, as well as normal monolayers. The morphology of the cells was observed and spheroid diameters measured. mRNA expression was compared between cells cultured under both culture conditions. The gene knockdown of endoglin (ENG) and SMAD4, components of the transforming growth factor-β (TGF-β) signaling system, using siRNAs was conducted in spheroids in order to identify affected protein signaling factors, determine the morphological changes occurring over time and to measure the invasive capacity of the cells constituting spheroids. The cell lines exhibited differences in their spheroid-forming abilities. The expression of SMAD4 and ENG concomitantly increased in the cells that formed spheroids. SMAD4 was transported into the nucleus when spheroids were formed. The expression of ENG was decreased in the cells in which SMAD4 was knocked down; by contrast, the expression of BMP and activin membrane-bound inhibitor (BAMBI) and noggin (NOG), further components of the TGF-β signaling system, increased. In the cells in which ENG was knocked down, the decreased mRNA expression of TGF-β receptor type 2 (TGFBR2) and SMAD9 was observed, as well as a change in the expression of pSMAD1/5/9, and a tendency of spheroids to decrease in size. Spheroids cultured on Matrigel exhibited a tendency towards a reduction in size over time, as well as a tendency to invade into the Matrigel. In particular, the cells in which ENG was knocked down exhibited spheroids which were reduced in size, and also exhibited an increase in invasiveness, and a decrease in adhesiveness. Thus, our data indicate that in pancreatic cancer cells, the expression of ENG may be controlled by a pathway mediated by SMAD4. In addition, ENG was found to be related to the spheroid-forming ability of cells and to be involved in the invasive capacity of pancreatic cancer cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Nucleus; Endoglin; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; Protein Transport; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Spheroids, Cellular; Transforming Growth Factor beta

Pancreatic stellate cells (PSCs) are the precursors of cancer-associated fibroblasts (CAFs), which potentiate pancreatic tumor growth and progression. In this study, we investigated whether Lipoxin A4 (LXA4), an endogenous bioactive lipid, can inhibit the differentiation of human PSCs (hPSCs) into CAF-like myofibroblasts and thereby hPSC-induced pro-tumorigenic effects. LXA4 significantly inhibited TGF-β-mediated differentiation of hPSCs by inhibiting pSmad2/3 signalling. Furthermore, treatment with LXA4 abolished the paracrine effects (proliferation and migration of Panc-1 tumor cells) of hPSCs in vitro. These data demonstrated that LXA4 can interrupt pro-tumoral paracrine signalling of hPSCs. Furthermore, LXA4 treatment significant decreased the size and growth rate of 3D-heterospheroids comprised of hPSC and Panc-1 and these effects were exhibited due to inhibition of hPSC-induced collagen1 expression. In vivo, we examined the therapeutic efficacy of LXA4 in a co-injection (Panc-1 and hPSCs) subcutaneous tumor model. Intriguingly, LXA4 significantly abolished the tumor growth (either injected intratumor or intraperitoneally), attributed to a significant reduction in fibrosis, shown with collagen1 expression. Altogether, this study proposes LXA4 as a potent inhibitor for hPSCs which can be applied to reprogram tumor stroma in order to treat pancreatic cancer.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cellular Reprogramming; Collagen Type I; Combined Modality Therapy; Gene Expression Regulation, Neoplastic; Humans; Lipoxins; Mice; Pancreatic Neoplasms; Pancreatic Stellate Cells; Paracrine Communication; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Tumor microenvironment consists of the extracellular matrix (ECM), stromal cells, such as fibroblasts (FBs) and cancer associated fibroblasts (CAFs), and a myriad of soluble factors. In many tumor types, including pancreatic tumors, the interplay between stromal cells and the other tumor microenvironment components leads to desmoplasia, a cancer-specific type of fibrosis that hinders treatment. Transforming growth factor beta (TGF-β) and CAFs are thought to play a crucial role in this tumor desmoplastic reaction, although the involved mechanisms are unknown.. Optical/fluorescence microscopy, atomic force microscopy, image processing techniques, invasion assay in 3D collagen I gels and real-time PCR were employed to investigate the effect of TGF-β on normal pancreatic FBs and CAFs with regard to crucial cellular morphodynamic characteristics and relevant gene expression involved in tumor progression and metastasis.. CAFs present specific myofibroblast-like characteristics, such as α-smooth muscle actin expression and cell elongation, they also form more lamellipodia and are softer than FBs. TGF-β treatment increases cell stiffness (Young's modulus) of both FBs and CAFs and increases CAF's (but not FB's) elongation, cell spreading, lamellipodia formation and spheroid invasion. Gene expression analysis shows that these morphodynamic characteristics are mediated by Rac, RhoA and ROCK expression in CAFs treated with TGF-β.. TGF-β modulates CAFs', but not FBs', cell shape, stiffness and invasion.. Our findings elucidate on the effects of TGF-β on CAFs' behavior and stiffness providing new insights into the mechanisms involved.

Topics: Cancer-Associated Fibroblasts; Cell Shape; Elastic Modulus; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Pseudopodia; Stress Fibers; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Pancreatic cancer is the fourth leading cause of cancer-associated mortality worldwide, with an overall 5-year survival rate <8%. We studied the therapeutic effect of itraconazole (ITZ), a commonly used broad-spectrum anti-fungal agent, in the treatment of pancreatic cancer, and to reveal the underlying anticancer mechanisms. Effects of ITZ on cell proliferation, apoptosis, invasion and migration were observed by MTT assays and colony formation assays, flow cytometry, wound scratch assays and transwell assays, respectively. Western blotting and immunofluorescence were performed to investigate the effect of ITZ on the epithelial to mesenchymal transition (EMT) of pancreatic cancer cells. Recombinant transforming growth factor-β (TGF-β) and TGF-β neutralizing antibody were used to study the effect of ITZ on the TGF-β/SMAD2/3 signaling. Transgenic engineered mice which harboring the spontaneous pancreatic cancer was applied to investigate the therapeutic role of ITZ in vivo. We report that ITZ inhibited the viability and induced apoptosis of pancreatic cancer cells. Furthermore, ITZ suppressed the invasion and migration of pancreatic cancer cells. We found that ITZ treatment was efficient in suppressing EMT and that the effect of ITZ was partially mediated by impaired TGF-β/SMAD2/3 signaling. The role of TGF-β/SMAD2/3 signaling in mediating the effect of ITZ was confirmed based on the results that recombinant TGF-β induced, but the TGF-β neutralizing antibody inhibited EMT as well as the invasion and migration of pancreatic cancer cells. Also, the anticancer effect of ITZ could be partially reversed by recombinant TGF-β. Furthermore, treatment with ITZ suppressed growth of tumor in vivo. Taken together, we suggest that ITZ may potentially serve as a new chemotherapeutic agent for the treatment of pancreatic cancer.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Itraconazole; Mice; Mice, Transgenic; Neoplasm Invasiveness; Pancreatic Neoplasms; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Recent studies have revealed that overexpression of long non‑coding RNA (lncRNA) PVT1 is correlated with several types of cancer. However, its role in pancreatic cancer development remains to be clarified. In the present study, we found that PVT1 promoted the growth and the epithelial‑mesenchymal transition (EMT) of pancreatic cancer cells. We first determined that PVT1 was upregulated in pancreatic cancer tissues compared with adjacent normal tissues. Knockdown of PVT1 inhibited viability, adhesion, migration and invasion. Furthermore, PVT1 knockdown reduced the expression of mesenchymal markers including Snail, Slug, β‑catenin, N‑cadherin and vimentin, while it increased epithelial marker expression of E‑cadherin. Finally, knockdown of PVT1 inhibited the TGF‑β/Smad signaling, including p‑Smad2/3 and TGF‑β1 but enhanced the expression of Smad4. In contrast, overexpression of PVT1 reversed the process. These findings revealed that PVT1 acts as an oncogene in pancreatic cancer, possibly through the regulation of EMT via the TGF‑β/Smad pathway and PVT1 may serve as a potential target for diagnostics and therapeutics in pancreatic cancer.

Topics: Biomarkers, Tumor; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; RNA, Long Noncoding; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced tumor responses even in patients with refractory diseases. However, the paucity of antigens that are tumor selective has resulted, on occasion, in "on-target, off-tumor" toxicities. To address this issue, we developed an approach to render T cells responsive to an expression pattern present exclusively at the tumor by using a trio of novel chimeric receptors. Using pancreatic cancer as a model, we demonstrate how T cells engineered with receptors that recognize prostate stem cell antigen, TGFβ, and IL4, and whose endodomains recapitulate physiologic T-cell signaling by providing signals for activation, costimulation, and cytokine support, produce potent antitumor effects selectively at the tumor site. In addition, this strategy has the benefit of rendering our cells resistant to otherwise immunosuppressive cytokines (TGFβ and IL4) and can be readily extended to other inhibitory molecules present at the tumor site (e.g., PD-L1, IL10, and IL13).

Topics: Animals; Antigens, Neoplasm; Cell Line, Tumor; Cell Survival; Genetic Engineering; GPI-Linked Proteins; Humans; Immunotherapy, Adoptive; Interleukin-4; Lymphocyte Activation; Mice; Neoplasm Proteins; Organ Specificity; Pancreatic Neoplasms; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

We studied the role of cytokines TGF-β and TNFα in reduction of the cytolytic activity of NK cells towards tumor cells. Exogenous TGF-β and TNFα reduced the sensitivity of MiaPaCa2 pancreatic adenocarcinoma cells to NK cytotoxicity, which was associated with reduction of ULBP-1 expression and increase of HLA-E, HLA-G, CD155, and CD112 expression on Mia-PaCa2 cells. Changes in the expression of ligands for NK receptors on tumor cells induced by TGF-β and TNFα may contribute to reduction of cytotoxicity of tumor-associated NK cells and thus prevent an adequate antitumor immune response leading to the disease progress.

Topics: Adenocarcinoma; Cell Line, Tumor; Cytotoxicity, Immunologic; Drug Interactions; Histocompatibility Antigens Class I; Humans; Killer Cells, Natural; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Distant metastasis to liver, lung, brain, or bone occurs by circulating tumor cells (CTC). We hypothesized that a subset of CTC had features that are more malignant than tumor cells at the primary site. We established a highly malignant cell line, Panc-1-CTC, derived from the human pancreatic cancer cell line Panc-1 using an in vivo selection method. Panc-1-CTC cells showed greater migratory and invasive abilities than its parent cell line in vitro. In addition, Panc-1-CTC cells had a higher tumor-forming ability than parent cells in vivo. To examine whether a difference in malignant phenotypes exists between Panc-1-CTC cells and parent cells, we carried out comprehensive gene expression array analysis. As a result, Panc-1-CTC significantly expressed transforming growth factor beta-induced (TGFBI), an extracellular matrix protein, more abundantly than did parent cells. TGFBI is considered to regulate cell adhesion, but its functions remain unclear. In the present study, knockdown of TGFBI reduced cell migration and invasion abilities, whereas overexpression of TGFBI increased both abilities. Moreover, elevated expression of TGFBI was associated with poor prognosis in patients with pancreatic cancer.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Transplantation; Neoplastic Cells, Circulating; Pancreatic Neoplasms; Prognosis; Survival Analysis; Transforming Growth Factor beta; Up-Regulation

Human surfactant protein-D (SP-D), an innate immune pattern recognition soluble factor, is known to modulate a range of cytokines and chemokines, such as TNF-α and TGF-β at mucosal surfaces during infection, allergy, and inflammation. A recent study has shown that treatment with a recombinant fragment of human SP-D (rfhSP-D) for 48 h induces apoptosis in pancreatic cancer cells. Our hypothesis is that at earlier time points, SP-D can also influence key cytokines as a part of its putative role in the immune surveillance against pancreatic cancer, where the inflammatory tumor microenvironment contributes to the epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. Here, we provide the first evidence that rfhSP-D can suppress the invasive-mesenchymal properties of highly aggressive pancreatic cancer cells. Mechanistically, rfhSP-D inhibited TGF-β expression in a range of pancreatic cancer cell lines, Panc-1, MiaPaCa-2, and Capan-2, thereby reducing their invasive potential. Smad2/3 expression diminished in the cytoplasm of rfhSP-D-treated cells as compared to the untreated control, suggesting that an interrupted signal transduction negatively affected the transcription of key mesenchymal genes. Thus, expressions of Vimentin, Zeb1, and Snail were found to be downregulated upon rfhSP-D treatment in the pancreatic cancer cell lines. Furthermore, blocking TGF-β with neutralizing antibody showed similar downregulation of mesenchymal markers as seen with rfhSP-D treatment. This study highlights yet another novel innate immune surveillance role of SP-D where it interferes with EMT induction by attenuating TGF-β pathway in pancreatic cancer.

Topics: Biomarkers; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Pulmonary Surfactant-Associated Protein D; Recombinant Proteins; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs), are the key effector cells in pancreatic ductal adenocarcinoma (PDAC), known to induce tumor growth and progression. Pancreatic stellate cells (PSCs) are the precursors of CAFs in PDAC that secrete abundant extracellular matrix, growth factors and cytokines. In this study, we targeted human relaxin-2 (RLX), an endogenous hormone, to PSCs to inhibit their differentiation into CAF-like myofibroblasts. RLX significantly inhibited TGF-β induced PSCs differentiation by inhibiting pSmad2 signaling pathway. In vitro in primary human PSCs (hPSCs), treatment with RLX dose-dependently inhibited the migration, contraction, and protein expression of alpha smooth muscle actin and collagen I These data demonstrate that RLX can regulate hPSCs activation in vitro. However, RLX has several drawbacks i.e. poor pharmacokinetics and systemic vasodilation, that limits its preclinical and clinical application. Thus, we designed and successfully synthesized a nanoparticle system by chemically conjugating RLX to superparamagnetic iron oxide nanoparticle (SPION) to improve its pharmacokinetics. Interestingly, we found RLX-SPION to be more efficacious compared to free RLX in vitro. Significantly, we observed RLX-SPION retarded the tumor growth by itself and also potentiated the effect of gemcitabine in a subcutaneous co-injection (Panc1 and hPSCs) tumor model. The treatment resulted in significant inhibition in tumor growth, which was attributed to reduced collagen I (ECM), desmin (hPSC marker) and CD31 (endothelial marker) expression. In contrast, free RLX showed no significant effects. Altogether, this study presents a novel therapeutic approach against tumor stroma using RLX-SPION to achieve an effective treatment against pancreatic tumor.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Differentiation; Cells, Cultured; Deoxycytidine; Ferric Compounds; Fibrosis; Gemcitabine; Humans; Male; Mice, SCID; Nanoparticles; Pancreatic Neoplasms; Pancreatic Stellate Cells; Receptors, G-Protein-Coupled; Receptors, Peptide; Relaxin; Transforming Growth Factor beta; Treatment Outcome

Pancreatic cancer remains a challenging disease with an overall cumulative 5-year survival rate around 6%. Though significant progress has been made in the availability of diagnostic techniques and treatment strategies, pancreatic cancer remains a disease of high mortality rate. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that governs the oncogenesis and metastasis process of pancreatic cancer. In the present study, by using the Cancer Genome Atlas (TCGA) dataset analysis, we demonstrated that sorting nexin 6 (SNX6) serves as a biomarker for predicting prognosis of pancreatic cancer. In vitro studies demonstrated that silencing of SNX6 expression reduced cell proliferation, colony formation, invasion, and metastasis. Higher level of SNX6 helps maintain the mesenchymal properties, which renders migration and invasive capacities to pancreatic cancer cells. Moreover, in the process of TGF-β-induced epithelial to mesenchymal transition (EMT), the expression level of SNX6 was increased, and silencing of SNX6 expression could inhibit the TGF-β-induced EMT program. These results collectively uncovered a novel predictive marker for pancreatic cancer and provided the possible underlying molecular mechanism.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Metastasis; Pancreatic Neoplasms; Prognosis; RNA Interference; Sorting Nexins; Survival Analysis; Transforming Growth Factor beta

The prognosis of pancreatic ductal adenocarcinoma (PDAC) remains poor due to the difficulty of disease diagnosis and therapy. Immunotherapy has had robust performance against several malignancies, including PDAC. In this study, we aim to analyze the expression of CD8 and FoxP3 on T lymphocytes and TGF-β expression in tumor tissues, and then analyze the possible clinical significance of these finding in order to find a novel effective immunotherapy target in PDAC using a murine model.. A tissue microarray using patient PDAC samples was stained and analyzed for associations with clinicopathological characteristics. A preclinical murine model administrated with various immunotherapies were analyzed by growth inhibitor, flow cytometry, enzyme-linked immuno sorbent assay and immunohistochemistry.. The infiltrating FoxP3. The combination of CD25, TGF-β and PD-1 blockade plays a potentially effective role in inhibiting tumor formation and progression. Our results also provide a strong rational strategy for use of IIR in future immunotherapy clinical trials.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Animals; B7-H1 Antigen; Carcinoma, Pancreatic Ductal; CD8-Positive T-Lymphocytes; Cell Proliferation; Disease Models, Animal; Female; Forkhead Transcription Factors; Humans; Immunotherapy; Interleukin-2 Receptor alpha Subunit; Lymphocytes, Tumor-Infiltrating; Male; Mice, Inbred C57BL; Middle Aged; Multivariate Analysis; Pancreatic Neoplasms; Prognosis; Programmed Cell Death 1 Receptor; Survival Analysis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Burden

While TGFβ signals are anti-proliferative in benign and well-differentiated pancreatic cells, TGFβ appears to promote the progression of advanced cancers. To better understand dysregulation of the TGFβ pathway, we first generated mouse models of neoplastic disease with TGFβ receptor deficiencies. These models displayed reduced levels of pERK irrespective of KRAS mutation. Furthermore, exogenous TGFβ led to rapid and sustained TGFBR1-dependent ERK phosphorylation in benign pancreatic duct cells. Similar to results that our group has published in colon cancer cells, inhibition of ERK phosphorylation in duct cells mitigated TGFβ-induced upregulation of growth suppressive pSMAD2 and p21, prevented downregulation of the pro-growth signal CDK2 and ablated TGFβ-induced EMT. These observations suggest that ERK is a key factor in growth suppressive TGFβ signals, yet may also contribute to detrimental TGFβ signaling such as EMT. In neoplastic PanIN cells, pERK was not necessary for either TGFβ-induced pSMAD2 phosphorylation or CDK2 repression, but was required for upregulation of p21 and EMT indicating a partial divergence between TGFβ and MEK/ERK in early carcinogenesis. In cancer cells, pERK had no effect on TGFβ-induced upregulation of pSMAD2 and p21, suggesting the two pathways have completely diverged with respect to the cell cycle. Furthermore, inhibition of pERK both reduced levels of CDK2 and prevented EMT independent of exogenous TGFβ, consistent with most observations identifying pERK as a tumor promoter. Combined, these data suggest that during carcinogenesis pERK initially facilitates and later antagonizes TGFβ-mediated cell cycle arrest, yet remains critical for the pathological, EMT-inducing arm of TGFβ signaling.

Topics: Animals; Blotting, Western; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunoprecipitation; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Pancreas; Pancreatic Neoplasms; Transforming Growth Factor beta

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.

Topics: Animals; Benzamides; Cell Tracking; Dioxoles; Disease Models, Animal; Fluorescence; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Luminescent Proteins; Mice; Mice, Transgenic; Pancreatic Neoplasms; Red Fluorescent Protein; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

This paper aimed to investigate the effect of long non-coding RNA TUG1 (lncRNA TUG1) on cell proliferation, as well as cell migration in pancreatic cancer.. The mRNA levels of Taurine-up-regulated gene 1 (TUG1) in three kinds of pancreatic cancer cells BxPC3, PaTu8988 and SW1990 was detected by RT-qPCR. Meantime, RT-qPCR was used to examine the mRNA levels of TUG1 in 20 cases of human pancreatic cancer tissues and its para-carcinoma tissues. pCDH-TUG1 plasmid and its empty plasmid pCDH were transfected into BxPC3 and PaTu8988 cells to up-regulate TUG1 expression. siRNA targeting TUG1 and the control siRNA were transfected into SW1990 cells to down-regulate TUG1 expression. Cell clone formation and CCK-8 assay were used to detect the cell proliferation capacity. Transwell assay was used to evaluate cell migration capacity. Western blot was applied to examine the protein expressions of MMP2, MMP9, E-cadherin, Smad 2, Smad 3, p-Smad 2, p-Smad 3, TGF-β and TGF-βR. RT-qPCR was used to detect the levels of MMP2 and MMP9.. The results showed that TUG1 was differentially expressed in the three kinds of pancreatic cancer cells, among which the expression level of SW1990 was relatively high, and the expression levels of BxPC3 and PaTu8988 were relatively low. TUG1 had more expression in pancreatic cancer tissues than that in para-carcinoma tissues. After the up-regulation of TUG1, cell proliferation and migration capacities were increased, protein levels of MMP2 and MMP9 were increased and protein level of E-cadherin was declined. Conversely, after down-regulation of TUG1 expression, cell proliferation and migration capacities were weakened, protein levels of MMP2 and MMP9 were decreased and protein level of E-cadherin was increased. In addition, over-expressed TUG1 could promote Smad2 and Smad3 phosphorylation, but Smad2 and Smad3 phosphorylation were weakened after down-regulated expression of TUG1. The protein expression of TGF-β and TGF-β receptor were more in the TUG1 overexpression group than that in the control group, while the result was just opposite after TUG1 expression was down-regulated.. These data suggest that lncRNA TUG1 may enhance the proliferation and migration of pancreatic cancer cells through EMT pathway.

Topics: Antigens, CD; Cadherins; Carcinoma; Cell Movement; Cell Proliferation; Down-Regulation; Humans; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Long Noncoding; RNA, Messenger; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Transfection; Transforming Growth Factor beta; Up-Regulation

Pancreatic ductal adenocarcinoma (PDAC) has single-digit 5-year survival rates at <7%. There is a dire need to improve pre-malignant detection methods and identify new therapeutic targets for abrogating PDAC progression. To this end, we mined our previously published pseudopodium-enriched (PDE) protein/phosphoprotein datasets to identify novel PDAC-specific biomarkers and/or therapeutic targets. We discovered that integrin alpha 1 (ITGA1) is frequently upregulated in pancreatic cancers and associated precursor lesions. Expression of ITGA1-specific collagens within the pancreatic cancer microenvironment significantly correlates with indicators of poor patient prognosis, and depleting ITGA1 from PDAC cells revealed that it is required for collagen-induced tumorigenic potential. Notably, collagen/ITGA1 signaling promotes the survival of ALDH1-positive stem-like cells and cooperates with TGFβ to drive gemcitabine resistance. Finally, we report that ITGA1 is required for TGFβ/collagen-induced EMT and metastasis. Our data suggest that ITGA1 is a new diagnostic biomarker and target that can be leveraged to improve patient outcomes.

Topics: Adenocarcinoma; Aldehyde Dehydrogenase 1 Family; Animals; Antimetabolites, Antineoplastic; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Collagen; Deoxycytidine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha Chains; Isoenzymes; Pancreatic Neoplasms; Prognosis; Retinal Dehydrogenase; RNA, Small Interfering; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta; Tumor Microenvironment

Psychological stress has recently been described as a risk factor in the development of pancreatic cancer. Here, we reported that increased neurotransmitter adrenaline was associated with the poor survival in pancreatic cancer patients. Moreover, in the cell model study, we found adrenaline promoted pancreatic cell PANC-1 migration in a dose dependent manner. Block of the β2-adrenoreceptor with ICI118,551, significantly reduced cell migration. Further study found that adrenaline induced a cytoplasmic translocation of RNA binding protein HuR, which in turn activated TGFβ, as shown by the SBE luciferase assay and phosphorylation of Smad2/3. Either HuR knockdown or TGFβ inhibition reduced cell migration induced by adrenaline. Taken together, our study here revealed that adrenaline-HuR-TGFβ regulatory axis at least partially contributes to the psychological stress induced metastasis in PANC-1 cells, shedding light on therapeutic targeting psychological stress in improving the prognosis of pancreatic cancer.

Topics: Adrenergic beta-Antagonists; Aged; Cell Line, Tumor; Cell Movement; ELAV-Like Protein 1; Epinephrine; Epithelial-Mesenchymal Transition; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Pancreatic Neoplasms; Propanolamines; Receptors, Adrenergic, beta-2; Transforming Growth Factor beta

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; Humans; Neoplasm Staging; Pancreatic Neoplasms; Phosphoproteins; Proto-Oncogene Proteins c-akt; RNA Interference; RNA-Binding Proteins; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; YAP-Signaling Proteins

Topics: Animals; Disease Models, Animal; Humans; Liver Neoplasms; Mice; Mice, Transgenic; Neoplasm Metastasis; Neoplasms, Experimental; Pancreas; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Earlier results from our group have shown that in pancreatic ductal adenocarcinoma (PDAC)-derived cells transforming growth factor (TGF)-β1-dependent epithelial-mesenchymal transition (EMT) and cell motility was inhibited by the Src inhibitors PP2 and PP1 both of which targeted the TGF-β receptors for inhibition.. In this study we evaluated the impact of another Src inhibitor, AZM475271, on various TGF-β responses in PDAC cells.. The effect of AZM475271 on TGF-β1-induced random cell migration (chemokinesis), the expression of EMT and migration/invasion-associated genes, TGF-β-induced luciferase activity, and C-terminal phosphorylation of Smad2 and Smad3 was measured in the PDAC-derived Panc-1 and Colo357 cell lines using real-time cell migration assays, quantitative real-time PCR, luciferase reporter gene assays and phosphoimmunoblotting, respectively.. AZM475271 effectively blocked TGF-β1-induced chemokinesis of Panc-1 cells in a dose-dependent fashion and inhibited the high chemokinetic activity of Panc-1 cells with ectopic expression of a constitutively active ALK5T204D mutant. AZM475271 but not another Src inhibitor, SU6656, partially relieved the suppressive effect of TGF-β1 on E-cadherin and inhibited TGF-β1-induced upregulation of the MMP2, MMP9, N-cadherin and vimentin genes, activity of a TGF-β1-dependent reporter gene, and activation of Smad2 and Smad3.. Our data suggest that AZM475271 cross-inhibits tumor-promoting TGF-β signaling and may thus function as an inhibitor of both TGF-β and Src in both experimental and clinical therapies against metastatic dissemination in late-stage PDAC.

Topics: Cell Line, Tumor; Cell Movement; Humans; Pancreas; Pancreatic Neoplasms; Piperazines; Protein Kinase Inhibitors; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

Methyl-CpG-binding domain 3 (MBD3) is an aberrant expression in human malignancies. However, the role of MBD3 in pancreatic cancer progression remains to be clarified. In this study, we investigated the effects of MBD3 on the epithelial-mesenchymal transition (EMT), and the underlying mechanism in pancreatic cancer cells.. The abilities of migration and invasion were examined by transwell and BD Matrigel invasion assays. EMT and TGF-β/Smad signalling were evaluated.. First, we find that MBD3 expression is lower in pancreatic cancer tissues than that in non-tumour tissues, and patients with lower MBD3 levels survive significantly less than those with higher levels. Subsequently, we find that MBD3 knockdown promotes the abilities of migration and invasion, while MBD3 overexpression inhibits the above abilities. Also, MBD3 knockdown remarkably increases mesenchymal markers expression of Vimentin, α-SMA, Snail, N-cadherin, β-catenin, and downregulates epithelial markers expression of E-cadherin. On the contrary, MBD3 overexpression results in the opposite effects. Further evidence reveals that MBD3 knockdown up-regulates expression of TGF-β, and then activates p-Smad2 and p-Smad3, while MBD3 overexpression results in downregulation of TGF-β, p-Smad2, and p-Smad3.. MBD3 inhibits EMT in pancreatic cancer cells probably via TGF-β/Smad signalling, and may be a new candidate target for diagnostics and prognosis of pancreatic cancer.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Line, Tumor; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; HEK293 Cells; Humans; Pancreatic Neoplasms; Prognosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Krüpple-like factor 10 (Klf10), an early response gene of TGFβ, was reported to be a prognostic biomarker for pancreatic cancer survival. The role of Klf10 in predicting tumor response to cancer treatment is unknown.. Genetically manipulated MiaPaCa and Panc-1 cells were established to evaluate clonogenic survival, autophagy, apoptosis and DNA repair after radiation. The interaction between Klf10 and UV radiation resistance-associated gene (UVRAG) was demonstrated by ChiP-PCR and luciferase reporter assay. Orthotopic murine tumor model and clinical specimens were used to evaluate radio-sensitivity of pancreatic cancer.. We found Klf10 silencing correlates with enhanced pancreatic cancer clonogenic survival and murine tumor growth after radiation. UVRAG was an essential down-stream mediator transcriptionally suppressed by Klf10. Silencing UVRAG mRNA in Klf10 depleted Panc-1 cells reversed the radio-resistant phenotypes including decreased apoptosis and enhanced DNA repair as well as autophagy. Metformin, an anti-diabetic agent, was found to increase Klf10 and suppress UVRAG expression to improve radiation cytotoxicity in pancreatic cancer. The predictive value of Klf10 in radiation response and the inverse correlation with UVRAG were confirmed in cohorts of pancreatic cancer patients.. Klf10 is a potential biomarker in predicting and sensitizing radiation effect in pancreatic cancer.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Cell Line, Tumor; DNA Repair; Early Growth Response Transcription Factors; Humans; Kruppel-Like Transcription Factors; Metformin; Mice; Pancreatic Neoplasms; Radiation Tolerance; Transforming Growth Factor beta

Topics: Actins; Cells, Cultured; Humans; Myocytes, Smooth Muscle; Pancreatic Neoplasms; Pancreatic Stellate Cells; Pancreatitis, Chronic; Plasma Kallikrein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Embigin is a member of the immunoglobulin superfamily and encodes a transmembrane glycoprotein. There have been reports of Embigin involvement in neuromuscular junction formation and plasticity; however, the molecular functions of Embigin in other organs are unknown. Our aim was to investigate the possible role of Embigin in pancreatic cancer. In pancreatic ductal adenocarcinoma tissues, Embigin expression was higher than that in normal pancreatic tissues. Immunohistochemical analysis revealed expression of Embigin in pancreatic cancer cells, as well as expression of monocarboxylate transporter 2 (MCT2) in cancer tissues. To gain further insight, we transfected BxPC-3 and HPAC pancreatic cancer cells with siRNA or shRNA targeting Embigin and observed reductions in cell proliferation, migration, invasion, wound healing, and reduced levels of matrix metalloproteinases-2 and -9. Silencing of Embigin increased intracellular L-lactate concentration by 1.5-fold and decreased MCT2 levels at the plasma membrane. Furthermore, Embigin silencing led to a reduced expression of PI3K, GSK3-β, and Snail/Slug. Upon treating BxPC-3 cells with transforming growth factor-β (TGF-β), we observed elevated expression of Snail/Slug, Embigin, and Vimentin; meanwhile, when treating cells with SB-216763, a GSK3-β inhibitor, we noted decreases in GSK3-β, Snail/Slug, and Embigin expression, suggesting that the TGF-β signaling cascade, comprising PI3K, GSK3-β, Snail/Slug, and Embigin signals, mediates epithelial to mesenchymal transition (EMT) in pancreatic cancer cells. These findings indicate the involvement of Embigin in EMT in pancreatic cancer progression and suggest Embigin as a putative target for the detection and/or treatment of pancreatic cancer.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Mice; Molecular Chaperones; Neoplasm Transplantation; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

To investigate the effects of small-interfering RNA (siRNA)-mediated silencing of SET binding factor 2 (SBF2) on pancreatic cancer cells and its underlying molecular mechanisms.. Five siRNAs, namely, siRNA-1, siRNA-2, siRNA-3, siRNA-4, and siRNA-5, were designed to silence SBF2 in pancreatic cancer PANC-1 cells. The relative expression levels of SBF2 after siRNA-mediated SBF2 silencing were detected by real-time polymerase chain reaction (RT-PCR). The inhibition and apoptosis of cells were detected by methyl thiazolyl tetrazolium and flow cytometry, respectively. The protein concentrations of SBF2, SMAD-2, phosphorylated-SMAD-2 (p-SMAD-2), SMAD-3, p-SMAD-3, and SMAD-7 were also measured via Western blot.. The relative expression levels of SBF2 in the siRNA-mediated SBF2-silenced groups decreased significantly (P < .05), and the proliferation of PANC-1 cells was significantly inhibited (P < .01) after SBF2 silencing using the 5 siRNAs. The relative expression level of SBF2 was the lowest (0.52 ± 0.05) and the cell inhibition rate was the highest (36.7 ± 4.0%) in the SBF2-silenced PANC-1 cells using siRNA-3 compared to the 5 siRNA-mediated SBF2-silenced groups. The cell apoptosis rate in the siRNA-3 transfection group was significantly higher than that in the control group (P < .05). The concentrations of p-SMAD-2 and p-SMAD-3 were reduced, while the concentrations of SMAD-2, SMAD-3, and SMAD-7 were increased in the siRNA-3-mediated SBF2-silenced PANC-1 cells.. SiRNA-mediated SBF2 silencing could significantly inhibit the proliferation and promote the apoptosis of pancreatic cancer cells possibly via attenuation of transforming growth factor β/SMAD signaling pathway. And SBF2 silencing can be a potential approach for treatment of pancreatic cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Pancreatic Neoplasms; Protein Tyrosine Phosphatases, Non-Receptor; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin-1 (IL1), CXC receptors (CXCR)1/2 ligands, and transforming growth factor β (TGFβ) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the antivascular endothelial growth factor (anti-VEGF) antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse this anti-VEGF resistance. Bevacizumab-resistant FGBR pancreatic cancer cells were treated in vitro with bevacizumab, the recombinant human IL1 receptor antagonist anakinra, the monoclonal antibody against TGFβ receptor type II TR1, and a novel recombinant antibody binding CXCR1/2 ligands. The FGBR cells treated with these agents in combination had significantly higher levels of E-cadherin and lower levels of vimentin, IL6, phosphorylated p65, and SMAD2, and showed significantly lower migration rates than did their controls treated with the same agents without bevacizumab or with a single agent bevacizumab as a control. Consistently, the combination of these agents with bevacizumab reduced the FGBR tumor burden and significantly prolonged mice survival compared with bevacizumab in monotherapy. Tumors from mice receiving the combination treatment showed significantly lower expression of IL6 and phosphorylated SMAD2, higher expression of E-cadherin and lower levels of vimentin, and a significantly lower infiltration by CD11b cells compared with bevacizumab-treated controls. This study suggests that inhibition of IL1, CXCR1/2, and TGFβ signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing epithelial-mesenchymal transition and inhibiting CD11b proangiogenic myeloid cells' tumor infiltration.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Bevacizumab; CD11b Antigen; Cell Line, Tumor; Chemokine CXCL1; Chemokine CXCL2; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Mice, Nude; Myeloid Cells; Neoplasm Transplantation; Pancreatic Neoplasms; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Long noncoding RNAs (lncRNAs) have been shown to play critical roles in the development and progression of diseases. lncRNA activated by transforming growth factor beta (TGF-β) (lncRNA-ATB) was discovered as a prognostic factor in hepatocellular carcinoma, gastric cancer, and colorectal cancer. However, little is known about the role of lncRNA-ATB in pancreatic cancer. This study aimed to assess lncRNA-ATB expression in pancreatic cancer and explore its role in pancreatic cancer pathogenesis. Quantitative real-time polymerase chain reaction was performed to detect lncRNA-ATB expression in 150 pancreatic cancer tissues and five pancreatic cancer cell lines compared to paired adjacent normal tissues and normal human pancreatic ductal epithelial cell line HPDE6c-7. The correlations between lncRNA-ATB expression and clinicopathological characteristics and prognosis were also analyzed. We found that lncRNA-ATB expression was decreased in pancreatic cancer tissues and pancreatic cancer cell lines. Low lncRNA-ATB expression levels were significantly correlated with lymph node metastases (yes vs. no, P = 0.009), neural invasion (positive vs. negative, P = 0.049), and clinical stage (early stage vs. advanced stage, P = 0.014). Moreover, patients with low lncRNA-ATB expression levels exhibited markedly worse overall survival prognoses (P < 0.001). Multivariate analysis indicated that decreased lncRNA-ATB expression was an independent predictor of poor prognosis in pancreatic cancer patients (P = 0.005). In conclusion, lncRNA-ATB may play a critical role in pancreatic cancer progression and prognosis and may serve as a potential prognostic biomarker in pancreatic cancer patients.

Topics: Cell Line; Cell Line, Tumor; Disease Progression; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Pancreatic Neoplasms; Prognosis; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA, Long Noncoding; Transforming Growth Factor beta

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, but overexpress pro-angiogenic factors and exhibit regions of microvasculature. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we previously reported that ~12% of PDACs have an angiogenesis gene signature with increased expression of multiple pro-angiogenic genes. By analyzing the recently expanded TCGA dataset, we now report that this signature is present in ~35% of PDACs but that it is mostly distinct from an angiogenesis signature present in pancreatic neuroendocrine tumors (PNETs). These PDACs exhibit a transcriptome that reflects active TGF-β signaling, and up-regulation of several pro-inflammatory genes, and many members of JAK signaling pathways. Moreover, expression of SMAD4 and HDAC9 correlates with endothelial cell abundance in PDAC tissues. Concomitantly targeting the TGF-β type I receptor (TβRI) kinase with SB505124 and JAK1-2 with ruxolitinib suppresses JAK1 phosphorylation and blocks proliferative cross-talk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs), and these anti-proliferative effects were mimicked by JAK1 silencing in ECs. By contrast, either inhibitor alone does not suppress their enhanced proliferation in 3D co-cultures. These findings suggest that targeting both TGF-β and JAK1 signaling could be explored therapeutically in the 35% of PDAC patients whose cancers exhibit an angiogenesis gene signature.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cells, Cultured; Cluster Analysis; Coculture Techniques; Endothelial Cells; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Immunoblotting; Inflammation Mediators; Janus Kinase 1; Neovascularization, Pathologic; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad4 Protein; Survival Analysis; Transforming Growth Factor beta

Periampullary adenocarcinomas can be of two histological subtypes, intestinal or pancreatobiliary. The latter is more frequent and aggressive, and characterized by a prominent desmoplastic stroma, which is tightly related to the biology of the cancer, including its poor response to chemotherapy. Whereas miRNAs are known to regulate various cellular processes and interactions between cells, their exact role in periampullary carcinoma remains to be characterized, especially with respect to the prominent stromal component of pancreatobiliary type cancers. The present study aimed at elucidating this role by miRNA expression profiling of the carcinomatous and stromal component in twenty periampullary adenocarcinomas of pancreatobiliary type. miRNA expression profiles were compared between carcinoma cells, stromal cells and normal tissue samples. A total of 43 miRNAs were found to be differentially expressed between carcinoma and stroma of which 11 belong to three miRNA families (miR-17, miR-15 and miR-515). The levels of expression of miRNAs miR-17, miR-20a, miR-20b, miR-223, miR-10b, miR-2964a and miR-342 were observed to be higher and miR-519e to be lower in the stromal component compared to the carcinomatous and normal components. They follow a trend where expression in stroma is highest followed by carcinoma and then normal tissue. Pathway analysis revealed that pathways regulating tumor-stroma interactions such as ECM interaction remodeling, epithelial-mesenchymal transition, focal adhesion pathway, TGF-beta, MAPK signaling, axon guidance and endocytosis were differently regulated. The miRNA-mRNA mediated interactions between carcinoma and stromal cells add new knowledge regarding tumor-stroma interactions.

Topics: Adenocarcinoma; Adult; Aged; Common Bile Duct Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; MicroRNAs; Middle Aged; Pancreatic Neoplasms; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

Analysis using Universal exPress Codes (UPCs) with the public microarray database GEO indicates significantly higher mRNA expressions of VEGF-A, bFGF, and bFGFR2 in pancreatic cancers than those in normal pancreatic tissues. Human pancreatic cancer cell line CFPAC-1 and SNU-213 had relatively differential sensitivity to exogenous VEGF-A, bFGF, and TGF-β1 in migration property. Treatment of quercetin-3-O-glucoside suppressed the migratory activity induced by TGF-β and VEGF-A even at relatively low dosages in CFPAC-1, but not in bFGF-activated SNU-213 cells. However, high dosages of quercetin-3-O-glucoside sufficiently suppressed the migratory activity induced by bFGF in SNU-213 cells. Furthermore, co-treatment with low dose of gemcitabine plus quercetin-3-O-glucoside showed synergistic inhibition effects on the infiltrate activity induced by bFGF in CFPAC-1 and SNU-213 cells. These results collectively suggested that quercetin-3-O glucoside could act as an inhibitor of local metastasis induced by various growth factors in pancreatic cancers and be an effective adjuvant to boost chemotherapeutic efficacy of gemcitabine, currently used in pancreatic cancers.

Topics: Cell Line, Tumor; Cell Movement; Databases, Genetic; Deoxycytidine; Dose-Response Relationship, Drug; Drug Synergism; Fibroblast Growth Factor 2; Flavonoids; Gemcitabine; Gene Expression Regulation, Neoplastic; Glucosides; Humans; In Vitro Techniques; Pancreatic Neoplasms; Quercetin; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

TGF-β signaling can be pro-tumorigenic or tumor suppressive. We investigated this duality in pancreatic ductal adenocarcinoma (PDA), which, with other gastrointestinal cancers, exhibits frequent inactivation of the TGF-β mediator Smad4. We show that TGF-β induces an epithelial-mesenchymal transition (EMT), generally considered a pro-tumorigenic event. However, in TGF-β-sensitive PDA cells, EMT becomes lethal by converting TGF-β-induced Sox4 from an enforcer of tumorigenesis into a promoter of apoptosis. This is the result of an EMT-linked remodeling of the cellular transcription factor landscape, including the repression of the gastrointestinal lineage-master regulator Klf5. Klf5 cooperates with Sox4 in oncogenesis and prevents Sox4-induced apoptosis. Smad4 is required for EMT but dispensable for Sox4 induction by TGF-β. TGF-β-induced Sox4 is thus geared to bolster progenitor identity, whereas simultaneous Smad4-dependent EMT strips Sox4 of an essential partner in oncogenesis. Our work demonstrates that TGF-β tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network.

Topics: Adenocarcinoma; Animals; Apoptosis; Carcinoma, Ductal; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Kruppel-Like Transcription Factors; Mice; Organoids; Pancreatic Neoplasms; Smad4 Protein; SOXC Transcription Factors; Transforming Growth Factor beta

TGF-β is long considered a "protean" cytokine in cancer, changing its role from anti- to pro-tumorigenic in a context-dependent manner. In this issue of Cell, David et al. use mouse models of pancreatic cancer to shed light on the mechanistic basis of how TGF-β-induced EMT is coupled to either apoptosis or tumor progression.

Topics: Animals; Carcinoma, Ductal; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Pancreatic Neoplasms; Transforming Growth Factor beta

Topics: Animals; Carcinoma, Ductal; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Pancreatic Neoplasms; Transforming Growth Factor beta

Advances made in pancreatic cancer therapy have been far from sufficient and have allowed only a slight improvement in global survival of patients with pancreatic ductal adenocarcinoma (PDA). Recent progresses in chemotherapy have offered some hope for an otherwise gloomy outlook, however, only a limited number of patients are eligible because of important cytotoxicity. In this context, enhancing our knowledge on PDA initiation and evolution is crucial to highlight certain weaknesses on which to specifically target therapy. We found that loss of transcriptionally active p73 (TAp73), a p53 family member, impacted PDA development. In two relevant and specific engineered pancreatic cancer mouse models, we observed that TAp73 deficiency reduced survival and enhanced epithelial-to-mesenchymal transition (EMT). Through proteomic analysis of conditioned media from TAp73 wild-type (WT) and deficient pancreatic tumor cells, we identified a secreted protein, biglycan (BGN), which is necessary and sufficient to mediate this pro-EMT effect. Interestingly, BGN is modulated by and modulates the transforming growth factor-β (TGF-β) pathway, a key regulator of the EMT process. We further examined this link and revealed that TAp73 impacts the TGF-β pathway by direct regulation of BGN expression and Sma and Mad-related proteins (SMADs) expression/activity. Absence of TAp73 leads to activation of TGF-β signaling through a SMAD-independent pathway, favoring oncogenic TGF-β effects and EMT. Altogether, our data highlight the implication of TAp73 in the aggressiveness of pancreatic carcinogenesis through modulation of the TGF-β signaling. By suggesting TAp73 as a predictive marker for response to TGF-β inhibitors, our study could improve the classification of PDA patients with a view to offering combined therapy involving TGF-β inhibitors.

Topics: Animals; Biglycan; Carcinoma, Pancreatic Ductal; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Humans; Male; Mice; Mice, Knockout; Mice, Nude; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nuclear Proteins; Pancreatic Neoplasms; RNA Interference; Signal Transduction; Smad Proteins; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

In early pancreatic carcinogenesis, TGFβ acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFβ appears to promote tumor progression. Therefore, to better understand the contributions of TGFβ signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFβ signals facilitated pancreatic tumorigenesis, whereas global loss of TGFβ signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFβ1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFβ-induced inactivation ex vivo, and adoptive transfer of TGFBR-deficient CD8(+) T cells led to enhanced infiltration and granzyme B-mediated destruction of developing tumors. These findings paralleled our observations in human patients, where TGFβ expression correlated with increased fibrosis and associated negatively with expression of granzyme B. Collectively, our findings suggest that, despite opposing the proliferation of some epithelial cells, TGFβ may promote pancreatic cancer development by affecting stromal and hematopoietic cell function. Therefore, the use of TGFBR inhibition to target components of the tumor microenvironment warrants consideration as a potential therapy for pancreatic cancer, particularly in patients who have already lost tumor-suppressive TGFβ signals in the epithelium. Cancer Res; 76(9); 2525-39. ©2016 AACR.

Topics: Adoptive Transfer; Animals; Blotting, Western; Carcinogenesis; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fibrosis; Flow Cytometry; Humans; Immunohistochemistry; Mice; Mice, Mutant Strains; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

Topics: Animals; Carcinoma, Pancreatic Ductal; Chromatography, Liquid; Collagen; Disease Models, Animal; Disease Progression; Extracellular Matrix; Fibrosis; Genotype; Humans; Integrin beta Chains; Mice; Microscopy, Atomic Force; Mutation; Pancreatic Neoplasms; Prognosis; Proteomics; Proto-Oncogene Proteins p21(ras); Real-Time Polymerase Chain Reaction; Signal Transduction; Smad4 Protein; STAT3 Transcription Factor; Survival Rate; Tandem Mass Spectrometry; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is lethal cancer whose primary tumor is characterized by dense composition of cancer cells, stromal cells, and extracellular matrix (ECM) composed largely of collagen. Within the PDAC tumor microenvironment, activated pancreatic stellate cells (PSC) are the dominant stromal cell type and responsible for collagen deposition. Lumican is a secreted proteoglycan that regulates collagen fibril assembly. We have previously identified that the presence of lumican in the ECM surrounding PDAC cells is associated with improved patient outcome after multimodal therapy and surgical removal of localized PDAC.. Lumican expression in PDAC from 27 patients was determined by IHC and quantitatively analyzed for colocalization with PSCs. In vitro studies examined the molecular mechanisms of lumican transcription and secretion from PSCs (HPSCs and HPaSteC), and cell adhesion and migration assays examined the effect of lumican on PSCs in a collagen-rich environment.. Here we identify PSCs as a significant source of extracellular lumican production through quantitative IHC analysis. We demonstrate that the cytokine, TGF-β, negatively regulates lumican gene transcription within HPSCs through its canonical signaling pathway and binding of SMAD4 to novel SBEs identified within the promoter region. In addition, we found that the ability of HPSCs to produce and secrete extracellular lumican significantly enhances HPSCs adhesion and mobility on collagen.. Our results demonstrate that activated pancreatic stellate cells within PDAC secrete lumican under the negative control of TGF-β; once secreted, the extracellular lumican enhances stellate cell adhesion and mobility in a collagen-rich environment. Clin Cancer Res; 22(19); 4934-46. ©2016 AACR.

Topics: Animals; Carcinoma, Pancreatic Ductal; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Lumican; Mice; Pancreatic Neoplasms; Pancreatic Stellate Cells; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-β and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-β and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-β1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro. Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2-/- mice displayed a greatly attenuated sensitivity to TGF-β1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-β1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-β1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-β-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Movement; Cells, Cultured; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Metastasis; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, PAR-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, G-Protein-Coupled; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Pancreatic ductal adenocarcinomas (PDACs) are aggressive with frequent lymphatic spread. By analysis of data from The Cancer Genome Atlas, we determined that ~35% of PDACs have a pro-angiogenic gene signature. We now show that the same PDACs exhibit increased expression of lymphangiogenic genes and lymphatic endothelial cell (LEC) markers, and that LEC abundance in human PDACs correlates with endothelial cell microvessel density. Lymphangiogenic genes and LECs are also elevated in murine PDACs arising in the KRC (mutated Kras; deleted RB) and KIC (mutated Kras; deleted INK4a) genetic models. Moreover, pancreatic cancer cells (PCCs) derived from KRC tumors express and secrete high levels of lymphangiogenic factors, including the EGF receptor ligand, amphiregulin. Importantly, TGF-β1 increases lymphangiogenic genes and amphiregulin expression in KRC PCCs but not in murine PCCs that lack SMAD4, and combinatorial targeting of the TGF-β type I receptor (TβRI) with LY2157299 and EGFR/HER2 with lapatinib suppresses tumor growth and metastasis in a syngeneic orthotopic model, and attenuates tumor lymphangiogenesis and angiogenesis while reducing lymphangiogenic genes and amphiregulin and enhancing apoptosis. Therefore, this combination could be beneficial in PDACs with lymphangiogenic or angiogenic gene signatures.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Movement; Cyclin-Dependent Kinase Inhibitor p16; ErbB Receptors; Female; Gene Expression Profiling; Genes, Retinoblastoma; Genetic Predisposition to Disease; Humans; Lapatinib; Lymphangiogenesis; Male; Mice, Transgenic; Molecular Targeted Therapy; Mutation; Neoplasm Invasiveness; Neovascularization, Pathologic; Pancreatic Neoplasms; Phenotype; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Pyrazoles; Quinazolines; Quinolines; Receptor, ErbB-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

A recent study finds that impaired TGFβ signaling can initiate a positive feedback loop between increasing ECM stiffness and epithelial cell contractility in pancreas cancer. Even more surprising is the possibility that this phenotype can liberate the epithelium from dependence on the genetic events that transformed it.

Topics: Animals; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Genotype; Humans; Pancreatic Neoplasms; Phenotype; Signal Transduction; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma is characterized by a prominent fibroinflammatory stroma with both tumor-promoting and tumor-suppressive functions. The pancreatic stellate cell (PSC) is the major cellular stromal component and the main producer of extracellular matrix proteins, including collagens, which are degraded by metalloproteinases (MMPs). PSCs interact with cancer cells through various factors, including transforming growth factor (TGF)β and interleukin (IL)-1α. The role of TGFβ in the dual nature of tumor stroma, i.e., protumorigenic or tumor suppressive, is not clear. We aimed to investigate the roles of TGFβ and IL-1α in the regulation of MMP profiles in PSCs and the subsequent effects on cancer cell migration. Human PSCs isolated from surgically resected specimens were cultured in the presence of pancreatic cancer cell lines, as well as IL-1α or TGFβ. MMP production and activities in PSCs were quantified by gene array transcripts, mRNA measurements, fluorescence resonance energy transfer-based activity assay, and zymography. PSC-conditioned media and pancreatic cancer cells were included in a collagen matrix cell migration model. We found that production of IL-1α by pancreatic cancer cells induced alterations in MMP and tissue inhibitors of matrix metalloproteinase (TIMP) profiles and activities in PSCs, upregulated expression and activation of MMP1 and MMP3, and enhanced migration of pancreatic cancer cells in the collagen matrix model. TGFβ counteracted the effects of IL-1α on PSCs, reestablished PSC MMP and TIMP profiles and activities, and inhibited migration of cancer cells. This suggests that tumor TGFβ has a role as a suppressor of stromal promotion of tumor progression through alterations in PSC MMP profiles with subsequent inhibition of pancreatic cancer cell migration.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Enzyme Activation; Fluorescence Resonance Energy Transfer; Humans; Interleukin-1alpha; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatic Stellate Cells; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-3; Transforming Growth Factor beta; Tumor Suppressor Proteins

The most abundant cells in the extensive desmoplastic stroma of pancreatic adenocarcinomas are the pancreatic stellate cells, which interact with the carcinoma cells and strongly influence the progression of the cancer. Tumor stroma interactions induced by IL-1α/IL-1R1 signaling have been shown to be involved in pancreatic cancer cell migration. TGFβ and its receptors are overexpressed in pancreatic adenocarcinomas. We aimed at exploring TGFβ and IL-1α signaling and cross-talk in the stellate cell cancer cell interactions regulating pancreatic adenocarcinoma cell migration.. Human pancreatic stellate cells were isolated from surgically resected pancreatic adenocarcinomas and cultured in the presence of TGFβ or pancreatic adenocarcinoma cell lines. The effects of TGFβ were blocked by inhibitors or amplified by silencing the endogenous inhibitor of SMAD signaling, SMAD7. Pancreatic stellate cell responses to IL-1α or to IL-1α-expressing pancreatic adenocarcinoma cells (BxPC-3) were characterized by their ability to stimulate migration of cancer cells in a 2D migration model.. In pancreatic stellate cells, IL-1R1 expression was found to be down-regulated by TGFβ and blocking of TGFβ signaling re-established the expression. Endogenous inhibition of TGFβ signaling by SMAD7 was found to correlate with the levels of IL-1R1, indicating a regulatory role of SMAD7 in IL-1R1 expression. Pancreatic stellate cells cultured in the presence of IL-1α or in co-cultures with BxPC-3 cells enhanced the migration of cancer cells. This effect was blocked after treatment of the pancreatic stellate cells with TGFβ. Silencing of stellate cell expression of SMAD7 was found to suppress the levels of IL-1R1 and reduce the stimulatory effects of IL-1α, thus inhibiting the capacity of pancreatic stellate cells to induce cancer cell migration.. TGFβ signaling suppressed IL-1α mediated pancreatic stellate cell induced carcinoma cell migration. Depletion of SMAD7 upregulated the effects of TGFβ and reduced the expression of IL-1R1, leading to inhibition of IL-1α induced stellate cell enhancement of carcinoma cell migration. SMAD7 might represent a target for inhibition of IL-1α induced tumor stroma interactions.

Topics: Cell Communication; Cell Line, Tumor; Cell Movement; Cells, Cultured; Coculture Techniques; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1alpha; Pancreatic Neoplasms; Pancreatic Stellate Cells; Receptors, Interleukin-1 Type I; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta

Inhibitors of bromodomain and extraterminal domain (BET) proteins, a family of chromatin reader proteins, have therapeutic efficacy against various malignancies. However, the detailed mechanisms underlying the anti-tumor effects in distinct tumor types remain elusive. Here, we show a novel antitumor mechanism of BET inhibition in pancreatic ductal adenocarcinoma (PDAC). We found that JQ1, a BET inhibitor, decreased desmoplastic stroma, a hallmark of PDAC, and suppressed the growth of patient-derived tumor xenografts (PDX) of PDACs. In vivo antitumor effects of JQ1 were not always associated with the JQ1 sensitivity of respective PDAC cells, and were rather dependent on the suppression of tumor-promoting activity in cancer-associated fibroblasts (CAFs). JQ1 inhibited Hedgehog and TGF-β pathways as potent regulators of CAF activation and suppressed the expression of α-SMA, extracellular matrix, cytokines, and growth factors in human primary CAFs. Consistently, conditioned media (CM) from CAFs promoted the proliferation of PDAC cells along with the activation of ERK, AKT, and STAT3 pathways, though these effects were suppressed when CM from JQ1-treated CAFs was used. Mechanistically, chromatin immunoprecipitation experiments revealed that JQ1 reduced TGF-β-dependent gene expression by disrupting the recruitment of the transcriptional machinery containing BET proteins. Finally, combination therapy with gemcitabine plus JQ1 showed greater efficacy than gemcitabine monotherapy against PDAC in vivo. Thus, our results reveal BET proteins as the critical regulators of CAF-activation and also provide evidence that stromal remodeling by epigenetic modulators can be a novel therapeutic option for PDAC.

Topics: Actins; Animals; Azepines; Cancer-Associated Fibroblasts; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cytokines; Disease Progression; Epigenesis, Genetic; Hedgehog Proteins; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred NOD; Mice, SCID; Pancreas; Pancreatic Neoplasms; Primary Cell Culture; Proteins; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic; Transforming Growth Factor beta; Triazoles; Xenograft Model Antitumor Assays

The deposition of extracellular matrix (ECM) is a defining feature of pancreatic ductal adenocarcinoma (PDA), where ECM signaling can promote cancer cell survival and epithelial plasticity programs. However, ECM signaling can also limit PDA tumor growth by producing cytotoxic levels of reactive oxygen species. For example, excess fibronectin stimulation of α5β1 integrin on stromal cells in PDA results in reduced angiogenesis and increased tumor cell apoptosis because of oxidative stress. Fibulin-5 (Fbln5) is a matricellular protein that blocks fibronectin-integrin interaction and thus directly limits ECM-driven reactive oxygen species production and supports PDA progression. Compared with normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a TGF-β- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor, PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-β receptor and PI3K/AKT inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression, whereas pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Hypoxia; Extracellular Matrix; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Integrin alpha5beta1; Mice; Mice, Mutant Strains; Neoplasms, Experimental; NIH 3T3 Cells; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta

The objective of this study was to investigate the effect of silencing gene protein phosphatase 1H (PPM1H) on malignant phenotype of human pancreatic cancer cell line BxPC-3. In order to explore the function of PPM1H in pancreatic cancer cells, real-time PCR and western blotting were used to detect the expression of PPM1H in different pancreatic cancer cell lines. Human pancreatic cancer cell line BxPC-3 was treated with 10 ng/ml TGF-β1 and 200 ng/ml BMP2 for 72 h, respectively, and the mRNA and protein expression levels of PPM1H and EMT-related markers (E-cadherin, vimentin) were detected by real-time PCR and western blotting, respectively. Using exogenous RNA interference technology to silence the PPM1H gene, the expression of PPM1H and EMT-related markers at mRNA and protein levels were detected by real-time PCR and western blotting. The cell migration and invasion were measured using Transwell assays. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the effect of PPM1H on cell proliferation and apoptosis of BxPC-3 cells. The expression levels of PPM1H in all of the examined pancreatic cancer cell lines (BxPC-3, MIA-PACA2, PANC-1, SW1990, PANC-03.27) were lower than that of normal pancreatic ductal epithelial cells (HPDE6-C7) at both mRNA and protein levels. Both TGF-β1 and BMP2 treatment induced EMT and downregulation of PPM1H in BxPC-3 cells. By using RNA interference to transiently knock down PPM1H expression in BxPC-3 cells, we found that the expression of E-cadherin was downregulated while vimentin was up-regulated. The data suggested that silencing PPM1H gene can induce EMT in BxPC-3 cells. In addition, Transwell migration assays showed that silencing PPM1H gene can promote the invasion and metastasis of BxPC-3 cells. Cell proliferation and apotosis detection demonstrated that silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. In conclusion, PPM1H is aberrantly expressed in human pancreatic cancer cell lines and can be downregulated when EMT is induced by cytokine stimulation. Silencing PPM1H gene can induce EMT in BxPC-3 cells, and promote the invasion and metastasis of BxPC-3 cells. Moreover, silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. PPM1H may be a new tumor-suppressor factor for pancreatic cancer and provides new insight into molecular targets for gene therapy of pancreatic cancer.

Topics: Antigens, CD; Apoptosis; Biomarkers, Tumor; Bone Morphogenetic Protein 2; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Pancreatic Neoplasms; Phosphoprotein Phosphatases; RNA Interference; Transforming Growth Factor beta; Vimentin

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-β (TGF-β) induces the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad2/3 signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-β-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Carcinoma, Pancreatic Ductal; Case-Control Studies; Cell Line, Tumor; Coculture Techniques; Cysteine-Rich Protein 61; Deoxycytidine; Down-Regulation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Equilibrative Nucleoside Transporter 1; Gemcitabine; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Membrane Transport Proteins; Pancreatic Neoplasms; Pancreatic Stellate Cells; Signal Transduction; Statistics, Nonparametric; Survival Analysis; Transforming Growth Factor beta; Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) has a low overall survival rate, which is approximately 20% during the first year and decreases to less than 6% within five years of the disease. This is due to premature dissemination accompanied by a lack of disease-specific symptoms during the initial stages. Additionally, to date there are no biomarkers for an early prognosis available.A growing number of studies indicate that epithelial to mesenchymal transition (EMT), triggered by WNT-, TGF-β- and other signaling pathways is crucial for the initiation of the metastatic process in PDAC. Here we show, that BCL9L is up-regulated in PDAC cell lines and patient tissue compared to non-cancer controls. RNAi-induced BCL9L knockdown negatively affected proliferation, migration and invasion of pancreatic cancer cells. On a molecular basis, BCL9L depletion provoked an increment of E-cadherin protein levels, with concomitant increase of β-catenin retention at the plasma membrane. This is linked to the induction of a strong epithelial phenotype in pancreatic cancer cells upon BCL9L knockdown even in the presence of the EMT-inducer TGF-β. Finally, xenograft mouse models of pancreatic cancer revealed a highly significant reduction in the number of liver metastases upon BCL9L knockdown. Taken together, our findings underline the key importance of BCL9L for EMT and thus progression and metastasis of pancreatic cancer cells. Direct targeting of this protein might be a valuable approach to effectively antagonize invasion and metastasis of PDAC.

Topics: Adherens Junctions; Animals; beta Catenin; Cadherins; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; Mice; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; Protein Transport; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-β family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Animals; Endoglin; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 2; Humans; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neovascularization, Pathologic; Neuroendocrine Tumors; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Burden

Many solid cancers including pancreatic ductal adenocarcinoma (PDAC) are characterized by an extensive stromal reaction that is accompanied by infiltrating tumor-associated macrophages (TAMs). The role of TAMs in malignant tumors is only partially understood. Previously, we identified the transcription factor CUX1 as an important mediator of tumor progression in PDAC. Interestingly, we found that CUX1 is highly expressed not only in tumor cells but also in TAMs. On the basis of these data, we aimed to elucidate the effects of CUX1 in TAMs in vitro and in vivo. We analyzed the effects of CUX1 on cytokine expression using overexpression and knockdown strategies. The cytokine regulation by CUX1 was further assessed by reporter assays, DNA pulldown experiments and chromatin-immunoprecipitation. CUX1 expression in TAMs was analyzed in human pancreatic cancer tissues and in a genetic mouse model. Immunohistochemical analysis revealed strong expression levels of CUX1 in a distinct subset of TAMs in human PDAC tissues. Furthermore, its expression increased during tumor progression in a genetic mouse model of PDAC. Profiling experiments showed that CUX1 downregulates several NF-κB-regulated chemokines such as CXCL10, which have been associated with M1 polarization and inhibition of angiogenesis and tumor progression. We could demonstrate that CUX1 interacts with NF-κB p65, leading to reduced binding of NF-κB p65 to the chemokine promoters. In addition, CUX1 reduces acetylation of NF-κB p65 at K310 by recruiting HDAC1. Functionally, CUX1 expression in TAMs antagonizes T-cell attraction and enhances angiogenesis in vitro. We identified CUX1 as an important modulator of the TAMs phenotype and function by modulating NF-κB-dependent cytokines.

Topics: Animals; Carcinoma, Pancreatic Ductal; Coculture Techniques; Disease Progression; Homeodomain Proteins; Humans; Macrophages; Mice; NF-kappa B; Nuclear Proteins; Pancreatic Neoplasms; Repressor Proteins; Signal Transduction; Transcription Factor RelA; Transcription Factors; Transfection; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) receptor signaling plays a paradoxical effect in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC), in which its tumor-inhibitory role at early stages turns into a tumor-promoting role at later stages. The underlying mechanism remains far from clear. Here we provide strong evidence that the activation of TGFβ receptor signaling in PDAC cells increased SMAD3 phosphorylation and nuclear translocation to inhibit cell growth. Meanwhile, it also activated SMAD7 to induce nuclear translocation and retention of β-catenin, which not only attenuated the inhibition of cell growth by nuclear SMAD3 but also activated vascular endothelial growth factor A (VEGF-A) to promote vascularization. Our data thus support a model involving crosstalk of the TGFβ and Wnt signaling pathways, for regulating the complicated effect of TGFβ signaling on the tumorigenesis of PDAC.

Topics: beta Catenin; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Humans; Pancreatic Neoplasms; Phosphorylation; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wnt Proteins

The hallmarks of pancreatic cancer are limitless replicative potential as well as tissue invasion and metastasis, leading to an extremely aggressive disease with shockingly high lethality. However, the molecular mechanisms underlying these characteristics remain largely unclear. Herein, we report the results of a differential miRNA expression screen that compared pancreatic cancer tissues and normal pancreatic tissues, where the pancreatic cancer tissues had highly downregulated miR-29c with relative Wnt cascade hyperactivation. MiR-29c directly suppressed the following Wnt upstream regulators: frequently rearranged in advanced T-cell lymphomas 2 (FRAT2), low-density lipoprotein receptor-related protein 6 (LRP6), Frizzled-4 (FZD4) and Frizzled-5 (FZD5). Furthermore, transforming growth factor-β (TGF-β) inhibited miR-29c expression, leading to Wnt activation. Significantly, our results were consistent with an important correlation between miR-29c levels and TGF-β hyperactivation and the activated Wnt cascade in human pancreatic cancer specimens. These findings reveal a novel mechanism for Wnt hyperactivation in pancreatic cancer and may suggest a new target for clinical intervention in pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Cell Movement; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplastic Stem Cells; Pancreatic Neoplasms; Phenotype; Prognosis; Smad3 Protein; Time Factors; Transfection; Transforming Growth Factor beta; Wnt Signaling Pathway

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths with no effective therapeutics. Invasion and metastasis are the major characteristics of PDAC. However, mechanisms underlying PDAC invasion and metastasis are elusive. In this report, we found that Kindlin-2 is a target protein of transforming growth factor β (TGF-β) signaling and is upregulated by TGF-β1 in PDAC cells. TGF-β1-upregulated Kindlin-2 promotes PDAC cell growth, migration and invasion, whereas Kindlin-2 upregulates transforming growth factor receptor I (TβRI), a key component of TGF-β signaling. Thereby Kindlin-2 and TGF-β signaling constitute a positive feedback loop. Mechanistically, Kindlin-2 promotes PDAC progression by downregulation of HOXB9 and E-cadherin. For clinical relevance, enhanced expression of Kindlin-2 predicts a poor overall survival for PDAC patients. Gene expression levels of Kindlin-2, TGF-β, TβRI and HOXB9 are all correlated with the overall survival of PDAC patients in an Oncomine dataset. Taken together, our findings demonstrated that TGF-β1-induced Kindlin-2 expression promotes PDAC progression by downregulation of HOXB9 and E-cadherin.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Pancreatic Ductal; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Disease Progression; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Muscle Proteins; Neoplasm Proteins; Pancreatic Neoplasms; Prognosis; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Cell Line, Tumor; Chromatin Immunoprecipitation; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Pancreatic Neoplasms; Retinal Dehydrogenase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Smad4 Protein; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Binding of α5β1 and αvβ3/β5 integrin receptors on the endothelium to their fibronectin substrate in the extracellular matrix has been targeted as a possible means of blocking tumor angiogenesis and tumor growth. However, clinical trials of blocking antibodies and peptides have been disappointing despite promising preclinical results, leading to questions about the mechanism of the inhibitors and the reasons for their failure. Here, using tissue-specific and inducible genetics to delete the α5 and αv receptors in the endothelium or their fibronectin substrate, either in the endothelium or globally, we show that both are dispensable for tumor growth, in transplanted tumors as well as spontaneous and angiogenesis-dependent RIP-Tag-driven pancreatic adenocarcinomas. In the nearly complete absence of fibronectin, no differences in vascular density or the deposition of basement membrane laminins, ColIV, Nid1, Nid2, or the TGFβ binding matrix proteins, fibrillin-1 and -2, could be observed. Our results reveal that fibronectin and the endothelial fibronectin receptor subunits, α5 and αv, are dispensable for tumor angiogenesis, suggesting that the inhibition of angiogenesis induced by antibodies or small molecules may occur through a dominant negative effect, rather than a simple functional block.

Topics: Adenocarcinoma; Animals; Basement Membrane; Calcium-Binding Proteins; Cell Adhesion Molecules; Endothelium; Extracellular Matrix; Fibrillin-1; Fibrillins; Fibronectins; Integrin alpha5beta1; Integrins; Membrane Glycoproteins; Mice; Microfilament Proteins; Neovascularization, Pathologic; Pancreatic Neoplasms; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant. Efficient control of cancer growth may substantially improve the survival of PDAC patients. However, no efficient treatments are so far available. Here, we inhibited transforming growth factor β (TGFβ) receptor signaling by overexpression of a key inhibitor of this pathway, SMAD7, in the mouse pancreas, using a recently developed intraductal infusion method. Overexpression of SMAD7 significantly increased growth of both implanted PDAC and PDAC by K-ras modification. Our data thus suggest that TGFβ receptor signaling restrains growth of PDAC, and modulation of TGFβ receptor signaling may be an effective treatment for PDAC.

Topics: Animals; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred NOD; Mice, SCID; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad7 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Topics: Animals; Base Sequence; Bone Marrow Cells; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Exosomes; Female; Fibronectins; Gene Expression Regulation, Neoplastic; Hepatic Stellate Cells; Humans; Liver; Liver Neoplasms; Macrophage Migration-Inhibitory Factors; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Neoplasms; Precancerous Conditions; RNA Interference; RNA, Small Interfering; Sequence Analysis, RNA; Signal Transduction; Transforming Growth Factor beta

Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression, and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability, and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-β1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Survival; Enzyme Activation; Extracellular Matrix; Fibroblasts; Fibrosis; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Pancreas; Pancreatic Neoplasms; Pancreatic Stellate Cells; Proto-Oncogene Proteins p21(ras); Smad3 Protein; Transforming Growth Factor beta; Tumor Microenvironment

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We report here that silencing SMURF2 (SMAD specific E3 ubiquitin protein ligase 2) promoted EMT in HPDE6c7 normal pancreas cells and overexpression of SMURF2 inhibited TGF-β-mediated EMT in the cells. Subsequent studies showed that SMURF2 was downregulated in pancreatic cancer tissues and it promoted mesenchymal-epithelial transition (MET) in pancreatic cancer cells as well as its expression negatively associated with gemcitabine-resistance, but it did not alter cell viability, cell cycle and cell senescence. In addition, we demonstrated that miR‑15b degraded SMURF2 and its overexpression promoted EMT in pancreatic cancer, and its expression was associated with metastasis in the disease. Elucidating molecular mechanism of EMT in pancreatic cancer not only will help us to further understand the pathogenesis and progression of the disease, but also offers new targets for effective therapies.

Topics: Cell Line; Deoxycytidine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Metastasis; Pancreatic Neoplasms; Promoter Regions, Genetic; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Cancer cachexia is a debilitating condition characterized by a combination of anorexia, muscle wasting, weight loss, and malnutrition. This condition affects an overwhelming majority of patients with pancreatic cancer and is a primary cause of cancer-related death. However, few, if any, effective therapies exist for both treatment and prevention of this syndrome. In order to develop novel therapeutic strategies for pancreatic cancer cachexia, appropriate animal models are necessary. In this study, we developed and validated a syngeneic, metastatic, murine model of pancreatic cancer cachexia. Using our model, we investigated the ability of transforming growth factor beta (TGF-β) blockade to mitigate the metabolic changes associated with cachexia. We found that TGF-β inhibition using the anti-TGF-β antibody 1D11.16.8 significantly improved overall mortality, weight loss, fat mass, lean body mass, bone mineral density, and skeletal muscle proteolysis in mice harboring advanced pancreatic cancer. Other immunotherapeutic strategies we employed were not effective. Collectively, we validated a simplified but useful model of pancreatic cancer cachexia to investigate immunologic treatment strategies. In addition, we showed that TGF-β inhibition can decrease the metabolic changes associated with cancer cachexia and improve overall survival.

Topics: Animals; Antibodies; Body Composition; Cachexia; Cell Line, Tumor; Disease Models, Animal; Immunotherapy; Male; Mice; Mice, Inbred C57BL; Muscular Atrophy; Neoplasm Metastasis; Pancreatic Neoplasms; Survival Analysis; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is associated with accumulation of particular oncogenic mutations and recent genetic sequencing studies have identified ataxia telangiectasia-mutated (ATM) mutations in PDAC cohorts. Here we report that conditional deletion of ATM in a mouse model of PDAC induces a greater number of proliferative precursor lesions coupled with a pronounced fibrotic reaction. ATM-targeted mice display altered TGFβ-superfamily signalling and enhanced epithelial-to-mesenchymal transition (EMT) coupled with shortened survival. Notably, our mouse model recapitulates many features of more aggressive human PDAC subtypes. Particularly, we report that low expression of ATM predicts EMT, a gene signature specific for Bmp4 signalling and poor prognosis in human PDAC. Our data suggest an intimate link between ATM expression and pancreatic cancer progression in mice and men.

Topics: Adult; Aged; Aged, 80 and over; Animals; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Bone Morphogenetic Protein 4; Carcinoma, Pancreatic Ductal; Cell Survival; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Mice; Middle Aged; Pancreatic Neoplasms; Prognosis; Proto-Oncogene Proteins p21(ras); Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Pancreatic fibrosis is a prominent histopathological characteristic of chronic pancreatitis and plausibly a dynamic process of transition to the development of pancreatic ductal adenocarcinoma. Conversely, the activation of pancreatic stellate cells (PSCs) has been recently suggested as the key initiating step in pancreatic fibrosis. As natural polyphenols had been largely applied in complementary therapies in the past decade, in this study, we aimed to investigate which groups of phenolic compounds exert promising inhibitory actions on fibrogenesis as there are few effective strategies for the treatment of pancreatic fibrosis to date.. We examined the anti-fibrotic effects of a variety of herbal constituents using a cellular platform, the LTC-14 cells, which retained essential characteristics and morphologies of primary PSCs, by means of various biochemical assays including cell viability test, real-time polymerase chain reaction and Western blotting analysis.. Among a number of commonly used herbal constituents, we found that the application of rhein, emodin, curcumin and resveratrol significantly suppressed the mRNA and protein levels of several fibrotic mediators namely alpha-smooth muscle actin, type I collagen and fibronectin in LTC-14 cells against transforming growth factor-beta stimulation. Though the values of cytotoxicity varied, the mechanism of the anti-fibrotic action of these four phenolic compounds was principally associated with a decrease in the activation of the nuclear factor-kappaB signaling pathway.. Our findings suggest that the mentioned phenolic compounds may serve as anti-fibrotic agents in PSC-relating disorders and pathologies, particularly pancreatic fibrosis.

Topics: Actins; Cell Line; Cell Survival; Collagen Type I; Fibronectins; Fibrosis; Humans; Pancreatic Neoplasms; Pancreatic Stellate Cells; Phenols; Signal Transduction; Transforming Growth Factor beta

Emodin is a traditional Chinese medicine, which has been demonstrated to inhibit the growth of pancreatic cancer cells. However, the underlying molecular mechanisms remain to be elucidated. The present study investigated whether emodin suppresses angiogenesis in pancreatic cancer. A nude mouse pancreatic cancer xenograft model was established using SW1990 human pancreatic cancer cells by surgical orthotopic implantation. Different doses of emodin were injected into the abdominal cavities of the tumor‑bearing mouse models and controls three times each week for 2 weeks. The tumors were measured and weighed, the expression of cluster of differentiation 34 was detected using immunochemistry, and microvessel densities were calculated. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting were performed to determine the mRNA and protein expression levels of transforming growth factor (TGF)‑β and drosophila mothers against decapentaplegic (Smad) homologs. The angiogenesis‑associated microRNAs (miR), miR‑20, miR‑155 and miR‑210 were assessed by RT‑qPCR. A negative dose‑dependent association was revealed between treatment with emodin and the volume and weight of tumors and microvessel density. Emodin was associated with lower mRNA and protein expression levels of TGF‑β1 and its downstream target, angiopoietin‑like 4, and higher mRNA and protein expression levels of TGF‑β receptor (TβR)I, TβRII and Smad4. Notably, treatment with emodin was associated with lower expression levels of miR‑155 and miR‑210 and higher expression levels of miR‑20b. The present study suggested that treatment with emodin may repress angiogenesis in pancreatic cancer by altering the activities of the TGF-β/Smad pathway and angiogenesis-associated miR-20b, miR-155, and miR-210.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Disease Models, Animal; Emodin; Female; Gene Expression; Heterografts; Humans; Mice; MicroRNAs; Neovascularization, Pathologic; Pancreatic Neoplasms; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Burden

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States. The TGF-β signaling protein SMAD family member 4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. However, the mechanisms by which SMAD4 loss promotes PDAC development are not fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and CFPAC1 by selection of stable clones containing an inducible SMAD4 tetracycline inducible expression system construct. After 24 hours of SMAD4 expression, TGF-β signaling-dependent G1 arrest was observed in BxPC3 cells with an increase in the G1 phase fraction from 48.9% to 71.5%. Inhibition of cyclin-dependent kinase inhibitor 1A by small interfering RNA eliminated the antiproliferative effect, indicating that up-regulation of cyclin-dependent kinase inhibitor 1A/p21 by TGF-β signaling is necessary for the phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced migration. Microarray analysis of gene expression at 8, 24, and 48 hours after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the novel TGF-β targets identified are anthrax toxin receptor 2 (3.58× at 8 h), tubulin, β-3 class III (7.35× at 8 h), cell migration inducing protein, hyaluronan binding (8.07× at 8 h), IL-1 receptor-like 1 (0.403× at 8 h), regulator of G protein signaling 4 (0.293× at 8 h), and THAP domain containing 11 (0.262× at 8 h). The gene expression changes we observed upon restoration of TGF-β signaling provide numerous new targets for future investigations into PDAC biology and progression.

Topics: Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genotype; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Transfection; Transforming Growth Factor beta; Up-Regulation

Steps in the genetic basis of pancreatic cancer (PC) have been recently identified, however, Studies focusing on the relationship between Jab1 and Smad4 in PC are rarely reported. This study was performed to examine the expression patterns and association of Jab1 and Smad4 in PC cells for gaining a further understanding of PC pathogenesis.. Human pancreatic cancer cell line PANC-1 cells were infected with retrovirus vector containing GFP, HA-Jab1, siGFP, and siJab1 respectively. The expression of Jab1 and Smad4 in PANC-1 cells was analyzed by Western blot and immunocytochemistry. Subsequently, the effect of overexpression of Jab1 on cell proliferation inhibition mediated by TGF-β was examined with MTT colorimetry.. The expression of Smad4 in PANC-1 cells was inhibited after the overexpression of Jab1. Inversely, the expression of Smad4 was increased after the down-regulation of Jab1 silenced by SiRNA. Smad4 expression in PANC-1 cells was negatively correlated with Jab1 expression. In addition, the cell proliferation inhibitory effect induced by TGF-β in PANC-1 cells was attenuated after the overexpression of Jab1.. The reverse correlation of Jab1 and Smad4 in PANC-1 cells may be involved in the Pathogenesis of PC. Jab1 can cause degradation of Smad4 via TGF-β signal pathway, consequently contributing to the proliferation of PC cells.

Topics: Cell Line, Tumor; Cell Proliferation; COP9 Signalosome Complex; Humans; Intracellular Signaling Peptides and Proteins; Pancreatic Neoplasms; Peptide Hydrolases; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Our neoadjuvant clinical trial of a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) revealed the development of tertiary lymphoid aggregates (TLAs) within the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment 2 weeks after GVAX treatment. Microarray studies revealed that multiple components of the TGF-β pathway were suppressed in TLAs from patients who survived greater than 3 years and who demonstrated vaccine-enhanced mesothelin-specific T cell responses. We tested the hypothesis that combining GVAX with TGF-β inhibitors will improve the anti-tumor immune response of vaccine therapy. In a metastatic murine model of pancreatic cancer, combination therapy with GVAX vaccine and a TGF-β blocking antibody improved the cure rate of PDA-bearing mice. TGF-β blockade in combination with GVAX significantly increased the infiltration of effector CD8+ T lymphocytes, specifically anti-tumor-specific IFN-g producing CD8+ T cells, when compared to monotherapy controls (all p < 0.05). TGF-β blockade alone did not deplete T regulatory cells (Tregs), but when give in combination with GVAX, GVAX induced intratumoral Tregs were depleted. Therefore, our PDA preclinical model demonstrates a survival advantage in mice treated with an anti-TGF-β antibody combined with GVAX therapy and provides strong rational for testing this combinational therapy in clinical trials.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antineoplastic Combined Chemotherapy Protocols; Cancer Vaccines; Carcinoma, Pancreatic Ductal; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Lymphocytes, Tumor-Infiltrating; Mesothelin; Mice; Pancreatic Neoplasms; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Autophagy is a cellular process to regulate the turnover of misfolded/aggregated proteins or dysfunctional organelles such as damaged mitochondria. Microtubule-associated protein MAP1S (originally named C19ORF5) is a widely-distributed homologue of neuronal-specific MAP1A and MAP1B with which autophagy marker light chain 3 (LC3) was originally co-purified. MAP1S bridges autophagic components with microtubules and mitochondria through LC3 and positively regulates autophagy flux from autophagosomal biogenesis to degradation. The MAP1S-mediated autophagy suppresses tumorigenesis as suggested in a mouse liver cancer model and in prostate cancer patients. The TGFβ signaling pathway plays a central role in pancreatic tumorigenesis, and high levels of TGFβ suggest a tumor suppressive function and predict a better survival for some patients with resectable pancreatic ductal adenocarcinoma. In this study, we try to understand the relationship between TGFβ and MAP1S-mediated autophagy in pancreatic ductal adenocarcinoma.. We collected the tumor and its adjacent normal tissues from 33 randomly selected patients of pancreatic ductal adenocarcinomas to test the association between TGFβ and autophagy markers MAP1S and LC3. Then we tested the cause and effect relation between TGFβ and autophagy markers in cultured pancreatic cancer cell lines.. Here we show that levels of TGFβ and autophagy markers MAP1S and LC3 are dramatically elevated in tumor tissues from patients with pancreatic ductal adenocarcinomas. TGFβ increases levels of MAP1S protein and enhances autophagy flux.. TGFβ may suppress the development of pancreatic ductal adenocarcinomas by enhancing MAP1S-mediated autophagy.

Topics: Animals; Autophagy; Carcinoma, Pancreatic Ductal; Cell Line; Follow-Up Studies; HeLa Cells; Humans; Mice; Microtubule-Associated Proteins; Pancreatic Neoplasms; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been shown to associate with a better outcome. However, the underlying mechanisms of this benefit remain unclear. Metformin has been found to modulate the activity of stellate cells in other disease settings. In this study, we examine the effect of metformin on PSC activity, fibrosis and inflammation in PDACs.. In overweight, diabetic PDAC patients and pre-clinical mouse models, treatment with metformin reduced levels of tumor extracellular matrix (ECM) components, in particular hyaluronan (HA). In vitro, we found that metformin reduced TGF-ß signaling and the production of HA and collagen-I in cultured PSCs. Furthermore, we found that metformin alleviates tumor inflammation by reducing the expression of inflammatory cytokines including IL-1β as well as infiltration and M2 polarization of tumor-associated macrophages (TAMs) in vitro and in vivo. These effects on macrophages in vitro appear to be associated with a modulation of the AMPK/STAT3 pathway by metformin. Finally, we found in our preclinical models that the alleviation of desmoplasia by metformin was associated with a reduction in ECM remodeling, epithelial-to-mesenchymal transition (EMT) and ultimately systemic metastasis.. Metformin alleviates the fibro-inflammatory microenvironment in obese/diabetic individuals with pancreatic cancer by reprogramming PSCs and TAMs, which correlates with reduced disease progression. Metformin should be tested/explored as part of the treatment strategy in overweight diabetic PDAC patients.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Interleukin-1beta; Macrophages; Male; Metformin; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Pancreatic Stellate Cells; Prognosis; STAT3 Transcription Factor; Transforming Growth Factor beta

Increased microRNA-10b (miR-10b) expression in the cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a marker of disease aggressiveness. In the present study, we determined that plasma miR-10b levels are significantly increased in PDAC patients by comparison with normal controls. By gene profiling, we identified potential targets downregulated by miR-10b, including Tat-interacting protein 30 (TIP30). Immunoblotting and luciferase reporter assays confirmed that TIP30 was a direct miR-10b target. Downregulation of TIP30 by miR-10b or siRNA-mediated silencing of TIP30 enhanced epidermal growth factor (EGF)-dependent invasion. The actions of miR-10b were abrogated by expressing a modified TIP30 cDNA resistant to miR-10b. EGF-induced EGF receptor (EGFR) tyrosine phosphorylation and extracellular signal-regulated kinase phosphorylation were enhanced by miR-10b, and these effects were mimicked by TIP30 silencing. The actions of EGF in the presence of miR-10b were blocked by EGFR kinase inhibition with erlotinib and by dual inhibition of PI3K (phosphatidylinositol 3'-kinase) and MEK. Moreover, miR-10b, EGF and transforming growth factor-beta (TGF-β) combined to markedly increase cell invasion, and this effect was blocked by the combination of erlotinib and SB505124, a type I TGF-β receptor inhibitor. miR-10b also enhanced the stimulatory effects of EGF and TGF-β on cell migration and epithelial-mesenchymal transition (EMT) and decreased the expression of RAP2A, EPHB2, KLF4 and NF1. Moreover, miR-10b overexpression accelerated pancreatic cancer cell (PCC) proliferation and tumor growth in an orthotopic model. Thus, plasma miR-10b levels may serve as a diagnostic marker in PDAC, whereas intra-tumoral miR-10b promotes PCC proliferation and invasion by suppressing TIP30, which enhances EGFR signaling, facilitates EGF-TGF-β cross-talk and enhances the expression of EMT-promoting genes, whereas decreasing the expression of several metastasis-suppressing genes. Therefore, therapeutic targeting of miR-10b in PDAC may interrupt growth-promoting deleterious EGF-TGF-β interactions and antagonize the metastatic process at various levels.

Topics: Acetyltransferases; Animals; Antineoplastic Agents; Base Sequence; Binding Sites; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Epidermal Growth Factor; Erlotinib Hydrochloride; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Factor 4; Male; Mice; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Transplantation; Pancreatic Neoplasms; Quinazolines; RNA Interference; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.. We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.. The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).. Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Carcinoma, Pancreatic Ductal; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Survival Rate; Transforming Growth Factor beta

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Carcinoma, Pancreatic Ductal; Cell Adhesion; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha2; Integrins; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; Nerve Tissue Proteins; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatic Stellate Cells; Promoter Regions, Genetic; Proto-Oncogene Proteins c-met; Receptors, Immunologic; RNA, Messenger; Roundabout Proteins; Sequence Analysis, DNA; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Pancreatic cancer is one of the most deadly types of cancer and has extremely poor prognosis. This malignancy typically induces only limited cellular immune responses, the magnitude of which can increase with the number of encountered cancer cells. On the other hand, pancreatic cancer is highly effective at evading immune responses by inducing polarization of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, and promoting expansion of myeloid derived suppressor cells, which block the killing of cancer cells by cytotoxic T cells. These factors allow immune evasion to predominate, promoting metastasis and poor responsiveness to chemotherapies and immunotherapies. In this paper we develop a mathematical model of pancreatic cancer, and use it to qualitatively explain a variety of biomedical and clinical data. The model shows that drugs aimed at suppressing cancer growth are effective only if the immune induced cancer cell death lies within a specific range, that is, the immune system has a specific window of opportunity to effectively suppress cancer under treatment. The model results suggest that tumor growth rate is affected by complex feedback loops between the tumor cells, endothelial cells and the immune response. The relative strength of the different loops determines the cancer growth rate and its response to immunotherapy. The model could serve as a starting point to identify optimal nodes for intervention against pancreatic cancer.

Topics: Cell Proliferation; ErbB Receptors; Gene Silencing; Humans; Immunotherapy; Models, Immunological; Pancreatic Neoplasms; Prognosis; RNA, Messenger; Transforming Growth Factor beta; Tumor Escape

Drug-resistance to gemcitabine chemotherapy in pancreatic cancer is still an unsolved problem. Combinations of other chemotherapy drugs with gemcitabine have been shown to increase the efficacy of gemcitabine-based treatment. In this study, the effect of berbamine on the antitumor activity of gemcitabine was evaluated in human pancreatic cancer cell lines Bxpc-3 and Panc-1, and the underlying mechanisms were explored. Our results demonstrated that berbamine exhibited a time- and dose-dependent inhibitory effect in the pancreatic cancer cell lines. Berbamine enhanced gemcitabine-induced cell growth inhibition and apoptosis in these cells. Combined treatment of berbamine and gemcitabine resulted in down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL) and up-regulation of pro-apoptotic proteins (Bax, Bid). More importantly, berbamine treatment in combination with gemcitabine activated the transforming growth factor-β/Smad (TGF-β/Smad) signaling pathway, as a result of a decrease in Smad7 and an increase in transforming growth factor-β receptor II (TβRII) expression. Changes in downstream targets of Smad7, such as up-regulation of p21 and down-regulation of c-Myc and Cyclin D1 were also observed. Therefore, berbamine could enhance the antitumor activity of gemcitabine by inhibiting cell growth and inducing apoptosis, possibly through the regulation of the expression of apoptosis-related proteins and the activation of TGF-β/Smad signaling pathway. Our study indicates that berbamine may be a promising candidate to be used in combination with gemcitabine for pancreatic cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Benzylisoquinolines; Cell Cycle; Cell Line, Tumor; Cell Survival; Deoxycytidine; Down-Regulation; Drug Interactions; Gemcitabine; Humans; Pancreatic Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines.. NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFβ1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFβ1 canonical signaling pathway. TGFβ1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFβ1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFβ1 (MALDI-TOF/MS and co-immuno-precipitation).. The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFβ1 on cell signaling, and the formation of complexes between TGFβ1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

Topics: Calcium Signaling; Calgranulin A; Calgranulin B; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Inflammation; NF-kappa B; Pancreatic Neoplasms; Peptide Fragments; Protein Binding; Proteolysis; Proto-Oncogene Proteins c-akt; Smad4 Protein; Transforming Growth Factor beta

Micro RNAs (miRNAs) are small noncoding RNAs that have gained attention as key molecules in the malignant characteristics of cancers, and several recent investigations also have identified some miRNAs as potential key regulators to inhibit the malignant characteristics of tumors. MiRNA-373 (miR-373) has recently been reported to induce E-cadherin, which is a key regulator of epithelial-mesenchymal transition (EMT). However, the role of miR-373 in the characteristics of cancer cells is not still well known.. We investigated the expression levels of miR-373 in pancreatic cancer cell lines and its effect on the invasiveness of pancreatic cancer by using in vitro and in vivo models. We also analyzed the expression of miR-373 using formalin-fixed paraffin-embedded (n = 152) and microdissected frozen (n = 57) samples from pancreatic tissues.. The levels of miR-373 expression were low in pancreatic cancer cell lines. In formalin-fixed paraffin-embedded and microdissected frozen samples, miR-373 expression was significantly down-regulated in pancreatic cancer compared with that in healthy pancreas (P < 0.001 and P = 0.005, respectively). We also found that reexpression of miR-373 repressed transforming growth factor-β-induced EMT, leading to inhibition of invasiveness of cancer cells. Furthermore, reexpression of miR-373 significantly inhibited peritoneal dissemination in vivo (P < 0.001).. MiR-373 is down-regulated in pancreatic cancer, and its reexpression represses the invasiveness of pancreatic cancer cells.

Topics: Animals; Cadherins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Hyaluronan Receptors; Mice; MicroRNAs; Neoplasm Invasiveness; Pancreas; Pancreatic Neoplasms; Peritoneal Neoplasms; RNA, Messenger; Transfection; Transforming Growth Factor beta; Vimentin

Progression of pancreatic ductal adenocarcinoma (PDAC) is promoted by desmoplasia induced by pancreatic stellate cells (PSC). Contributory to this progression is epithelial mesenchymal transition (EMT), which shares many characteristics with the cancer stem cell (CSC) hypothesis. We investigated the role of these processes on the radioresponse and tumorigenicity of pancreatic cancer cells.. We used an in vitro sphere model and in vivo xenograft model to examine the role of PSC in EMT and CSC processes.. We demonstrated that PSC enhanced the CSC phenotype and radioresistance of pancreatic cancer cells. Furthermore, the expression of several EMT and CSC markers supported enhanced processes in our models and that translated into remarkable in vivo tumorigenicity. Multi-dose TGFβ neutralizing antibody inhibited the EMT and CSC processes, sensitized cells to radiation and reduced in vivo tumorigenicity. A proteomic screen identified multiple novel factors that were regulated by PSC in pancreatic cells.. These results are critical in highlighting the role of PSC in tumor progression and radioresistance by manipulating the EMT and CSC processes. TGFβ and the novel factors identified are important targets for better therapeutic outcome in response to PSC mediated mechanisms.

Topics: Adenocarcinoma; Antibodies, Neutralizing; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Survival; Epithelial-Mesenchymal Transition; Fibroblasts; Humans; Neoplastic Stem Cells; Pancreatic Neoplasms; Pancreatic Stellate Cells; Phenotype; Radiation Tolerance; Transforming Growth Factor beta; Tumor Cells, Cultured

Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial-mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor-stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.

Topics: Benzamides; Cell Line; Cell Line, Tumor; Coculture Techniques; Collagen; Dioxoles; Epithelial-Mesenchymal Transition; Fibroblasts; Hepatocyte Growth Factor; Humans; Models, Statistical; Neoplasm Invasiveness; Pancreatic Neoplasms; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor β (TGFβ)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFβ-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFβ-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Enzyme Activation; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; MicroRNAs; Organ Specificity; Pancreatic Neoplasms; Phenotype; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Elevated levels of TGFβ are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result, the TGFβ pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGFβ remains challenging because TGFβ has tumor suppressor functions in many epithelial malignancies, including pancreatic cancer. In fact, direct neutralization of TGFβ promotes tumor progression of genetic murine models of pancreatic cancer. Here, we report that neutralizing the activity of murine TGFβ receptor 2 using a monoclonal antibody (2G8) has potent antimetastatic activity in orthotopic human tumor xenografts, syngeneic tumors, and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density, and vascular function. These stromal-specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGFβ signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGFβ-dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors. Cancer Res; 74(18); 4996-5007. ©2014 AACR.

Topics: Animals; Antibodies, Monoclonal; Antimetabolites, Antineoplastic; Cell Differentiation; Cell Line, Tumor; Deoxycytidine; Gemcitabine; Humans; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; NIH 3T3 Cells; Pancreatic Neoplasms; Random Allocation; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell-like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow-derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.

Topics: Animals; Arginase; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Disease Progression; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Inflammation; Interleukin-4; Macrophages; Mice; Neoplasm Metastasis; Pancreatic Neoplasms; Phenotype; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Transforming Growth Factor beta

SnoN is a negative regulator of TGF-β signaling and also an activator of the tumor suppressor p53 in response to cellular stress. Its role in human cancer is complex and controversial with both pro-oncogenic and anti-oncogenic activities reported. To clarify its role in human cancer and provide clinical relevance to its signaling activities, we examined SnoN expression in normal and cancerous human esophageal, ovarian, pancreatic and breast tissues. In normal tissues, SnoN is expressed in both the epithelium and the surrounding stroma at a moderate level and is predominantly cytoplasmic. SnoN levels in all tumor epithelia examined are lower than or similar to that in the matched normal samples, consistent with its anti-tumorigenic activity in epithelial cells. In contrast, SnoN expression in the stroma is highly upregulated in the infiltrating inflammatory cells in high-grade esophageal and ovarian tumor samples, suggesting that SnoN may potentially promote malignant progression through modulating the tumor microenvironment in these tumor types. The overall levels of SnoN expression in these cancer tissues do not correlate with the p53 status. However, in human cancer cell lines with amplification of the snoN gene, a strong correlation between increased SnoN copy number and inactivation of p53 was detected, suggesting that the tumor suppressor SnoN-p53 pathway must be inactivated, either through downregulation of SnoN or inactivation of p53, in order to allow cancer cell to proliferate and survive. These data strongly suggest that SnoN can function as a tumor suppressor at early stages of tumorigenesis in human cancer tissues.

Topics: Adult; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Progression; Esophageal Neoplasms; Esophagus; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Middle Aged; Ovarian Neoplasms; Ovary; Pancreas; Pancreatic Neoplasms; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53

mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling.

Topics: Apoptosis; Cell Line, Tumor; Eukaryotic Initiation Factor-4E; Gene Knockdown Techniques; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Pancreatic Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Smad4 Protein; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Vasohibin-1 has been detected in endothelial cells as an intrinsic angiogenesis inhibitor. Both tumor-associated macrophages (TAMs) and transforming growth factor-β (TGF-β)/bone morphogenic protein (BMP) signaling have been reported to promote angiogenesis in cancer. However, whether vasohibin-1 expression is regulated by TGF-β/BMP signaling between TAMs and cancer cells remains unclear. The expression of TGF-β1, TGF-β2, BMP-4, and BMP-7 in TAMs and the expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and VEGF-C in two pancreatic cancer cell lines (a nonmetastatic cell line Panc-1 and a distant metastatic cell line HPAF-II) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The TGF-β receptor 1 and BMP receptor 1 were inhibited by the inhibitor SB-431542 and LDN193189, respectively. Thereafter, vasohibin-1, VEGF-A, and VEGF-C expression was detected by real-time RT-PCR. We found that the expression of TGF-β1, TGF-β2, BMP-4, and BMP-7 was upregulated in TAMs cocultured with pancreatic cancer cells. Vasohibin-1, VEGF-A, and VEGF-C mRNA expression in pancreatic cancer cells was upregulated by TAMs. Vasohibin-1 expression in pancreatic cancer cells cocultured with TAMs was upregulated significantly when TGF-β receptors or BMP receptors were inhibited, but VEGF-C expression was downregulated. Therefore, Vasohibin-1 expression is regulated by the TGF-β/BMP signaling between TAMs and pancreatic cancer cells. These results might shed a new light on the antiangiogenesis therapy in the pancreatic cancer.

Topics: Benzamides; Blotting, Western; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Dioxoles; Gene Expression; Humans; Macrophages; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor C

The expression of SMAD4, the central component of the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathways, is lost in 50% of pancreatic cancers and is associated with a poor survival. Although the TGF-β pathway has been extensively studied and characterised in pancreatic cancer, there is very limited data on BMP signalling, a well-known tumour-suppressor pathway. BMP signalling can be lost not only at the level of SMAD4 but also at the level of BMP receptors (BMPRs), as has been described in colorectal cancer.. We performed immunohistochemical analysis of the expression levels of BMP signalling components in pancreatic cancer and correlated these with survival. We also manipulated the activity of BMP signalling in vitro.. Reduced expression of BMPRIA is associated with a significantly worse survival, primarily in a subset of SMAD4-positive cancers. In vitro inactivation of SMAD4-dependent BMP signalling increases proliferation and invasion of pancreatic cancer cells, whereas inactivation of BMP signalling in SMAD4-negative cells does not change the proliferation and invasion or leads to an opposite effect.. Our data suggest that BMPRIA expression is a good prognostic marker and that the BMP pathway is a potential target for future therapeutic interventions in pancreatic cancer.

Topics: Angiopoietin-1; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Biomarkers, Tumor; Bone Morphogenetic Protein Receptors, Type I; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neovascularization, Pathologic; Pancreatic Neoplasms; Prognosis; Pyrazoles; Pyrimidines; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Survival; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Resveratrol (trans-3,4',5-trihydroxystilbene), a natural polyphenolic compound detected in grapes, berries, and peanuts, possesses a wide spectrum of pharmacological properties, including anti-tumor metastasis activities. However, the underlying mechanisms through which resveratrol inhibits the metastasis of pancreatic cancer are still not fully elucidated. As epithelial-to-mesenchymal transition (EMT) is a key player for metastasis in tumor, the aim of this study is to determine whether resveratrol affects EMT in pancreatic cancer cells and the related mechanism. The results showed that resveratrol not only inhibited cell proliferation, migration, and invasion in a dose-dependent manner, but also mediated the expression of EMT-related genes (E-cadherin, N-cadherin, vimentin, MMP-2, and MMP-9) which are important for cancer cellular motility, invasiveness and metastasis during tumorigenesis. In addition, the levels of phospho-Akt and phospho- NF-κB in BxPC-3 and Panc-1 cells were reduced by both resveratrol and LY294002 (a PI3-K inhibitor). Furthermore, transforming growth factor-β (TGF-β)-induced alterations in cell morphology that are characteristic of EMT as well as increased cell invasive ability could also be reversed by resveratrol. Taken together, these data indicate that resveratrol suppresses pancreatic cancer migration and invasion through the inhibition of the PI-3K/Akt/NF-κB signaling pathway. This study suggests that resveratrol may be a potential anticancer agent for pancreatic cancer.

Topics: Antineoplastic Agents; Cadherins; Cell Line, Tumor; Cell Movement; Chromones; Epithelial-Mesenchymal Transition; Humans; Matrix Metalloproteinases; Morpholines; NF-kappa B; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Stilbenes; Transforming Growth Factor beta; Vimentin

Pancreatic ductal adenocarcinoma (PDAC) elicits a dense stromal response that blocks vascular access because of pericyte coverage of vascular fenestrations. In this way, the PDAC stroma contributes to chemotherapy resistance in addition to causing other problems. In order to improve the delivery of gemcitabine, a first-line chemotherapeutic agent, a PEGylated drug-carrying liposome was developed, using a transmembrane ammonium sulfate gradient to encapsulate the protonated drug up to 20% w/w. However, because the liposome was precluded from entering the xenograft site due to the stromal interference, we developed a first-wave nanocarrier that decreases pericyte coverage of the vasculature through interference in the pericyte recruiting TGF-β signaling pathway. This was accomplished using a polyethyleneimine (PEI)/polyethylene glycol (PEG)-coated mesoporous silica nanoparticle (MSNP) for molecular complexation to a small molecule TGF-β inhibitor, LY364947. LY364947 contains a nitrogen atom that attaches, through H-bonding, to PEI amines with a high rate of efficiency. The copolymer coating also facilitates systemic biodistribution and retention at the tumor site. Because of the high loading capacity and pH-dependent LY364947 release from the MSNPs, we achieved rapid entry of IV-injected liposomes and MSNPs at the PDAC tumor site. This two-wave approach provided effective shrinkage of the tumor xenografts beyond 25 days, compared to the treatment with free drug or gemcitabine-loaded liposomes only. Not only does this approach overcome stromal resistance to drug delivery in PDAC, but it also introduces the concept of using a stepwise engineered approach to address a range of biological impediments that interfere in nanocancer therapy in a spectrum of cancers.

Topics: Animals; Cell Line, Tumor; Collagen; Deoxycytidine; Drug Carriers; Drug Combinations; Endothelial Cells; Female; Gemcitabine; Humans; Laminin; Liposomes; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; Nanomedicine; Nanoparticles; Neoplasm Transplantation; Pancreatic Neoplasms; Polyethylene Glycols; Polyethyleneimine; Proteoglycans; Signal Transduction; Silicon Dioxide; Smad2 Protein; Transforming Growth Factor beta

Cancer-associated fibroblasts contribute to cancer progression that is caused by epithelial-mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor-promoting cancer stroma. Here we report that α-smooth muscle actin-positive myofibroblast-like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC-derived myofibroblasts function to maintain tumor-initiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere- forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ-secretase inhibitor and siRNA directed against Jagged-1 attenuated MSC-associated E-cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC-derived myofibroblasts play important roles in regulating EMT and tumor-initiating stem cell-like properties of pancreatic cancer cells through an intermediating Notch signal.

Topics: Actins; Amyloid Precursor Protein Secretases; Animals; Calcium-Binding Proteins; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Male; Membrane Proteins; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Myofibroblasts; Pancreatic Neoplasms; Receptors, Notch; RNA, Small Interfering; Serrate-Jagged Proteins; Transforming Growth Factor beta

Pancreatic cancer is a devastating disease with historically limited success in treatment and a poor prognosis. Pancreatic cancer appears to have a progressive pathway of development, initiating from well-described pancreatic intraepithelial neoplasia lesions and concluding with invasive carcinoma. These early lesions have been shown to harbor-specific alterations in signaling pathways that remain throughout this tumorigenesis process. Meanwhile, new alterations occur during this process of disease progression to have a cumulative effect. This series of events not only impacts the epithelial cells comprising the tumor, but they may also affect the surrounding stromal cells. The result is the formation of complex signaling networks of communication between the tumor epithelial cell and the stromal cell compartments to promote a permissive and cooperative environment. This article highlights some of the most common pathway aberrations involved with this disease, and how these may subsequently affect one or both cellular compartments. Consequently, furthering our understanding of these pathways in terms of their function on the tumoral epithelial and stromal compartments may prove to be crucial to the development of targeted and more successful therapies in the future.

Topics: Adenocarcinoma; Cell Communication; Clinical Trials as Topic; Epithelial Cells; Genes, p16; Genes, p53; Hedgehog Proteins; Humans; Molecular Targeted Therapy; Pancreatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptors, Notch; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Wnt Proteins

Gene therapy is an important means for the comprehensive treatment of pancreatic cancer. Challenges associated with gene therapy include control of vector security and effective genetic screening. In this paper, a CEA promoter-regulated oncolytic adenovirus vector was constructed. The reporter gene assay demonstrated that the viral vector was confirmed to have tumor-specific replication features. In vitro cytology studies showed that the CEA promoter regulated the proliferation of the adenovirus vector carrying the Hsp70 gene (AdCEAp-Hsp70), which significantly increased the expression levels of Hsp70 in the CEA-positive pancreatic cancer cells, resulting in an overall reduction in the survival of cancer cells. In the human pancreatic cancer Panc-1 xenograft model in immune deficient nude mice, the CEA promoter-regulated adenovirus AdCEAp-Hsp70 significantly inhibited tumor growth. In the rat pancreatic cancer DSL-6A/C1 xenograft model in rats, the viral proliferation and high expression levels of Hsp70 promoted the interstitial infiltration of CD4+, CD8+ and gamma/delta T cells into tumors, induced host secretion of the cytokines TGF-β, INF-γ, and IL-6 and had a dual anti-tumor effects that completely inhibited the growth of pancreatic cancer. The results demonstrated that the oncolytic adenovirus under the control of CEA promoter provides additional assurances regarding the safety and efficiency of cancer gene therapy. This gene therapy model improves anti-cancer efficiency and has broad applications and developmental prospects.

Topics: Adenoviridae; Animals; Carcinoembryonic Antigen; Cell Line, Tumor; Genetic Therapy; Genetic Vectors; HSP70 Heat-Shock Proteins; Humans; Interleukin-6; Male; Mice; Mice, Nude; Oncolytic Virotherapy; Pancreatic Neoplasms; Promoter Regions, Genetic; Rats; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFβ1 were increased significantly in tumors grown in SPARC-null mice. TGFβ1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFβ1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFβ1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFβ induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFβ1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFβ availability and activation.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Disease Progression; Extracellular Matrix; Losartan; Mice; Mice, Knockout; Neoplasm Invasiveness; Neoplasm Metastasis; Osteonectin; Pancreatic Neoplasms; Survival Rate; Transforming Growth Factor beta; Treatment Outcome

The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-β) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-β-dependent Lefty up-regulation.. Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-β-mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal-regulated kinase (ERK) inhibitor LY294002.. Transforming growth factor β upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-β-Lefty pathway.. Our data suggest that Lefty is a novel TGF-β target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-β-Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-β.

Topics: Butadienes; Cell Line, Tumor; Cell Proliferation; Chromones; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Left-Right Determination Factors; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Pyrazoles; Pyridazines; ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transforming Growth Factor beta; Tumor Suppressor Proteins; Up-Regulation

Response gene to complement-32 (RGC-32) is comprehensively expressed in many kinds of tissues and has been reported to be expressed abnormally in different kinds of human tumors. However, the role of RGC-32 in cancer remains controversial and no reports have described the effect of RGC-32 in pancreatic cancer. The present study investigated the expression of RGC-32 in pancreatic cancer tissues and explored the role of RGC-32 in transforming growth factor-beta (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human pancreatic cancer cell line BxPC-3.. Immunohistochemical staining of RGC-32 and E-cadherin was performed on specimens from 42 patients with pancreatic cancer, 12 with chronic pancreatitis and 8 with normal pancreas. To evaluate the role of RGC-32 in TGF-β-induced EMT in pancreatic cancer cells, BxPC-3 cells were treated with TGF-β1, and RGC-32 siRNA silencing and gene overexpression were performed as well. The mRNA expression and protein expression of RGC-32 and EMT markers such E-cadherin and vimentin were determined by quantitative reverse transcription-PCR (qRT-PCR) and western blot respectively. Finally, migration ability of BxPC-3 cells treated with TGF-β and RGC-32 siRNA transfection was examined by transwell cell migration assay.. We found stronger expression of RGC-32 and higher abnormal expression rate of E-cadherin in pancreatic cancer tissues than those in chronic pancreatitis tissues and normal pancreatic tissues. Immunohistochemical analysis revealed that both RGC-32 positive expression and E-cadherin abnormal expression in pancreatic cancer were correlated with lymph node metastasis and TNM staging. In addition, a significant and positive correlation was found between positive expression of RGC-32 and abnormal expression of E-cadherin. Furthermore, in vitro, we found sustained TGF-β stimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of siRNA silencing and gene overexpression, we further demonstrated that RGC-32 mediated TGF-β-induced EMT and migration in BxPC-3 cells.. The results above indicated that RGC-32 might be a novel metastasis promoting gene in pancreatic cancer and it enhances metastatic phenotype by mediating TGF-β-induced EMT in human pancreatic cancer cell line BxPC-3.

Topics: Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Muscle Proteins; Neoplasm Metastasis; Nerve Tissue Proteins; Pancreatic Neoplasms; Phenotype; RNA Interference; Transforming Growth Factor beta

The basic helix loop helix (bHLH) transcription factor DEC2 is associated with the regulation of apoptosis, circadian rhythm and the response to hypoxia. However, the significance of DEC2 in pancreatic cancer remains unknown. Here, we showed for the first time that DEC2 inhibits the progression of human pancreatic cancer. Human pancreatic cancer BxPC-3 cells were treated with or without transforming growth factor-β (TGF-β), siRNA against DEC2, or a combination of TGF-β and DEC2 siRNA or DEC2 overexpression. The cells were analyzed by RT-PCR, real-time PCR, western blotting, immunofluorescent staining and ChIP assay. We also performed immunohistochemical analyses of DEC2 expression in surgically-resected pancreatic cancers. The expression of DEC2 was increased in TGF-β-treated BxPC-3 cells. In the presence of TGF-β, DEC2 overexpression decreased the migration and invasion of BxPC-3 cells. Knockdown of DEC2 by siRNA in the presence of TGF-β significantly increased the expression and nuclear concentration of slug. We also showed that DEC2 siRNA decreased the binding of DEC2 to the E-box of the slug promoter. Immunohistochemically, little DEC2 was detected in pancreatic cancer tissues, whereas significant amounts were detected in the adjacent non-cancerous pancreatic tissues. These results indicate that DEC2 has inhibitory effects against human pancreatic cancer that involve TGF-β and slug.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cell Movement; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Pancreatic Neoplasms; RNA Interference; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways.

Topics: ErbB Receptors; Gene Knockdown Techniques; Gene Silencing; Genes, erbB-2; Humans; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; Transforming Growth Factor beta

The TGF-β pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-β inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-β in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-β inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvβ6 integrin acted as a key upstream activator of TGF-β in evolving pancreatic cancers. In addition, TGF-β or αvβ6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-β signaling. Therefore, our findings indicate that αvβ6 and TGF-β act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.

Topics: Animals; Antigens, Neoplasm; Disease Models, Animal; Disease Progression; Immunohistochemistry; Integrins; Mice; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

DEC1 (BHLHE40/Stra13/Sharp2) is a basic helix-loop-helix (bHLH) transcription factor that is involved in the regulation of apoptosis and cell proliferation and the response to hypoxia. Epithelial-mesenchymal transition (EMT) is an important step leading to invasion and migration of various tumor cells, and TGF-β treatment has been shown to induce cancer cells to undergo EMT. However, the significance of DEC1 in TGF-β-induced EMT remains unknown. We examined the role of DEC1 in EMT of PANC-1 cells, a human pancreatic cancer cell line. As a result, we found that DEC1 was upregulated by TGF-β in PANC-1 cells, and regulated the expression and the levels of nuclear, cytoplasmic or membrane localization of EMT-related factors, including phosphorylated Smad3 (pSmad3), snail, claudin-4 and N-cadherin. In the presence of TGF-β, DEC1 knockdown by siRNA inhibited morphological changes during EMT processes, while TGF-β induced PANC-1 cells to taken on a spindle-shaped morphology. Furthermore, a combination treatment of DEC1 expression with TGF-β was closely linked to the migration and invasion of PANC-1 cells. Immunohistochemically, DEC1 and pSmad3 were detected within pancreatic cancer tissues, whereas claudin-4 expression was weaker in the cancer tissues compared with the adjacent non-cancer pancreatic tissues. These findings suggest that DEC1 plays an important role in the regulation of these EMT-related factors in pancreatic cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Claudin-4; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; RNA, Small Interfering; Smad3 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins

Aberrant activation of Cyclin D-Cdk4/6 signaling pathway is commonly found in pancreatic ductal adenocarcinoma (PDAC). Here, we show that PD-0332991, a highly specific inhibitor for Cdk4 and Cdk6, exerted growth inhibitory effects on three human PDAC cell lines. Microarray analysis revealed that PD-0332991 downregulated cell-cycle-related genes, but upregulated genes implicated in extracellular matrix (ECM) remodeling and pancreatic cancer cell invasion and metastasis. Moreover, PD-0332991 enhanced invasion in TGF-β-responsive PDAC cell lines that harbor a wild-type SMAD4 gene (COLO-357, PANC-1), but not in TGF-β-resistant AsPC-1 cells that harbor a mutated SMAD4. PD-0332991 also induced epithelial-mesenchymal transition (EMT) in COLO-357 and PANC-1, but not in AsPC-1 cells. Inhibition of CDK4/6 using shRNA mimicked the effects of PD-0332991 on EMT induction. Furthermore, PD-0332991 increased Smad transcriptional activity in luciferase readout assays and activated TGF-β signaling. SB-505124, an inhibitor of the type-I TGF-β receptor (TβRI) kinase, completely blocked EMT induction by PD-0332991. When combined with PD-0332991, SB-505124 inhibited the growth of COLO-357 and PANC-1 cells. Taken together, these data suggest that anti-Cdk4/6 therapy could induce EMT and enhance pancreatic cancer cell invasion by activating Smad-dependent TGF-β signaling, and that combining PD-0332991 and SB-505124 may represent a novel therapeutic strategy in PDAC.

Topics: Benzodioxoles; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Screening Assays, Antitumor; Drug Synergism; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Imidazoles; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Piperazines; Pyridines; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Stem Cell Assay

Pancreatic cancer is the fourth leading cause of cancer death in the United States because most patients are diagnosed too late in the course of the disease to be treated effectively. Thus, there is a pressing need to more clearly understand how gene expression is regulated in cancer cells and to identify new biomarkers and therapeutic targets. Translational regulation is thought to occur primarily through non-SMAD directed signaling pathways. We tested the hypothesis that SMAD4-dependent signaling does play a role in the regulation of mRNA entry into polysomes and that novel candidate genes in pancreatic cancer could be identified using polysome RNA from the human pancreatic cancer cell line BxPC3 with or without a functional SMAD4 gene. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for most of the remaining polysome RNA, levels are regulated via a "tagging" of the RNAs in the nucleus for rapid entry into the polysomes; (iv) a SMAD4-dependent pathway appears to indeed play a role in regulating mRNA entry into polysomes; and (v) a gene list derived from differentially expressed polysome RNA in BxPC3 cells generated new candidate genes and cell pathways potentially related to pancreatic cancer.

Topics: Cell Nucleus; Cytoplasm; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Polyribosomes; RNA; RNA, Messenger; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

The aim of this study was to evaluate whether apoptosis-resistant cancer cells have cancer stem cell (CSC)-like properties.. Panc-1 pancreatic cancer cells were incubated in the presence of 5-fluorouracil (5-FU) for 24 h, and further incubated without 5-FU for 28 days. To assess the capacity of self-renewal, surviving cells were planted for sphere-forming assay. Epithelial-to-mesenchymal transition (EMT) was induced with TGF-β, then mRNA expression was evaluated by real-time PCR for E-cadherin, SNAIL, and vimentin. The E-Cadherin protein levels were also examined by immunoblot analysis. The Local invasion ability was analyzed by Matrigel invasion assay.. The frequency of cells that were capable of initiating spheres was higher in 5-FU-pre treated cells, which also overexpressed stem cell marker genes, OCT4 and NANOG. Matrigel invasion activity of apoptosis-resistant Panc-1 cells was greater than that of control Panc-1 cells.. Apoptosis-resistant cancer cells have CSC-like properties, i.e., able to initiate sphere formation, express stem cell genes, and respond to EMT stimulation.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Fluorouracil; Homeodomain Proteins; Humans; Nanog Homeobox Protein; Neoplastic Stem Cells; Octamer Transcription Factor-3; Pancreatic Neoplasms; Transforming Growth Factor beta

Epithelial to mesenchymal transition (EMT) promotes cellular motility, invasiveness and metastasis during embryonic development and tumorigenesis. Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of EMT. A lot of evidences suggest that this process is Smad3-dependent. Herein we showed that exposure of aspc-1 and panc-1 pancreatic cancer cells to TGF-β1 resulted in characteristic morphological alterations of EMT, and enhancement of cell motility and gemcitabine (Gem) resistance along with an up-regulation of EMT markers genes such as vimentin, N-cadherin, MMP2 and MMP9. Naringenin (Nar) down-regulated EMT markers expression in both mRNA and protein levels by inhibiting TGF-β1/Smad3 signal pathway in the pancreatic cancer cells. Consequently, Nar suppressed the cells migration and invasion and reversed their resistance to Gem.

Topics: Apoptosis; Blotting, Western; Cell Movement; Cell Proliferation; Deoxycytidine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Estrogen Antagonists; Flavanones; Gemcitabine; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; Phosphorylation; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

Epithelial to mesenchymal transition (EMT) plays a dual role in tumor progression. It enhances metastasis of tumor cells by increasing invasive capacity and promoting survival, and it decreases tumor cell sensitivity to epithelial cell-targeting agents such as epithelial growth factor receptor kinase inhibitors. In order to study EMT in tumor cells, we have characterized 3 new models of ligand-driven EMT: the CFPAC1 pancreatic tumor model and the H358 and H1650 lung tumor models. We identified a diverse set of ligands that drives EMT in these models. Hepatocyte growth factor and oncostatin M induced EMT in all models, while transforming growth factor-β induced EMT in both lung models. We observed morphologic, marker and phenotypic changes in response to chronic ligand treatment. Interestingly, stimulation with 2 ligands resulted in more pronounced EMT compared with single-ligand treatment, demonstrating a spectrum of EMT states induced by parallel signaling, such as the JAK and PI3K pathways. The EMT changes observed in response to the ligand were reversed upon ligand withdrawal, demonstrating the 'metastable' nature of these models. To study the impact of EMT on cell morphology and invasion in a 3D setting, we cultured cells in a semisolid basement membrane extract. Upon stimulation with EMT ligands, the colonies exhibited changes to EMT markers and showed phenotypes ranging from modest differences in colony architecture (CFPAC1) to complex branching structures (H358, H1650). Collectively, these 3 models offer robust cell systems with which to study the roles that EMT plays in cancer progression.

Topics: Blotting, Western; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Microscopy, Confocal; Oncostatin M; Pancreatic Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor beta

Both transforming growth factor (TGF)-ß and the non-receptor tyrosine kinase Src play major roles during tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis, but little is known about the signaling crosstalk between them. To interfere with Src function in vitro and in vivo many studies have employed the pharmacologic Src inhibitors PP2 and PP1. Both agents have recently been shown to be powerful inhibitors of TGF-ß receptor type I/ALK5 and type II. As this situation prohibited any definite conclusions with respect to the relative contribution of TGF-ß vs. Src signaling, we decided to reappraise a potential role of Src in TGF-ß1-mediated cellular responses using RNA and dominant-negative (dn) interference to block Src expression and function, respectively. In TGF-ß-responsive pancreatic ductal adenocarcinoma (PDAC) cells, we show that Src is activated by TGF-ß1 and that its specific inhibition strongly attenuated basal proliferation and enhanced TGF-ß1-mediated growth arrest. However, Src inhibition was unable to impair TGF-ß1-controlled EMT as evidenced by cell morphology and regulation of the epithelial marker E-cadherin. Despite its dispensibility for TGF-ß-induced EMT, specific inhibition of Src dramatically reduced basal and TGF-ß1-induced cell migration in Panc-1 cells as measured with a novel real-time migration assay (xCELLigence DP system). Biochemically, dnSrc inhibition failed to block TGF-ß1/ALK5-induced activation of Smad2 and Smad3, but partially inhibited transcriptional activation of TGF-ß/Smad-responsive reporter genes, and effectively blocked basal and TGF-ß1-induced activation of p38 MAPK. Together, the data provide evidence for a role of Src in the regulation of basal proliferation as well as in basal and TGF-ß1-mediated cell motility but not EMT in TGF-ß-responsive pancreatic (tumor) cells.

Topics: Carcinoma, Pancreatic Ductal; Cell Cycle; Cell Growth Processes; Cell Movement; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Phosphorylation; RNA, Small Interfering; Smad Proteins; src-Family Kinases; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Trop2 is a recently discovered cell surface glycoprotein overexpressed in pancreatic cancer which could potentially be used as an immunotherapeutic target. Enveloped virus-like particles (VLPs) are highly immunogenic and versatile immune stimulatory agents which can be modified to incorporate exogenous proteins on their membrane envelope to use as cancer vaccines. In this study, we investigated the effects of murine Trop2 (mTrop2) VLP immunization in a pancreatic cancer syngeneic murine model. VLPs incorporating mTrop2 were used to immunize C57BL/6 tumor-bearing mice. Immunization with mTrop2 VLPs led to a significant reduction in tumor growth accompanied by a broad activation and tumor infiltration of CD4(+) and CD8(+) T cells as well as natural killer and natural killer T cells. VLP immunization generated mTrop2-specific cytotoxic T lymphocytes and antibodies with no measurable induction of autoimmunity. Importantly, VLP immunization decreased the population of regulatory T cells and myeloid-derived suppressor cells inside the tumor tissue resulting in decreased levels of immunosuppressive cytokines like interleukin-10 and transforming growth factor-β while promoting the activation of immature macrophages and dendritic cells. Furthermore, combination of VLP immunization with gemcitabine treatment showed an improved effect significantly increasing the survival of tumor bearing mice. Our results demonstrate that mTrop2 VLP immunization can activate broad antitumor immune responses and affect key players in the tumor microenvironment overcoming a major barrier, which has limited the efficacy of cancer vaccines. This study presents a novel immunotherapeutic approach which could potentially be used as an alternative treatment option in combination therapies to treat pancreatic cancer patients.

Topics: Adenocarcinoma; Animals; Antibodies; Antigens, Neoplasm; Cancer Vaccines; Cell Adhesion Molecules; Combined Modality Therapy; Dendritic Cells; Deoxycytidine; Female; Gemcitabine; Humans; Immunization; Immunotherapy, Active; Interferon-gamma; Interleukin-10; Macrophages; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Proteins; Recombinant Proteins; Survival Rate; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vaccines, Virus-Like Particle

TIEG1 (TGF-β inducible early gene 1) plays a significant role in regulating cell proliferation and apoptosis in various cell types. Previous studies have shown a close relationship between the expression level of TIEG1 and various cancers, including breast, prostate, colorectal and pancreatic cancer. In this study, we up-regulated the gene expression of TIEG1 in SW1990 pancreatic cancer cell line by a lentivirus transfection system and investigated its potential as a therapeutic target for pancreatic cancer. The results showed that lentivirus-mediated overexpression of TIEG1 gene inhibited human pancreatic cancer SW1990 cell proliferation and caused the cell cycle arrest at the G1-phase in vitro. SW1990 cells transduced with lenti-TIEG1 showed significant inhibition of colony formation and cancer cell growth in 3-D culture model. Moreover, overexpression of TIEG1 gene significantly slowed the growth of SW1990 xenografts in nude mice. Taken together, these data provided evidence that overexpression of TIEG1 gene by a lentivirus transfection system led to suppressed human pancreatic cancer cell growth and might therefore be a feasible approach in the clinical management of pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Humans; Lentivirus; Male; Mice; Mice, Nude; Pancreatic Neoplasms; RNA, Messenger; Transfection; Transforming Growth Factor beta

Transforming Growth Factor-β (TGF-β) exerts cell type-specific and context-dependent effects. Understanding the intrinsic effects of TGF-β on cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a prerequisite for rationalized clinical implementation of TGF-β targeting therapies. Since the tumor microenvironment can affect how cancer cell respond to TGF-β, we employed a novel three-dimensional (3D) culturing system to recapitulate stromal and extracellular matrix interactions. We show here that TGF-β stimulates growth of human and murine pancreatic cancer cell lines (PCCs) when embedded in a 3% collagen IV/laminin-rich gelatinous medium (Matrigel™) over a solidified layer of soft agar. Moreover, in this novel 3D model, concomitant treatment with TGF-β1 and epidermal growth factor (EGF) enhanced PCC growth to a greater extent than either growth factor alone, and conferred increased chemoresistance to cytotoxic compounds. These cooperative growth-stimulatory effects were blocked by pharmacological inhibition of TGF-β type I receptor with SB431542 or the EGF receptor with erlotinib. Co-incubation with SB431542 and erlotinib enhanced the efficacy of gemcitabine and cisplatin in PCCs and in primary cell cultures established from pancreata of genetically-engineered mouse models of PDAC. These findings suggest that concomitant inhibition of TGF-β and EGF signaling may represent an effective therapeutic strategy in PDAC, and that this 3D culturing system could be utilized to test ex vivo the therapeutic response of pancreatic tumor biopsies from PDAC patients, thereby providing a functional assay to facilitate personalized targeted therapies.

Topics: Animals; Benzamides; Carcinoma, Pancreatic Ductal; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Cisplatin; Culture Media; Deoxycytidine; Dioxoles; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Extracellular Matrix; Gemcitabine; Humans; Mice; Mice, Transgenic; Pancreatic Neoplasms; Quinazolines; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

To discover novel markers for improving the efficacy of pancreatic cancer (PC) diagnosis, the secretome of two PC cell lines (BxPC-3 and MIA PaCa-2) was profiled. UL16 binding protein 2 (ULBP2), one of the proteins identified in the PC cell secretome, was selected for evaluation as a biomarker for PC detection because its mRNA level was also found to be significantly elevated in PC tissues.. ULBP2 expression in PC tissues from 67 patients was studied by immunohistochemistry. ULBP2 serum levels in 154 PC patients and 142 healthy controls were measured by bead-based immunoassay, and the efficacy of serum ULBP2 for PC detection was compared with the widely used serological PC marker carbohydrate antigen 19-9 (CA 19-9).. Immunohistochemical analyses revealed an elevated expression of ULPB2 in PC tissues compared with adjacent non-cancerous tissues. Meanwhile, the serum levels of ULBP2 among all PC patients (n = 154) and in early-stage cancer patients were significantly higher than those in healthy controls (p<0.0001). The combination of ULBP2 and CA 19-9 outperformed each marker alone in distinguishing PC patients from healthy individuals. Importantly, an analysis of the area under receiver operating characteristic curves showed that ULBP2 was superior to CA 19-9 in discriminating patients with early-stage PC from healthy controls.. Collectively, our results indicate that ULBP2 may represent a novel and useful serum biomarker for pancreatic cancer primary screening.

Topics: Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Gene Expression Profiling; GPI-Linked Proteins; Humans; Immunoassay; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Pancreatic Neoplasms; Transforming Growth Factor beta

TGF-β-activated kinase-1 (TAK1), a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor κB (NF-κB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer.. NF-κB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n = 40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. The results of in vitro proliferation were analyzed for statistical significance of differences by nonlinear regression analysis; differences in mouse survival were determined using a log-rank test. All statistical tests were two-sided.. AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-κB activity than did their respective control cell lines (relative luciferase activity: AsPc-1, mean = 0.18, 95% confidence interval [CI] = 0.10 to 0.27; control, mean = 3.06, 95% CI = 2.31 to 3.80; MDAPanc-28, mean = 0.30, 95% CI = 0.13 to 0.46; control, mean = 4.53, 95% CI = 3.43 to 5.63; both P < .001). TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC(50) between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells (P < .001). In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden (gemcitabine vs gemcitabine plus LYTAK1, P = .03) and prolonged survival duration (median survival: gemcitabine, 82 days vs gemcitabine plus LYTAK1, 122 days; hazard ratio = 0.334, 95% CI = 0.027 to 0.826, P = .029).. The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Camptothecin; Cell Line, Tumor; Deoxycytidine; Drug Administration Schedule; Drug Resistance, Neoplasm; Electrophoretic Mobility Shift Assay; Gemcitabine; Gene Silencing; Humans; Irinotecan; MAP Kinase Kinase Kinases; Mice; Mice, Nude; NF-kappa B; Organoplatinum Compounds; Oxaliplatin; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Burden; Xenograft Model Antitumor Assays

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer.

Topics: Cell Hypoxia; Cell Line, Tumor; Claudins; Down-Regulation; Electric Impedance; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression; Hepatocyte Nuclear Factor 3-beta; Humans; MARVEL Domain Containing 2 Protein; Membrane Proteins; Nerve Tissue Proteins; Occludin; Pancreatic Ducts; Pancreatic Neoplasms; Permeability; Protein Isoforms; RNA-Binding Proteins; RNA, Small Interfering; Snail Family Transcription Factors; Sphingomyelins; Telomerase; Tight Junctions; Transcription Factors; Transfection; Transforming Growth Factor beta

Pancreatic cancer is one of the most lethal of human malignancies, and potent therapeutic options are lacking. Inhibition of cell cycle progression through pharmacological blockade of cyclin-dependent kinases (CDK) has been suggested as a potential treatment option for human cancers with deregulated cell cycle control. Dinaciclib (SCH727965) is a novel small molecule multi-CDK inhibitor with low nanomolar potency against CDK1, CDK2, CDK5 and CDK9 that has shown favorable toxicity and efficacy in preliminary mouse experiments, and has been well tolerated in Phase I clinical trials. In the current study, the therapeutic efficacy of SCH727965 on human pancreatic cancer cells was tested using in vitro and in vivo model systems. Treatment with SCH727965 significantly reduced in vitro cell growth, motility and colony formation in soft agar of MIAPaCa-2 and Pa20C cells. These phenotypic changes were accompanied by marked reduction of phosphorylation of Retinoblastoma (Rb) and reduced activation of RalA. Single agent therapy with SCH727965 (40 mg/kg i.p. twice weekly) for 4 weeks significantly reduced subcutaneous tumor growth in 10/10 (100%) of tested low-passage human pancreatic cancer xenografts. Treatment of low passage pancreatic cancer xenografts with a combination of SCH727965 and gemcitabine was significantly more effective than either agent alone. Gene Set Enrichment Analysis identified overrepresentation of the Notch and Transforming Growth Factor-β (TGF-β) signaling pathways in the xenografts least responsive to SCH727965 treatment. Treatment with the cyclin-dependent kinase inhibitor SCH727965 alone or in combination is a highly promising novel experimental therapeutic strategy against pancreatic cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic N-Oxides; Cyclin-Dependent Kinases; Deoxycytidine; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gemcitabine; Humans; Indolizines; Male; Mice; Mice, Nude; Pancreatic Neoplasms; Phosphorylation; Pyridinium Compounds; ral GTP-Binding Proteins; Receptors, Notch; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

A major goal in cancer research is to develop carriers that can deliver drugs effectively and without side effects. Liposomal and particulate carriers with diameters of ∼100 nm have been widely used to improve the distribution and tumour accumulation of cancer drugs, but so far they have only been effective for treating highly permeable tumours. Here, we compare the accumulation and effectiveness of different sizes of long-circulating, drug-loaded polymeric micelles (with diameters of 30, 50, 70 and 100 nm) in both highly and poorly permeable tumours. All the polymer micelles penetrated highly permeable tumours in mice, but only the 30 nm micelles could penetrate poorly permeable pancreatic tumours to achieve an antitumour effect. We also showed that the penetration and efficacy of the larger micelles could be enhanced by using a transforming growth factor-β inhibitor to increase the permeability of the tumours.

Topics: Animals; Antineoplastic Agents; Drug Carriers; Humans; Liposomes; Mice; Mice, Inbred BALB C; Micelles; Organoplatinum Compounds; Pancreatic Neoplasms; Particle Size; Permeability; Polyethylene Glycols; Pyrazoles; Pyrroles; Transforming Growth Factor beta

The elucidation of biological concepts enriched with differentially expressed genes has become an integral part of the analysis and interpretation of genomic data. Of additional importance is the ability to explore networks of relationships among previously defined biological concepts from diverse information sources, and to explore results visually from multiple perspectives. Accomplishing these tasks requires a unified framework for agglomeration of data from various genomic resources, novel visualizations, and user functionality.. We have developed ConceptGen, a web-based gene set enrichment and gene set relation mapping tool that is streamlined and simple to use. ConceptGen offers over 20,000 concepts comprising 14 different types of biological knowledge, including data not currently available in any other gene set enrichment or gene set relation mapping tool. We demonstrate the functionalities of ConceptGen using gene expression data modeling TGF-beta-induced epithelial-mesenchymal transition and metabolomics data comparing metastatic versus localized prostate cancers.

Topics: Animals; Computational Biology; Databases, Genetic; Gene Expression Profiling; Gene Regulatory Networks; Humans; Male; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Pattern Recognition, Automated; Software; Transforming Growth Factor beta

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

Topics: Amino Acid Sequence; Animals; Cadherins; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Cell Line, Tumor; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Proteins; Pancreatic Neoplasms; Phosphorylation; Proteomics; Sequence Homology, Amino Acid; Serine Endopeptidases; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Topics: Cadherins; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Humans; Hydrolases; Intracellular Signaling Peptides and Proteins; Mutation; Neoplasm Proteins; Pancreatic Neoplasms; Phosphorylation; RNA Interference; Serine; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Recent studies suggest that transforming growth factor beta (TGF-beta) inhibits telomerase activity by repression of the telomerase reverse transcriptase (TERT) gene. In this report, we show that TGF-beta induces TERT repression-dependent apoptosis in pancreatic tumor, vascular smooth muscle, and cervical cancer cell cultures. TGF-beta activates Smad3 signaling, induces TERT gene repression and results in G1/S phase cell cycle arrest and apoptosis. TERT over-expression stimulates the G1/S phase transition and alienates TGF-beta-induced cell cycle arrest and apoptosis. Our data suggest that telomere maintenance is a limiting factor of the transition of the cell cycle. TGF-beta triggers cell cycle arrest and death by a mechanism involving telomerase deregulation of telomere maintenance.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Nucleus; G1 Phase; Humans; Pancreatic Neoplasms; Phosphorylation; Protein Transport; Rats; S Phase; Signal Transduction; Smad3 Protein; Telomerase; Transforming Growth Factor beta

The transcription factor NF-kappaB is constitutively active in pancreatic adenocarcinoma. Here we explore the contribution of NF-kappaB to the malignant phenotype of pancreatic cancer cells in addition to its anti-apoptotic role. Block of NF-kappaB signalling by non-destructible IkappaBalpha rendered cells resistant to TGF-beta-induced epithelial-mesenchymal transition (EMT). In contrast, NF-kappaB activation by TNF-alpha or expression of constitutively active IKK2 induced an EMT-phenotype with up-regulation of vimentin and ZEB1, and down-regulation of E-cadherin. EMT could also be induced in cells with defective TGF-beta signalling. Functional assays demonstrated reduced or strongly enhanced migration and invasion upon NF-kappaB inhibition or activation, respectively.

Topics: Cell Line, Tumor; Cell Movement; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Homeodomain Proteins; Humans; MAP Kinase Signaling System; Matrix Metalloproteinases; Mesoderm; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; NF-kappa B; Pancreatic Neoplasms; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Zinc Finger E-box-Binding Homeobox 1

Epithelial-to-mesenchymal transdifferentiation (EMT) mediated by transforming growth factor-β (TGF-β) signaling leads to aggressive cancer progression. In this study, we identified zinc-α2-glycoprotein (AZGP1, ZAG) as a tumor suppressor in pancreatic ductal adenocarcinoma whose expression is lost due to histone deacetylation. In vitro, ZAG silencing strikingly increased invasiveness of pancreatic cancer cells accompanied by the induction of a mesenchymal phenotype. Expression analysis of a set of EMT markers showed an increase in the expression of mesenchymal markers (vimentin (VIM) and integrin-α5) and a concomitant reduction in the expression of epithelial markers (cadherin 1 (CDH1), desmoplakin and keratin-19). Blockade of endogenous TGF-β signaling inhibited these morphological changes and the downregulation of CDH1, as elicited by ZAG silencing. In a ZAG-negative cell line, human recombinant ZAG (rZAG) specifically inhibited exogenous TGF-β-mediated tumor cell invasion and VIM expression. Furthermore, rZAG blocked TGF-β-mediated ERK2 phosphorylation. PCR array analysis revealed that ZAG-induced epithelial transdifferentiation was accompanied by a series of concerted cellular events including a shift in the energy metabolism and prosurvival signals. Thus, epigenetically regulated ZAG is a novel tumor suppressor essential for maintaining an epithelial phenotype.

Topics: Adipokines; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Transdifferentiation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Genes, Tumor Suppressor; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Mesoderm; Neoplasm Invasiveness; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-beta1 is associated with drug resistance in pancreatic cancer.. Pancreatic cancer BxPC3 cells were stably transfected with TGF-beta1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-beta1. Western blotting and immunohistochemistry were used to detect expression of TGF-beta1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-beta1 plus PKCalpha inhibitor Gö6976. TGF-beta1 type II receptor, TbetaRII was also knocked down using TbetaRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay.. Overexpression of TGF-beta1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-beta1 protein increases expression of PKCalpha in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-beta1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCalpha. However, blockage of PKCalpha with Gö6976 and TbetaRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-beta1. Immunohistochemical data show that pancreatic cancers overexpress TGF-beta1 and P-gp relative to normal tissues. In addition, TGF-beta1 expression is associated with P-gp and membranous PKCalpha expression in pancreatic cancer.. TGF-beta1-induced drug resistance in pancreatic cancer cells was associated with PKCalpha expression. The PKCalpha inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Cycle; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Extracellular Matrix Proteins; Humans; Immunoenzyme Techniques; Pancreatic Neoplasms; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Subtraction Technique; Transforming Growth Factor beta; Tumor Cells, Cultured

We recently reported that transforming growth factor-β (TGF-β) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-β mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [Ca(2+)](cyt) in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-β induced both intracellular Ca(2+) release and extracellular Ca(2+) entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate (IP(3)) receptor and TRPC], LaCl(3) (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the Ca(2+) entry mode of NCX) markedly inhibited the TGF-β-induced increase in [Ca(2+)](cyt). 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKCα induced by TGF-β. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKCα serine phosphorylation induced by TGF-β. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-β-induced pancreatic cancer cell motility. Therefore, TGF-β induces Ca(2+) entry likely via TRPC1 and NCX1 and raises [Ca(2+)](cyt) in pancreatic cancer cells, which is essential for PKCα activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer.

Topics: Boron Compounds; Calcium; Carbazoles; Cell Line; Cell Movement; Enzyme Inhibitors; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate Receptors; Pancreatic Ducts; Pancreatic Neoplasms; Phosphorylation; Protein Kinase C-alpha; Sodium-Calcium Exchanger; Thiourea; Transforming Growth Factor beta; Transient Receptor Potential Channels

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.

Topics: Antibodies, Neutralizing; Biomarkers, Tumor; Cadherins; Cell Line, Tumor; Cell Movement; Coculture Techniques; Epithelial-Mesenchymal Transition; Fibroblasts; Humans; Pancreatic Neoplasms; Pancreatic Stellate Cells; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) inducible early gene 1 (TIEG1) is known to induce apoptosis in TGF-beta sensitive pancreatic cancer cells, yet its effect on TGF-beta resistant cancer cells remains unclear. In this study, TIEG1 was found to induce apoptosis in TGF-beta resistant cancer cells and concurrently enhanced gemcitabine chemosensitivity. Down-regulation of stathmin was noted to associate with TIEG1 expression, whilst ectopic overexpression of stathmin prevented TIEG1 mediated growth inhibition of tumor cells. Small interfering RNAs targeting stathmin inhibited pancreatic cancer cell growth. These suggest that stathmin is a downstream target of TIEG1.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Cell Proliferation; Deoxycytidine; Down-Regulation; Drug Resistance, Neoplasm; Early Growth Response Transcription Factors; Gemcitabine; Humans; Kruppel-Like Transcription Factors; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotide Reductases; RNA, Messenger; RNA, Small Interfering; Stathmin; Transforming Growth Factor beta; Tumor Cells, Cultured

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh-Ptch-Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh-Ptch-Smo signaling and is regulated by TGF-beta and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Mice; Pancreatic Ducts; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Transforming Growth Factor beta; Zinc Finger Protein GLI1

Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-associated proteinase inhibitor that potently inhibits a variety of serine proteinases, including those that are membrane bound. Although HAI-1/SPINT1 is widely expressed by epithelial cells and cancer cells, its functional role is still unclear, particularly in cancer. Here, we show that stable knockdown of HAI-1/SPINT1 in the human pancreatic cancer cell line SUIT-2 induces an elongated spindle-like morphology associated with accelerated invasion, thereby mimicking an epithelial to mesenchymal transition (EMT). We found that HAI-1/SPINT1 knockdown significantly reduced the expression of E-cadherin and was accompanied by up-regulation of Smad-interacting protein 1 (SIP1), an E-cadherin transcriptional repressor. In addition, matrix metalloproteinase-9 (MMP-9) was up-regulated. Similar results were obtained in the HLC-1 lung carcinoma cell line. Moreover, a metastatic variant of SUIT-2 (S2-CP8) that showed loss of E-cadherin expression also showed a significantly reduced level of HAI-1/SPINT1. Engineered overexpression of HAI-1/SPINT1 in S2-CP8 resulted in reversion of E-cadherin expression and SIP1 down-regulation, which accompanied reestablishment of epithelial morphology in culture. The EMT caused by HAI-1/SPINT1 knockdown seemed to be mediated, at least partly, by membrane-bound serine proteinases, matriptase/ST14 and TMPRSS4, as knockdown of matriptase/ST14 or TMPRSS4 in HAI-1/SPINT1 knockdown SUIT-2 cells and HLC-1 cells resulted in reversion of SIP1 and/or MMP-9 expression levels. We suggest that interactions between HAI-1/SPINT1 and membrane-bound serine proteinases contribute to transcriptional and functional changes involved in EMT in certain carcinoma cells.

Topics: Animals; Cadherins; Cell Line, Tumor; Epithelial Cells; Humans; Male; Mesoderm; Mice; Mice, Inbred BALB C; Neoplasms; Nerve Tissue Proteins; Pancreatic Neoplasms; Proteinase Inhibitory Proteins, Secretory; RNA-Binding Proteins; Serine Endopeptidases; Transforming Growth Factor beta

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up-regulated E-cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF-beta, SP cells changed their shape into mesenchymal-like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E-cadherin expression level. TGF-beta induced EMT-associated gene alteration such as reduction of E-cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)-2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF-beta treatment, whereas MP cells did not respond to TGF-beta-mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro-invasion, and in vivo metastasis, as compared to MP cells. Because micro-invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Transdifferentiation; Epithelial Cells; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Immunoblotting; Liver Neoplasms; Matrix Metalloproteinase 2; Mesoderm; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplastic Stem Cells; Pancreatic Neoplasms; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

CD4+25+Foxp3+ regulatory T cells (Treg) play a critical role in the induction of tolerance to tumor-associated antigens and suppression of antitumor immunity. How Treg are induced in cancer is poorly understood. We reported previously that Treg are significantly elevated in the peripheral blood of patients with pancreas cancer and that in a murine pancreas cancer model induction of Treg seems to be transforming growth factor (TGF)-beta dependent. Here we provide additional evidence that Treg are increased locally within the tumor microenvironment by a mechanism that seems dependent on TGF-beta receptor expression and the presence of tumor derived TGF-beta. The murine pancreas cancer cell line Pan02 produces high levels of TGF-beta both in vitro and in vivo. In contrast, the esophageal murine cancer cell line, Eso2, does not. Immunohistochemical staining of Foxp3 in explanted tumors shows an identifiable population of Treg in the Pan02 (TGF-beta positive) tumors but not Eso2 (TGF-beta negative). Naive CD4+25-Foxp3- T cells, when adoptively transferred into Rag-/- mice, are converted into Foxp3+ Treg in the presence of Pan02 but not Eso2 tumors. Induction of Treg in Pan02 mice is blocked by systemic injection of an anti-TGF-beta antibody. If Rag-/- mice are instead reconstituted with naive CD4+25- T cells expressing a mutated TGF-beta receptor, induction of Foxp3+ Treg in Pan02 bearing mice is blocked. Collectively, these observations further support the role of TGF-beta in the induction of Treg in pancreas adenocarcinoma.

Topics: Animals; CD4 Antigens; Cell Line, Tumor; Esophageal Neoplasms; Female; Forkhead Transcription Factors; Homeodomain Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Evidence suggests that multiple tumors, including pancreatic adenocarcinoma, display heterogeneity in parameters that are critical for tumor formation, progression and metastasis. Understanding heterogeneity in solid tumors is increasingly providing a plethora of new diagnostic and therapeutic approaches. In this study, a particular focus was put on identifying a subpopulation of stem cell-like, slow cycling tumor cells in a pancreas adenocarcinoma cell lines. Using a label retention technique a subpopulation of slow cycling cells (DiI+/SCC) was identified and further evaluated in the BxPC-3 and Panc03.27 cell lines. These slowly cycling cells managed to retain the lipophilic labeling dye DiI, while the bulk of the cells (>94%) did not. The DiI+/SCC population, showed only a partial overlap with the CSC markers CD24(+)/CD44(+), CD133(+) and ALDH but they survived chemotherapeutic treatment, and were able to recreate the initial heterogeneous tumor cell population. DiI+/SCCs exhibited an increased invasive potential as compared with their non-label retaining, faster cycling cells (DiI-/FCC). They also had increased tumorigenic potential and morphological changes resembling cells that have undergone an epithelial to mesenchymal transition (EMT). Analysis of DiI+/SCC cells by real time PCR revealed a selective up-regulation of tell tale components of the Hedgehog/TGFbeta pathways, as well as a down-regulation of EGFR, combined with a shift in crucial components implied in EMT. The presented findings offer an expanded mechanistic understanding that associates tumor initiating potential with cycling speed and EMT in pancreatic cancer cell lines.

Topics: Adenocarcinoma; Animals; Antigens, CD; Antimetabolites, Antineoplastic; Cell Line, Tumor; ErbB Receptors; Female; Flow Cytometry; Fluorouracil; Hedgehog Proteins; Humans; Mice; Mice, SCID; Neoplasm Metastasis; Neoplastic Stem Cells; Pancreatic Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor beta

Early detection of solid tumors, particularly pancreatic cancer, is of substantial importance in clinics. Enhanced magnetic resonance imaging (MRI) with iron oxide nanoparticles is an available way to detect the cancer. The effective and selective accumulation of these nanoparticles in the tumor tissue is needed for improved imaging, and in this regard, their longevity in the blood circulation time is crucial. We developed here block copolymer-coated magnetite nanoparticles for pancreatic cancer imaging, by means of a chelation between the carboxylic acid groups in poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-PAsp) and Fe on the surface of the iron oxide nanoparticles. These nanoparticles had considerably narrow distribution, even upon increased ionic strength or in the presence of fetal bovine serum. The PEG-PAsp-coated nanoparticles were further shown to be potent as a contrast agent for enhanced MRI for an experimental pancreatic cancer, xenografts of the human-derived BxPC3 cell line in BALB/c nude mice, with combined administration of TGF-beta inhibitor. Iron staining of tumor tissue confirmed the accumulation of the nanoparticles in tumor tissue. Use of the PEG-PAsp-coated magnetite nanoparticles, combined with the TGF-beta inhibitor, is of promising clinical importance for the detection of intractable solid cancers, including pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Drug Delivery Systems; Electrochemistry; Female; Ferrosoferric Oxide; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Light; Magnetic Resonance Imaging; Magnetics; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Pancreatic Neoplasms; Polyethylene Glycols; Scattering, Radiation; Transforming Growth Factor beta

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-beta (TGF-beta)-induced growth arrest. As TGF-beta has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-beta-induced transcriptional activity, which is associated with increased TGF-beta-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-beta-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial-mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Pancreatic Neoplasms; Prognosis; Proto-Oncogene Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

To dissect the role of constitutively altered Tgfbr1 signaling in pancreatic cancer development, we crossed Elastase-Kras(G12D) (EL-Kras) mice with Tgfbr1 haploinsufficient mice to generate EL-Kras/Tgfbr1(+/-) mice. Mice were euthanized at 6 to 9 months to compare the incidence, frequency, and size of precancerous lesions in the pancreas. Only 50% of all EL-Kras/Tgfbr1(+/-) mice developed preinvasive lesions compared with 100% of EL-Kras (wild-type Tgfbr1) mice. The frequency of precancerous lesions was 4-fold lower in haploinsufficient than in control mice. Paradoxically, the precancerous lesions of EL-Kras/Tgfbr1(+/-) mice were considerably larger than those in EL-Kras mice. Yet, the mitotic index of precancerous cells and the observable levels of fibrosis, lipoatrophy, and lymphocytic infiltration were reduced in EL-Kras/Tgfbr1(+/-) mice. We conclude that Tgfbr1 signaling promotes the development of precancerous lesions in mice. These findings suggest that individuals with constitutively decreased TGFBR1 expression may have a decreased risk of pancreatic cancer.

Topics: Animals; Apoptosis; Cell Growth Processes; Female; Haploidy; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pancreatic Neoplasms; Precancerous Conditions; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The role of Smad4 in transforming growth factor beta (TGFbeta)-mediated epithelial-mesenchymal transition (EMT), invasion, and metastasis was investigated using isogenically matched pancreatic cancer cell lines that differed only in expression of Smad4. Cells expressing Smad4 showed an enhanced TGFbeta-mediated EMT as determined by increased expression of vimentin and decreased expression of beta-catenin and E-cadherin. TGFbeta-mediated invasion was suppressed in Smad4-intact cells as determined by in vitro assays, and these cells showed a reduced metastasis in an orthotopic model of pancreatic cancer. Interestingly, TGFbeta inhibited STAT3(Tyr705) phosphorylation in Smad4-intact cells. The decrease in STAT3(Tyr705) phosphorylation was linked to a TGFbeta/Smad4-dependent and enhanced activation of extracellular signal-regulated kinases, which caused an increase in serine phosphorylation of STAT3(Ser727). Down-regulating signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA in Smad4-deficient cells prevented TGFbeta-induced invasion. Conversely, expressing a constitutively activated form of STAT3 (STAT3-C) in Smad4-intact cells enhanced invasion. This study indicates the requirement of STAT3 activity for TGFbeta-induced invasion in pancreatic cancer cells and implicates Smad4-dependent signaling in regulating STAT3 activity. These findings further suggest that loss of Smad4, leading to aberrant activation of STAT3, contributes to the switch of TGFbeta from a tumor-suppressive to a tumor-promoting pathway in pancreatic cancer.

Topics: Blotting, Western; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Phosphorylation; Smad4 Protein; STAT3 Transcription Factor; Transforming Growth Factor beta; Tyrosine

Functional inactivation of the tumor suppressor Smad4 in colorectal and pancreatic carcinogenesis occurs coincident with the transition to invasive growth. Breaking the basement membrane (BM) barrier, a prerequisite for invasive growth, can be due to tumor induced proteolytic tissue remodeling or to reduced synthesis of BM molecules by incipient tumor cells. Laminin-332 (laminin-5), a heterotrimeric BM component composed of alpha 3-, beta 3- and gamma 2-chains, has recently been identified as a target structure of Smad4 and represents the first example for expression control of an essential BM component by a tumor and invasion suppressor. Biochemically Smad4 is a transmitter of signals of the TGFbeta superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGFbeta-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2.. Promoter-reporter constructs harboring 4 kb upstream regions, each of the three genes encoding Laminin-322 as well as deletion and mutations constructs were established. Promoter activities and TGFbeta induction were assayed through transient transfections in Smad4-negative human cancer cells and their stable Smad4-positive derivatives. Functionally relevant binding sites were subsequently confirmed through chromatin immunoprecipitation.. Herein, we report that Smad4 mediates transcriptional regulation through three different mechanisms, namely through Smad4 binding to a functional SBE site exclusively in the LAMA3 promoter, Smad4 binding to AP1 (and Sp1) sites presumably via interaction with AP1 family components and lastly a Smad4 impact on transcription of AP1 factors. Whereas Smad4 is essential for positive regulation of all three genes, the molecular mechanisms are significantly divergent between the LAMA3 promoter as compared to the LAMB3 and LAMC2 promoters.. We hypothesize that this divergence in modular regulation of the three promoters may lay the ground for uncoupled regulation of Laminin-332 in Smad4-deficient tumor cells in response to stromally expressed cytokines acting on budding tumor cells.

Topics: Basement Membrane; Carcinoma; Cell Adhesion Molecules; Cell Line, Tumor; Colorectal Neoplasms; Cytokines; Gene Expression Regulation, Neoplastic; Humans; Kalinin; Laminin; Pancreatic Neoplasms; Promoter Regions, Genetic; Smad4 Protein; Transcription Factor AP-1; Transcription, Genetic; Transforming Growth Factor beta

The growth of LNCaP, a human prostate adenocarcinoma cell line, and MCF-7, a human breast adenocarcinoma cell line, is initially hormone dependent. We previously demonstrated that LNrho0-8 and MCFrho0, derived from LNCaP and MCF-7 by depleting mitochondrial DNA (mtDNA), exhibited hormone-independent growth that led to progressed phenotypes. Here, we demonstrate that LNrho0-8 and MCFrho0 have invasive characters as evaluated by the ability of invasion through the extracellular matrix (ECM) in vitro. In addition, the induction of vimentin and the repression of E-cadherin expression in rho0 cells indicate that they are mesenchymal cells. Since LNrho0-8 and MCFrho0 were derived from epithelial cancer cell lines, LNCaP and MCF-7 must have lost epithelial features and gained the mesenchymal phenotype by epithelial-mesenchymal transition (EMT) during the mtDNA depletion. In the rho0 cell lines, the Raf/MAPK signaling cascade was highly activated together with the expressions of transforming growth factor-beta (TGF-beta) and type I TGF-beta receptor (TGF-betaRI). EMT requires cooperation of TGF-beta signaling with activation of the Raf/MAPK cascade, suggesting that EMT was induced in mtDNA depleted cells resulting in the acquisition of progressive tumor features, such as higher invasiveness and loss of hormone dependent growth. Our results indicate that decreasing mtDNA content induces EMT, enabling the progressive phenotypes observed in cancer.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; DNA, Mitochondrial; Epithelial Cells; Female; Humans; In Vitro Techniques; Male; Mesoderm; Pancreatic Neoplasms; Phenotype; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Vimentin

Transforming growth factorbeta (TGF-beta) signals through Smad-dependent and Smad-independent pathways. However, Smad signaling is altered by allelic deletion or intragenic mutation of the Smad4 gene in more than half of pancreatic ductal adenocarcinomas. We show here that loss of Smad4-dependent signaling leads to aberrant expression of RON, a phosphotyrosine kinase receptor, and that signaling by RON cooperates with Smad4-independent TGF-beta signaling to promote cell motility and invasion. Restoring Smad4 expression in a pancreatic ductal adenocarcinoma cell line that is deficient in Smad4 repressed RON expression. Conversely, small interference RNA knock down of Smad4 or blocking TGF-beta signaling with a TGF-beta type I receptor kinase inhibitor in Smad4-intact cell lines induced RON expression. TGF-beta-induced motility and invasion were inhibited in cells that express Smad4 and that have low levels of RON compared with isogenically matched cells that were deficient in Smad4. Furthermore, knocking down RON expression in Smad4-deficient cells suppressed TGF-beta-mediated motility and invasion. We further determined that Smad4-dependent signaling regulated RON expression at the transcriptional level by real-time reverse transcription PCR and RON promoter luciferase reporter assays. Functional inactivation by site-directed mutations of two Smad binding sites on the RON promoter inhibited TGF-beta-mediated repression of RON promoter activity. These studies indicate that loss of Smad4 contributes to aberrant RON expression and that cross-talk of Smad4-independent TGF-beta signaling and the RON pathway promotes an invasive phenotype.

Topics: Alleles; Cell Line; Cell Movement; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype; Receptor Protein-Tyrosine Kinases; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

In the last several years, many laboratories have tried to unravel the complex signaling mechanisms activated by TGF-beta(1) in transformed cells. Smad proteins are the principal mediators of the transforming growth factor beta (TGF-beta) response, but this factor can also activate Smad-independent pathways in different cell types. Our previous studies in murine keratinocytes led to the identification of a cooperation between oncogenic Ras and Smad4 inactivation during malignant progression. We further investigated the function of Smad4 in human pancreatic cancer, in which loss-of-function mutations affecting Smad4 occur with a 50% frequency. Expression of a dominant-negative Smad4 construct in the adenocarcinoma cell line PANC-1 led to increased ubiquitination and proteasomal degradation of beta-catenin. Moreover, loss of Smad4 abrogated beta-catenin-signaling activity and was associated with a reduction of the tumorigenic potential of PANC-1 cells in scid mice. Although the expression of the dominant-negative Smad4 blocked TGF-beta(1)/Smad2,3-signaling activity, the above-mentioned effects of Smad4 on beta-catenin stability were independent of the TGF-beta1/Smad2,3-signaling pathway. These findings provide evidence for a cross talk between Smad4 and the Wnt/beta-catenin pathway in pancreatic carcinoma cells, suggesting a new role for Smad4 as an attenuator of beta-catenin proteasomal degradation.

Topics: Animals; beta Catenin; Cell Line, Tumor; Genes, ras; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Pancreatic Neoplasms; RNA, Neoplasm; Signal Transduction; Smad4 Protein; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous

Lymphangiogenesis is induced by various growth factors, including VEGF-C. Although TGF-beta plays crucial roles in angiogenesis, the roles of TGF-beta signaling in lymphangiogenesis are unknown. We show here that TGF-beta transduced signals in human dermal lymphatic microvascular endothelial cells (HDLECs) and inhibited the proliferation, cord formation, and migration toward VEGF-C of HDLECs. Expression of lymphatic endothelial cell (LEC) markers, including LYVE-1 and Prox1 in HDLECs, as well as early lymph vessel development in mouse embryonic stem cells in the presence of VEGF-A and C, were repressed by TGF-beta but were induced by TGF-beta type I receptor (TbetaR-I) inhibitor. Moreover, inhibition of endogenous TGF-beta signaling by TbetaR-I inhibitor accelerated lymphangiogenesis in a mouse model of chronic peritonitis. Lymphangiogenesis was also induced by TbetaR-I inhibitor in the presence of VEGF-C in pancreatic adenocarcinoma xenograft models inoculated in nude mice. These findings suggest that TGF-beta transduces signals in LECs and plays an important role in the regulation of lymphangiogenesis in vivo.

Topics: Animals; Cell Line; Disease Models, Animal; Lymphangiogenesis; Mice; Mice, Nude; Pancreatic Neoplasms; Peritonitis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor C

Radiotherapy is an important treatment for many malignant tumors, but there are recent reports that radiation may increase the malignancy of cancer cells by stimulating expression of type IV collagenases. In this study, we examined changes in matrix metalloproteinase (MMP) inhibitors, such as the tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and RECK, in response to irradiation in Panc-1 pancreatic cancer cells. Irradiation increased RECK protein levels but not mRNA levels, whereas no significant changes were found in TIMP-1 and TIMP-2. The enhanced RECK protein levels were associated with an increase in MMP inhibitory activity. However, irradiation slightly but reproducibly increased the invasiveness of the Panc-1 cells. Like irradiation, treatment of Panc-1 cells with transforming growth factor (TGF)-Beta1 led to a 2-fold increase in RECK protein levels. Transient transfection with Smad3 also increased RECK protein levels, but transfection with Smad7 markedly reduced them. Stable expression of Smad7 and treatment with SB431542, an inhibitor of TGF-Beta receptor I kinase, abolished TGF-Beta1- and radiation-mediated effects on RECK. Furthermore, irradiation increased levels of phosphorylated Smad3. We conclude that radiation post-transciptionally enhances RECK protein levels in Panc-1 cells, at least in part, via TGF-Beta signaling, and that irradiation increases Panc-1 invasiveness via a mechanism that may not be linked to MMP-2 activity.

Topics: Benzamides; Cell Line, Tumor; Dioxoles; Gamma Rays; Gene Expression Profiling; GPI-Linked Proteins; Humans; Matrix Metalloproteinase 2; Membrane Glycoproteins; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad7 Protein; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Topics: Animals; Anoikis; Antimetabolites, Antineoplastic; Apoptosis; Cell Movement; Deoxycytidine; Drug Therapy, Combination; Gemcitabine; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Invasiveness; Neovascularization, Pathologic; Pancreatic Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Ribonucleotide Reductases; Signal Transduction; Smad2 Protein; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Alteration in transforming growth factor-beta signalling pathway is one of the main causes of pancreatic cancer. The human runt-related transcription factor 3 gene (RUNX3) is an important component of this pathway. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including pancreatic cancer. Therefore, we examined genetic and epigenetic alterations of RUNX3 in human pancreatic cancer. Thirty-two patients with pancreatic cancer were investigated in this study. We examined the methylation status of RUNX3 promoter region, loss of heterozygosity (LOH) at 1p36, and conducted a mutation analysis. The results were compared with clinicopathological data. Promoter hypermethylation was detected in 20 (62.5%) of 32 pancreatic cancer tissues, confirmed by sequence of bisulphite-treated DNA. Loss of heterozygosity was detected in 11 (34.3%) of 32 pancreatic cancers. In comparison with clinicopathological data, hypermethylation showed a relation with a worse prognosis (P=0.0143). Hypermethylation and LOH appear to be common mechanisms for inactivation of RUNX3 in pancreatic cancer. Therefore, RUNX3 may be an important tumour suppressor gene related to pancreatic cancer.

Topics: Adult; Aged; Chromosomes, Human, Pair 1; Core Binding Factor Alpha 3 Subunit; DNA Methylation; DNA Mutational Analysis; Epigenesis, Genetic; Female; Gene Silencing; Humans; Loss of Heterozygosity; Male; Middle Aged; Multivariate Analysis; Pancreatic Neoplasms; Polymorphism, Single-Stranded Conformational; Predictive Value of Tests; Prognosis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

Activation of the Raf/MEK/ERK pathway, often by gain-of-function mutations of RAS or RAF, is observed in many human cancers. The extracellular signal-regulated kinase (ERK) pathway is required for the proliferation of cancer cell lines harboring activating BRAF or, to a lesser extent, activating RAS mutations. It is still unclear, however, whether the pathway is required in vivo for tumor development, particularly in tumors in which B-Raf is not mutationally activated. During embryonic development, B-Raf is essential for angiogenesis in the placenta. To address the question of whether B-Raf contributed to tumor angiogenesis in vivo we conditionally ablated B-Raf in a model of pancreatic islet carcinoma driven by the functional inactivation of tumor suppressors (RIP1Tag2), which critically depends on angiogenesis for growth. We find that B-Raf is dispensable for the proliferation of tumor cells in culture, but necessary for ERK activation and for the expression of angiogenic factors by tumor cells in vivo and in vitro. In vivo, these defects result in the formation of hollow tumors with decreased vessel density and strongly reduced proliferation. The progression from adenoma to carcinoma is also significantly impaired. Thus, endogenous B-Raf contributes to the development of RIP1Tag2 tumors by supporting the stromal response and tumor progression.

Topics: Animals; Base Sequence; Cell Transformation, Neoplastic; Disease Models, Animal; Disease Progression; DNA Primers; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Immunohistochemistry; Islets of Langerhans; Mice; Mice, Knockout; Pancreatic Neoplasms; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.

Topics: Adenocarcinoma; Adenoma; Basement Membrane; Cell Adhesion Molecules; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kalinin; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

In this study, the authors investigated the expression of activin receptor-like kinase 1 (ALK1) in pancreatic carcinoma and evaluated its potential role as a tumor suppressor in vitro and in vivo. Endogenous ALK1 expression was demonstrated by immunohistochemistry in both pancreatic tumor tissue and peritumoral normal tissue from 6 patients and by RT-PCR in 8/12 established pancreatic cancer cell lines. Ectopic expression of a constitutively active (ca) ALK1 mutant in TGF-beta sensitive PANC-1 and COLO-357 cells augmented transcriptional activation of a Smad2/3 responsive reporter, and slowed down basal growth in vitro. Both effects were further enhanced by TGF-beta/ALK5 stimulation, suggesting largely independent nuclear Smad signaling by both type I receptors. Upon orthotopic transplantation of PANC-1-caALK1 into immunodeficient mice, tumor size was strongly reduced and was associated with a lower microvessel density in the PANC-1-caALK1-derived tumors. In vitro, this mutant efficiently blocked TGF-beta-induced epithelial-to-mesenchymal transdifferentiation and suppressed TGF-beta/ALK5-mediated activation of the p38 MAPK pathway. Mechanistically, caALK1 silenced MyD118, an immediate TGF-beta target gene whose protein product, GADD45beta, couples Smad signaling to p38 activation. These results show that ALK1 activation in pancreatic tumor cells is antioncogenic by inducing ALK5-independent growth inhibition and by blocking TGF-beta/ALK5-mediated epithelial-to-mesenchymal transdifferentiation and, possibly, invasion and metastatic progression.

Topics: Activin Receptors, Type I; Adenocarcinoma; Animals; Antigens, Differentiation; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunoblotting; Immunohistochemistry; Mesoderm; Mice; Mice, Inbred BALB C; Mice, SCID; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Promoter Regions, Genetic; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad Proteins; Transcriptional Activation; Transfection; Transforming Growth Factor beta

We investigated the immune status in 32 pancreatic cancer patients (PC) in comparison with healthy controls (HC).. Using flow cytometry, peripheral blood lymphocytes (PBL) were characterized by the expression of surface markers for T helper cells (CD4), T suppressor cells (CD8), B cells (CD19) and NK cells (CD56). The blastogenic response of PBL was analyzed after stimulation with concavalin A (ConA), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and anti-CD3 antibodies. The serum levels of TNF-alpha, IL-1beta, IL-2, IL-10, IL-12, IL-18, IL-1RA, sIL-2R and TGF-beta were determined by ELISA.. No differences in the distribution of peripheral immunocytes in PC were found, whereas the blastogenic response of peripheral blood lymphocytes (PBL) after stimulation with PHA or anti-CD3 antibodies was significantly decreased in PC. In PC, we found reduced serum levels of IL-2 and significantly elevated levels of TNF-alpha, TGF-beta1, IL-10, IL-2R, IL-1beta and IL-1RA.. These data provide evidence for a systemic immune dysfunction in pancreatic cancer patients characterized by a shift towards a T helper cell type 2 cytokine profile, a significant elevation of substances related to T cell suppression and a reduced blastogenic response to PHA and anti-CD3 antibodies of PBL.

Topics: Aged; Aged, 80 and over; Case-Control Studies; Cytokines; Female; Humans; Immunity, Cellular; Immunophenotyping; Interleukins; Lymphocytes; Male; Middle Aged; Mitogens; Pancreatic Neoplasms; Prospective Studies; Transforming Growth Factor alpha; Transforming Growth Factor beta; Treatment Outcome

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.

Topics: Activin Receptors, Type I; Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Line; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression; Humans; Immunoblotting; Mice; Mice, SCID; Mutation; Neoplasm Metastasis; Neoplasms, Experimental; Pancreatic Neoplasms; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins; Transfection; Transforming Growth Factor beta; Tumor Burden

Smad4 is a tumour suppressor gene frequently deleted in pancreatic cancer. To investigate the roles of Smad4 deficiency in invasive and matastatic capabilities of pancreatic cancer, we examined the effects of Smad4 deficiency on regulation of the invasion suppressor E-cadherin in pancreatic cancer cell line PANC-1. TGF-beta decreased expression of E-cadherin and beta-catenin proteins at the plasma membrane, increased Snail and Slug mRNA expression, and induced fibroblastoid morphology in PANC-1 cells. These effects of TGF-beta were abrogated in Smad4-knocked-down PANC-1 cells. We also found that TGF-beta-induced down-regulation of E-cadherin expression was partially inhibited in Snail- and Slug-knocked-down PANC-1 cells. Thus, Smad4 mediates down-regulation of E-cadherin induced by TGF-beta in PANC-1 cells, at least in part, through Snail and Slug induction. These results suggest that Smad4 deficiency observed in invasive and metastatic pancreatic cancer might not be linked to the loss of E-cadherin.

Topics: Cadherins; Cell Line, Tumor; Down-Regulation; Humans; Pancreatic Neoplasms; Smad3 Protein; Smad4 Protein; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transition from a sessile epithelial phenotype to a migrating mesenchymal phenotype is a crucial step in transforming growth factor-beta (TGF-beta)-induced pancreatic cancer cell migration and invasion. These profound morphologic and functional alterations are associated with characteristic changes in TGF-beta-regulated gene expression, defined by rapid repression of epithelial markers and a strong and sustained transcriptional induction of mesenchymal markers such as the intermediate filament vimentin. In this study, we have analyzed the role of the transcription factor Sp1 in TGF-beta-induced and Smad-mediated gene regulation during epithelial to mesenchymal transition (EMT) and migration of pancreatic cancer cells. Here, we show that Sp1 is required for TGF-beta-induced EMT, and that this function is especially mediated through transcriptional induction of vimentin. Our results emphasize the functional relevance of vimentin in TGF-beta-induced EMT because prevention of its induction strongly reduces cell migration. Altogether, this study helps to better understand the role of Sp1 in TGF-beta-induced progression of pancreatic cancer. It suggests that Sp1, via transcriptional induction of vimentin, cooperates with activated Smad complexes in mesenchymal transition and migration of pancreatic cancer cells upon TGF-beta stimulation.

Topics: Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Small Interfering; Sp1 Transcription Factor; Transfection; Transforming Growth Factor beta; Vimentin

Oncogenic Kras initiates pancreatic tumorigenesis, while subsequent genetic events shape the resultant disease. We show here that concomitant expression of Kras(G12D) and haploinsufficiency of the Smad4/Dpc4 tumor suppressor gene engenders a distinct class of pancreatic tumors, mucinous cystic neoplasms (MCNs), which culminate in invasive ductal adenocarcinomas. Disease evolves along a progression scheme analogous to, but distinct from, the classical PanIN-to-ductal adenocarcinoma sequence, and also portends a markedly different prognosis. Progression of MCNs is accompanied by LOH of Dpc4 and mutation of either p53 or p16. Thus, these distinct phenotypic routes to invasive adenocarcinoma nevertheless share the same overall mutational spectra. Our findings suggest that the sequence, as well as the context, in which these critical mutations are acquired helps determine the ensuing pathology.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cyclin-Dependent Kinase Inhibitor p16; Cystadenoma, Mucinous; Disease Models, Animal; Genes, ras; Genetic Predisposition to Disease; Haplotypes; Humans; Mice; Mice, Mutant Strains; Mutation; Neoplasm Invasiveness; Pancreatic Neoplasms; Prognosis; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Transforming growth factor beta (TGFbeta) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGFbeta pathway in tumor cells often leads to resistance to the antiproliferative effects of TGFbeta while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGFbeta receptor I kinase (TGFbetaRI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGFbeta-dependent Smad2 phosphorylation and expression of TGFbeta-inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGFbeta-regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD-208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGFbeta-stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGFbeta signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGFbetaRI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer.

Topics: Activin Receptors, Type I; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Proliferation; Genes, myc; Humans; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Pteridines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor C

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Topics: Blotting, Western; Cell Line; Flavonoids; Humans; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad4 Protein; Transforming Growth Factor beta

Connective tissue growth factor (CTGF/CCN2) is thought to play a role in normal wound repair and bone remodeling, but also promotes fibrosis in several disease processes including diabetic nephropathy, sclerodoma and pancreatitis. A contribution to desmoplasia associated with pancreatic cancer progression has also been proposed. CTGF is induced by TGFbeta in diverse cell types, but TGFbeta receptor mediated signaling is impaired in pancreatic cancers and cell lines, usually due to DPC4/Smad4 mutations which arise during the later stages of intraepithelial neoplastic progression. Therefore, in order to define signaling pathways that mediate basal and TGFbeta-induced CTGF expression in normal and transformed cells, we compared CTGF gene regulation in pancreatic cancer cells and fibroblasts by measuring the effects of small molecule inhibitors and dominant negative mutants of signaling proteins on CTGF promoter reporter activity, message, and protein expression. We determined that the previously identified TEF-1 cis element is essential for CTGF promoter reporter activity in pancreatic cancer cell lines. Whereas p38 mediated CTGF induction by TGFbeta in fibroblasts, MEK/ERK signaling mediated TGFbeta-induced CTGF expression in pancreatic cancer cells and was also responsible for basal CTGF expression in pancreatic cancer cell lines with defective Smad signaling. Since activating Ras mutations occur in the earliest stages of pancreatic cancer, CTGF may be induced independent of Smad4 in pancreatic cancer cells.

Topics: 3T3 Cells; Animals; Blotting, Western; Cells, Cultured; Connective Tissue Growth Factor; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mice; Nuclear Proteins; Pancreatic Neoplasms; Phosphorylation; Promoter Regions, Genetic; Response Elements; RNA, Messenger; Signal Transduction; Smad Proteins; Sp1 Transcription Factor; TEA Domain Transcription Factors; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-beta signals that block cell cycle progression in G(1) are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-beta signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467-sites on Smads that get phosphorylated by the TGF-beta receptor and positively regulate TGF-beta signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-beta. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255-sites that are phosphorylated by MAP kinase and negatively regulate TGF-beta signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-beta. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-beta signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-beta signals that promote G(1) arrest.

Topics: Breast Neoplasms; Cell Line, Tumor; G1 Phase; Humans; Pancreatic Neoplasms; Phospholipase D; Phosphorylation; Signal Transduction; Transforming Growth Factor beta

Gata-3 has been shown to specifically alter its expression patterns in different types of cancers. Recent evidence suggests that an interference of Gata-3 exists in the TGF-beta signaling pathway. To determine the role of Gata-3 in pancreatic cancer, pancreatic cancer samples were analyzed in comparison to normal pancreatic tissues. Furthermore, four different pancreatic cancer cell lines with different alterations of the TGF-beta pathway were studied. To evaluate if a potential relationship with TGF-beta signaling pathway exists, we correlated mRNA expression levels with the expression of TGF-betas, TGF-beta receptors, and Smad-3. Finally, we analyzed the influence of TGF-beta on Gata-3 expression in vitro. All pancreatic cancer samples demonstrated a marked overexpression of Gata-3 mRNA and protein. Immunohistochemical staining revealed strong and persistent cytoplasmic Gata-3 immunoreactivity in cancer cells. In an electrophoretic mobility shift assay, a disturbed nuclear translocation was confirmed. The expression of Gata-3 showed a significant correlation with the expression of TGF-betas, TGF-beta receptors, and Smad-3. TGF-beta responsive cell lines showed a downregulation of Gata-3 mRNA upon TGF-beta exposure, whereas in TGF-beta-unresponsive cell lines, Gata-3 mRNA expression persisted at high levels. Furthermore, strong specific upregulation of Gata-3 impaired nuclear translocation and its cooperative action with the TGF-beta pathway, suggesting that Gata-3 plays a central role in human pancreatic cancer.

Topics: Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Electrophoretic Mobility Shift Assay; Female; GATA3 Transcription Factor; Humans; Immunohistochemistry; Male; Microscopy, Confocal; Middle Aged; Neoplasm Staging; Pancreas; Pancreatic Neoplasms; Protein Transport; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Dendritic cells (DCs) are important for an immune surveillance. Myeloid DCs (DC1) are important for an effective antitumor immune system. The function and count of circulating DC1 (cDC1) in hosts with a malignant tumor would be defective. This study focused on analyzing the immunological features of cDC1 in patients with pancreatic cancer during the perioperative period.. Thirty-two pancreatic cancer patients who underwent pancreatectomy and 18 age-matched healthy individuals as controls were enrolled in this study. The perioperative cDC count, the stimulatory capacity of cDC1 against allogeneic T cells and TGF-beta1 level in the serum were measured. The cDC count was measured at 12 months after the operation.. The preoperative cDC1/cDC2 ratio, cDC1 count, and stimulatory capacity of cDC1 were impaired in patients in comparison to controls (P<0.05). The serum TGF-beta1 level was significantly higher in patients than controls (P<0.001). The stimulatory capacity of cDC1 recovered after pancreatectomy (P<0.05). The serum TGF-beta1 level significantly decreased after the operation (P<0.05); however, they were still significantly higher than controls (P<0.05). Although the cDC1/cDC2 ratio and the cDC1 count did not increase after the pancreatectomy, they recovered as the controls' level at 12 months after the pancreatectomy in disease-free patients (P<0.05) and the serum TGF-beta1 level in those patients at 12 months after the operation significantly decreased compared with those at the postoperative period (P<0.05).. Surgical resection of pancreatic cancer could be associated with improved cDC1 function. When a patient remained disease free, the recovery of cDC1 counts was observed approximately 12 months after pancreatectomy. Further strategy will be needed to improve immune function in patients with pancreatic cancer.

Topics: Aged; Aged, 80 and over; Blood Cell Count; Case-Control Studies; Dendritic Cells; Disease-Free Survival; Female; Follow-Up Studies; Humans; Killer Cells, Natural; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Male; Middle Aged; Pancreatectomy; Pancreatic Neoplasms; Phytohemagglutinins; Postoperative Period; T-Lymphocytes; Transforming Growth Factor beta; Transforming Growth Factor beta1

Tenascin C (TNC) is a component of the provisional extracellular matrix (ECM) that characterizes solid tumours. Cell surface annexin II is a high-affinity receptor for large TNC splice variants. The aim of this study was to analyse whether TNC and annexin II play a role in the development of pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by a rich ECM populated by pancreatic stellate cells, which play a crucial role in pancreatic desmoplasia. The mRNA and protein levels of TNC and of annexin II were analysed in pancreatic tissues by DNA array, quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and immunohistochemistry. TNC large splice variants were detected by RT-PCR. Enzyme linked immunosorbent assay (ELISA) was used to measure TNC levels in serum and culture supernatants. TNC and annexin II mRNA levels were significantly higher in pancreatic cancer tissues than in the normal pancreas. TNC expression was detected with increased frequency in the progression from PanIN-1 lesions to PDAC, and a parallel switch from cytoplasmic to cell surface expression of annexin II was observed. Large TNC transcripts were found in pancreatic cancer and in chronic pancreatitis, but not in the normal pancreas. TNC expression was demonstrated in pancreatic stellate cells, where it could be induced by tumour necrosis factor alpha (TNFalpha), transforming growth factor beta1 (TGF-beta1) and by cancer cell supernatants supplemented with TGF-beta1. In conclusion, the expression of TNC and cell surface annexin II increases in the progression from low-grade PanIN lesions to pancreatic cancer. Pancreatic stellate cells are identified as a source of TNC in pancreatic tissues, possibly under the influence of soluble factors released by the tumour cells.

Topics: Adenocarcinoma; Annexin A2; Blotting, Western; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Neoplasm Proteins; Pancreatic Neoplasms; Pancreatitis, Chronic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tenascin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Myofibroblasts are considered to play central roles in pancreatic fibrosis. The potent fibrogenic capacities of transforming growth factor betas (TGF-betas) have been emphasized in vitro and in animal studies. However, the roles of TGF-betas in human chronic pancreatitis have not been fully clarified.. To investigate whether expressions of TGF-betas are related to myofibroblast distribution in chronic, cancer-associated, obstructive pancreatitis (COP).. Histopathologic studies using hematoxylin-eosin and Elastica-Masson trichrome and immunohistochemical studies using antibodies against alpha-smooth muscle actin (SMA); CD68; TGF-beta1, -beta2, and -beta3; and TGF-beta soluble receptor type II were performed in 19 COP cases and 6 controls. By classifying COP tissues into 3 fibrosis phases by the amount of collagen deposits, immunoreactivities for TGF-betas, histopathologic changes, and myofibroblast distribution were examined for each fibrosis phase.. Six cases were categorized in the early stage of fibrosis, 8 in the intermediate stage, and 5 in the advanced stage. Immunoreactivities for all 3 isoforms of TGF-beta were observed in occasional myofibroblasts. In the early and intermediate stages, TGF-beta1-expressing macrophages and neutrophils were distributed in the midst of myofibroblasts. TGF-beta2 and TGF-beta3 expressions were observed in ductal structures, sometimes even in sites where no or few myofibroblasts were seen. TGF-beta soluble receptor type II was immunoreactive for myofibroblasts, endothelium, and ductal structures.. All 3 isoforms of TGF-betas may contribute to fibrosis in COP. Macrophages and neutrophils may be sources of fibrogenic TGF-beta1. Infiltration of these cells appears to play an important role in the progression of COP fibrosis.

Topics: Aged; Chronic Disease; Constriction, Pathologic; Female; Fibroblasts; Fibrosis; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Protein Isoforms; Transforming Growth Factor beta

Proteinase-activated receptor-2 (PAR-2) is expressed in various tissues, including cancer lesions. However, the functional consequences of PAR-2 expression in cancer cells, especially in pancreatic cancer cells, are poorly understood. To clarify the biological significance of PAR-2 signaling in pancreatic cancer, we examined the production of growth factors and cytokines, such as IL-6, IL-8, bFGF, TGF-beta1, and VEGF, by specific ELISAs. Two human pancreatic cancer cell lines, SUIT2 and MiaPaCa2, which have been shown to express PAR-2, were stimulated by trypsin and PAR-2 activating peptide (PAR-2AP: SLIGKV-NH2). After 24 h, the culture supernatants were collected and specific ELISAs were performed. Although no significant changes were observed in the release of IL-6, bFGF, TGF-beta1, or VEGF, that of IL-8 was significantly up-regulated by PAR-2 agonists in a dose-dependent manner. In addition, IL-8 receptor expression was found in pancreatic cancer cells and fibroblasts. These results suggest that the PAR-2 signal up-regulates IL-8 release from pancreatic cancer cells. This up-regulated IL-8 has an effect on the pancreatic cancer cells in an autocrine manner and on the fibroblasts in a paracrine manner. Thus, this signal might contribute to tumor progression and characteristic fibrosis in pancreatic cancer.

Topics: Amino Acid Sequence; Blotting, Western; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Models, Biological; Oligopeptides; Pancreatic Neoplasms; Receptor, PAR-2; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin; Up-Regulation; Vascular Endothelial Growth Factor A

Pancreatic ductal adenocarcinoma (PDAC) is characterized by multiple alterations in the TGF-beta signaling pathway. Yes-associated protein (YAP65) interacts with Smad7 thereby influencing TGF-beta signaling. In the present study, the expression of YAP65 in PDAC was analyzed in order to elucidate the potential role of this molecule in the pathogenesis of pancreatic cancer. YAP65 mRNA expression levels in human pancreatic tissue samples and cell lines were analyzed by Northern blotting and quantitative RT-PCR. Immunohistochemistry was carried out to localize and quantify YAP65 expression in relation to Smad7 expression and Smad4 mutations. The effects of TGF-beta1 on Smad7 and YAP65 mRNA expression were analyzed by quantitative RT-PCR. Enhanced expression of YAP65 mRNA was identified by Northern blotting and quantitative RT-PCR in PDAC in comparison to the normal pancreas (2.5-fold increase) and to chronic pancreatitis (1.3-fold increase). In the normal pancreas, YAP65 was absent in acinar cells, large ducts and islet cells, but exhibited moderate to strong immunoreactivity in centroacinar cells and ductules. Tubular complexes in CP and CP-like lesions in PDAC also exhibited strong staining. In contrast, weak to moderate YAP65 immunoreactivity was present in the cancer cells. There was no correlation between YAP65 immunostaining and Smad7 staining or Smad4 mutations in the cancer samples. TGF-beta1 strongly induced Smad7 mRNA in Colo-357 and in Panc-1 cells, but only slightly induced YAP65 mRNA in Colo-357 cells. In conclusion, YAP65 is expressed mainly in centroacinar and small ductal cells in the normal pancreas. In PDAC, YAP65 is present in tubular complexes and to a lesser extent in cancer cells. Together with the known function of YAP65 in different growth and differentiation regulating pathways, it is suggested that this gene plays a role in the normal and diseased pancreas.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adolescent; Adult; Aged; Aged, 80 and over; Blotting, Northern; Blotting, Western; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Mutation; Pancreatic Neoplasms; Phosphoproteins; RNA, Messenger; Smad4 Protein; Smad7 Protein; Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins

Cytokine concentration in pancreatic juice of patients with pancreatic disease is unknown. Secretin stimulation allows endoscopic collection of pancreatic juice secreted into the duodenum. We aimed to evaluate the cytokine concentrations in pancreatic juice of patients with abdominal pain to discriminate presence from absence of pancreatic disease.. From January 2003-December 2004, consecutive patients with abdominal pain compatible with pancreatic origin were enrolled. Patients underwent upper endoscopy. Intravenous secretin (0.2 mug/kg) was given immediately before scope intubation. Pancreatic juice collected from the duodenum was immediately snap-frozen in liquid nitrogen until assays were performed. Pancreatic juice levels of interleukin-8, interleukin-6, intercellular adhesion molecule 1, and transforming growth factor-beta 1 were measured by modified enzyme-linked immunosorbent assays. The final diagnosis was made by the primary gastroenterologist on the basis of medical history; laboratory, endoscopic, and imaging studies; and clinical follow-up. Fisher exact test and Kruskal-Wallis rank sum test were used for statistical analysis.. Of 130 patients screened, 118 met the inclusion criteria. Multivariate analysis revealed that only interleukin-8 was able to discriminate between normal pancreas and chronic pancreatitis (P = .011), pancreatic cancer (P = .044), and the presence of pancreatic diseases (P = .007). Individual cytokine concentrations were not significantly different in chronic pancreatitis compared with pancreatic cancer.. Cytokine levels can be measured in pancreatic juice obtained from the duodenum without direct cannulation of the pancreatic duct. Interleukin-8 concentration in pancreatic juice can be used to discriminate between normal pancreas and patients with pancreatic disease. This is a relatively simple and noninvasive method to aid in the diagnosis of pancreatic diseases.

Topics: Abdominal Pain; Adult; Aged; Aged, 80 and over; Biomarkers; Cytokines; Diagnosis, Differential; Endoscopy, Gastrointestinal; Female; Humans; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Lipase; Male; Middle Aged; Pancreatic Diseases; Pancreatic Juice; Pancreatic Neoplasms; Pancreatitis, Chronic; Secretin; Transforming Growth Factor beta

The transcription factor Sp1 has been implicated in cell-type-specific activation of transforming growth factor-beta (TGFbeta) target genes in normal epithelial cells as well as in aberrant gene activation by TGFbeta in epithelial tumor cells. Here, we have examined the interaction of Sp1 with components of the Smad signaling cascade and its role in TGFbeta-induced early gene expression in pancreatic cancer cells. Gene expression profiling was carried out in mithramycin-A-treated cells to identify Sp1-regulated TGFbeta early response genes. We found that in pancreatic cancer cells Smad proteins and Sp1 cooperatively regulate expression of a distinct set of TGFbeta target genes potentially involved in tumor progression, including MMP-11, cyclin D1 and Smad7. Mechanistically, TGFbeta rapidly induces nuclear translocation of Smad proteins and subsequently stimulates Smad-Sp1 complex formation. Using the Smad7 promoter as a model for Smad-/Sp1-induced early gene activation, we demonstrated that this interaction increases Sp1 binding to GC-rich promoter boxes and results in superinduction of Sp1-mediated transcription. Moreover, inhibition of Sp1-DNA binding or transfection of Sp1-specific siRNA prevents TGFbeta-induced Smad7 expression and consequently enhances Smad signaling in pancreatic cancer cells, as indicated by increased receptor-mediated phosphorylation of Smad3. We thus conclude that Sp1 strongly contributes to the aberrant transcriptional response of transformed epithelial cells to TGFbeta stimulation.

Topics: Base Sequence; Cell Line, Tumor; DNA Primers; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Molecular Sequence Data; Pancreatic Neoplasms; Plasmids; Smad7 Protein; Sp1 Transcription Factor; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is associated with an intense fibrotic reaction around the tumor known as desmoplastic reaction. This tissue is composed of interstitial matrix, predominantly type I collagen, together with proliferating fibroblastic cells. Despite the recognized importance of tumor-stromal interactions, very little is known about the interactions among pancreatic cells, myofibroblasts, and the interstitial matrix. The current study was undertaken to test the hypothesis that the desmoplastic reaction alters PDAC gene expression and cellular behavior. Evaluation of human pancreatic specimens showed increased fibrosis and enhanced membrane type 1-matrix metalloproteinase (MT1-MMP) expression in tumor specimens compared with normal pancreas. Using an in vitro model of tumor cell-stromal interactions, type I collagen and the extracellular matrix deposited by pancreatic fibroblasts and PDAC cells regulated motility of human papillomavirus-immortalized human pancreatic ductal epithelial (HPDE) cells. These "stromal" matrices also regulated MT1-MMP expression by HPDE cells, without affecting the expression of tissue inhibitor of metalloproteinase 2. Treatment with transforming growth factor-beta1 (TGF-beta1) type I receptor kinase inhibitors and function-blocking anti-TGF-beta1 antibody abrogated matrix-mediated MT1-MMP induction. TGF-beta1 also promoted MT1-MMP-dependent migration by HPDE cells. Moreover, compared with normal tissue, there was increased TGF-beta1 signaling in grade 3 tumor specimens as shown by increased phospho-Smad2 staining. These data show that the crosstalk between cancer cells and stromal elements mediated by TGF-beta1 influences cell surface- and pericellular matrix-degrading potential in vitro and may contribute to pancreatic cancer progression in vivo.

Topics: Carcinoma, Pancreatic Ductal; Cell Movement; Collagen Type I; Fibrosis; Humans; Immunohistochemistry; Matrix Metalloproteinases; Matrix Metalloproteinases, Membrane-Associated; Pancreatic Neoplasms; Precancerous Conditions; RNA, Messenger; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

Glypican1 (GPC1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors as well as for members of the TGF-beta family. GPC1 plays a role in pancreatic cancer by regulating growth factor responsiveness. In view of the importance of members of the TGF-beta family in pancreatic cancer, in the present study, the role of GPC1 in TGF-beta, BMP and activin signaling was analyzed. Quantitative RT-PCR and immunohistochemistry were utilized to analyze GPC1 and TGF-beta, BMP and activin receptor expression levels. Panc-1 and T3M4 pancreatic cancer cells were transfected in a stable manner with a GPC1 antisense expression construct. Anchorage-dependent and -independent growth was determined by MTT and soft agar assays. TGF-beta1, activin-A and BMP-2 responsiveness was determined by MTT assays and immunoblotting with p21, p-Smad1, and p-Smad2 antibodies. QRT-PCR demonstrated increased GPC1 mRNA levels in pancreatic ductal adenocarcinoma (PDAC) compared to normal pancreatic tissues (NPT), as described previously. There was a significant correlation between GPC1 mRNA levels and TbetaRII, act-R1a, act-R1b, act-R2a, BMP-R1a, and BMP-R2 mRNA expression in NPT. In contrast, GPC1 mRNA expression correlated directly with act-R1a and BMP-R1a in N0 PDAC cases and with act-R2a and BMP-R1a in lymph node positive cases. Down-regulation of GPC1 resulted in increased doubling time in Panc-1 but not in T3M4 cells, and decreased anchorage-independent growth in both cell lines. GPC1 down-regulation resulted in a slightly altered response towards TGF-beta1, activin-A and BMP-2 in terms of growth, p21 induction and Smad2 phosphorylation. In conclusion, enhanced GPC1 expression correlates with BMP and activin receptors in pancreatic cancer. GPC1 down-regulation suppresses pancreatic cancer cell growth and slightly modifies signaling of members of the TGF-beta family of growth factors.

Topics: Activin Receptors; Adult; Aged; Aged, 80 and over; Bone Morphogenetic Protein Receptors; Carcinoma, Pancreatic Ductal; Down-Regulation; Female; Glypicans; Humans; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

The human MUC4 mucin plays an important role in the pathogenesis of pancreatic cancer. Recently, we have demonstrated that MUC4 expression in pancreatic tumor cells is regulated by interferon-gamma (IFNgamma) and by retinoic acid via transforming growth factor beta 2 (TGFbeta-2). In the present study, we established the pathobiological association of various cytokines and MUC4 in pancreatic tumor tissues and tumor cell lines.. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemical analyses, we examined the expression of MUC4, IFNgamma, TGFbetas, and several immunologically relevant cytokines in a panel of 11 pancreatic adenocarcinomas (PA), three normal pancreatic (NP) tissue specimens, and 11 pancreatic tumor cell lines.. Our data revealed that both MUC4 and IFNgamma were expressed at moderate to high levels in the majority of PA, while being undetectable in NP. Moreover, transcript for interleukin 2 (IL-2), a known marker of activated T helper 1 (TH1) lymphocytes, exhibited an expression profile similar to IFNgamma, suggesting a role of these immune effector cells as a potential source of IFNgamma in PA. Of note, IFNgamma protein was detected in the inflamed tissues neighboring tumor areas. Furthermore, TGFbetas were expressed by most cell lines and frequently upregulated in PA compared with NP. Interestingly, both IL-12 and IL-10, two key cytokines of the TH1 and TH2 pathways, respectively, were expressed at higher levels in PA relative to NP.. These observations support the potential implication of IFNgamma and TGFbetas in MUC4 regulation in vivo and suggest a complex interaction of TH1 and TH2 signaling in the pancreatic tumor microenvironment. These findings may provide useful insights into the pathobiology of pancreatic cancer.

Topics: Adenocarcinoma; Case-Control Studies; Cell Line, Tumor; Humans; Interferon-gamma; Interleukins; Mucin-4; Mucins; Pancreatic Neoplasms; Transforming Growth Factor beta

c-myc promoter silencing is a key step in epithelial cell growth inhibition by transforming growth factor beta (TGFbeta). During carcinogenesis, however, epithelial cells escape from c-myc repression and consequently become refractory to TGFbeta-mediated antiproliferation. Here, we assessed the role of the repressor, KLF11, in TGFbeta-induced growth inhibition in normal epithelial as well as pancreatic carcinoma cells. Endogenous KLF11 was stably down-regulated by RNA interference technology, and the functional consequences were studied by proliferation assays, reporter assays, DNA binding studies, and expression analyses. Coimmunoprecipitation and glutathione S-transferase pulldown assays were conducted to define KLF11-Smad3 interaction and U0126 was administered to examine the effects of the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase on complex formation and c-myc promoter binding of KLF11 and Smad3 in pancreatic cancer cells. In TGFbeta-stimulated normal epithelial cells, nuclear KLF11, in concert with Smad3, binds to and represses transcription from the core region of the TGFbeta-inhibitory element (TIE) of the c-myc promoter. Disruption of KLF11-Smad3 interaction or small interfering RNA-mediated knockdown of endogenous KLF11 strongly diminishes Smad3-TIE promoter binding and repression, and consequently impairs TGFbeta-mediated growth inhibition. In pancreatic cancer cells with oncogenic Ras mutations, hyperactive ERK counteracts TGFbeta-induced c-myc repression and growth inhibition through at least two mechanisms, i.e., via disruption of KLF11-Smad3 complex formation and through inhibition of KLF11-Smad3 binding to the TIE element. Together, these results suggest a central role for KLF11 in TGFbeta-induced c-myc repression and antiproliferation and identifies a novel mechanism through which ERK signaling antagonizes the tumor suppressor activities of TGFbeta in pancreatic cancer cells with oncogenic Ras mutations.

Topics: Apoptosis Regulatory Proteins; Base Sequence; Cell Cycle Proteins; Cell Proliferation; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Repressor Proteins; Response Elements; RNA, Small Interfering; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

Topics: Animals; Carcinoma, Pancreatic Ductal; Genes, Tumor Suppressor; Mice; Pancreatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC-alone or in combination with PDAC--relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRAS(G12D) allele, SMAD4 deficiency enabled rapid progression of KRAS(G12D)-initiated neoplasms. While KRAS(G12D) alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRAS(G12D) and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRAS(G12D) INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-beta (TGF-beta) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-beta-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRAS(G12D)-initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-beta-dependent growth.

Topics: Alleles; Animals; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Disease Progression; Gene Deletion; Genes, ras; Mice; Pancreas; Pancreatic Neoplasms; Phenotype; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p14ARF

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease in humans. Transforming growth factor-beta (TGF-beta) signaling plays an important role in PDAC progression, as indicated by the fact that Smad4, which encodes a central signal mediator downstream from TGF-beta, is deleted or mutated in 55% and the type II TGF-beta receptor (Tgfbr2) gene is altered in a smaller subset of human PDAC. Pancreas-specific Tgfbr2 knockout mice have been generated, alone or in the context of active Kras (Kras(G12D)) expression, using the Cre-loxP system driven by the endogenous Ptf1a (pancreatic transcription factor-1a) locus. Pancreas-selective Tgfbr2 knockout alone gave no discernable phenotype in 1.5 yr. Pancreas-specific Kras(G12D) activation alone essentially generated only intraepithelial neoplasia within 1 yr. In contrast, the Tgfbr2 knockout combined with Kras(G12D) expression developed well-differentiated PDAC with 100% penetrance and a median survival of 59 d. Heterozygous deletion of Tgfbr2 with Kras(G12D) expression also developed PDAC, which indicated a haploinsufficiency of TGF-beta signaling in this genetic context. The clinical and histopathological manifestations of the combined Kras(G12D) expression and Tgfbr2 knockout mice recapitulated human PDAC. The data show that blockade of TGF-beta signaling and activated Ras signaling cooperate to promote PDAC progression.

Topics: Adenocarcinoma; Aging; Animals; Carcinoma, Pancreatic Ductal; Connective Tissue Growth Factor; Disease Progression; Epithelium; Gene Expression; Genes, ras; Heterozygote; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Neoplasm Invasiveness; Pancreas; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Stromal Cells; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

Topics: Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; RNA Interference; RNA, Messenger; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Dendritic cells (DCs) are important for immune surveillance and play a central role in protection against infection and malignancy. DCs comprise two subsets: DC1 (myeloid DC) and DC2 (lymphoid DC). The aim of this study is to determine whether the number and/or function of circulating DCs were decreased in patients with pancreatic cancer and to evaluate the effects of these changes in patients with locally advanced pancreatic cancer before and after chemoradiotherapy (CRT). We examined the circulating DC number and function using the peripheral blood from 29 patients with pancreatic cancer and 20 healthy control subjects. In patients who underwent CRT (n = 20), blood samples were taken before and after CRT. DCs were tested for the ability to stimulate allogeneic T lymphocytes in mixed leukocyte reaction (MLR). CD4/8, NK activity, PHA, and TGF-beta1 were also measured.. The number and allostimulatory activity of circulating DC1s in patients were significantly lower than those in controls. In the patients who underwent CRT, the allostimulatory activity of DC1s at post-CRT was significantly increased as compared to those at pre-CRT. The level of TGF-beta1 was also significantly decreased at post-CRT as compared to pre-CRT. There were no changes in CD4/8, NK activity and proliferative response of T lymphocytes at the peri-CRT period.. These data indicate that the number and function of circulating DCs were impaired in patients with pancreatic cancer. Chemoradiotherapy, however, improved DC function, which might be related to decreased immunosuppressive cytokine levels.

Topics: Aged; Antineoplastic Agents; Cell Count; Combined Modality Therapy; Dendritic Cells; Female; HLA-DR Antigens; Humans; Interleukin-12; Lymphocyte Subsets; Male; Middle Aged; Pancreatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1

Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 protein degradation in pancreatic cancer cells. We found that F-box protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Carcinoma, Ductal, Breast; DNA-Binding Proteins; Drug Stability; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Point Mutation; Proteasome Endopeptidase Complex; SKP Cullin F-Box Protein Ligases; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Ubiquitin

CUTL1, also known as CDP, Cut, or Cux-1, is a homeodomain transcriptional regulator known to be involved in development and cell cycle progression. Here we report that CUTL1 activity is associated with increased migration and invasiveness in numerous tumor cell lines, both in vitro and in vivo. Furthermore, we identify CUTL1 as a transcriptional target of transforming growth factor beta and a mediator of its promigratory effects. CUTL1 activates a transcriptional program regulating genes involved in cell motility, invasion, and extracellular matrix composition. CUTL1 expression is significantly increased in high-grade carcinomas and is inversely correlated with survival in breast cancer. This suggests that CUTL1 plays a central role in coordinating a gene expression program associated with cell motility and tumor progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease-Free Survival; DNA-Binding Proteins; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasms; NIH 3T3 Cells; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Repressor Proteins; RNA, Double-Stranded; Signal Transduction; Smad4 Protein; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Up-Regulation

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Topics: Carcinoma, Pancreatic Ductal; Cell Growth Processes; Cell Transformation, Viral; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunohistochemistry; Laminin; Oligonucleotide Array Sequence Analysis; Oncogene Proteins, Viral; Pancreatic Ducts; Pancreatic Neoplasms; Papillomaviridae; Papillomavirus E7 Proteins; ras Proteins; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Our previous investigations showed that retinoids, at specific concentrations, can inhibit cell proliferation. In this investigation, we hypothesize that high concentrations of retinoids can induce phenotypic changes (differentiation) and late apoptosis in pancreatic cancer cells in vitro.. To test our hypothesis, retinoid-induced differentiation was assessed: (1) phenotypically by light and electron microscopy and (2) biochemically by measuring carbonic anhydrase, aerobic metabolic and mucin producing activities. Modulation of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) autocrine pathways were utilized as mechanistic and differentiation markers.. The extensive differentiation-indicative phenotypic changes correlated with several folds increase in the aerobic metabolism (MTT reduction and Mitochondrial mass), carbonic anhydrase activity and mucin production. There was a marked increase in TGF-beta (Bioassay and ELISA) and TGF-beta (RIA) secretion. EGF receptor density (Receptor binding assay) was reduced by 50% within six hours and was reflected on abolishment of EGFR ligand-induced proliferation. Cotreatment with the RAR-alpha antagonist, Ro41-5253 or pan-TGF-beta neutralizing antibody abolished the phenotypic and antiproliferative effects of all-trans retinoic acid. Apoptosis (TUNEL assay) was undetectable after three days of treatment with the maximum concentration used. However, apoptosis was extensively induced after six days of treatment.. High concentrations of retinoids were able to induce phenotypic changes (differentiation) and late apoptosis in pancreatic cancer cells in vitro. The clinical ramifications of these observations await further investigations.

Topics: Apoptosis; Carbonic Anhydrases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Induction; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Microscopy, Electron; Pancreatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; Time Factors; Transforming Growth Factor beta; Tretinoin

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.

Topics: Biglycan; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cytochalasin D; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Models, Biological; Mutation; NADPH Oxidases; Nucleic Acid Synthesis Inhibitors; Onium Compounds; Osteosarcoma; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Proteoglycans; rac1 GTP-Binding Protein; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

The purpose of our study was to investigate the immunohistochemical expression of TGF-beta1 and p27 in pancreatic adenocarcinomas and to compare the findings with the clinicopathological features and survival. We also aimed to evaluate the expression of TGF-beta1 and p27 in the context of other cell cycle and proliferation markers such as cyclin D1 and Ki-67.. We examined TGF-beta1 and p27 expression immunohistochemically in 63 cases of invasive ductal adenocarcinoma of the pancreas. Standard streptavidin-biotin immunperoxidase method was used for immunostaining and the stained slides were examined microscopically using semiquantitative criteria.. TGF-beta1 stained the cytoplasms of the tumor cells in 43 cases [68.3%]. There was a statistically significant difference among TGF-beta1 staining scores in terms of clinicopathologic factors such as blood vessel invasion, stage and distant metastasis [p < 0.05]. Of the 63 tumors evaluated 23 [36.5%] were positive for p27 within the nucleus. An inverse correlation was found between p27 immunoreactivity and grade [p < 0.05]. But no significant correlation was found between p27 and other parameters. Among the patients with survival data 27 patients had RO resections and these cases were considered in survival analysis. In the univariate analysis, neither TGF-beta1 nor p27 expression was related with patient survival.. Our findings suggest that in pancreatic carcinoma, TGF-beta1 expression is related to tumor growth and metastasis. But it is not associated with cell cycle proteins. p27 expression is reduced in pancreatic adenocarcinomas and decreased protein levels of p27 may play a role in the differentiation of pancreatic cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biotin; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Cytoplasm; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Streptavidin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome

In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. Mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.

Topics: Cell Cycle; Cell Differentiation; Cell Line, Transformed; Cell Movement; DNA-Binding Proteins; Epithelial Cells; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Keratinocytes; Mesoderm; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Small Interfering; Smad4 Protein; Tetracycline; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

Topics: Adenocarcinoma; Antimetabolites, Antineoplastic; Antineoplastic Agents, Alkylating; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Dacarbazine; Fluorouracil; Gene Expression; Gene Expression Profiling; Humans; Methionine; Neoplasm Proteins; O(6)-Methylguanine-DNA Methyltransferase; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Phosphorylation; Retinoblastoma Protein; Signal Transduction; Temozolomide; Transforming Growth Factor beta

Plasminogen activator inhibitor-1 (PAI-1) is a unique type of serine protease inhibitor and one of the key regulators of tumor invasion and metastasis. The purpose of this study was to elucidate the effect of PAI-1 gene transfection on liver metastasis and its mechanism by using the human high liver metastasis pancreatic cancer cell line, SW1990. PAI-1-transfected SW1990 (SW/PAI-1) produced a significantly higher level of PAI-1 in supernatant than parental cells. While no difference was observed for the production of u-PA and u-PA activity in the supernatant, cell proliferation of SW/PAI-1 was slightly suppressed on the 7th day of incubation compared to parental cells. Cellular invasion, in vivo tumorigenesis in xenograft and liver metastasis were significantly suppressed in SW/PAI-1 cells compared to parental cells. The angiogenesis of xenograft by detecting microvascular density and the production of metastasis-related factors, such as VEGF and TGF-beta1, were also decreased in SW/PAI-1 cells. These findings suggested that PAI-1 gene transfection might have the ability to prevent the liver metastasis of pancreatic cancer by modulating angiogenesis.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Platelet Endothelial Cell Adhesion Molecule-1; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which the transforming growth factor beta (TGF-beta) signal transducer, Smad4, is commonly mutated or deleted. BxPC3 human pancreatic cancer cells exhibit a homozygous deletion of the Smad4 gene, yet are growth inhibited by TGF-beta1. In the present study, we sought to determine whether reintroduction of Smad4 into BxPC3 cells alters their behavior in vitro and in vivo. Sham transfected and Smad4 expressing BxPC3 cells exhibited similar responses to TGF-beta1 with respect to p21 upregulation, hypophosphorylation of the RB protein, Smad2 phosphorylation, and Smad2/3 nuclear translocation. TGF-beta1 did not alter p27 expression, and silencing of p21 with an appropriate siRNA markedly attenuated TGF-beta1-mediated growth inhibition. Nonetheless, the presence of Smad4 was associated in vitro with a more prolonged doubling time, enhanced sensitivity to the growth inhibitory actions of exogenous TGF-beta1, and a more flattened cellular morphology. In vivo, Smad4 expression resulted in delayed tumor growth and decreased cellular proliferation, without effects on either apoptosis or angiogenesis. These findings indicate that, in spite of the absence of Smad4, growth inhibition in BxPC3 cells by TGF-beta1 is dependent on p21 upregulation and maintenance of RB in a hypophosphorylated, active state. Moreover, the presence of a functional Smad4 attenuates the capacity of BxPC3 cells to proliferate in vivo. However, this effect is transient, indicating that Smad4 growth inhibitory actions are circumvented in the later stages of pancreatic tumorigenicity.

Topics: Active Transport, Cell Nucleus; Animals; Carcinoma, Pancreatic Ductal; Cell Nucleus; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Humans; Mice; Mice, Nude; Neovascularization, Pathologic; Pancreatic Neoplasms; Phosphorylation; Retinoblastoma Protein; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Up-Regulation

Expression profiling analyses were used to elucidate the functional relevance of RAS proteins in mediating the effect of TGFB1 on the transcriptional phenotype of the pancreatic cancer cell line PANC-1. Despite the presence of one mutated KRAS2 allele in parental PANC-1 pancreatic cancer cells, RAS-dependent signal transduction remained susceptible to stimulation by EGF and TGFB1. To analyze the impact of RAS proteins on the TGFB1-induced transcriptional phenotype, we used PANC-1 cells stably transfected with a dominant negative HRAS(S17N) mutant or with a constitutively active KRAS2(G12V) mutant. TGFB1 treatment of mock-transfected PANC-1 cells led to an expression profile suggestive of epithelial-mesenchymal transdifferentiation (EMT). Profiling of the HRAS(S17N)-expressing clone demonstrated that induction of endogenous RAS activity by TGFB1 is required for the development of the TGFB1-induced transcriptional phenotype of PANC-1 cells. The expression of the KRAS2(G12V) mutant by itself repressed transcription of markers of epithelial differentiation and induced transcription of several extracellular matrix-associated genes. This effect was not enhanced further by TGFB1 treatment. In contrast, transcript levels of genes associated with proliferation and cell cycle progression did not appear to be the primary targets of the synergism between the RAS- and TGFB1-dependent cascades. The introduction of the dominant negative and the constitutively active RAS mutants induced partly overlapping and partly inverse effects on the TGFB1-induced expression profile of PANC-1 cells. Additional mechanisms such as the induction of autocrine loops and the use of different RAS isoforms or alternate, ERK-independent signaling pathways may be involved in the interaction between the RAS- and the TGFB1-dependent signaling cascades.

Topics: Amino Acid Substitution; Cell Line, Tumor; Cluster Analysis; Culture Media, Serum-Free; DNA, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Mutation; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Recombinant Proteins; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

MUC4 is highly expressed in human pancreatic tumours and pancreatic tumour cell lines, but is minimally or not expressed in normal pancreas or chronic pancreatitis. Here, we investigated the aberrant regulation of MUC4 expression in vivo using clonal human pancreatic tumour cells (CD18/HPAF) grown either orthotopically in the pancreas (OT) or ectopically in subcutaneous tissue (SC) in the nude mice. Histological examination of the OT and SC tumours showed moderately differentiated and anaplastic morphology, respectively. The OT tumour cells showed metastases to distant lymph nodes and faster tumour growth (P<0.01) compared to the SC tumours. The MUC4 transcripts in OT tumours were very high compared to the undetectable levels in SC tumours. The SC tumour cells regained their ability to express MUC4 transcripts after in vitro culture. Immunohistochemical analysis using MUC4-specific polyclonal antiserum confirmed the results obtained by Northern blot analysis. Interestingly, the OT tumours showed expression of TGFbeta2 compared to no expression in SC, suggesting a possible link between MUC4 and TGFbeta2. The MUC4 expression, morphology, and metastasis of human pancreatic tumour cells are regulated by a local host microenvironment. TGFbeta2 may serve as an interim regulator of this function.

Topics: Animals; Biomarkers, Tumor; Blotting, Northern; Choristoma; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Immunosuppressive Agents; Mice; Mice, Nude; Mucin-4; Mucins; Pancreatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta2; Transplantation, Heterologous; Tumor Cells, Cultured

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

Topics: Active Transport, Cell Nucleus; Cell Division; Cell Line, Tumor; Cell Nucleus; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; DNA, Complementary; Endoribonucleases; Enzyme Induction; Gene Deletion; Gene Expression Regulation; Genes, Tumor Suppressor; Humans; Keratinocytes; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta

Runx transcription factors are important regulators of lineage specific gene expression, cell proliferation, and differentiation. Runx3 expression is lost in a high proportion of gastric cancers, suggesting a tumour suppressive role in this malignancy. This study investigates the expression and localisation of Runx3 in pancreatic tissues.. Quantitative polymerase chain reaction was used to measure Runx3 mRNA. Immunohistochemistry was carried out to localise Runx3 in normal pancreatic tissues, and in primary and metastatic pancreatic ductal adenocarcinoma (PDAC). Basal and transforming growth factor beta1 (TGFbeta1) induced Runx3 expression was analysed in cultured pancreatic cancer cell lines.. Runx3 expression was low to absent in normal pancreatic tissues, but increased in a third of cancer tissues. Runx3 was present only in islets in normal pancreas, whereas in pancreatic cancers, Runx3 was detected in the cancer cells of seven of 24 samples analysed. In addition, it was expressed by lymphocytes in six of the 16 cases with lymphocyte infiltration. In pancreatic cancer cell lines, Runx3 mRNA was present in Colo-357 and T3M4 cells, but was low to absent in the other cell lines tested. TGFbeta1 repressed Runx3 mRNA expressed in Colo-357 cells, and had no effect on Runx3 expression in the other pancreatic cancer cell lines.. Runx3 expression is restricted to islets in the normal pancreas. In contrast, a considerable proportion of pancreatic tumours express Runx3, and its expression is localised in the tumour cells and in the infiltrating lymphocytes. Thus, Runx3 might play a role in the pathogenesis of PDAC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Blotting, Western; Core Binding Factor Alpha 3 Subunit; DNA-Binding Proteins; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Islets of Langerhans; Lymphocytes; Male; Middle Aged; Neoplasm Metastasis; Pancreas; Pancreatic Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-beta-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-beta stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-beta. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-beta signal pathway that did not involve Smad4.

Topics: Blotting, Western; Cell Line, Tumor; DNA-Binding Proteins; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Flavonoids; Gene Silencing; Humans; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Phosphorylation; Protein Biosynthesis; Proteins; Proteome; RNA Interference; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Pancreatic cancer is an aggressive malignancy that exhibits a number of genetic and epigenetic alterations. Indian hedgehog (Ihh) and its 2 signaling receptors, patched (Ptc) and smoothened (Smo), are involved in pancreatic development and regulation of beta-cell function as well as in certain human tumors. In the current study, we analyzed the expression, distribution and function of Ihh and its receptors in pancreatic cancer. Quantitative RT-PCR and immunohistochemistry were utilized to analyze the expression, localization and transcriptional regulation of Ihh, Ptc and Smo. The effects of inhibition and stimulation of the hedgehog signaling pathway on pancreatic cancer cell growth were examined by the MTT cell growth assay. By quantitative RT-PCR, Ihh, Ptc and Smo mRNA levels were increased 35-, 1.2- and 1.6-fold, respectively, in pancreatic cancer tissues in comparison to normal pancreatic tissues. By immunohistochemistry, Ihh, Ptc and Smo were expressed in the islet cells of normal and cancerous tissues and in pancreatic cancer cells. The growth of pancreatic cancer cells was dose-dependently inhibited by the hedgehog antagonist cyclopamine through G0/G1 arrest. In contrast, Ihh agonists exhibited no significant effect on pancreatic cancer cell growth. TGF-beta1 repressed Ihh transcription in a TGF-beta1-responsive pancreatic cancer cell line, but had no effect on the other tested cell lines. In conclusion, Ihh and its receptors Ptc and Smo are expressed in pancreatic cancer, and blockage of hedgehog signaling results in inhibition of pancreatic cancer cell growth, suggesting that aberrant activation of the Ihh signaling pathway contributes to tumor development in this malignancy.

Topics: Adult; Aged; Blotting, Western; Cell Line, Tumor; DNA, Complementary; Dose-Response Relationship, Drug; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Immunohistochemistry; Lasers; Middle Aged; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

MUC4: encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-beta, a pleiotropic cytokine, regulates numerous genes involved in pancreatic carcinogenesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-beta in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4+/Smad4-), CAPAN-2 (MUC4+/Smad4+) and PANC-1 (MUC4-/Smad4+), were used. By RT-PCR, transfection assays and immunohistochemistry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-beta, (ii) Smad2 positively cooperates with Smad4 to activate the promoter, (iii) activation of Smad4 by exogenous TGF-beta induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-beta activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-beta is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-beta as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.

Topics: Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Enzyme Inhibitors; Gastric Mucins; Gene Expression Regulation, Neoplastic; Histones; Humans; Ligands; Mitogen-Activated Protein Kinase 1; Mucin-4; Mucins; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Protein Kinase C; Receptor, ErbB-2; Regulatory Sequences, Nucleic Acid; Signal Transduction; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors.. We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)beta, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines.. N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change.. This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

Topics: Aged; Blotting, Western; Cadherins; Cell Adhesion; Cell Proliferation; Epithelium; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; Mesoderm; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreas; Pancreatic Neoplasms; Time Factors; Transforming Growth Factor beta; Up-Regulation; Vimentin

The heparan sulfate proteoglycan glypican-1 is essential as a co-receptor for heparin binding growth factors, such as HB-EGF and FGF-2, in pancreatic cancer cells. In the present study, the role of glypican-1 in the regulation of TGF-beta signaling was investigated. Colo-357 pancreatic cancer cells were stably transfected with a full-length glypican-1 antisense construct. Cell growth was determined by MTT and soft agar assays. TGF-beta1 induced p21 expression and Smad2 phosphorylation were analyzed by immunoblotting. PAI-1 promoter activity was determined by luciferase assays. Down-regulation of glypican-1 expression by stable transfection of a full-length glypican-1 antisense construct resulted in decreased anchorage-dependent and -independent cell growth in Colo-357 pancreatic cancer cells and attenuated TGF-beta1 induced cell growth inhibition, Smad2 phosphorylation, and PAI-1 promoter activity. There was, however, no significant difference in TGF-beta1 induced p21 expression and Smad2 nuclear translocation. In conclusion, glypican-1 is required for efficient TGF-beta1 signaling in pancreatic cancer cells.

Topics: Cell Division; Cell Line, Tumor; DNA, Antisense; Gene Expression Regulation, Neoplastic; Heparan Sulfate Proteoglycans; Humans; Pancreatic Neoplasms; Signal Transduction; Transfection; Transforming Growth Factor beta

Tumor-associated trypsinogen (TAT), urokinase-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2), and MMP-9 each play a dominant role in the degradation of extracellular matrix (ECM) during the invasion process of pancreatic cancer. Transforming growth factor beta1 (TGF-beta1) is a multifunctional poly-peptide that regulates cell growth and differentiation, ECM deposition, cellular adhesion properties, angiogenesis, and also immune functions. We previously reported that TGF-beta1 up-regulated vascular endothelial growth factor (VEGF) production and protease production of MMP-2 and of u-PA in the highly metastatic pancreatic cancer cell lines SW1990 and CAPAN-2. In this study, we examined the inhibitor effects of a protease inhibitor, gabexate mesilate (GM), on cell invasion, cell proliferation, growth factor production, and ECM degradation. We also examined the effect of GM on the production of growth factor and ECM degradation by these cell proteases and enzymatic activities.. GM down-regulated the invasiveness and liver metastasis potential of SW1990 and CAPAN-2 cells, but it did not affect the proliferation of these cells. GM inhibited not only the enzymatic activities of TAT and u-PA but also the production of MMP-2, and u-PA, all of which have been known to be secondarily down-regulated by TGF-beta1.. These findings suggested that GM has very good potential for use in the treatment against invasion and metastasis of pancreatic cancer.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Line, Tumor; Collagen; Drug Combinations; Enzyme Induction; Female; Gabexate; Gene Expression Regulation, Neoplastic; Humans; Laminin; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Protease Inhibitors; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A

Transforming growth factor-beta (TGF-beta) suppresses tumor formation by blocking cell cycle progression and maintaining tissue homeostasis. In pancreatic carcinomas, this tumor suppressive activity is often lost by inactivation of the TGF-beta-signaling mediator, Smad4. We found that human pancreatic carcinoma cell lines that have undergone deletion of MADH4 constitutively expressed high endogenous levels of phosphorylated receptor-associated Smad proteins (pR-Smad2 and pR-Smad3), whereas Smad4-positive lines did not. These elevated pR-Smad levels could not be attributed to a decreased dephosphorylation rate nor to increased expression of TGF-beta type I (TbetaR-I) or type II (TbetaR-II) receptors. Although minimal amounts of free bioactive TGF-beta1 and TGF-beta2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-beta3 specific) TGF-beta-neutralizing antibody and with anti-alpha(V)beta(6) integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-beta-inducible reporter gene in neighboring cells, respectively. Thus, activation of TGF-beta at the cell surface was responsible for the increased autocrine endogenous and paracrine signaling. Blocking TbetaR-I activity using a selective kinase inhibitor (SD-093) strongly decreased the in vitro motility and invasiveness of the pancreatic carcinoma cells without affecting their growth characteristics, morphology, or the subcellular distribution of E-cadherin and F-actin. Moreover, exogenous TGF-beta strongly stimulated in vitro invasiveness of BxPC-3 cells, an effect that could also be blocked by SD-093. Thus, the motile and invasive properties of Smad4-deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF-beta signaling. Therefore, targeting the TbetaR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.

Topics: Activin Receptors, Type I; Antigens, Neoplasm; Cadherins; Cell Movement; DNA-Binding Proteins; Enzyme Inhibitors; Gene Deletion; Humans; Integrins; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Tumor Cells, Cultured; Wound Healing

Recent studies have demonstrated that transforming growth factor beta 1 (TGF-beta1) expression is markedly enhanced in invasive ductal pancreatic adenocarcinomas, although the precise role of TGF-beta1 in pancreatic carcinogenesis remains unclear. We analyzed TGF-beta1 expression in pancreatic intraepithelial neoplasias (PanINs) and the effects of chronic TGF-beta1 exposure on conditionally immortalized pancreatic epithelial (IMPE) cells.. Sixty-one PanIN lesions were immunohistochemically stained with a polyclonal rabbit antibody against human TGF-beta1. Growth-inhibitory effects of short-term exposure to TGF-beta1 were examined in IMPE cells. IMPE cells resistant to TGF-beta1 (IMPE-Tr cells) were generated by continuous exposure to 1 ng/mL of TGF-beta1 for more than 50 days. Phenotypic alterations of IMPE-Tr cells were examined by soft agar and Matrigel assay and Western blot analysis. IMPE and IMPE-Tr cells were injected subcutaneously into nude mice for an in vivo tumorigenicity assay.. Forty-six percent of PanINs (28/61) were positive for TGF-beta1 expression, whereas all the epithelia of normal pancreatic ducts were negative. TGF-beta1 treatment showed the marked growth-inhibitory effects (>75%) in IMPE cells, whereas its effects were not observed in IMPE-Tr cells. IMPE-Tr cells were more spindle shaped compared with IMPE cells. In soft agar and Matrigel, formations of many colonies were observed in IMPE-Tr cells, but not in IMPE cells. Interestingly, the expression of p21(WAF1/CIP1) was induced by short-term exposure to TGF-beta1 in IMPE cells, whereas the induction was decreased in IMPE-Tr cells. All of the IMPE-Tr cell-injected mice (5/5) had subcutaneous tumors, although no tumor was found in the IMPE cell-injected mice.. TGF-beta1 expression in PanINs and neoplastic transformation of IMPE cells by long-term exposure to TGF-beta1 suggest that TGF-beta1 may act as a tumor promoter in the early stage of pancreatic carcinogenesis.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Collagen; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Down-Regulation; Drug Combinations; Epithelial Cells; Humans; Laminin; Mice; Mice, Nude; Pancreas; Pancreatic Neoplasms; Proteoglycans; Transforming Growth Factor beta; Transforming Growth Factor beta1

Smad-regulated transcription plays a central role in transforming growth factor (TGF)-beta-induced cell growth inhibition and tumor suppression. Like the Smads, KLF11 is an early response transcription factor that mediates TGF-beta-induced growth inhibition in untransformed epithelial cells. Here, we investigated the functional implications of KLF11 in TGF-beta signaling and transcription in normal epithelial as well as pancreatic cancer cells.. The effects of KLF11 on TGF-beta signaling and transcription were examined on the levels of reporter transactivation, Smad2 phosphorylation, and expression of endogenous TGF-beta-regulated genes. Promoter analysis, real-time polymerase chain reaction, and coimmunoprecipitation studies were performed to study KLF11-induced and mSin3A corepressor-mediated repression of Smad7. Erk-induced KLF11 phosphorylation was examined in vitro and in vivo, and its impact on KLF11-mSin3A-mediated Smad7 repression was verified in pancreatic cancer cells using site-directed mutagenesis.. KLF11 potentiates TGF-beta signaling by terminating the inhibitory Smad7 loop. Mechanistically, KLF11 represses TGF-beta-induced transcription from the Smad7 promoter by recruiting mSin3a via GC-rich sites. This function is inhibited in pancreatic cancer cells with oncogenic Ras mutations, in which Erk/mitogen-activated protein kinase phosphorylates KLF11, leading to disruption of KLF11-mSin3a interaction. Expression of an Erk-insensitive KLF11 mutant restores both mSin3a binding and Smad7 repression and results in enhanced TGF-beta signaling in pancreatic cancer cells.. These results define a novel mechanism in TGF-beta-regulated gene expression. KLF11 potentiates Smad-signaling activity in normal epithelial cells through termination of the negative feedback loop imposed by Smad7. The fact that this function of KLF11 is inhibited by oncogenic Erk/mitogen-activated protein kinase in pancreatic cancer cells emphasizes the importance of this mechanism for oncogenesis.

Topics: Animals; Apoptosis Regulatory Proteins; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; CHO Cells; COS Cells; Cricetinae; DNA-Binding Proteins; Epithelial Cells; GC Rich Sequence; Gene Expression Regulation, Neoplastic; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nuclear Proteins; Pancreatic Neoplasms; Phosphorylation; Promoter Regions, Genetic; ras Proteins; Repressor Proteins; Sin3 Histone Deacetylase and Corepressor Complex; Smad2 Protein; Smad7 Protein; Trans-Activators; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

Pancreatic cancer is often associated with an intense production of interstitial collagens, known as the desmoplastic reaction. To understand more about desmoplasia in pancreatic cancer, the expression of mRNA for type I and III collagens and potent desmoplastic inducing growth factors transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), acidic and basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) A and C and epidermal growth factor (EGF) was analysed by quantitative RT-PCR. Expression of both collagens in 23 frozen primary pancreatic cancer nodules was significantly higher than that in 15 non-neoplastic pancreatic tissues. The expressions of mRNAs for TGF-beta, acidic FGF, basic FGF and PDGF C were likewise higher in surgical cancer nodules, while that of CTGF, PDGF A and EGF were not. Among these growth factors, the expression of TGF-beta mRNA showed the most significant correlation with that of collagens (P<0.0001). By immunohistochemistry, TGF-beta showed faint cytoplasmic staining in cancer cells. In contrast, isolated cells, mainly located on the invasive front surrounding cancerous nests, were prominently and strongly stained. These TGF-beta-positive cells contained a segmented nucleus, were negative for anti-macrophage (CD68) and positive for anti-granulocyte antibodies, indicating their granulocytic nature. In conclusion, TGF-beta seemed to play a major role among the various growth factors in characteristic overproduction of collagens in pancreatic cancer. Moreover, the predominant cells that express TGF-beta were likely to be infiltrated granulocytes (mostly are neutrophils) and not pancreatic cancer cells.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Collagen; Female; Gene Expression Regulation, Neoplastic; Granulocytes; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Connective tissue growth factor (CTGF), which is regulated by transforming growth factor-ss (TGFss), has recently been implicated in the pathogenesis of fibrotic diseases and tumor stroma. Inasmuch as generation of desmoplastic tissue is characteristic for pancreatic cancer, it is not known whether it gives pancreatic cancer cells a growth advantage or is a reaction of the body to inhibit cancer cell progression. In the present study we analyzed the expression and localization of CTGF and evaluated whether it influences the prognosis of pancreas cancer. Tissue samples were obtained from 25 individuals (6 women, 19 men) undergoing pancreatic resection for pancreatic cancer. Tissue samples from 13 previously healthy organ donors (5 women, 8 men) served as controls. Expression of CTGF was studied by Northern blot analysis. In situ hybridization and immunohistochemistry localized the respective mRNA moieties and proteins in the tissue samples. Northern blot analysis revealed that pancreatic cancer tissue samples exhibited a 46-fold increase in CTGF mRNA expression ( p < 0.001) over that of normal controls. In vitro studies confirmed that pancreatic stellate cells are the major source of CTGF mRNA expression and revealed a large variance in basal and TGFss-induced CTGF expression in cultured pancreatic cancer cells. This could also be confirmed by in situ hybridization, indicating that CTGF mRNA signals were located principally in fibroblasts, with only weak signals in the cancer cells. High CTGF mRNA levels in the tissue samples correlated with better tumor differentiation ( p < 0.03). In addition, patients whose tumors exhibited high CTGF mRNA levels (> onefold increase above normal controls) lived significantly longer than those whose tumors expressed low CTGF mRNA levels (none to onefold) ( p < 0.04 multivariate analysis). Our present data indicate that CTGF, as a downstream mediator of TGFss, is overexpressed in connective tissue cells and to a lesser extent in pancreatic cancer cells. Because patients with high CTGF mRNA expression levels have a better prognosis, our findings indicate that the desmoplastic reaction provides a growth disadvantage for pancreatic cancer cells.

Topics: Adenocarcinoma; Aged; Cell Division; Cell Transformation, Neoplastic; Connective Tissue Growth Factor; Female; Fibroblasts; Fibrosis; Follow-Up Studies; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Neoplasm Staging; Pancreas; Pancreatectomy; Pancreatic Neoplasms; Prognosis; RNA, Messenger; Survival Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured

The TGF-beta signaling pathway has potent anti-mitogenic effects in epithelial cells and loss of negative growth regulation is often associated with increased tumorigenicity. The human pancreatic ductal adenocarcinoma cell line, UK Pan-1, which expresses DPC4, is not highly responsive to TGF-beta due to transcriptional repression of TGF-beta type II receptor (RII). Here, we show that UK Pan-1 cells transfected with a plasmid to overexpress rap1 protein (UK/rap1) causes an increase in RII transcription and restores sensitivity to TGF-beta growth inhibition. The overexpression of rap1 was associated with diminished ras signaling as measured by ras binding domain (RBD)-binding assays. Electrophoretic mobility shift assays (EMSA) analysis revealed increased binding of nuclear proteins to a previously identified positive regulatory element (PRE1) of the RII promoter in rap1 transfected cells. Competition with an oligo containing the AP-1 consensus site was able to inhibit this binding of nuclear proteins to the PRE1 region. Further EMSA analysis using antibodies to various AP-1 components revealed that junB antibodies partially depleted the increase in binding to the PRE1 seen in UK/rap1 cells while antibodies to other AP-1 constituents such as c-jun, c-fos, and ATF-1 had no effect on binding. Consistent with this data, transient transfection of UK Pan-1 cells with junB resulted in greater RII transcription (twofold) as measured by RII-luciferase assay. Mutation of the AP-1 site inhibited junB-mediated or rap1-mediated increases in RII promoter activity. These data suggest that rap1 signaling may mediate an increase in RII transcription via increased binding of nuclear factors including junB to the PRE1 region of the RII promoter.

Topics: Carcinoma; Cell Division; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Genes, Regulator; Humans; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; rap1 GTP-Binding Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Repressor Proteins; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Smad4 is a tumor suppressor gene that is commonly lost or mutated in colorectal and pancreatic cancers. The activated transforming growth factor-beta (TGF-beta) receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus. Smad4 mutations when detected as present in some human cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe a colon cancer cell line, VACO-9M, that is Smad4 null when analysed by multiple assays. To study the role of Smad4 in TGF-beta-induced translocation of the receptor-activated Smads to the nucleus, we analysed by immunofluorescence the cellular localization of endogenous Smad2 and Smad3 after TGF-beta treatment of VACO-9M, plus four additional Smad4 null cell lines of breast (MDA-MB-468), or pancreatic (BxPC3, Hs766T, CFPAC-1) origin. In each cell line, TGF-beta treatment resulted in both Smad2 and Smad3 moving to the nucleus in a Smad4-independent fashion. Nuclear translocation of Smad2 and Smad3 was, however, not sufficient to activate reporters for TGF-beta-induced transcriptional responses, which were however restored by transient transfection of wild-type Smad4. We conclude that Smad4 is not required for nuclear translocation of Smad2 and Smad3, but is needed for activation of at least certain transcriptional responses.

Topics: Active Transport, Cell Nucleus; Adenocarcinoma; Breast Neoplasms; Cell Nucleus; Colonic Neoplasms; DNA-Binding Proteins; Genes, Reporter; Humans; Microscopy, Fluorescence; Neoplasm Proteins; Pancreatic Neoplasms; Recombinant Fusion Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta-1 (TGFB1)-induced gene expression was studied in five pancreatic carcinoma cell lines and one known TGFB1-sensitive cell line (HaCaT) by use of high-density filter-based cDNA microarrays representing over 4,000 human genes. The results indicate a complex cellular response to TGFB1 with 10% of the investigated genes showing altered expression after 3 or 48 hr of TGFB1 exposure. The tumor cell lines displayed a gradually inversed gene expression pattern, which correlated with reduced sensitivity to TGFB1, as compared to the HaCaT cell line. In the HaCaT cells, several proapoptotic genes showed increased expression in response to TGFB1, whereas the expression of antiapoptotic genes was decreased. In contrast, two pancreatic carcinoma cell lines, previously found to be growth stimulated by TGFB1, displayed an expression pattern opposite to that of these genes. Similarly, the expression of other functional groups of genes, such as cell cycle and transcription factor related genes, was almost completely reversed in these two tumor cell lines. Importantly, three of the five investigated pancreatic carcinoma cell lines responded to TGFB1, although they had SMAD4 inactivations, suggesting that the observed gene expression changes in these cell lines must be accomplished by SMAD-independent pathways.

Topics: Cluster Analysis; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Neoplasm; Humans; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; RNA, Neoplasm; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

We have investigated the mechanism whereby tumor cells become resistant to the antiproliferative effects of transforming growth factor (TGF)-beta, while maintaining other responses that can lead to increased malignancy and invasiveness. TGF-beta signaling results in nuclear accumulation of active Smad complexes which regulate transcription of target genes. Here we show that in two pancreatic carcinoma cell lines, PT45 and Panc-1, that are resistant to TGF-beta-induced growth arrest, the TGF-beta-Smad signaling pathway is attenuated compared with epithelial cells that are sensitive to the antiproliferative effects of TGF-beta (HaCaT and Colo-357). In PT45 and Panc-1 cells, active Smad complexes remain nuclear for only 1-2 h compared with more than 6 h in HaCaT and Colo-357 cells. The attenuated pathway in PT45 and Panc-1 cells correlates with low levels of TGF-beta type I receptor and results in an altered expression profile of TGF-beta-inducible genes required for cell cycle arrest. Most significantly, expression of the CDK inhibitor, p21(Cip1/WAF1), which is required for TGF-beta-induced growth arrest in these cells, is not maintained. Moreover, we show that artificially attenuating the TGF-beta-Smad signaling pathway in HaCaT cells is sufficient to prevent TGF-beta-induced growth arrest. Our results demonstrate that the duration of TGF-beta-Smad signaling is a critical determinant of the specificity of the TGF-beta response.

Topics: Activin Receptors, Type I; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Humans; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve diagnosis and treatment, key mechanisms of deregulated molecular functions have to be identified. Using microarray analysis, the expression patterns of 5600 human genes were assessed in PDAC by comparison with the normal pancreas and chronic pancreatitis (CP). The expression of 467 of 5600 genes was increased in PDAC in comparison to the normal pancreas, and the expression of 120 of these genes was not increased in CP. In addition, 341 of 5600 genes were expressed at decreased levels in PDAC tissues, of which 96 were decreased in comparison to both normal and CP tissues. Thus, a total of 808 of 5600 human genes were differentially expressed in pancreatic cancer. The identification of a large panel of altered genes in PDAC will stimulate additional studies that will lead to improved understanding of the molecular mechanisms underlying pancreatic malignant growth.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Female; Gene Expression Profiling; Humans; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Pancreatitis; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Carcinoma, Neuroendocrine; DNA-Binding Proteins; Gastrointestinal Neoplasms; Humans; Neoplasm Proteins; Pancreatic Neoplasms; Proto-Oncogene Proteins; Rats; Receptors, Transforming Growth Factor beta; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

The role of transforming growth factor beta-1 (TGFbeta-1) in neuroendocrine tumour biology is currently unknown. We therefore examined the expression and biological significance of TGFbeta signalling components in neuroendocrine tumours (NETs) of the gastroenteropancreatic (GEP) tract.. Expression of TGFbeta-1 and its receptors, Smads and Smad regulated proteins, was examined in surgically resected NET specimens and human NET cell lines by immunohistochemistry, reverse transcriptase-polymerase chain reaction, immunoblotting, and ELISA. Activation of TGFbeta-1 dependent promoters was tested by transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. The role of endogenous TGFbeta was assessed by a TGFbeta neutralising antibody and stable transfection of a dominant negative TGFbetaR II receptor construct.. Coexpression of TGFbeta-1 and its receptors TGFbetaR I and TGFbetaR II was detected in 67% of human NETs and in all three NET cell lines examined. NET cell lines expressed the TGFbeta signal transducers Smad 2, 3, and 4. In two of the three cell lines, TGFbeta-1 treatment resulted in transactivation of a TGFbeta responsive reporter construct as well as inhibition of c-myc and induction of p21((WAF1)) expression. TGFbeta-1 inhibited anchorage dependent and independent growth in a time and dose dependent manner in TGFbeta-1 responsive cell lines. TGFbeta-1 mediated growth inhibition was due to G1 arrest without evidence of induction of apoptosis. Functional inactivation of endogenous TGFbeta revealed the existence of an autocrine antiproliferative loop in NET cells.. Neuroendocrine tumour cells of the gastroenteropancreatic tract are subject to paracrine and autocrine growth inhibition by TGFbeta-1, which may account in part for the low proliferative index of this tumour entity.

Topics: Autocrine Communication; Blotting, Western; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gastrointestinal Neoplasms; Humans; Neoplasm Proteins; Neuroendocrine Tumors; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

SMAD4 is a critical cofactor in signal transduction pathways activated in response to transforming growth factor-beta (TGF-beta)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumours highlighted it as a tumour-suppressor gene. However, restoration of the TGF-beta antiproliferative pathway following SMAD4 gene transfer in null-tumour cell lines is controversial. Herein, we report the inhibitory effects of SMAD4 on pancreatic tumour invasion and angiogenesis. Adenoviral transfer of this gene in a panel of SMAD4 homozygous-deleted human pancreatic tumour cell lines restored SMAD4 protein expression and function. Although it did not affect proliferation significantly in vitro, SMAD4 inhibited in vivo tumour growth in immunodeficient mice. In this xenograft setting, differential suppression of tumour growth in vivo was mediated, at least in part, through downregulation of vascular endothelial growth factor and expression of gelatinases. We documented the reduced invasion and angiogenesis histologically and by intravital microscopy, and gained mechanistic insight at the messenger and protein level. Finally, we found a negative reciprocal regulation between SMAD4 and ETS-1. ETS-1 is considered a marker for tumour invasion. Upon SMAD4 deletion, we detected high expression levels of ETS-1 in pancreatic tumour cells, suggesting the shift of the pancreatic tumour toward an invasive phenotype.

Topics: Adenocarcinoma; Adenoviridae; Animals; Biomarkers, Tumor; Cell Transplantation; DNA-Binding Proteins; Down-Regulation; Gelatinases; Gene Deletion; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Genes, Tumor Suppressor; Genetic Therapy; Ligands; Mice; Mice, SCID; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; RNA, Messenger; Smad4 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Claudin-4 has been identified as an integral constituent of tight junctions and has been found to be highly expressed in pancreatic cancer. The aim of the present study was to elucidate the effect of claudin-4 on growth and metastatic potential in pancreatic cancer cells, as well as the regulation of claudin-4 by oncogenic pathways. Claudin-4 was stably overexpressed in SUIT-2 pancreatic cancer cells, and its effect on invasion and growth in vitro was examined by using two-chamber invasion assays, soft agar assays, and fluorescence-activated cell sorter analysis. Claudin-4 localization was characterized by light and electron microscopy, and pulmonary colonization was analyzed in vivo after injection of claudin-4 overexpressing cells into the tail vein of nude mice. Overexpression of claudin-4 was associated with significantly reduced invasive potential in vitro and inhibited colony formation in soft agar assays. In vivo, tail vein-injected claudin-4 overexpressing cells formed significantly less pulmonary metastases in comparison with mock-transfected cells. These effects were not caused by changes in proliferation, cell cycle progression, or matrix metalloproteinase gelatinolytic activity, but were paralleled by increased cell contact formation. Moreover, proinvasive transforming growth factor beta was able to down-regulate claudin-4 in PANC-1 cells. Inhibition of Ras signaling by using dominant-negative Ras and specific inhibitors of both downstream effectors mitogen-activated protein/extracellular signal-regulated kinase kinase and phosphatidylinositol 3'-kinase also decreased claudin-4 expression. Our findings identify claudin-4 as a potent inhibitor of the invasiveness and metastatic phenotype of pancreatic cancer cells, and as a target of the transforming growth factor beta and Ras/Raf/extracellular signal-regulated kinase pathways.

Topics: Carcinoma, Pancreatic Ductal; Cell Adhesion; Cell Cycle; Cell Division; Cell Line, Tumor; Claudin-4; Humans; Immunohistochemistry; Lung Neoplasms; Matrix Metalloproteinase 2; Membrane Proteins; Neoplasm Invasiveness; Pancreatic Neoplasms; ras Proteins; Receptors, Cell Surface; Signal Transduction; Transforming Growth Factor beta

p8 is a stress-induced protein with multiple functions and biochemically related to the architectural factor HMG-I/Y. We analyzed the expression and function of p8 in pancreatic cancer-derived cells.. Expression of p8 was silenced in the human pancreatic cancer cell lines Panc-1 and BxPc-3 by infection with a retrovirus expressing p8 RNA in the antisense orientation. Cell growth was measured in control and p8-silenced cells. Influence on p8 expression of the induction of intracellular pathways promoting cellular growth or growth arrest was monitored.. p8-silenced cells grew more rapidly than control cells transfected with the empty retrovirus. Activation of the Ras-->Raf-->MEK-->ERK and JNK intracellular pathways down-regulated p8 expression. In addition, the MEK1/2 inhibitor U0126 and the JNK inhibitor SP600125 up-regulates expression of p8. Conversely, p38 or TGFbeta-1 induced p8 expression whereas the specific p38 inhibitor SB203580 down-regulated p8 expression. Finally, TGFbeta-1 induction was in part mediated through p38.. p8 inhibits the growth of human pancreatic cancer cells. p8 expression is induced through pathways involved in growth inhibition and repressed by factors that promote cell growth. These results suggest that p8 belongs to a pathway regulating the growth of pancreatic cancer cells.

Topics: Anthracenes; Basic Helix-Loop-Helix Transcription Factors; Butadienes; Cell Line, Tumor; Cell Proliferation; Culture Media; Culture Media, Serum-Free; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Vectors; Growth Inhibitors; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Pancreatic Neoplasms; Proto-Oncogene Proteins c-raf; Pyridines; ras Proteins; Retroviridae; RNA, Antisense; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

Overexpression of the small leucine-rich proteoglycan biglycan (BGN) in fibrosis and desmoplasia results from enhanced activity of transforming growth factor-beta (TGF-beta). In pancreatic adenocarcinoma, the tumor cells themselves may contribute to BGN synthesis in vivo, since 8 of 18 different pancreatic carcinoma cell lines constitutively expressed BGN mRNA, as shown by reverse transcription-PCR analysis. In PANC-1 cells, TGF-beta1 dramatically stimulated BGN mRNA accumulation through a BGN transcription-independent, cycloheximide-sensitive mechanism and strongly increased the synthesis and release of the proteoglycan form of BGN. The ability of TGF-beta1 to induce BGN mRNA was critically dependent on Smad signaling, since 1) the up-regulation of BGN mRNA was preceded by a marked increase in Smad2 phosphorylation in TGF-beta1-treated PANC-1 cells, 2) TGF-beta1 was unable to induce BGN mRNA in pancreatic carcinoma cell lines that carry homozygous deletions of the Smad4/DPC4 gene, 3) inhibition of the Smad pathway in PANC-1 cells by transfection with a dominant negative Smad4/DPC4 mutant significantly reduced TGF-beta1-induced BGN mRNA expression, 4) stable reintroduction of wild type Smad4/DPC4 into Smad4-null CFPAC-1 cells restored the TGF-beta1 effect, and 5) overexpression of Smad2 and Smad3 in PANC-1 cells augmented TGF-beta1 induction of BGN mRNA, whereas forced expression of Smad7, an inhibitory Smad, effectively blocked it. These results clearly show that a functional Smad pathway is crucial for TGF-beta regulation of BGN mRNA expression. Since BGN has been shown to inhibit growth of pancreatic cancer cells, the Smad4/DPC4 mediation of the TGF-beta effect may represent a novel tumor suppressor function for Smad4/DPC4: antiproliferation via expression of autoinhibitory BGN.

Topics: Biglycan; Cell Division; Chondroitin ABC Lyase; Cycloheximide; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Humans; Immunoblotting; Mutation; Pancreatic Neoplasms; Phosphorylation; Protein Synthesis Inhibitors; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Smad3 Protein; Smad4 Protein; Smad7 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Based upon molecular allelotyping and comparative genomic hybridization studies, chromosome 15q is the likely location of a tumor suppressor gene important in the pathogeneses of sporadic enteropancreatic endocrine tumors and parathyroid adenomas. Interest has focused on Smad3 as a candidate endocrine tumor suppressor gene because 1) it is localized to 15q and 2) it encodes a TGF beta signaling molecule that has been identified as a binding partner of the multiple endocrine neoplasm type 1 gene product menin, itself involved in enteropancreatic and parathyroid neoplasia. To determine whether Smad3 plays a primary role in development of these tumors, 20 enteropancreatic tumors and 67 parathyroid adenomas were investigated for loss of heterozygosity at DNA markers surrounding Smad3. Twenty percent of enteropancreatic tumors and 24% of parathyroid adenomas showed loss. All 9 coding exons and intron-exon boundaries of the Smad3 gene were then sequenced in genomic DNA from all 20 enteropancreatic and 25 parathyroid tumors, including every case with loss of heterozygosity. No acquired clonal mutations, insertions, or microdeletions in Smad3 were detected in any tumors. Because inactivating somatic mutation is the hallmark of an authentic tumor suppressor, Smad3 is unlikely to function as a classical tumor suppressor gene in the pathogenesis of sporadic parathyroid or enteropancreatic endocrine tumors.

Topics: Adenoma; DNA Mutational Analysis; DNA-Binding Proteins; Genes, Tumor Suppressor; Humans; Islets of Langerhans; Neoplasm Proteins; Pancreatic Neoplasms; Parathyroid Neoplasms; Polymorphism, Genetic; Proto-Oncogene Proteins; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

betaig-h3 (TGFBI, keratoepithelin) was first identified as a transforming growth factor-beta1 (TGF-beta1)-inducible gene in a human lung adenocarcinoma cell line. It encodes for a secreted extracellular matrix (ECM) protein, which is thought to act on cell attachment and ECM composition. Mutations of the betaig-h3 gene are involved in several corneal dystrophies. Pancreatic cancers display multiple alterations in the TGF-beta signaling pathway and in TGF-beta response genes, such as overexpression of all three TGF-beta isoforms and Smad4 mutations. In this report, we determined that betaig-h3 mRNA levels were induced by TGF-beta1 in two out of five examined pancreatic cancer cell lines (CAPAN-1, PANC-1). In CAPAN-1 cells, which harbor a Smad4 mutation, betaig-h3 but not PAI-1 was induced by TGF-beta1, whereas in PANC-1 cells that express wild-type Smad4, TGF-beta1 induced both PAI-1 and betaig-h3. In human pancreatic tissues, there was a 32.4-fold increase in betaig-h3 mRNA levels in pancreatic cancers in comparison to normal control tissues. In situ hybridization analysis revealed that betaig-h3 mRNA was expressed mainly in the cancer cells within the pancreatic tumor mass. These findings suggest that betaig-h3 is induced by TGF-betas in pancreatic cancer cells even in the presence of Smad4 mutations, which might explain, in part, the increased betaig-h3 mRNA levels observed in pancreatic cancer cells in vivo.

Topics: Adolescent; Adult; Aged; Blotting, Northern; Cloning, Molecular; DNA-Binding Proteins; Extracellular Matrix Proteins; Female; Gene Expression Regulation; Humans; In Situ Hybridization; Male; Middle Aged; Mutation; Neoplasm Proteins; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Activated ras is known to dysregulate TGF-beta signaling by altering the expression of TGF-beta type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-beta signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 microM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 microM concentrations (P>0.004). The TGF-beta effector gene p21(waf1/cip1) was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-beta responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-beta signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT-PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-beta signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

Topics: Adenocarcinoma; Alkyl and Aryl Transferases; Apoptosis; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Induction; Enzyme Inhibitors; Farnesyltranstransferase; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Methionine; Neoplasm Proteins; Pancreatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Radiation Tolerance; Radiation-Sensitizing Agents; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Transforming Growth Factor beta

The tumor suppressor gene Smad4/DPC4, a key transcription factorin transforming growth factor beta (TGF-beta) signaling cascades,is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-beta-mediated expression of cell-cycle regulatory genes p15(ink4b) and p21(waf1).. Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGF-beta downstream target gene p21(waf1), regulation of the p15(ink4b) promoter, anchorage-independent growth, and tumorigenesis were examined.. We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15(ink4b), p21(waf1), and TGF-beta-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between -356 and -329 bp of the p15(ink4b) promoter. The p15(ink4b) promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15(ink4b) is one of the downstream target genes regulated by Smad/DPC4.. These results explain the role of Smad4/DPC4 in TGF-beta-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-beta-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.

Topics: Adenoviridae; Animals; Blotting, Northern; Blotting, Western; Cell Cycle Proteins; Cell Division; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Gene Deletion; Genes, Reporter; Homozygote; Humans; Immunoblotting; Luciferases; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Promoter Regions, Genetic; Signal Transduction; Smad4 Protein; Time Factors; Trans-Activators; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that frequently metastasizes and that overexpresses transforming growth factor-beta s (TGF-beta s). To determine whether TGF-beta s can act to enhance the metastatic potential of PDAC, PANC-1 human pancreatic cancer cells were transfected with an expression construct encoding a soluble type II TGF-beta receptor (sT beta RII) that blocks cellular responsiveness to TGF-beta 1. When injected s.c. in athymic mice, PANC-1 clones expressing sT beta RII exhibited decreased tumor growth in comparison with sham-transfected cells and attenuated expression of plasminogen activator inhibitor 1 (PAI-1), a gene associated with tumor growth. When tested in an orthotopic mouse model, these clones formed small intrapancreatic tumors that exhibited a suppressed metastatic capacity and decreased expression of plasminogen activator inhibitor 1 and the metastasis-associated urokinase plasminogen activator. These results indicate that TGF-beta s act in vivo to enhance the expression of genes that promote the growth and metastasis of pancreatic cancer cells and suggest that sT beta RII may ultimately have a therapeutic benefit in PDAC.

Topics: Animals; Blotting, Northern; Carcinoma, Pancreatic Ductal; Female; Gene Expression; Genetic Vectors; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Serine Proteinase Inhibitors; Signal Transduction; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

To detect the relations of c-erbB-2 oncogene protein, epidermal growth factor receptor (EGFR) and transforming growth factor-beta1 (TGF-beta1) to the progression or metastasis of pancreatic carcinoma.. Using streptavidinbiotin complex (SABC) method, c-erbB-2 oncongene protein, we examined immunohistochemically EGFR and TGF-beta1 expressions in wax-tissue sections from 10 individuals with normal pancreas (NP), 13 patients with chronic pancreatitis (CP) and 36 patients with pancreatic ductal adenocarcinoma (PC).. The positive expression rates of c-cerbB-2 oncogene protein, EGFR and TGF-beta1 in the NP, CP and PC groups were 0, 0, 10%; 7.7%, 7.7%, 7.7%; and 41.7%, 50.0%, 44.4%, respectively. The positive expression rates of the three specific proteins increased more significantly in the PC group than in the NP and CP groups (P<0.05). The individual expression of c-erbB-2, EGFR and TGF-beta1 was not related to the age and sex of the patients as well as the site, size and histopathological grade of tumors (P>0.05), but to the clinical stage of tumors (P<0.01). The coexpression rate of the three proteins was 27.8% (10/36). This coexpression in the PC group was correlated with the histopathological grades and clinical stages of tumors (P<0.01).. Detection of c-erbB-2 oncogene protein, EGFR, and TGF-beta1 expressions in pancreatic tissue is helpful to judge the malignancy, progression, and metastasis of PC.

Topics: Adenocarcinoma; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Pancreatic Ducts; Pancreatic Neoplasms; Receptor, ErbB-2; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the relation of transfer growth factor (TGF-beta1) and beta-glucuronidase (beta-GCD) on the occurrence and progress of pancreatic cancer.. The expression of TGF-beta1 and beta-GCD in the pancreatic cancer tissue and normal pancreatic tissue was determined synchronously using ABC method of immunohistochemistry.. The percentage of TGF-beta1 positive cells was significantly higher in pancreatic cancer tissue (43.8%+/-5.2%) than in adjacent pancreatic tissue (28.7%+/-3.6%, P<0.01). The worse the cancer cells differentiated and lymph nodes metastasis, the more over-expression of TGF-beta1. The percentage of beta-GCD positive cells was also significantly higher in the pancreatic cancer tissue (62.5%+/-4.1%) than in the adjacent pancreatic tissue (33.5%+/-2.8%, P<0.01). The degree of over-expression of beta-GCD was related to the degree of cancer cells differentiation, but not to the lymph nodes metastasis. The expression of TGF-beta1 was significantly correlated with the expression of beta-GCD in pancreatic cancer tissue.. The genesis of pancreatic cancer results from multi-factor, multi-step and multi-gene variation. The synchronous detection of TGF-beta1 and beta-GCD helps to determine the malignant degree of tumors and the prognosis of patients with such disease.

Topics: Cell Differentiation; Disease Progression; Glucuronidase; Humans; Lymphatic Metastasis; Pancreatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1

In this study, we investigated whether lack of transforming growth factor beta (TGF-beta) type II receptor (RII) expression and loss of TGF-beta signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a TGF-beta-responsive reporter construct. The RII transfectants (MIA PaCa-2/RII) showed a significant increase in sensitivity to radiation when compared with MIA PaCa-2/vector cells. The increase in sensitivity to radiation was reversed by neutralizing antibodies to TGF-beta, indicating that these changes were dependent on TGF-beta signaling. Compared with MIA PaCa-2/vector cells, MIA PaCa-2/RII cells showed a greater than 3-fold increase in apoptosis after radiation. Enhanced radiation sensitivity of MIA PaCa-2/RII cells was associated with an induction of Bax mRNA and protein that was followed by a release of cytochrome c and activation of caspase-3 and poly(ADP-ribose) polymerase cleavage after radiation exposure. Overexpression of Bcl-x(L) or treatment with antisense oligodeoxynucleotides targeted against Bax significantly inhibited radiation-induced apoptosis in MIA PaCa-2/RII but not in MIA PaCa-2/Vector cells, suggesting that Bax induction is necessary for radiation-induced TGF-beta signaling-mediated apoptosis. Thus, restoration of TGF-beta signaling sensitized these cells to ionizing radiation, although these cells possess a mutated p53 gene. In addition, disruption of RII function by dominant negative mutant of RII inhibited the radiation-induced TGF-beta signaling and apoptosis in primary cultures of mouse embryonic fibroblasts. Together, these observations imply that RII is an important component of radiation-induced TGF-beta signaling, and loss of function of RII may enhance resistance to radiation-induced apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytochrome c Group; Humans; Mutation; Pancreatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The cell type responsible for the desmoplastic reaction surrounding human pancreatic carcinoma is unknown. Hepatic stellate cells, which activate to a myofibroblast-like form, are responsible for collagen deposition in cirrhosis and around hepatocellular carcinomas. Recently, pancreatic stellate cells have been described and implicated in the fibrosis of chronic pancreatitis. We sought to determine whether these cells are responsible for the scirrhous reaction surrounding pancreatic adenocarcinomas.. Archival formalin-fixed, paraffin-embedded pancreatic tissues from 10 patients undergoing pancreaticoduodenectomy for ductal adenocarcinoma and from 2 patients with pancreatic islet cell tumors were examined immunohistochemically for alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin heavy chain (SMMHC), procollagen I, collagen IV, and endothelial cell markers, von Willebrand factor and cluster of differentiation 31.. In non-neoplastic areas, staining for alpha-SMA and SMMHC was confined to interlobular septal regions. In contrast, the desmoplastic reaction surrounding all 10 pancreatic adenocarcinoma specimens displayed intense interstitial staining for alpha-SMA, SMMHC, and collagen IV but no staining for von Willebrand factor and cluster of differentiation 31. Procollagen I staining localized intracellularly to fibroblast-shaped cells within this alpha-SMA/SMMHC-positive scirrhous region. Islet cell tumors demonstrated an increase in alpha-SMA staining, although this was not as marked as in ductal adenocarcinomas.. A massive increase in myofibroblast activity, compatible with the activation of stellate cells, is associated with the deposition of collagen types I and IV in the desmoplastic reaction around pancreatic adenocarcinomas.

Topics: Actins; Aged; Collagen Type IV; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Middle Aged; Myosin Light Chains; Pancreatic Neoplasms; Transforming Growth Factor beta

PTEN is a candidate tumour suppressor gene and frequently mutated in multiple cancers, however, not in pancreatic cancer. Recently, it has been demonstrated that PTEN expression is regulated by TGF-beta1. Using TGF-beta1 transgenic mice (n=7) and wildtype littermates (n=6), as well as pancreatic tissues obtained from organ donors (n=10) and patients with pancreatic cancer (n=10), we assessed the expression of PTEN by means of immunohistochemistry and semiquantitative PCR analysis. In addition, PANC-1 cells were treated with TGF-beta1 in vitro and the levels of PTEN mRNA were determined in these cells. In human pancreatic cancers PTEN mRNA levels were significantly decreased (P<0.05). In addition, in the pancreas of TGF-beta1 transgenic mice the expression of PTEN was significantly reduced (P<0.01), as compared to wildtype littermates and incubation of PANC-1 cells with TGF-beta1 decreased PTEN mRNA levels after 24 h. Inasmuch as TGF-beta1 decreases PTEN expression in human pancreatic cancer cells and human pancreatic cancers overexpress TGF-beta1, the reduced expression of PTEN in pancreatic cancer may be mediated by TGF-beta1 overexpression. Thus, although PTEN is not mutated in pancreatic cancers, the reduction of its expression may give pancreatic cancer cells an additional growth advantage.

Topics: Animals; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Pancreatic Neoplasms; Phosphoric Monoester Hydrolases; Polymerase Chain Reaction; PTEN Phosphohydrolase; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

DPC4/Smad4 is inactivated in about 50% of pancreatic ductal cancers. It has been recently reported that this gene is also inactivated in neoplasms arising from pancreatic islet cells, a phenomenon suggested to be related to similar progressions of malignancy found in common ductal cancers. To evaluate this possibility, we analysed 20 metastases of pancreatic endocrine carcinomas and their corresponding primary lesion for inactivation of DPC4 using immunohistochemical staining. In fact, immunohistochemical labelling has been shown to correlate with DPC4 gene status with high sensitivity and specificity. The cancers included 18 nonfunctioning tumours, one gastrinoma and one ViPoma all with liver, nodal and/or adrenal metastases. Seventeen were well-differentiated and three poorly differentiated endocrine carcinomas. Dpc4 expression was absent in only one primary well-differentiated endocrine cancer and its liver metastasis, while all the remaining 19 primary tumours and their metastases stained positive for the protein. All positively staining cases showed diffuse cytoplasmic and nuclear staining in virtually all neoplastic cells. Our data suggest that DPC4 is only rarely involved in pancreatic endocrine tumourigenesis and give further weight to the hypothesis that tumours arising from pancreatic exocrine and endocrine epithelia are genetically distinct.

Topics: Adult; Aged; Carcinoma, Islet Cell; DNA-Binding Proteins; Female; Follow-Up Studies; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Pancreatic Neoplasms; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

We characterized four pancreatic carcinoma cell lines (designated SNU-213, SNU-324, SNU-410, and SNU-494) established from histopathologically varied primary or liver metastatic tumor samples of Korean patients. Three cell lines grew as adherent monolayers and one as adherent and floating cell clumps. All lines had: (1) relatively high viability; (2) an absence of mycoplasma or bacterial contamination; (3) genetic heterogeneity as assessed by DNA-fingerprinting analysis; (4) an absence of MADH4 mutation. Among the lines, three lines had mutations in codon 12 in K- ras, two lines harbored p53 mutations within the DNA-binding domain; two lines had homozygous deletions in both p16 and p15 genes; and one line had a missense mutation. Two lines (SNU-324 and SNU-410) had genetic alterations in the TGFBR2 gene: the SNU-324 line had a -1-bp or +1-bp mutation in 10-bp polydeoxyadenine repeat tracts; the SNU-410 line had a genomic deletion in this gene. Mutation analysis of mismatch repair genes demonstrated that SNU-324 has two heterozygous missense mutations in different exons of the hMLH1 gene. In addition, this line showed microsatellite instability and harbored frameshift mutations in simple repeated sequences of the coding regions of the TGFBR2, BAX, and hMSH3 genes. These defects of microsatellite instability and mismatch repair genes suggest the possibility of a new mutator phenotype for pancreatic carcinogenesis. These cell lines should be very useful for studying the biology of pancreatic carcinoma, particularly those related to mutator phenotype and genetic alterations in the TGFBR2 gene.

Topics: Base Pair Mismatch; Carcinoma; Cell Line; Cloning, Molecular; DNA Fingerprinting; DNA Mutational Analysis; DNA Repair; DNA, Complementary; DNA, Neoplasm; Genes, p53; Humans; Microsatellite Repeats; Pancreatic Neoplasms; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured

Mutations in the tumour suppressor genes SMAD4 (DPC4, deleted in pancreatic cancer locus 4) and adenomatous polyposis coli (APC) have been implicated in the development of pancreatic cancer in humans. Treatment of wild-type, Smad4(+/-), Apc(Min/+) or Apc(Min/+)Smad4(+/-) mice with N-Nitroso-N-Methyl Urea (NMU) results in abnormal foci in pancreatic acinar cells characterized by increased levels of beta-catenin. Previously such foci have been shown to be the precursors of pancreatic neoplasia. Interestingly, only NMU-treated Apc(Min/+)Smad4(+/-) mice exhibit a significant increase in abnormal pancreas, which was found to be due to increased number of abnormal foci rather than increased focus size. A range of foci sizes were analysed, but only smaller abnormal foci were characterized by morphological nuclear atypia. These studies suggest functional co-operation between TGF-beta and Wnt signalling pathways in the suppression of pancreatic tumorigenesis in the mouse.

Topics: Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Carcinogens; Cell Division; Cytoskeletal Proteins; DNA-Binding Proteins; Genotype; Immunohistochemistry; In Situ Nick-End Labeling; Methylnitrosourea; Mice; Mutation; Pancreatic Neoplasms; Proto-Oncogene Proteins; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

Proliferation of fibrotic tissue (desmoplasia) is one of the hallmarks of several epithelial tumors including pancreatic adenocarcinoma. This tissue reaction may be deleterious or advantageous to the host or tumor. In a systematic analysis, we identified two growth factors expressed by human pancreatic carcinoma cells that are positively correlated with the ability to induce fibroblast proliferation both in vitro and in vivo, i.e., transforming growth factor (TGF)-beta1 and fibroblast growth factor-2. Here we demonstrate that the overexpression of TGF-beta1 induced up-regulation of matrix proteins and growth factors in the TGFbeta1-transfected pancreatic tumor cells. Furthermore, transfection of PANC-1 cells induces the same change in fibroblasts in either cocultivation experiments or when they are grown in conditioned medium from TGF-beta1-transfected PANC-1 cells. TGF-beta1-transfected pancreatic tumor cells induced a rich stroma after orthotopical transplantation in the nude mouse pancreas. The transfer of a single growth factor, TGF-beta1, conveys the ability to induce a fibroblast response similar to that seen in desmoplasia in human pancreatic adenocarcinoma. This effect cannot only be attributed to direct effects of TGF-beta1 but also results from the up-regulation of several other factors including collagen type I, connective tissue growth factor, and platelet-derived growth factor.

Topics: Animals; Cell Division; Coculture Techniques; Disease Models, Animal; Extracellular Matrix Proteins; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous; Tumor Cells, Cultured; Up-Regulation

The sst2 somatostatin receptor is an inhibitory G protein-coupled receptor, which exhibits anti-tumor properties. Expression of sst2 is lost in most human pancreatic cancers. We have cloned 2090 base pairs corresponding to the genomic DNA region upstream of the mouse sst2 (msst2) translation initiation codon (ATG). Deletion reporter analyses in mouse pituitary AtT-20 and human pancreatic cancer PANC-1, BxPC-3, and Capan-1 cells identify a region from nucleotide -260 to the ATG codon (325 base pairs) showing maximal activity, and a region between nucleotides -2025 and -260 likely to comprise silencer or transcriptional suppressor elements. In PANC-1 and AtT-20 cells, transforming growth factor (TGF)-beta up-regulates msst2 transcription. Transactivation is mediated by Smad4 and Smad3. The cis-acting region responsible for such regulation is comprised between nucleotides -1115 and -972 and includes Sp1 and CAGA-box sequences. Expression of Smad4 in Smad4-deficient Capan-1 and BxPC-3 cells restores TGF-beta-dependent and -independent msst2 transactivation. Expression of Smad4 in BxPC-3 cells reestablishes both endogenous sst2 expression and somatostatin-mediated inhibition of cell growth. These findings demonstrate that msst2 is a new target gene for TGF-beta transcription regulation and underlie the possibility that loss of Smad4 contributes to the lack of sst2 expression in human pancreatic cancer, which in turn may contribute to a stimulation of tumor growth.

Topics: 5' Untranslated Regions; Animals; Base Sequence; Cloning, Molecular; Codon; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genomic Library; Humans; Luciferases; Mice; Molecular Sequence Data; Pancreatic Neoplasms; Pituitary Neoplasms; Promoter Regions, Genetic; Receptors, Somatostatin; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Pancreatic cancer is characterized by an extremely poor prognosis. For the development of more effective immunotherapies, the systemic and local immunological escape mechanisms need to be further elaborated. These mechanisms may include the secretion of immunosuppressive cytokines, the local hindrance of tumor-infiltrating lymphocytes (TILs), or the loss of the signal transducing CD3 zeta-chain of TILs. In this study, we have analyzed these parameters in 116 patients suffering from pancreatic ductal adenocarcinoma. Mean concentrations of interleukin (IL)-10 and transforming growth factor-beta1/2 were considerably higher than in control sera (P < 0.0001). Disseminated tumor cells were found in 16 of 39 cases. In 28 of 33 surgical specimens, TILs did not reach tumor cells in significant numbers, being "trapped" in the peritumoral tissues. We suggest this as a simple but highly effective tumor escape mechanism. In cases of a TIL/tumor cell contact, CD3 zeta was mostly lost. Overall, 27 of 33 surgical specimens, 9 of 19 peritumoral lymph nodes, and 13 of 25 peritoneal lavage specimens showed significant loss of CD3 zeta (P < 0.02). Elevated concentrations of IL-10/TGF-beta1/2 were, in all but one of three cases, correlated with a CD3 zeta loss in corresponding specimens. Patients with disseminated tumor cells also showed a CD3 zeta loss in all but two corresponding tumor specimens. These results present strong evidence for an active systemic immunosuppression in pancreatic cancer, as shown by elevated IL-10 and TGF-beta1/2 serum levels as well as the presence of disseminated tumor cells. Killing of tumor cells by potentially cytotoxic TILs is obviously suppressed by the prevention of a direct TIL/tumor cell contact and the inactivation of TILs, as shown by a severe loss of CD3 zeta. In addition to active immunization strategies, successful immunotherapies have to focus on restoring in vivo T-cell function to improve the almost always fatal prognosis of pancreatic cancer.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Pancreatic Ductal; CD3 Complex; CD4 Antigens; CD8 Antigens; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Immunosuppression Therapy; Immunotherapy; Interleukin-10; Lymphatic Metastasis; Lymphocytes; Male; Middle Aged; Pancreatic Neoplasms; Signal Transduction; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

To screen a library of small chemicals for compounds that activate the DPC4 signal transduction pathway in a human pancreatic cancer cell line.. Various tumor-suppressor genes are mutated in all human cancers. Specifically, DPC4 (deleted in pancreatic carcinoma, locus 4 or MADH4/SMAD4) is a tumor-suppressor gene mutated in approximately 50% of human pancreatic adenocarcinomas. DPC4 plays an important role in the well-studied transforming growth factor-beta (TGFbeta) signaling pathway. It would be useful to identify therapies that augment or restore the downstream functions of this critical signal transduction pathway, in hopes that such therapy would have a rational role in anticancer therapy.. Using a commercially available plasmid vector with a luciferase reporter gene already incorporated, a DPC4-specific reporter construct was genetically engineered. This was done by inserting six copies of the palindromic Smad binding element (6SBE), which is a DNA binding element specific for DPC4, in front of the minimal promoter in the plasmid. This construct was then stably integrated into the genome of a human pancreatic cancer cell line (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGFbeta, which is known to activate the DPC4 signal transduction pathway. A single transfected clone was chosen for the drug screen based on basal luciferase (reporter) expression and TGFbeta inducibility. A systematic screen of the chemical library was then performed, using luciferase activity to detect DPC4 activity and induction of the signaling pathway.. A high-throughput system based on this stably integrated reporter system was used to screen a library of 16,320 random compounds to identify agents that conferred robust augmentation of the DPC4 signal transduction pathway. Of the 16,320 compounds screened, 11 were associated with a 2- to 5-fold induction of luciferase activity, and one with a 12-fold activation. The latter compound was shown to be a novel histone deacetylase inhibitor and was further characterized.. These results confirm the feasibility of a specific high-throughput reporter system to screen a large compound library in human cells efficiently. The screening identified several compounds capable of augmenting DPC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our understanding of complex tumor-suppressive signaling pathways and may identify other potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifying drugs or lead compounds for potential therapeutic use.

Topics: Adenocarcinoma; DNA-Binding Proteins; Drug Evaluation, Preclinical; Gene Library; Genes, Reporter; Genes, Tumor Suppressor; Humans; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Transforming growth factor-beta1 (TGF-beta1) inhibits the growth of a variety of epithelial cells; however, in many types of tumors it loses its inhibitory effect. p21(WAF1/CIP1), one of the cyclin-dependent kinase (Cdk) inhibitors induced by TGF-beta1, is considered a downstream effector of the growth-inhibitory function of TGF-beta1. We assessed the clinicopathologic significance of TGF-beta1 and p21 expression in resectable invasive ductal carcinoma (IDC) of the pancreas. Immunohistochemical examination of the expression of TGF-beta1 and p21 in 62 patients revealed positive expression of TGF-beta1 in 28 (45%) and of p21 in 25 (40%) of the 62 patients, and a significant correlation between the two expressions. The survival curve of patients with TGF-beta1(+) tumors was significantly higher than that of patients with TGF-beta1(-) tumors; p21(+) patients showed a higher survival curve than did p21(-) patients, but the difference was not statistically significant. Simultaneous analysis of TGF-beta1 and p21 expression showed that the patients with TGF-beta1(+)/p21(+) tumors had a significantly better prognosis than the others. Multivariate analysis showed that TGF-beta1 was a significantly low risk factor for death due to IDC. The concurrent evaluation of TGF-beta1 and p21 expression would be an effective tool in the prediction of the prognosis of patients with pancreatic cancer.

Topics: Adult; Aged; Carcinoma, Pancreatic Ductal; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatectomy; Pancreatic Neoplasms; Prognosis; Survival Rate; Transforming Growth Factor beta

The aim of this study was to examine the effects of transforming growth factor (TGF) beta1 on the phenotype and the biological behavior of pancreatic cancer cell lines with and without mutations in the TGF-beta signaling pathway and to elucidate whether the Ras signaling cascade participates in mediating these effects of TGF-beta1. TGF-beta-responsive (PANC-1, COLO-357, and IMIM-PC1) and nonresponsive (CAPAN1 and IMIM-PC2) pancreatic cancer cell lines with activating mutations of the Ki-Ras oncogene were treated with 10 ng/ml TGF-beta1 over time. Phenotypic alterations were studied by electron and phase contrast microscopy and by immunohistochemistry and expression analyses of differentiation markers. The influence of TGF-beta on tumor cell scattering, migration, and invasion was determined. The role of the Ras-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade in mediating TGF-beta-induced morphological and functional effects were studied by pretreatment with the MEK1 inhibitor PD 98059 and by measuring ERK2 activation using immune complex kinase assays. TGF-beta1 led to a reversible and time-dependent epithelial-mesenchymal transdifferentiation (EMT) in TGF-beta-responsive pancreatic cancer cell lines, characterized by a fibroblastoid morphology and an up-regulation of mesenchymal markers and a down-regulation of epithelial markers. EMT was associated with an increase in tumor cell migration, invasion, and scattering. In the responsive cell lines, TGF-beta1 induced a moderate but sustained activation of ERK2. EMT, the concomitant changes in gene expression, and the invasive and migratory potential were reduced or abolished by pretreatment with the selective MEK1 inhibitor. Thus, in TGF-beta-responsive pancreatic cancer cells with activating Ki-Ras mutations, TGF-beta1 treatment caused an EMT associated with a more invasive phenotype. Cross-talk with the Ras-MEK-ERK-signaling cascade appears to be essential for mediating these effects of TGF-beta1.

Topics: Cell Differentiation; Cell Movement; Enzyme Activation; Epithelial Cells; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mesoderm; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; ras Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Alteration of the transforming growth factor beta (TGFB) signalling pathway is important in pancreatic carcinogenesis, as shown by the frequent inactivation of the downstream target SMAD4. We recently analysed a series of pancreatic carcinoma cell lines with respect to alterations of five SMAD genes involved in TGFB signalling, and showed that SMAD4 was structurally rearranged in 42% of these. This pathway may, however, also be affected by alterations of genes whose products regulate the activation of TGFB as well as of TGFB receptor genes. We therefore studied the expression of UPA, UPAR, IGF2R, ALK5 (TGFBR1), TGFBR2, TGFBR3, ENG, ALK1, TGFB1, TGFB2, and TGFB3 in a series of 14 pancreatic carcinoma cell lines. We also analysed ALK5 and TGFBR2 for mutations, cell surface localisation of TGFBR2 and ENG, and TGFB1 response. No mutations of ALK5 or TGFBR2 were found. However, 4 cell lines were methylated within the ALK5 promoter region. ALK5 expression was strongly reduced in 9 cases, whereas TGFBR2 expression was increased in 12 of the cell lines. The TGFB signalling associated receptors ENG and ALK1 were co-expressed in 4 of the cell lines. There was no evidence for disruption of the UPAR-IGF2R TGFB activating pathway. The response to TGFB1 was analysed in 12 cell lines, and 6 of these (50%) showed increased proliferation. The cell lines stimulated by TGFB showed frequent mutations of SMAD4, KRAS2, and TP53, as well as frequent absence of CDKN2B expression. These results suggest that the ALK5-SMAD4 part of the TGFB signalling pathway is a major target for inactivation in pancreatic carcinomas, that the expression of TGFBR2, TGFBR3, and receptors involved in TGFB activation are maintained, and that alterations of components of the TGFB signalling pathway may be accompanied by a positive effect of TGFB on cell growth.

Topics: Activin Receptors, Type I; Carcinoma, Pancreatic Ductal; Cell Division; DNA Primers; DNA, Neoplasm; Gene Expression; Genes, p53; Humans; Male; Middle Aged; Mutation; Neoplasm Proteins; Pancreatic Neoplasms; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

TGF-beta strongly promotes local tumor progression in advanced epithelial tumors, though the underlying mechanisms are poorly understood. In the present study, we demonstrate the potential of TGF-beta to increase the invasiveness of the pancreatic cancer cell lines PANC-1 and IMIM-PC1. TGF-beta-induced tumor cell invasion occurred in a time-dependent manner, started after 12 hr and continued to increase even 48 hr after a single application of the growth factor. Blocking of secreted TGF-beta1 by application of neutralizing antibodies 24 hr after TGF-beta treatment completely prevented the sustained effects of TGF-beta on tumor cell invasion. Together with our previous observation that TGF-beta1 up-regulates its own expression in both cell lines, our data suggest that TGF-beta1 acts in an autocrine manner to maintain tumor cell invasion. As measured by Northern blot hybridization and zymography, TGF-beta treatment of PANC-1 and IMIM-PC1 cells resulted in strong up-regulation of expression and activity of both matrix metalloproteinase-2 (MMP-2) and the urokinase plasminogen activator (uPA) system. Treatment with MMP inhibitors or inhibitors of the uPA system caused significant reduction of TGF-beta-induced invasiveness in both cell lines. In contrast, expression and activity of MMP-2 and uPA as well as tumor cell invasiveness remained unaffected in cell lines with defects of the TGF-beta type II receptor (MiaPaca2) or the Smad4 gene (IMIM-PC2 and CAPAN-1). In these cell lines, TGF-beta also failed to auto-induce its own expression. In conclusion, our results suggest that TGF-beta1 is a strong promotor of pancreatic cancer progression. TGF-beta thereby acts in an autocrine manner to induce tumor cell invasion, which is mediated by MMP-2 and the uPA system.

Topics: Enzyme Activation; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Pancreatic Neoplasms; Protease Inhibitors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The expression of several growth factors and K-ras gene mutation in bile were studied to better understand the pathogenesis and improve early diagnosis of bile duct cancers. Bile samples were collected from 12 cholangiocarcinomas (CLC), 10 ampullary cancers (APC), 3 gallbladder cancers (GBC), 7 pancreatic cancers (PNC), 9 biliary tract infection (BTI), 8 biliary stone disease (ST), and 5 normal controls (NC). The highest mean value of TGF-beta in bile was in patients with BTI; the mean levels of bFGF and PDGF were highest in CLC, and patients with APC and CLC had higher expression of HER2/Neu than other groups. In bile, a K-ras gene codon 12 mutation was found in 5 of 6 (83%) cases of CLC by the PCR-RFLP method. The results suggest overexpression of bFGF, PDGF, and HER2/Neu and the presence of K-ras mutation are important for carcinogenesis of bile duct cancers, and detection of the above abnormalities in bile is helpful for early diagnosis.

Topics: Ampulla of Vater; Bile; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biliary Tract Diseases; Biliary Tract Neoplasms; Biomarkers; Biomarkers, Tumor; Cholangiocarcinoma; Cholelithiasis; Common Bile Duct Neoplasms; Enzyme-Linked Immunosorbent Assay; Gallbladder Neoplasms; Humans; Infections; Pancreatic Neoplasms; Platelet-Derived Growth Factor; Polymerase Chain Reaction; ras Proteins; Receptor, ErbB-2; Transforming Growth Factor beta

TGF-betas are multifunctional polypeptides that regulate cell growth and differentiation, extracellular matrix deposition, cellular adhesion properties, angiogenesis and immune functions. In this study, we investigated the effect of TGF-beta1 on liver metastasis and its mechanism by using human pancreatic cancer cell lines Panc-1, Capan-2, and SW1990. Capan-2 and SW1990 cells demonstrated enhanced liver metastatic potential by in vivo splenic injection with TGF-beta1. Consequently, we examined the role of TGF-beta1 on in vitro angiogenesis and received cytotoxicity by peripheral blood mononuclear leukocytes (PBMLs). While TGF-beta1 slightly decreased cell proliferation, it also upregulated VEGF production in all cancer cells examined. The binding of PBMLs to cancer cells and cancer cell cytotoxicity during co-culture with PBMLs were remarkably decreased by treatment with TGF-beta1. Panc-1 cells revealed no liver metastasis despite their high immunogenetic and angiogenetic abilities, which was attributed to a lack of expression of the cell surface carbohydrates that induce attachment to endothelial cells. We concluded that the presence of TGF-beta1 in the microenvironment of tumour site might play an important role in enhancing liver metastasis of pancreatic cancer by modulating the capacity of angiogenesis and immunogenicity.

Topics: Animals; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Liver Neoplasms; Lymphokines; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transforming growth factor beta (TGF-beta) because of a lack of expression of the TGF-beta type II receptor (RII). We show that the lack of RII expression is caused by a deficit of the transcription factor Sp1. Nuclear run-off assays and Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine, resulted in an increase in the rate of Sp1 transcription, in Sp1 protein expression, and in the binding of Sp1 to the RII promoter. Ectopic expression of Sp1 cDNA in MIA PaCa-2 cells led to an increase in RII promoter-chloramphenicol acetyltransferase activity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of responsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MIA PaCa-2 Sp1 transfectants) when treated with mithramycin, an inhibitor of Sp1 binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltransferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the Sp1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivation of the RII gene and thus lack of responsiveness to TGF-beta.

Topics: Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Azacitidine; Cell Division; Chloramphenicol O-Acetyltransferase; Decitabine; DNA Modification Methylases; DNA, Complementary; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Pancreatic Neoplasms; Plicamycin; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Sp1 Transcription Factor; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Biglycan (PG-I), a component of the extracellular matrix (ECM), is overexpressed in pancreatic cancer. To determine possible matrix-tumor interactions, we investigated the effects of PG-I on pancreatic cancer.. PG-I expression in cell lines and tissue samples was examined by Northern blot and immunofluorescence. The effect of PG-I on proliferation was determined by measuring activity of Ras, ERK, Rb, [(3)H]-thymidine incorporation, and cell cycle analysis. Expression of cyclin A, B1, D1, E1, G1, PCNA, p21, and p27 was analyzed by Northern and Western blots.. PG-I was overexpressed in the ECM of pancreatic cancer samples compared with normal pancreas or chronic pancreatitis tissues. Addition of transforming growth factor (TGF)-beta induced PG-I expression in HFL and HFFF2 fibroblasts as well as in the pancreatic cancer cell line PANC-1. PG-I inhibited growth of both TGF-beta-responsive and TGF-beta-unresponsive pancreatic cancer cells by inducing G1-arrest, which is accompanied by an increase of p27 and reduction of cyclin A and proliferating cell nuclear antigen. Furthermore, endogenous Ras and ERK activation was partly reduced by PG-I in vitro.. The ECM protein PG-I inhibits growth by arresting pancreatic cancer cells in G1 and may be part of a host defense mechanism aimed at slowing down pancreatic tumor progression.

Topics: Adult; Aged; Animals; Biglycan; Cell Cycle Proteins; Cyclin A; Cyclin-Dependent Kinase Inhibitor p27; Extracellular Matrix Proteins; Female; G1 Phase; G2 Phase; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Pancreatic Neoplasms; Phosphorylation; Proliferating Cell Nuclear Antigen; Proteoglycans; Proto-Oncogene Proteins p21(ras); Retinoblastoma Protein; RNA, Messenger; S Phase; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Proteins

Recently, several members of the claudin family have been identified as integral constituents of tight junctions. Using expression profiling, we previously found claudin-4 to be overexpressed in pancreatic cancer. Because claudin-4 has been described as a receptor for the cytotoxic Clostridium perfringens enterotoxin (CPE), we investigated the effect of CPE on pancreatic cancer cells.. Expression of claudin-4 was analyzed by Northern blots. In vitro toxicity of CPE was determined by trypan blue exclusion and the (86)Rb-release assay. The in vivo effect of CPE was studied in claudin-4-expressing nude mouse xenografts of the Panc-1 cell line.. Expression analyses showed that claudin-4 was overexpressed in most pancreatic cancer tissues and cell lines and several other gastrointestinal tumors. CPE led to an acute dose-dependent cytotoxic effect, restricted to claudin-4-expressing cells and dependent on claudin-4 expression levels. Furthermore, transforming growth factor beta was identified as a negative modulator of both claudin-4 expression and susceptibility to CPE. In vivo, intratumoral injections of CPE in Panc-1 xenografts led to large areas of tumor cell necrosis and significant reduction of tumor growth.. Our findings suggest that targeting claudin-4-expressing tumors with CPE represents a promising new treatment modality for pancreatic cancer and other solid tumors.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Claudin-4; Dose-Response Relationship, Drug; Enterotoxins; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Membrane Proteins; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; RNA, Messenger; Tight Junctions; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

At early stages of tumorigenesis, the transforming growth factor-beta (TGF-beta) signaling pathway is thought to have tumor suppressor activity as a result of its ability to arrest the growth of epithelial cells. Smad4 plays a pivotal role in the TGF-beta signaling pathway and has been identified as a tumor suppressor, being mutated or deleted in approximately 50% of pancreatic carcinomas and 15% of colorectal cancers. A nonsense mutation generating a C-terminal truncation of 38 amino acids in the Smad4 protein has been identified in a pancreatic adenocarcinoma (Hahn, S. A., Schutte, M., Hoque, A. T., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E. (1996) Science 271, 350-353), and here we investigate the functional consequences of this mutation. We demonstrate that the C-terminal truncation prevents Smad4 homomeric complex formation and heteromeric complex formation with activated Smad2. Furthermore, the mutant protein is unable to be recruited to DNA by transcription factors and hence cannot form transcriptionally active DNA-binding complexes. These observations are supported by molecular modeling, which indicates that the truncation removes residues critical for homomeric and heteromeric Smad complex formation. We go on to show that the mutant Smad4 is highly unstable compared with wild type Smad4 and is rapidly degraded through the ubiquitin-proteasome pathway. Consistent with this, we demonstrate that the pancreatic adenocarcinoma harboring this mutated allele, in conjunction with loss of the other allele, expresses no Smad4 protein. Thus we conclude that these tumors completely lack Smad4 activity.

Topics: 3T3 Cells; Adenocarcinoma; Alleles; Amino Acids; Animals; Blotting, Western; Cell Line; Codon; Codon, Nonsense; Cycloheximide; DNA-Binding Proteins; Genes, Dominant; Humans; Loss of Heterozygosity; Mesoderm; Mice; Models, Molecular; Mutation; Nerve Growth Factors; Pancreatic Neoplasms; Plasmids; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Ribonucleases; RNA, Messenger; Signal Transduction; Smad Proteins; Smad2 Protein; Smad4 Protein; Time Factors; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Ubiquitin; Xenopus; Xenopus Proteins

Human pancreatic ductal adenocarcinomas overexpress transforming growth factor-betas (TGF-betas). This overexpression has been correlated with decreased patient survival. TGF-betas bind to a type II TGF-beta receptor (TbetaRII) dimer, which heterotetramerizes with a type I TGF-beta receptor (TbetaRI) dimer, thereby activating downstream signaling.. To determine whether blocking TGF-beta actions would suppress pancreatic cancer cell growth in vivo, we expressed a soluble TbetaRII, encoding amino acids 1-159 of the extracellular domain in COLO-357 human pancreatic cancer cells. This cell line expresses all of the three mammalian TGF-beta isoforms and is growth inhibited by TGF-beta in vitro.. COLO-357 clones expressing soluble TbetaRII were no longer growth inhibited by exogenous TGF-beta1 and exhibited a marked decrease in their invasive capacity in vitro. When injected s.c. into athymic mice, these clones exhibited attenuated growth rates and angiogenesis and decreased levels of plasminogen activator inhibitor-1 mRNA as compared with tumors formed by sham-transfected cells.. These results indicate that endogenous TGF-betas can confer a growth advantage in vivo to a pancreatic cancer cell line that is growth inhibited in vitro and suggest that a soluble receptor approach can be used to block these tumorigenic effects of TGF-betas.

Topics: Animals; Cell Division; Cell Movement; Female; Gene Expression; Genetic Vectors; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Solubility; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

We investigated the effect of TGF-beta1 on liver metastasis of pancreatic cancer using surgical specimens of pancreatic cancer and human pancreatic cancer cell lines Capan-2 and SW1990. Immunostaining of TGF-beta1 showed that TGF-beta1 positivity was significantly related to venous invasion and tumor staging, and also relatively associated with liver metastasis. Cellular invasion and protease production of MMP-2 and u-PA, and in vivo liver metastasis were significantly enhanced after treatment of cells with TGF-beta1. These findings suggest that TGF-beta1 might play an important role in enhancing liver metastasis of pancreatic cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Animals; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Matrix Metalloproteinases; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Staging; Neoplasm Transplantation; Pancreatic Neoplasms; Pancreaticoduodenectomy; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

To explore the relationships between Smad4, TGF beta1 and TbetaR II in the TGF-beta pathway and the possible mechanisms by which they effect pancreatic carcinoma.. The expression of Smad4, TGF beta1 and TbetaR II in paraffin embedded pancreatic carcinoma tissues was detected by using antibodies against Smad4, TGF beta1 and TbetaR II with EnVision immunohistochemistry.. The positive rates of Smad4, TGF beta1 and TbetaR II were 58.93% (33), 66.07% (37) and 60.71% (34), respectively. The positive rates of the above three proteins in matched normal pancreatic tissues were 89.29% (50), 25.00% (14) and 25.00% (14) respectively. There was a significant relationship between the expression of TGF beta1, clinical stage and the metastasis of the tumor (P < 0.05). There was also a significant relationship between the expressions of TGF beta1 and TbetaR II (P < 0.05).. The expression of Smad4 in pancreatic carcinoma tissue is significantly decreased but the expression of TGF beta1 and TbetaR II are increased. Smad4 may play an important role in the regulation of TGFbeta inducible gene expression and subsequent growth inhibition, but TGFbeta may also act in a Smad4-independent manner.

Topics: DNA-Binding Proteins; Humans; Pancreas; Pancreatic Neoplasms; Paraffin Embedding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Activation of the receptor c-met stimulates motility, mitosis, morphogenesis, processes involved in organ regeneration, or progression of malignancies. In the present study we investigated the expression of c-met protein in the regenerating pancreas and characterized the influence of cytokines on c-met expression.. Acute pancreatitis was induced in rats by cerulein injection. Rat acini and rat and human pancreatic cancer cells were stimulated with interleukin-1alpha (IL-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). C-met expression was analyzed by means of Western blotting and localization in pancreatic tissue by immunohistochemistry.. C-met protein expression was significantly upregulated in the regenerating pancreas and localized in areas of regenerating tissue. Stimulation with cytokines resulted in a two- to threefold increase of c-met expression in vitro.. Enhanced c-met expression after acute pancreatitis suggests that HGF/met has an important role in pancreatic regeneration, which is probably mediated by cytokines. This regulatory mechanism is also of importance in pancreatic cancer.

Topics: Acute Disease; Animals; Blotting, Western; Cells, Cultured; Ceruletide; Cytokines; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Interleukin-1; Interleukin-6; Male; Pancreas; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins c-met; Rats; Rats, Wistar; Regeneration; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In this study, we attempted to determine how transforming growth factor (TGF)-beta1 affects complement C3 secretion in the pancreatic cancer cell lines PANC-1 and BxPC-3. We also compared the responses in C3 secretion with those in interleukin (IL)-8 secretion. The C3 and IL-8 expression was evaluated at the protein and messenger RNA (mRNA) levels. The activation of nuclear factor-kappaB (NF-kappaB) was assessed by an electrophoretic gel mobility shift assay (EMSA). IL-1beta and tumor necrosis factor (TNF)-alpha both induced a marked increase in C3 and IL-8 secretion. However, TGF-beta1 potently decreased the IL-1beta- and TNF-alpha-induced C3 secretion, whereas the IL-8 secretion was weakly but significantly enhanced. These responses were also observed at the mRNA level. In PANC-1 cells, IL-1beta and TNF-alpha induced a rapid activation of nuclear factor (NF)-kappaB, and TGF-beta1 enhanced this activation slightly. The induction of Fos protein has been reported to be required for the inhibitory action of TGF-beta1, and the translocation of Fos protein into the nucleus was associated with TGF-beta1 stimulation in PANC-1 cells. Our results suggest that TGF-beta1 may act as a potent inhibitor of C3 secretion in pancreatic cancer cell lines under inflammatory conditions. This action of TGF-beta1 did not correlate with NF-kappaB activation, but associated with the translocation of Fos protein into the nucleus.

Topics: Antibodies; Binding, Competitive; Blotting, Northern; Complement C3; Humans; Immunoblotting; Interleukin-1; Interleukin-8; NF-kappa B; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Biological signals for transforming growth factor beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to a family of intracellular mediators known as Smads. Smad2 and Smad4 are important for transcriptional and antiproliferative responses to TGF-beta, and their inactivation in human cancers indicates that they are tumor suppressors. A missense mutation at a conserved arginine residue in the amino-terminal MH1 domain of both Smad2 and Smad4 has been identified in tumors from patients with colorectal and pancreatic cancers, respectively. However, the mechanism whereby this mutation interferes with Smad activity is uncertain. Here we show that these mutations do not disrupt activation of Smads, including receptor-mediated phosphorylation of Smad2, Smad2/Smad4 heteromeric complex formation, and Smad nuclear translocation. In contrast, we demonstrate that the mutant Smads are degraded rapidly in comparison with their wild-type counterparts. We show that this decrease in Smad protein stability occurs through induction of Smad ubiquitination by pathways involving the UbcH5 family of ubiquitin ligases. These studies thus reveal a mechanism for tumorigenesis whereby genetic defects in Smads induce their degradation through the ubiquitin-mediated pathway.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Arginine; Cell Nucleus; Colorectal Neoplasms; Conserved Sequence; Cysteine Endopeptidases; DNA-Binding Proteins; Genes, Tumor Suppressor; Humans; Multienzyme Complexes; Mutation, Missense; Pancreatic Neoplasms; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Recombinant Proteins; Signal Transduction; Smad2 Protein; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Ubiquitins

A highly tumorigenic cell line designated as UK Pan-1 was established in a surgically removed human pancreatic adenocarcinoma and characterized as having many of the genotypic and phenotypic alterations commonly found in pancreatic tumors.. The cell line was characterized by its morphology, growth rate in monolayer culture and soft agar, tumorigenicity in nude mice, and chromosomal analysis. Furthermore, the status of p53, Ki-ras mutation and transforming growth factor (TGF)-/receptor expression were determined. The characteristics of UK Pan-1 were compared with those of other commonly used pancreatic carcinoma cell lines.. Quiescent UK Pan-1 cells could be stimulated to proliferate in growth factor free nutrient media, indicating a growth factor independent phenotype. UK Pan- 1 cells grew in soft agar and rapidly formed tumors in nude mice. This cell line possesses a mutation at codon 12 of the c-Ki-ras-2 gene that is commonly found in pancreatic carcinoma. Fluorescence in situ hybridization showed that two alleles of p53 tumor suppressor gene were present in UK Pan-1. However, sequencing analysis revealed a mutation in one allele at exon 8, codon 273 (G to A; Arg to His). Additional growth assays indicated that the cell line was insensitive to negative growth regulation induced by exogenous TGF-beta. Molecular analysis of the TGF-beta signaling pathway showed that UK Pan-1 did not express appreciable levels of the TGF-beta receptor type I, II, or III mRNAs, but did express DPC4 mRNA. Karyotype analysis revealed an 18q21 deletion indicating a possible loss of heterozygosity for DPC4, as well as other chromosomal deletions and rearrangements.. This study indicates that UK Pan-1 is a highly tumorigenic cell line possessing a molecularly complex pattern of mutations that may be used as a model to further the understanding of the mechanisms responsible for the development of pancreatic carcinoma.

Topics: Adenocarcinoma; Alleles; Animals; Cell Division; Codon; Culture Media; DNA-Binding Proteins; Exons; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, ras; Genotype; Humans; Loss of Heterozygosity; Male; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Pancreatic Neoplasms; Phenotype; Point Mutation; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Tumor transplants into nude mice (NM) may reveal abnormal biological behavior compared with the original tumor. Despite this, human tumor xenografts in NM have been widely used to study the biology of tumors and to establish diagnostic and therapeutic modalities. Clearly, precise differences in the biology of a given tumor in human and in NM cannot be assessed. We compared the growth kinetics, differentiation pattern and karyotype of an anaplastic Syrian hamster pancreatic cancer cell line in NM and in allogenic hamsters. As with the original tumor, transplants in hamsters grew fast, were anaplastic and expressed markers related to tumor malignancy like galectin 3, TGF-alpha and its receptor EGFR at high levels. However, tumors in the NM were well-differentiated adenocarcinomas, grew slower, had increased apoptotic rate and had a high expression of differentiation markers such as blood group A antigen, DU-PAN-2, carbonic anhydrase II, TGF-beta(2) and mucin. Karyotypically, the tumors in the NM acquired additional chromosomal damage. Our results demonstrate significant differences in the morphology and biology of tumors grown in NM and the allogenic host, and call for caution in extrapolating data obtained from xenografts to primary cancer.

Topics: Animals; Antigens, Differentiation; Cricetinae; ErbB Receptors; Female; Galectin 3; Genes, ras; Humans; Immunohistochemistry; Karyotyping; Mice; Mice, Nude; Pancreatic Neoplasms; Protein Binding; Radioimmunoassay; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

To investigate whether transforming growth factor beta (TGFbeta) signaling is disrupted in human pancreatic cancer cells, and to study the role of TGFbeta receptors and Smad genes.. TGFbeta is a known inhibitor of pancreatic growth. Disruption of the TGFbeta signaling pathway may play a role in pancreatic cancer development.. The effect of TGFbeta on the BxPC-3, MiaPaCa-2, and PANC-1 pancreatic cancer cell lines was evaluated by [3H]thymidine incorporation and a TGFbeta-responsive reporter assay. Expression of TGFbeta receptors and Smads 2 and 3 was assessed by cross-linking assays and reverse transcriptase-polymerase chain reaction (RT-PCR). The ability to restore TGFbeta responsiveness was evaluated by transfection of TGFbeta signaling components.. TGFbeta produced little inhibition of DNA synthesis and did not activate a TGFbeta-responsive reporter in pancreatic cancer cell lines. 125TGFbeta cross-linking and RT-PCR confirmed the presence of TGFbeta receptors and Smad2 and Smad3 transcripts. Transfection of TGFbeta receptors or Smads 2 and 3 did not restore responsiveness. However, transfection of Smad4 into the BxPC-3 pancreatic cancer cell line restored TGFbeta responsiveness.. Pancreatic cancer cells show loss of TGFbeta responsiveness. Smads 2 and 3 and TGFbeta receptors are not defective in the cell lines studied. Transfection of Smad4 into one of the cell lines restored TGFbeta responsiveness, suggesting an important role for Smad4 in pancreatic cancer. It is likely that other, as yet unidentified genes are important in TGFbeta resistance in pancreatic cancer cells.

Topics: Animals; DNA-Binding Proteins; Humans; Pancreatic Neoplasms; Rats; Rats, Wistar; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The MUC4 mucin is considered as the homologue of rat sialomucin complex (SMC, rat Muc4) due to its similar structural organization. Like SMC, MUC4 may also exist as two subunits: a mucin type unit known as MUC4alpha and a growth factor-like transmembrane subunit, MUC4beta. The expression of MUC4 in normal human pancreas is not detectable, but it is highly expressed in pancreatic tumor cells. In the present study, we investigated the regulation of MUC4 expression in human pancreatic tumor cells CD18/HPAF, exhibiting a high level of MUC4 transcripts and protein. When these cells were adapted to grow in the serum-free medium (CD18/HPAF-SF), the MUC4 expression was undetectable. Among several serum constituents, all-trans-retinoic acid (RA) induced the expression of MUC4 transcripts in a concentration- and time-dependent manner. The RA-mediated increase in the level of the MUC4 transcript coincided with an increased expression of transforming growth factor-beta2 (TGF-beta2) transcript. The antagonist of the retinoic acid receptor (RAR)-alpha (Ro41-5253) abrogated the expression of MUC4 and TGF-beta2 induced by RA. The exogenous addition of TGF-beta2 also increased the MUC4 expression. The TGF-beta-neutralizing antibody blocked the RA-induced as well as TGF-beta2-mediated MUC4 expression. In conclusion, induction of MUC4 expression in pancreatic carcinoma by RA is mediated through the RAR-alpha signaling pathway, and TGF-beta2 may serve as an interim mediator of this regulated expression.

Topics: Humans; Mucin-4; Mucins; Pancreatic Neoplasms; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs. Smad4 encodes a key intracellular messenger in the transforming growth factor beta (TGF-beta) signaling cascade. TGF-beta is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation in vivo, yet it did not restore sensitivity to TGF-beta. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors in vivo, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Movement; DNA-Binding Proteins; Drug Resistance, Neoplasm; Endothelial Growth Factors; Endothelium, Vascular; Fibroblast Growth Factor 2; Genes, Tumor Suppressor; Humans; Lymphokines; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad4 Protein; Thrombospondin 1; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transforming growth factor beta (TGFbeta) is a tumor suppressor acting as inhibitor of cell cycle progression of epithelial cells. We show that treatment of the pancreatic carcinoma cell lines PANC-1 and BxPC-3 with TGFbeta1 inhibits both growth factor-induced activation of the extracellular signal-regulated kinase 2 (ERK2) and translocation of the kinase to the nucleus. TGFbeta1 causes a concentration-dependent reduction of cell proliferation in both cell lines. By measuring ERK activation, we can show that TGFbeta1 is able to repress ERK activation induced by mitogenic stimuli such as EGF. This inhibitory effect of TGFbeta1 is not mediated by suppression of Ras or c-Raf-1 activation, but mediated by TGFbeta1-induced activation of a serine-threonine phosphatase, as demonstrated by inhibition of phosphatases by treatment with okadaic acid. Results obtained in the Smad4-deficient pancreatic carcinoma cell line BxPC-3, demonstrate that TGFbeta1-induced growth inhibition is mediated by a Smad4-independent prevention of ERK2 activation. In contrast to the effects of TGFbeta1 on epithelial cells, mesenchymal NIH3T3 fibroblasts exhibit elevated ERK2 activation and increased cell proliferation in response to TGFbeta1 treatment. Smad4-independent phosphatase-mediated inhibition of mitogen-activated ERK2 represents a novel effector pathway contributing to suppression of epithelial pancreatic carcinoma cell proliferation by TGFbeta1, in addition to the well-known Smad-induced tumor suppressor activity of TGFbeta. Oncogene (2000) 19, 4531 - 4541.

Topics: 3T3 Cells; Animals; Biological Transport; Carcinoma; Cell Cycle; Cell Division; Cell Nucleus; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Humans; Mice; Mitogen-Activated Protein Kinase 1; Okadaic Acid; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; ras Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta1 (TGF-beta1) and matrix metalloproteinases (MMPs) produced by tumor cells play important roles in tumor invasion. PSK, a protein-bound polysaccharide, is widely used in Japan as an immunopotentiating biological response modifier for cancer patients. In this study, we focused on the effects of PSK on invasiveness, TGF-beta1 production, and MMPs expression in two human tumor cell lines, pancreatic cancer cell line (NOR-P1) and gastric cancer cell line (MK-1P3). PSK significantly decreased the invasiveness of both cell lines through Matrigel-coated filters but did not affect cell viability, proliferation, or adhesion. Decreased invasion was associated with the inhibition of TGF-beta1, MMP-2, and MMP-9 at both mRNA and protein levels as assessed by reverse transcriptase-polymerase chain reaction, gelatin zymography, and enzyme-linked immunosorbent assay. Antibody against TGF-beta1 neutralized the MMP activities of both cell lines. PSK also suppressed the expression of urokinase plasminogen activator (uPA) and uPA receptor but did not change plasminogen activator inhibitor-1 (PAI-1) expression. Western blot analysis showed that PSK reduced uPA protein expression but not PAI-1 expression in the both cell lines. These results indicate that PSK suppresses tumor cell invasiveness through down-regulation of several invasion-related factors including TGF-beta1, uPA, MMP-2, and MMP-9.

Topics: Adjuvants, Immunologic; Antibiotics, Antineoplastic; Cell Adhesion; Cell Division; Collagen; Down-Regulation; Drug Combinations; Drug Screening Assays, Antitumor; Humans; Laminin; Matrix Metalloproteinases; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Proteoglycans; Stomach Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

It is generally assumed that TGFbeta induces cell cycle arrest through the cooperative action of cell cycle inhibitors p15, p27 and p21. Here, we found that several pancreatic carcinoma cell lines exert TGFbeta-induced negative growth control in spite of the loss of p15 and p16 expression. In these cell lines, TGFbeta-induced growth control correlates with the upregulation of the p21 protein and active pRb expression. Conversely, cells without p21 and/or pRb expression are resistant to TGFbeta -induced growth inhibition. Moreover, overexpression of p21 in the p21-deficient cell line Panc Tu1 leads to growth arrest. Thus, TGFbeta-induced growth control correlates with p21 expression and pRb status independent of p15 and/or p16 expression.

Topics: Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; G1 Phase; Growth Inhibitors; Humans; Pancreatic Neoplasms; Retinoblastoma Protein; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

CTGF is an immediate early growth responsive gene that has been shown to be a downstream mediator of TGFbeta actions in fibroblasts and vascular endothelial cells. In the present study hCTGF was isolated as immediate early target gene of EGF/TGFalpha in human pancreatic cancer cells by suppression hybridization. CTGF transcripts were found in 13/15 pancreatic cancer cell lines incubated with 10% serum. In 3/7 pancreatic cancer cell lines EGF/TGFalpha induced a significant rise of CTGF transcript levels peaking 1-2 h after the start of treatment. TGFbeta increased CTGF transcript levels in 2/7 pancreatic cancer cell lines after 4 h of treatment and this elevation was sustained after 24 h. Only treatment with TGFbeta was accompanied by a parallel induction of collagen type I transcription. 15/19 human pancreatic cancer tissues were shown to overexpress high levels of CTGF transcripts. CTGF transcript levels in pancreatic cancer tissues and nude mouse xenograft tumors showed a good correlation to the degree of fibrosis. In situ hybridization and the nude mouse experiments revealed that in pancreatic cancer tissues, fibroblasts are the predominant site of CTGF transcription, whereas the tumor cells appear to contribute to a lesser extent. We conclude that CTGF may be of paramount importance for the development of the characteristic desmoplastic reaction in pancreatic cancer tissues.

Topics: Animals; Collagen; Connective Tissue Growth Factor; Epidermal Growth Factor; Fibroblasts; Growth Substances; Humans; Immediate-Early Proteins; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Mice; Mice, Nude; Neoplasm Proteins; Pancreatic Neoplasms; Time Factors; Transcription, Genetic; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Transforming growth factor (TGF)-beta signaling is initiated by heterodimerization of TGF-beta receptor type I (TbetaRI) and type II (TbetaRII). Subsequently, the signal is transduced via Smad proteins, which upon phosphorylation and heterodimerization translocate to the nucleus and regulate gene transcription. Smad6 functions as an intracellular antagonist of TGF-beta signaling. In the present study we demonstrate that Smad6 is overexpressed in vivo in human pancreatic cancer cells. We also show that stable transfection of a full-length Smad6 construct into COLO-357 pancreatic cancer cells abrogates TGF-beta1 induced growth inhibition, and leads to enhanced anchorage-independent growth. Thus, enhanced expression of the TGF-beta signaling inhibitor Smad6 in pancreatic cancer may present a novel mechanism of TGF-beta resistance, which might have the potential to enhance the transformed phenotype of human cancer cells.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Blotting, Northern; Cell Division; DNA-Binding Proteins; Female; Growth Inhibitors; Humans; Male; Middle Aged; Pancreatic Neoplasms; Signal Transduction; Smad6 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta (TGFbeta) activates transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene through a major TGFbeta-responsive region (-740 and -647) in the PAI-1 promoter. This process requires the Smad family of signaling molecules. Upon phosphorylation by the TGFbeta receptors, Smad2 and Smad3 homoligomerize and heteroligomerize with Smad4, translocate to the nucleus and activate transcription of TGFbeta responsive genes. Smad3 and Smad4 have been shown to bind to various sites in the PAI-1 promoter. To determine the number of Smad-binding sites within the 94-base pair major TGFbeta-responsive region and the mechanism of Smad-mediated transactivation, we systematically mapped the Smad-binding sites and show that Smad4 and Smad3 bind cooperatively to two adjacent DNA elements in this region. Both elements were required for TGFbeta-induced, Smad3- and Smad4-dependent activation of PAI-1 transcription. Contrary to previous reports, transactivation of the PAI-1 promoter was mediated by the amino- but not carboxyl-terminal domains of the Smads. Furthermore, oligomerization of Smad3 markedly enhanced its binding to the two binding sites. Finally, a Smad4 mutation identified in a human pancreatic carcinoma that inactivates Smad4 signaling abolished Smad4 DNA binding activity, hence preventing transactivation of TGFbeta-responsive genes. These results underscore the importance of the Smad4 DNA binding activity in controlling cell growth and carcinogenesis.

Topics: Base Sequence; Binding Sites; DNA; DNA-Binding Proteins; Humans; Molecular Sequence Data; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Point Mutation; Promoter Regions, Genetic; Smad3 Protein; Smad4 Protein; Structure-Activity Relationship; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta

A serious insulin resistance characterizes pancreatic cancer-associated diabetes mellitus. Elsewhere, we demonstrated that MIA PaCa2 cultured cells secrete a soluble factor responsible for reduced glucose tolerance induced in SCID mice. The intracellular mechanism of insulin resistance was investigated in isolated and perfused rat hepatocytes incubated with MIA PaCa2 conditioned medium. Lactate production was reduced compared to hepatocytes incubated with control medium while 1,2-DAG was increased and PKC was activated in the hepatocytes incubated with MIA PaCa2 conditioned medium. This behavior was not reproduced treating the hepatocytes with the growth factors EGF, interleukin Ibeta, interleukin-6, and TGF-beta1. In an attempt to make a biochemical identification of the hypothesized tumor associated-diabetogenic factors we observed a low molecular weight protein in the conditioned medium, absent in the nonconditioned one, that may be responsible for the described behaviors.

Topics: Animals; Biological Factors; Cell Membrane; Cells, Cultured; Culture Media, Conditioned; Cytosol; Diglycerides; Epidermal Growth Factor; Glucose; Humans; Insulin Resistance; Interleukins; Lactic Acid; Liver; Male; Molecular Weight; Pancreatic Neoplasms; Perfusion; Protein Kinase C; Rats; Rats, Wistar; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of signaling molecules. We characterized the expression of BMP-2 and its receptors in human pancreatic tissues and pancreatic cancer cell lines and examined the effects of BMP-2 on mitogenesis.. Expression of BMP-2 and its receptors was determined by Northern blot analysis using specific complementary DNA probes. Distribution of BMP-2 in pancreatic cancers was examined by immunohistochemistry and in situ hybridization. Effects of BMP-2 on mitogenesis were assessed by monitoring cell proliferation and activation of mitogen-activated protein kinase (MAPK).. Compared with the normal pancreas, pancreatic cancers showed a 12.5-fold (P < 0.01), 2-fold (P < 0.01), and 8-fold (P < 0.01) increase of BMP-2, BMP receptor (R)-IA, and BMPR-II messenger RNA levels, respectively. By immunohistochemistry and in situ hybridization, BMP-2 was expressed in the cancer cells within the tumor mass. There was a significant correlation between the presence of BMP-2 immunostaining in the tumors and shorter postoperative survival. Pancreatic cancer cell lines expressed variable levels of messenger RNA encoding BMP-2 and its receptors. BMP-2 stimulated the growth of two pancreatic cancer cell lines (ASPC-1 and CAPAN-1). This mitogenic effect was associated with MAPK activation and blocked by the MAPK inhibitor PD98059 in CAPAN-1 but not in ASPC-1 cells. In both cell lines, expression of wild-type Smad4 abolished the BMP-2-mediated growth stimulation. BMP-2 inhibited the growth of COLO-357 cells, an effect that was blocked by expressing a dominant negative Smad4. BMP-2 had no effect in three cell lines that underexpressed either the BMP receptors or Smad1.. These findings indicate that BMP-2 has the capacity to act as a mitogen when Smad4 is mutated and suggest that it might play a role in the pathobiology of human pancreatic cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Division; Cricetinae; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Pancreas; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Smad Proteins; Smad1 Protein; Survival Rate; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Apoptosis; Gene Transfer Techniques; Genetic Therapy; Humans; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Somatostatin; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The effects of transforming growth factor (TGF)-beta 1 on type I and type II TGF-beta receptor (T beta RI and T beta RII) expression were examined in five pancreatic cancer cell lines. In contrast to its actions in COLO-357, a TGF-beta-sensitive pancreatic cancer cell line, TGF-beta 1 did not significantly alter TGF-beta receptor expression in either the TGF-beta-sensitive BXPC-3 and PANC-1 cells or in the TGF-beta-resistant CAPAN-1 and T3M4 cells. Neutralizing anti-T beta RII antibodies blocked TGF-beta 1-dependent signaling in COLO-357 cells but exhibited an attenuated effect in COLO-357 cells preincubated with TGF-beta 1 for 48 h. Basal T beta RII expression levels were comparable in all five cell lines examined. In contrast, COLO-357 cells and BX-PC-3 cells expressed relatively high basal levels of T beta RI. However, COLO-357 cells harbored a normal Smad4 gene, whereas BX-PC-3 cells exhibited a complete deletion of this gene. We conclude that the TGF-beta 1-induced T beta RII upregulation serves to enhance TGF-beta 1 responsiveness in COLO-357 cells, and that this upregulation requires the presence of adequate levels of T beta RI and T beta RII, and a functional Smad4 gene product. Our findings also indicate that TGF-beta 1 may inhibit pancreatic cancer cell growth via a Smad4-independent pathway.

Topics: Cell Division; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Mutation; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The tumor suppressor gene deleted in pancreatic cancer locus 4 (Smad4/DPC4) is inactivated in about 50% of pancreatic adenocarcinomas. The role of DPC4 in the transforming growth factor-beta (TGF-beta) receptor-mediated signal transduction cascade in human pancreatic, colon, and breast carcinoma cell lines has been investigated by a number of laboratories. The results demonstrate that Smad4/DPC4 protein functions as a key transcription factor required in regulation of TGF-beta inducible gene expression and subsequent growth inhibition. Many transcription regulators that are involved in cell growth, differentiation, and oncogenesis have been identified and cloned. Yet paradoxically, it is much more difficult to identify the important downstream target genes responsible for the biological effects elicited by these transcription factors. Although numerous attempts have been made and different approaches have been used to identify the target genes, only limited success has been achieved. Our data show that p21waf1 is one of the Smad4/DPC4-regulated downstream target genes and suggest that overexpression of the Smad4/DPC4 gene can bypass TGF-beta receptor activation and reestablish one of the key regulatory controls of cell proliferation. Identification of the Smad-regulated downstream target genes responsible for diverse biological processes that they control will extend our understanding of the mechanism for cell cycle regulation and cell differentiation.

Topics: Adenocarcinoma; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

In this study, we report coexpression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) in pancreatic carcinoma tissue associated with significantly elevated levels of both cytokines in the sera of pancreatic carcinoma patients. Using conditioned media (CM) of pancreatic carcinoma cells, we further demonstrate that tumor cell-derived TGF-beta and IL-10 inhibited in an additive fashion both proliferation and the development of Th1-like responses in peripheral blood mononuclear cell (PBMC) preparations derived from normal donors. The antiproliferative and Th1-suppressive activities contained in CM of pancreatic carcinoma cells were due primarily to IL-10 and/or TGF-beta, as shown by the capacity of cytokine-specific neutralizing antibodies to reverse these effects. Finally, as compared to normal controls, PBMC derived from pancreatic carcinoma patients displayed a Th2-like cytokine expression pattern upon activation with either anti-CD3 antibody or Staphylococcus aureus strain Cowan I. Taken together, these results suggest that aberrant production of TGF-beta and IL-10 in pancreatic tumor patients skews T-cell cytokine production patterns in favor of a Th2 immunophenotype.

Topics: Adenocarcinoma; Aged; Antibodies, Monoclonal; Culture Media, Conditioned; Dose-Response Relationship, Drug; Female; Humans; Immunophenotyping; Interferon-gamma; Interleukin-10; Interleukin-12; Killer Cells, Lymphokine-Activated; Male; Middle Aged; Pancreatic Ducts; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

It has been shown that TGFBs, their receptors, or downstream targets show genetic alterations in pancreatic cancer. This study was designed to identify transcriptional alterations induced by prolonged treatment of pancreatic cancer cell lines with TGFB. The TGFB-responsive PANC-1 cell line was treated with 10-ng/ml TGFB1 for 24 hr. cDNA representational difference analysis was used to generate subtracted hybridization probes enriched for TGFB regulated genes. These probes were hybridized on gridded arrays of cDNA clones containing genes differentially expressed in pancreatic cancer. Twenty-seven distinct cDNA clones were shown to be TGFB target genes. Eleven genes were upregulated by TGFB and were associated with extracellular matrix composition and formation, including genes usually transcribed by cells of mesenchymal origin only. Transcript levels of 16 genes were downregulated by TGFB and could mainly be classified into markers of epithelial differentiation and genes involved in the transcriptional and translational machinery. In conclusion, a 24-hr treatment of PANC-1 cells with TGFB induced a loss of epithelial and a gain of mesenchymal markers. As in other tumors, this epithelial-mesenchymal transdifferentiation may be of general importance during pancreatic carcinogenesis, and may participate, e.g., in the development of the desmoplastic reaction or the acquisition of an invasive phenotype of pancreatic tumor cells. This study demonstrates the usefulness of cDNA RDA and gridded clone libraries to study the effect of signaling cascades on the expression profile of tumor cells. Similar approaches may be helpful in the context of the genome project for the characterization of novel genes. Genes Chromosomes Cancer 26:70-79, 1999.

Topics: Cell Division; DNA Probes; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genes; Humans; Nucleic Acid Hybridization; Pancreatic Neoplasms; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) inhibits cell growth in susceptible cells by interacting with a family of protein kinases that control cell cycle progression. In the present study, we investigated the effects of TGF-beta1 on cyclin D1 expression and activity in COLO-357 human pancreatic cancer cells. TGF-beta1 increased cyclin D1 mRNA and protein levels. Nuclear runoff transcription and protein synthesis inhibition by cycloheximide revealed that this increase was, in part, due to increased cyclin D1 mRNA synthesis. Despite its stimulatory effects on cyclin D1 levels, TGF-beta1 inhibited cyclin D1-associated kinase activity and the growth of COLO-357 cells. Furthermore, suppression of cyclin D1 expression with a cyclin D1 antisense cDNA resulted in loss of TGF-beta1-mediated growth inhibition in association with reduced induction of cyclin D1, p21(C)(ip)(1) and plasminogen activator inhibitor-1 (PAI-1). Concomitantly, there was a marked decrease in the levels of the type I TGF-beta receptor (TbetaRI). Our findings suggest that in some cell types cyclin D1 expression may be important for TGF-beta1-mediated signaling and that cyclin D1 may be involved in the transcriptional regulation of TbetaRI.

Topics: Activin Receptors, Type I; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Down-Regulation; Growth Inhibitors; Humans; Insulin-Like Growth Factor I; Oligonucleotides, Antisense; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

DPC4/SMAD4 is a candidate tumor suppressor gene with a strikingly high frequency of gene alterations in pancreatic cancer that suggests a discrete role for DPC4 in these tumors. DPC4 tumor-suppressive function has been implicated to mediate the transforming growth factor-beta (TGFbeta)-suppressive pathway; however, in a DPC4-null pancreatic cancer cell line, TGFbeta growth-inhibitory and transcriptional responses were found to be DPC4-independent. This was observed within native cells having a natural homozygous deletion and in clones engineered for stable expression of wild-type DPC4 integrated into the genome. This observation contrasted with the absolute DPC4 dependence of TGFbeta responses in a breast cancer cell line studied in parallel. This growth-inhibitory response to TGFbeta in DPC4-null cells relied on an intact ras effector pathway. These data further suggest a major categorization of TGFbeta responses into DPC4-dependent and -independent signaling pathways and specifically suggest that disruption of the TGFbeta-independent signal might be a basis of selection for the emergence of DPC4 alterations during tumorigenesis in the pancreas and other sites.

Topics: Alleles; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; DNA-Binding Proteins; Gene Deletion; Genes, Tumor Suppressor; Humans; Pancreatic Neoplasms; ras Proteins; Signal Transduction; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaRII) with the type I TGF-beta receptor (TbetaRI). Activated TbetaRI then mediates TGF-beta signals by inducing the phosphorylation of Smad2 and/or Smad3, which separately hetetorodimerize with Smad4 and translocate to the nucleus. Phosphorylation of Smad2/Smad3 by activated TbetaRI is inhibited by two newly discovered members of the Smad family, Smad6 and Smad7. We now report that Smad7 mRNA levels are increased in human pancreatic cancer by comparison with the normal pancreas, and that by in situ hybridization, Smad7 is over-expressed in the cancer cells within the tumor mass. Stable transfection of COLO-357 human pancreatic cancer cells with a full-length Smad7 construct leads to complete loss of the growth inhibitory response to TGF-beta1, without altering TGF-beta1-mediated induction of PAI-I. Furthermore, Smad7 transfected COLO-357 cells display enhanced anchorage-independent growth and accelerated growth in nude mice. These findings point to a previously unrecognized mechanism for selective suppression of TGF-beta-mediated growth inhibition in cancer cells that allows for continued activation of the PAI-I promoter by TGF-beta1, which may act to enhance the tumorigenicity of certain cancer cells.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Cell Division; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Pancreas; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; RNA, Messenger; Signal Transduction; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Smad2 belongs to a family of cytoplasmic molecules that are critical components in the transforming growth factor beta (TGF-beta) signaling pathway. Upon ligand binding, the type II TGF-beta receptor (TbetaRII) heterodimerizes with and activates TGF-beta receptor type I (TbetaRI). Activated TbetaRI phosphorylates Smad2, which then heterodimerizes with Smad4, translocates into the nucleus, and subsequently effects gene transcription. Previously we have shown that pancreatic cancers overexpress TGF-betas and TbetaRII. Here, we show by northern blot analysis that Smad2 mRNA levels are significantly increased in pancreatic cancer samples in comparison with normal pancreatic tissues. By immunohistochemistry, Smad2 is present in the cancer cells of 67% of the pancreatic cancer samples. Analysis of serial sections reveals coexpression of Smad2 and TGF-beta1 in the cancer cells. Furthermore, TGF-beta1 increases steady-state levels of Smad2 mRNA in the TGF-beta1-sensitive pancreatic cancer cell line COLO-357. It is suggested that pancreatic cancer cells have the capacity to up-regulate Smad2 expression, which may lead to excessive activation of specific components of the TGF-beta-signaling pathway.

Topics: Aged; Aged, 80 and over; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreas; Pancreatic Neoplasms; Reference Values; Smad2 Protein; Tissue Distribution; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Variations in cancer cell adhesion to extracellular matrix (ECM) proteins might underlie an enhanced metastatic potential. ECM binding is mediated by cell-adhesion molecules, the membrane expression of which might be influenced by soluble mediators, such as cytokines. The aims of our study were to ascertain whether epidermal growth factor (EGF), transforming growth factor beta1 (TGF-beta1), interleukin 1alpha (IL-1alpha), or interleukin 1beta (IL-1beta) can modify MIA PaCa 2 (pancreatic cancer cell line) and CAPAN-1 (metastatic pancreatic cancer cell line) adhesion to fibronectin, laminin, or type I collagen, and whether these cytokines can shift the membrane expression of the hyaluronic acid receptor (CD44). EGF significantly enhanced MIA PaCa 2, but not CAPAN-1, adhesion to fibronectin, laminin, and type I collagen. TGF-beta1 reduced MIA PaCa 2 adhesion to type I collagen, but enhanced CAPAN-1 adhesion to fibronectin and laminin. IL-1alpha was found to enhance MIA PaCa 2 adhesion to fibronectin, while reducing adhesion to type I collagen, whereas IL-1beta reduced the adhesion to laminin. IL-1alpha enhanced CAPAN-1 adhesion to laminin in a dose-dependent manner; IL-1beta slightly increased the adhesion of these cells to laminin at low dosage, and to type I collagen at high dosage. Both IL-1alpha and IL-1beta reduced CD44 membrane expression of MIA PaCa 2, while TGF-beta1 increased the percentage of CD44-positive CAPAN-1 cells. We suggest that the effects on cell adhesion induced by different cytokines depend on the status of the target pancreatic cancer cell. EGF and, in part, IL-1alpha can favor nonmetastatic pancreatic cancer cell adhesion to ECM, possibly favoring tumor spread. Metastatic cells seem to lose the responsiveness to EGF, while becoming hyperresponsive to IL-1alpha. TGF-beta1 might exert an antidiffusive effect on primary, and a prodiffusive effect on metastatic pancreatic cancer cells. Only IL-1alpha, IL-1beta, and TGF-beta1 seem to influence CD44 membrane expression. All the results presented in this study were obtained in vitro, and in vivo studies are needed to verify whether the studied cytokines can favor or counteract pancreatic cancer spread.

Topics: Cell Adhesion; Cell Membrane; Cytokines; Epidermal Growth Factor; Extracellular Matrix Proteins; Fibronectins; Humans; Hyaluronan Receptors; Interleukin-1; Laminin; Liver Neoplasms; Pancreatic Ducts; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-betas (TGF-betas) bind to the type II TGF-beta receptor (TbetaRII), which then heterodimerizes with the type I TGF-beta receptor (TbetaRI), thereby initiating a signaling cascade. TGF-betas are overexpressed in pancreatic cancer, and this overexpression is associated with more aggressive disease. Although TbetaRII also is overexpressed in pancreatic ductal adenocarcinoma cells in vivo, the biologic significance of this overexpression is not completely known. Therefore in this study, we characterized TbetaRII expression by Northern blot analysis in 32 normal and 42 cancerous pancreatic tissues and correlated the survival of the cancer patients with TbetaRII messenger RNA (mRNA) levels. Northern blot analysis revealed that, by comparison with the normal controls, TbetaRII expression was increased in 19 (45%) of 42 cancer samples. Densitometric analysis of all the pancreatic tissues revealed a 3.4-fold increase (p < 0.01) in TbetaRII mRNA levels in the cancer tissues by comparison with normal controls. There was a strong correlation between the expression of TbetaRII and the levels of two invasion-promoting genes, plasminogen-activator-inhibitor-1 (PAI-1) and matrix-metalloproteinase-9 (MMP9). Log-rank analysis of the Kaplan-Meier survival curves indicated that patients whose tumors overexpressed TbetaRII had a significantly shorter survival period than did patients whose cancers expressed low levels of TbetaRII. It is suggested that TbetaRII overexpression may be a marker that correlates with disease progression in pancreatic ductal adenocarcinoma.

Topics: Adolescent; Adult; Blotting, Northern; Female; Gene Expression; Humans; Male; Matrix Metalloproteinase 9; Middle Aged; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Prognosis; Receptors, Transforming Growth Factor beta; RNA, Messenger; Survival Rate; Transforming Growth Factor beta

In the present study we investigated the actions of transforming growth factor (TGF)-beta1 on gene induction and cyclin-dependent kinase inhibitors in relation to TGF-beta receptor modulation in COLO-357 pancreatic cancer cells. TGF-beta1 inhibited the growth of COLO-357 cells in a time- and dose-dependent manner and caused a rapid but transient increase in plasminogen activator inhibitor-I and insulin-like growth factor binding protein-3 mRNA levels. TGF-beta1 caused a delayed but sustained increase in the protein levels of the cyclin-dependent kinase inhibitors p15(Ink4B), p21(Cip1), and p27(Kip1) and a sustained increase in type I and II TGF-beta receptors (TbetaRI and TbetaRII) mRNA and protein levels. The protein synthesis inhibitor cycloheximide (10 microg/ml) completely blocked the TGF-beta1-mediated increase in TbetaRI and TbetaRII expression. Furthermore, a nuclear runoff transcription assay revealed that the increase in receptor mRNA levels was due to newly transcribed RNA. There was a significant increase in TbetaRI and TbetaRII mRNA levels in confluent cells in comparison to subconfluent (

Topics: Animals; Carrier Proteins; Cattle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Humans; Insulin-Like Growth Factor Binding Protein 3; Microtubule-Associated Proteins; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

Transforming growth factor beta1 (TGF beta1) is a multifunctional factor that regulates many aspects of cellular functions such as epithelial cell growth and synthesis of extracellular matrices. TGFbeta transduces signaling through a heterodimeric complex of type I and type II TGFbeta receptors (TbetaRI and TbetaRII). Recently, it has been shown that enhanced expression of TGFbeta1 is associated with the progress of pancreatic duct cell carcinoma (PDC) and chronic pancreatitis (CP). In this study, the expression of TGFbeta1 and its receptors, TbetaRI and TbetaRII, is examined in 21 cases of PDC by immunohistochemistry using specific antibodies, and the results are compared with those for 13 cases of CP. In the epithelial cells of PDC and CP, there are no significant differences in the expression of TGFbeta1, TbetaRI, and TbetaRII. In contrast, stromal expression of this cytokine and its receptors tends to be stronger in PDC than in CP; especially, the expression of TbetaRII is significantly stronger in PDC (p < 0.05). These findings suggest that there are some pathological differences in the properties of stromal reactions between PDC and CP, although the morphologies of their stroma resemble each other.

Topics: Aged; Aged, 80 and over; Carcinoma, Ductal, Breast; Chronic Disease; Epithelial Cells; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Pancreatitis; Receptors, Transforming Growth Factor beta; Stromal Cells; Transforming Growth Factor beta

Loss of p53 and p21WAF1 expression have previously been reported in pancreatic adenocarcinoma. Despite these findings in several reports of oncogene and tumor suppressor gene alterations in pancreatic cancer, the clinical significance of these changes is still poorly understood. In an attempt to detect molecular prognostic markers for pancreatic carcinoma, we studied the immunohistochemical expression of p53, p21WAF1, and TGF-beta1 proteins in 42 pancreatic adenocarcinomas of the ductal type. The results were correlated with clinicopathologic findings to identify the markers with prognostic significance. p53 nuclear immunoreactivity was seen in 20 (48%) of the cases, and it was strong to moderate in 14 (33%) of them. p21WAF1 cytoplasmic positivity was found in 16 (38%) of the tumors, with 72% staining strong to moderate. TGF-beta1 stained the cytoplasm of the tumor cells in 13 (31%). Of the p53-negative cases, 12 (54%) exhibited p21WAF1 expression. In 3 (30%) of cases, TGF-beta1 reactivity was seen in the absence of p53 and p21WAF1 p53 positivity identified tumors of higher grade, but did not correlate with stage or survival. TGF-beta1 expression, however, identified low-grade tumors and patients with longer survival. No correlation was found between the expression of any of these molecular markers and smoking history. We report a significant correlation between TGF-beta1 reactivity and low-grade tumors and between TGF-beta1 and better survival. This is a novel finding pointing to TGF-beta1 as a possible new stage-independent predictor of tumor survival in pancreatic ductal adenocarcinoma. In agreement with others, we also found p53 mutation in 20 (48%) of the tumors.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Ductal, Breast; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; Survival Rate; Transforming Growth Factor beta; Tumor Suppressor Protein p53

We have previously shown that thrombospondin-1 (TSP-1) and TGF-beta 1 upregulate the urokinase plasminogen activator (uPA) and its receptor (uPAR) and promote tumor cell invasion in breast cancer. To date, the effect of TSP-1 and TGF-beta 1 on the plasminogen/plasmin system in gastrointestinal epithelial malignancies has not been investigated. In this study, we determined the effect of TSP-1 and TGF-beta 1 on uPA and uPAR expression and on tumor cell invasion in pancreatic cancer. ASPC1 human pancreatic adenocarcinoma cells were incubated for 48 h on cell-conditioned media (CCM) either alone (Control) or with the addition of either TSP-1 (40 micrograms/ml) or TGF-beta 1 (5 ng/ml). uPA and uPAR expression were determined by ELISA. ASPC1 cell invasion was determined in a modified Boyden chamber type I collagen invasion assay. The upper chamber was treated with CCM either alone (Control) or with the addition of anti-uPA (10 micrograms/ml) or anti-uPAR (10 micrograms/ml). The lower chamber was treated with CCM either alone (Control) or with the addition of either TSP-1 (40 micrograms/ml) or TGF-beta 1 (5 ng/ml). TSP-1 and TGF-beta 1 induced a twofold increase on uPAR expression but only a slight increase on total uPA. Tumor cell invasion was upregulated 3.5 to 4.5-fold by TSP-1 and TGF-beta 1, respectively. Anti-uPA and anti-uPAR antibodies completely blocked the TSP-1 and TGF-beta 1-mediated pancreatic tumor cell invasion. We conclude that TSP-1 and TGF-beta 1 mediate pancreatic tumor cell invasion through upregulation of the plasminogen/plasmin system.

Topics: Adenocarcinoma; Enzyme Precursors; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Thrombospondin 1; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

Angiostatin, a proteolytic fragment of plasminogen, is a potent inhibitor of angiogenesis. We have previously shown that the human pancreatic cancer cell line ASPC-1 produces enzymatic activity capable of generating angiostatin. In this study we sought to determine whether angiostatin production by ASPC-1 cells was regulated by the growth factor transforming growth factor-beta 1 (TGF-beta 1), a key mediator of tumor angiogenesis.. ASPC-1 cells were grown to 70% to 80% confluence in 20% fetal calf serum-RPMI. Medium was changed to serum free. TGF-beta 1 was added at concentrations of 0, 1, 5, and 10 ng/mL with or without plasminogen activator inhibitor type-1 (PAI-1) at concentrations of 0, 5, 10, 50, and 100 micrograms/mL. Cells were then cultured for an additional 24 hours. The serum-free conditioned medium was obtained. Angiostatin generation was determined by incubating 20 micrograms of plasminogen with 100 microL of serum-free conditioned medium for 0, 1, 2, 3, 6, 12, and 24 hours. Samples were run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred. The membrane was probed with a monoclonal antibody to the kringle 1-3 fragment of plasminogen and developed using enhanced chemiluminescence.. TGF-beta 1 and PAI-1 inhibited the conversion of plasminogen into angiostatin in a time- and dose-dependent manner. Antibody to PAI-1 completely blocks TGF-beta 1 mediated angiostatin inhibition.. TGF-beta 1 inhibits the generation of the antiangiogenic molecule angiostatin by human pancreatic cancer cells in a time- and dose-dependent manner. This effect is mediated through modulation of the plasminogen/plasmin system.

Topics: Adenocarcinoma; Angiostatins; Antibodies; Antineoplastic Agents; Blotting, Western; Dose-Response Relationship, Drug; Humans; Neovascularization, Pathologic; Pancreatic Neoplasms; Peptide Fragments; Plasminogen; Plasminogen Activator Inhibitor 1; Serine Proteinase Inhibitors; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) signaling is initiated following heterodimerization of the type II TGF-beta receptor (TbetaRII) with the type I TGF-beta receptor (TbetaRI). Both receptors are required for TGF-beta responsiveness. In the present study, we characterized the actions of TGF-beta1 in T3M4 human pancreatic cancer cells, which express low levels of TbetaRI and high levels of TbetaRII. Cells were transiently transfected with p3TP-Lux, a TGF-beta-responsive luciferase reporter gene construct. TGF-beta1 was without effect in parental T3M4 cells, but caused a time- and dose-dependent increase in luciferase activity in T3M4 cells co-transfected with a TbetaRI cDNA expression vector. Co-transfection of TbetaRI with a truncated Smad4 cDNA that is known to block TGF-beta-dependent signaling, abrogated the TbetaRI-induced increase in luciferase activity. Sequencing of the TbetaRI and the Smad4 genes in T3M4 cells did not reveal any mutations. These findings indicate that one mechanism for TGF-beta resistance in pancreatic cancer is due to a quantitative decrease in TbetaRI expression.

Topics: Activin Receptors, Type I; Cell Division; DNA-Binding Proteins; Humans; Immunoblotting; Mutation; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Resistance to TGF-beta1 occurred in pancreatic cancer cells suggesting that inactivation of TGF-beta inhibitory signaling pathways may play an important role in human pancreatic cancer. The aim of our study was to determine the presence of alterations in the main putative components of the TGF-beta inhibitory signaling pathways (p15, Smad4, Smad2, TGFbeta-RII, CDC25A). A panel of human carcinomas of the exocrine pancreas orthotopically implanted and perpetuated in nude mice and pancreatic cancer cell lines were studied. p15 gene alterations, mainly homozygous deletions that involved exons 1 and/or 2, were found in the 62.5% (5 of 8) of pancreatic xenografts whereas Smad4 gene aberrations were found in one of eight xenografts and in two of seven cell lines. Additional aberrations in these genes were acquired during in vivo perpetuation and distal dissemination. Paradoxically, TGFbeta-RII overexpression and a decrease in CDC25A protein levels were found in all tumors and cell lines. In one cell line, resistance to TGF-beta1 occurred in the absence of alterations in the genes analysed so far. We conclude that all human pancreatic tumor cells analysed herein have non-functional TGF-beta pathways. The majority of cells harbor alterations in at least one of the putative components of TGF-beta pathways, mainly in p15 and Smad4 genes. These results suggest that inactivation of TGF-beta signaling pathways plays an important role in human pancreatic tumorigenesis.

Topics: Animals; Base Sequence; Carrier Proteins; cdc25 Phosphatases; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; DNA Primers; DNA Replication; DNA-Binding Proteins; Humans; Mice; Mice, Nude; Microtubule-Associated Proteins; Neoplasm Transplantation; Pancreatic Neoplasms; Point Mutation; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

We have recently reported that the geranylgeranyltransferase I inhibitor GGTI-298 arrests human tumor cells at the G1 phase of the cell cycle and increases the protein and RNA levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Here, we show that GGTI-298 acts at the transcriptional level to induce p21(WAF1/CIP1) in a human pancreatic carcinoma cell line, Panc-1. This upregulation of p21(WAF1/CIP1) promoter was selective, since GGTI-298 inhibited serum responsive element- and E2F-mediated transcription. A functional analysis of the p21(WAF1/CIP1) promoter showed that a GC-rich region located between positions -83 and -74, which contains a transforming growth factor beta-responsive element and one Sp1-binding site, is sufficient for the upregulation of p21(WAF1/CIP1) promoter by GGTI-298. Electrophoretic mobility shift assays showed a small increase in the amount of DNA-bound Sp1-Sp3 complexes. Furthermore, the analysis of Sp1 transcriptional activity in GGTI-298-treated cells by using GAL4-Sp1 chimera or Sp1-chloramphenicol acetyltransferase reporter revealed a significant increase in Sp1-mediated transcription. Moreover, GGTI-298 treatment also resulted in increased Sp1 and Sp3 phosphorylation. These results suggest that GGTI-298-mediated upregulation of p21(WAF1/CIP1) involves both an increase in the amount of DNA-bound Sp1-Sp3 and enhancement of Sp1 transcriptional activity. To identify the geranylgeranylated protein(s) involved in p21(WAF1/CIP1) transcriptional activation, we analyzed the effects of the small GTPases Rac1 and RhoA on p21(WAF1/CIP1) promoter activity. The dominant negative mutant of RhoA, but not Rac1, was able to activate p21(WAF1/CIP1). In contrast, constitutively active RhoA repressed p21(WAF1/CIP1). Accordingly, the ADP-ribosyl transferase C3, which specifically inhibits Rho proteins, enhanced the activity of p21(WAF1/CIP1). Taken together, these results suggest that one mechanism by which GGTI-298 upregulates p21(WAF1/CIP1) transcription is by preventing the small GTPase RhoA from repressing p21(WAF1/CIP1) induction.

Topics: Alkyl and Aryl Transferases; Benzamides; Binding Sites; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Enzyme Inhibitors; G1 Phase; Genes, Regulator; GTP-Binding Proteins; Humans; Nuclear Proteins; Pancreatic Neoplasms; Phosphorylation; Promoter Regions, Genetic; rhoA GTP-Binding Protein; Sp1 Transcription Factor; Sp3 Transcription Factor; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Transforming growth factor beta (TGF-beta) is an extracellular ligand that binds to a heterodimeric receptor, initiating signals that regulate growth, differentiation, and apoptosis. Many cancers, including pancreatic cancer, harbor defects in TGF-beta signaling and are resistant to TGF-beta-mediated growth suppression. Genetic alterations of DPC4, which encodes a DNA binding protein that is a downstream component of the pathway, most frequently occur in pancreatic and biliary carcinomas. We searched for other targets of mutation of the TGF-beta pathway in these cancers. We report somatic alterations of the TGF-beta type I receptor gene ALK-5. Homozygous deletions of ALK-5 were identified in 1 of 97 pancreatic and 1 of 12 biliary adenocarcinomas. A germ-line variant of ALK-5, presumably a polymorphism, was identified, but no somatic intragenic mutations were identified upon sequencing of all coding regions of ALK-5. Somatic alterations of the TGF-beta type II receptor gene (TGFBR2) were identified in 4 of 97 (4.1%) pancreas cancers, including a homozygous deletion in a replication error-negative cancer and three homozygous frameshift mutations of the poly(A) tract of the TGF-beta type II receptor in replication error-positive cancers. We also studied other related type I receptors of the TGF-beta superfamily. In a panel of pancreas cancers preselected for loss of heterozygosity at the ALK-1 locus, sequencing of all coding exons of the ALK-1 gene revealed no alterations. No homozygous deletions were detected in the ALK-1, ALK-2, ALK-3, or ALK-6 genes in a panel of 86 pancreatic cancer xenografts and 11 pancreatic cancer and 22 breast cancer cell lines. The rate of genetic inactivation of TGF-beta pathway members was determined in 45 pancreatic cancers. Eighty-two % of these pancreatic cancers had genetic inactivation of the DPC4, p15, ALK-5, or TGFBR2 genes. Our results indicate that the TGF-beta type I and type II receptor genes are selective targets of genetic inactivation in pancreatic and biliary cancers.

Topics: Activin Receptors; Adenocarcinoma; Aged; Biliary Tract Neoplasms; Carcinoma, Ductal, Breast; DNA-Binding Proteins; Female; Genes, p16; Humans; Loss of Heterozygosity; Male; Middle Aged; Mutation; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional polypeptide that is related to the progression of chronic pancreatitis. However, the mechanism of beta-cell damage by TGF-beta1 is unknown. Treatment with TGF-beta1 enhanced internucleosomal DNA cleavage caused by exogenous hydrogen peroxide in a hamster pancreatic beta-cell line (HIT). TGF-beta1 also induced protein oxidation, assessed by measuring carbonyl groups in proteins, and was involved in reactions that lead to lipid peroxidation. This eventually destructs membrane lipids and forms malondialdehyde. We have investigated its effects on two major antioxidative enzymes, catalase and glutathione peroxidase (GPx). TGF-beta1 suppressed mRNA expression as well as reduced the activities of catalase and GPx. The decrease in the catalase and GPx activities in TGF-beta1-treated cells resulted in an increase in intracellular peroxides as judged by flow cytometric analysis using a peroxide-sensitive dye, 2',7'-dichlorofluorescin diacetate. These data suggest that the augmented production of reactive oxygen species by TGF-beta1 through suppression of antioxidative enzymes may cause cellular damage and consequent apoptosis and induce pancreatitis or diabetes.

Topics: Adenoma, Islet Cell; Animals; Apoptosis; Catalase; Cricetinae; DNA; Flow Cytometry; Gene Expression; Glutathione Peroxidase; Hydrogen Peroxide; Oxidation-Reduction; Pancreatic Neoplasms; Peroxides; Reactive Oxygen Species; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

A family of structurally related proteins homologous to the Drosophila mothers against dpp (MAD) gene product have been implicated in signal transduction by members of the TGF-beta superfamily. One of these MAD related proteins (DPC4) has been cloned as a candidate tumour suppressor in pancreas carcinomas, suggesting a role for DPC4 in growth regulation by TGF-beta related proteins. The involvement of DPC4 in TGF-beta1 induced growth inhibition and transcriptional response is demonstrated here, by the introduction of DPC4 in the TGF-beta and activin insensitive breast tumour cell line MDA-MB-468, from which the DPC4 gene is deleted. Transfection of DPC4 in this cell line restores both growth inhibition and the induction of a TGF-beta sensitive reporter construct (3TPlux) by TGF-beta1. In contrast, a DPC4 splice variant lacking amino acid residues 223-301 and cloned from another TGF-beta and activin resistant breast tumour cell line (MDA-MB-231), does not restore the induction of the 3TPlux reporter by TGF-beta1. We also show that in this latter cell line activin resistance is partly due to the absence of a functional activin type IB receptor. These results indicate that DPC4 is part of the TGF-beta signalling cascade and mediates TGF-beta induced growth inhibition. Together with the deletion of DPC4 from pancreas carcinomas these results suggest a role for DPC4 as a tumour suppressor.

Topics: Activins; Alternative Splicing; Amino Acid Sequence; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Breast Neoplasms; Cell Division; DNA Primers; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Gene Deletion; Genes, Tumor Suppressor; Humans; Inhibins; Luciferases; Molecular Sequence Data; Pancreatic Neoplasms; Polymerase Chain Reaction; Recombinant Fusion Proteins; Repressor Proteins; Sequence Deletion; Sequence Homology, Amino Acid; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.

Topics: Adult; Amino Acid Sequence; Animals; Apoptosis; Cell Division; Cell Line; Consensus Sequence; DNA-Binding Proteins; Early Growth Response Transcription Factors; Epithelial Cells; Epithelium; Gene Expression; Gene Library; Humans; Kruppel-Like Transcription Factors; Male; Molecular Sequence Data; Organ Specificity; Pancreas; Pancreatic Neoplasms; Rats; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Zinc Fingers

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Fibronectins; Gene Expression; Homeostasis; Immunohistochemistry; Liver; Metallothionein; Mice; Mice, Transgenic; Mutation; Pancreas; Pancreatic Neoplasms; Phenotype; Proliferating Cell Nuclear Antigen; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Pancreatic neoplasms harbor different prognoses according to their histological type: a benign course for serous cystadenoma, a low malignant potential for intraductal papillary mucinous neoplasms (IPMN), and high aggressiveness for ductal adenocarcinoma (ADC). Transforming growth factor beta 1 (TGF beta 1) may regulate tumor growth. The present study analyzes and compares the expression of its precursor beta 1-latency-associated peptide (beta 1-LAP), its latent binding protein (LTBP), and its mRNA in ductal adenocarcinoma (n = 10), in IPMN (n = 8), in serous cystadenoma (n = 2), and in normal tissues (n = 5). LTBP is thought to play a strategic role in the processing and active secretion of latent TGF beta 1 and its stockage in the extracellular matrix. Localization of beta 1-LAP and LTBP was assessed by immunohistochemistry using specific antibodies and expression of TGF beta 1 mRNA by reverse-transcriptase polymerase chain reaction analysis. beta 1-LAP was only slightly expressed in normal specimens, while LTBP was not detected. beta 1-LAP was detected in the cytoplasm of neoplastic cells in 9 of 10 patients with ADC. An intense staining was present in stromal cells surrounding the neoplastic glands in all cases except in one carcinoma in situ. LTBP was detected only in stromal cells and in the surrounding extracellular matrix. In IPMN with mild-grade dysplasia and in cystadenoma, beta 1-LAP was strongly expressed in the epithelial cells, while it was poorly detected in invasive IPMN; stromal cells were poorly or not all stained by beta 1-LAP, except in invasive IPMN (n = 2). LTBP was detected in neoplastic cells of three cases with benign IPMN and two of two cases with cystadenoma, while stroma was not immunostained. TGF beta 1 mRNA was strongly expressed in most of the tumors and no difference in expression was observed between the different types of neoplasms. There is no quantitative difference in expression of TGF beta 1 in ADC and in IPMN or cystadenoma. However, the latter are able to secrete TGF beta 1 efficiently, in contrast to ductal ADC as shown by the ability of the neoplastic cells to express both beta 1-LAP and LTBP. Invasive stroma reaction was associated with enhanced beta 1-LAP and LTBP expression in stromal cells and could be mediated by TGF beta 1 via LTBP

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Carcinoma, Ductal, Breast; Carcinoma, Papillary; Carrier Proteins; Cystadenoma, Serous; DNA Primers; Female; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Male; Middle Aged; Pancreas; Pancreatic Neoplasms; Peptide Fragments; Protein Biosynthesis; Protein Precursors; Proteins; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: DNA-Binding Proteins; Genes, Tumor Suppressor; Models, Molecular; Pancreatic Neoplasms; Protein Binding; Protein Conformation; Signal Transduction; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta

The tumor suppressor gene deleted in pancreatic cancer locus 4 (DPC4) is inactivated in about 50% of pancreatic adenocarcinomas. DPC4 was found to be homologous to Smad4 and may function as a transcription factor in the transforming growth factor beta (TGF-beta) receptor-mediated signal transduction pathway. We have investigated the role of DPC4 in the TGF-beta receptor-mediated signal transduction cascade in five human pancreatic cancer cell lines (Panc-1, MDAPanc-28, HS766T, Capan-1, and MiaPaCa-2). Our results demonstrate that the loss of responsiveness to TGF-beta-induced growth inhibition correlates with the loss of expression of DPC4. We have shown that TGF-beta induces p21waf1 expression in Panc-1 cells, whereas no induction of p21waf1 expression by TGF-beta was detected in the other four cell lines lacking either DPC4 expression or the TGF-beta type II receptor. No increase in p21waf1 mRNA stability was observed after treatment with TGF-beta, which suggests that the induction of p21waf1 in Panc-1 cells is transcriptionally regulated by TGF-beta. Our data also demonstrate that the expression of DPC4 is directly involved in TGF-beta-mediated induction of the 3TP-lux reporter gene, which contains a known TGF-beta-inducible plasminogen activator inhibitor promoter. These data suggest that: (a) TGF-beta-mediated induction of p21waf1 and subsequent growth inhibition require the expression of DPC4; (b) p21waf1 is a downstream target gene of DPC4; and (c) transfection of the DPC4 gene restores the TGF-beta-inducible gene expression. Inactivation of the tumor suppressor gene DPC4 and other components of the TGF-beta signal cascades may abolish one of the key negative controls of cell proliferation in pancreatic adenocarcinomas.

Topics: Adenocarcinoma; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Pancreatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.

Topics: Alleles; Amino Acid Sequence; Animals; Base Sequence; Cell Division; Chromosome Mapping; Chromosomes, Human, Pair 18; DNA-Binding Proteins; Gene Deletion; Gene Expression; Genes, Tumor Suppressor; Genetic Markers; Humans; Mice; Molecular Sequence Data; Mutation; Neoplasm Transplantation; Pancreatic Neoplasms; Proteins; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Some monocytic cytokines are important immune regulators. We have investigated cytokine production by monocytes and the blood levels of IL-1 beta, IL-6, TNF alpha, and TGF beta, in patients with obstructive jaundice. The supernatant from LPS stimulated monocytes from jaundiced patients released significantly increased quantities of TNF alpha by both bioassay and radioimmunoassay (RIA) (12.4 +/- 2.5 fmol/mL and 32.6 +/- 8.3 fmol/mL, respectively, for jaundice, compared with 1.6 +/- 0.3 fmol/mL and 2.4 +/- 0.5 fmol/mL respectively for controls, and also of IL-6 (54.8 +/- 5.0 fmol/mL in jaundice compared with 35.6 +/- 5.0 fmol/mL for controls). The production of IL-1 beta and TGF beta by stimulated monocytes was unchanged. Jaundiced patients had significantly higher plasma TGF beta, but TNF alpha and IL-1 beta were below the limits of detection. The highest monocyte TNF alpha and IL-6 levels were seen in malignant disease patients, especially those with a poor immediate prognosis. We conclude that the production of some cytokines by monocytes is up-regulated in patients with obstructive jaundice.

Topics: Bile Duct Diseases; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Case-Control Studies; Cholangiocarcinoma; Cholelithiasis; Cholestasis; Cytokines; Female; Humans; Interleukin-1; Interleukin-6; Male; Middle Aged; Monocytes; Pancreatic Neoplasms; Prognosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Transforming growth factor-beta (TGF-beta) receptors constitute a family of transmembrane proteins that bind TGF-beta ligands. In this study we assessed the growth responsiveness to TGF-beta 1 in pancreatic cancer cell lines and characterized the levels of expression of TGF-beta receptors in these cell lines and in human pancreatic cancer tissues. COLO 357 cells were most sensitive to the growth inhibitory actions of TGF-beta 1, PANC-1 cells exhibited moderate sensitivity, Hs766T cells exhibited slight sensitivity and MIA PaCa-2 and T3M4 cells were resistant to TGF-beta 1. Only COLO 357 cells expressed high levels of ALK5, the major type I TGF-beta receptor (T beta RI). Hs766T and PANC-1 cells expressed high levels of SKR1, another T beta RI subtype. Only MIA PaCa-2 cells did not exhibit the type II TGF-beta receptor (T beta-RII) transcript, whereas type III TGF-beta receptor (T beta-RIII) mRNA levels were elevated in this cell line and in HS766T cells. All the cell lines expressed TGF-beta 1, but TGF-beta 2 and TGF-beta 3 mRNA levels were variable. ALK5 and SKR1 mRNA levels were 6.8- and 9-fold greater in the pancreatic tumors in comparison with the corresponding levels in the normal pancreas. However, in the cancer cells, ALK5 immunoreactivity was faint, whereas T beta RII immunoreactivity was focal and intense. Conversely, in ductal cells adjacent to cancer cells ALK5 immunoreactivity was strong, whereas T beta RII immunoreactivity was weak. Since ALK5 heterodimerization with T beta RII is crucial for TGF-beta-mediated signaling, our findings suggest that low levels of ALK5 in pancreatic cancer cells within a tumor may protect against growth inhibition.

Topics: Adolescent; Adult; Aged; Base Sequence; Cell Division; Female; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; Molecular Sequence Data; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Tumor spread may be favored by a reduced production and/or an enhanced degradation of extracellular matrix components (collagen, fibronectin, laminin). Most tumor cell behavior, from growth to spread, may be regulated by cytokines, the exact roles of which, however, are not yet fully understood. We here evaluate the effects of some cytokines (epidermal growth factor, transforming growth factor-beta 1, interleukin-1 alpha, and interleukin-1 beta) on both cell growth and the production of the aminoterminal peptide of type III procollagen, the urokinase plasminogen activator, and the plasminogen activator inhibitor-1 in neoplastic cell lines originating in the pancreas and colon. Cells were stimulated daily with the above cytokines and the aminoterminal peptide of type III procollagen, urokinase plasminogen activator, and plasminogen activator inhibitor-1 were measured in the conditioned media. Epidermal growth factor stimulated cell growth of both cell lines. Transforming growth factor-beta 1 counteracted cell proliferation and stimulated type III procollagen and plasminogen activator inhibitor-1 production only in the colon cancer cell line. Interleukin-1 alpha slightly stimulated cell growth, but inhibited plasminogen activator inhibitor-1 production in both cell lines; interleukin-1 beta did not affect cell growth, but stimulated plasminogen activator inhibitor-1 production by the colon cancer cell line. Our findings suggest that transforming growth factor-beta 1 and interleukin-1 beta may have an antidiffusive effect. These results confirm that cytokine-producing cells have a potential role in stimulating or counteracting tumor growth and spread and also confirm the pivotal role of host-tumor interactions in determining the outcome of a particular neoplasia.

Topics: Cell Division; Collagen; Colonic Neoplasms; Cytokines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Interleukin-1; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Serine Proteinase Inhibitors; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The growth characteristics associated with tumorigenicity were determined in clones of MIA PaCa-2 and PANC-1 pancreatic carcinoma cells. MIA PaCa-2 cells differed from PANC-1 cells in that they rapidly formed tumors in nude mice, formed colonies more rapidly and formed larger colonies in soft agar, and were cloned more efficiently when seeded at low density. MIA PaCa-2 cells but not PANC-1 cells were stimulated to escape quiescence and undergo DNA synthesis with nutrient media lacking growth factors. Both cell lines were stimulated to proliferate with serum-free media containing EGF, transferrin, and insulin. Antibody neutralization assays indicated that an IGF-1 autocrine loop was required for the nutrient stimulation of growth in MIA PaCa-2 cells and for the growth-factor stimulation in both MIA PaCa-2 and PANC-1 cells. Both cell lines were stimulated to proliferate with exogenous IGF-1 in basal media; this stimulation was specifically blocked by antibodies to IGF-1 or its receptor. MIA PaCa-2 and PANC-1 cells expressed similar levels of IGF-1 receptor mRNA and showed similar binding kinetics in receptor binding assays. In contrast to PANC-1 cells, MIA PaCa-2 cells were insensitive to TGF-beta 1 and did not express TGF-beta receptor type II. The results suggest that the growth-factor independence is representative of a more tumorigenic phenotype. We hypothesize that growth-factor independence of MIA PaCa-2 cells is mediated by an aberrant regulation of an IGF-1 autocrine loop. A decreased regulation of this IGF-1 loop may be potentiated by loss of response to TGF-beta.

Topics: Cell Division; Colonic Neoplasms; Gene Expression; Humans; In Vitro Techniques; Insulin-Like Growth Factor I; Pancreatic Neoplasms; Receptor, IGF Type 1; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The effects of transforming growth factor-beta (TGF-beta) on insulin secretion were investigated using a glucose-responsive clonal cell line, MIN6. One hundred pM TGF-beta stimulated insulin release during 0.5-24 h of incubation in the presence of 5.5 mM glucose, but not after 48 h; 1 nM TGF-beta also stimulated insulin release up to 2 h of exposure, but the effect was not seen after 6 h of exposure. When cells were incubated with 25 mM glucose for 24 h, 100 pM TGF-beta significantly inhibited glucose-stimulated insulin release, whereas insulin release was not altered at 0 or 2.8 mM glucose. On the contrary, forskolin- (10 microM) and tolbutamide- (40 microM) induced insulin release were not affected by TGF-beta. TGF-beta affected neither the cell growth nor the cellular insulin content. An addition of 1 microM nitrendipine abolished TGF-beta-induced insulin secretion at 5.5 mM glucose. The presence study shows that TGF-beta exerts a bimodal effect on glucose-induced insulin secretion from MIN6 cells, depending on dose, time of exposure and concentrations of coexisting glucose. These effects might be mediated by the Ca(2+)-dependent mechanism.

Topics: Animals; Colforsin; Dose-Response Relationship, Drug; Glucose; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Mice; Pancreatic Neoplasms; Tolbutamide; Transforming Growth Factor beta; Tumor Cells, Cultured

Our previous studies have shown that 3 human pancreatic carcinoma cell lines (PC-1, PC-2, PC-3) established in our laboratory express EGF receptor gene, EGF and its mRNA. In this study, gene expression of TGF alpha (a ligand of EGF receptor), TGF beta 1 and c-erbB2 oncogene were studied. The results showed that all 3 human pancreatic carcinoma cell lines expressed TGF alpha mRNA. PC-2, PC-3 cell lines also express TGF beta 1 mRNA. C-erbB2 oncogene (a homology of EGF receptor) mRNA was strongly expressed in PC-1, weakly in PC-2 and was not detectable in PC-3. The presence of TGF alpha and c-erbB2 in human pancreatic carcinoma cell lines further verifies the occurrence of an autocrine growth regulation of this cancer.

Topics: ErbB Receptors; Gene Expression; Humans; Pancreatic Neoplasms; Proto-Oncogene Proteins; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta s (TGF-beta s) constitute a family of bifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. Perturbations in TGF-beta expression and function may lead to loss of negative constraints on cell growth. In this study, we examined TGF-beta expression in human pancreatic cancer.. The distribution of TGF-beta isoforms in 60 human pancreatic cancers was examined using immunohistochemical, Northern blot, and in situ hybridization techniques.. Immunohistochemical analysis showed the presence of TGF-beta 1 (47% of tumors), TGF-beta 2 (42% of tumors), and TGF-beta 3 (40% of tumors) in the cancer cells. The presence of TGF-beta 2 was associated with advanced tumor stage (P < 0.05). Furthermore, there was a significant correlation between the absence of TGF-beta s in the tumors and longer postoperative survival. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas showed 11- (P < 0.001), 7- (P < 0.05), and 9-fold (P < 0.001) increases in the messenger RNA (mRNA) levels encoding TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively. By in situ hybridization, these mRNA moieties colocalized with their respective proteins in the cancer cells.. These findings show that human pancreatic cancers show increased levels of TGF-beta isoforms and enhanced TGF-beta mRNA expression and suggest that the presence of TGF-beta s in pancreatic cancer cells may contribute to disease progression.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Blotting, Northern; Cell Division; Child; Female; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Pancreatic Neoplasms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Survival Rate; Transforming Growth Factor beta

Studies of growth-regulation of neuroendocrine cells have been hampered by a lack of suitable in vitro models. We established and have maintained a functioning human pancreatic carcinoid cell (BON) line. BON cells synthesize and secrete several growth factors. Among those, we have found that serotonin stimulates growth of BON cells through specific receptors linked to cyclic AMP pathway. In this study, effects of other growth factors on growth and serotonin release, and the effects of serotonin and TGF-beta 1 on the polyamine biosynthetic pathway, were examined. TGF-beta 1 inhibited both growth and serotonin release in a dose-dependent fashion. Basic FGF stimulated growth, but failed to affect serotonin release. Other peptide growth factors had no effect on either growth or serotonin release. Serotonin stimulated ODC enzyme activity, but TGF-beta 1 failed to affect ODC enzyme activity. These findings suggest that growth of neuroendocrine cells can be delicately regulated by their own products in an autocrine fashion.

Topics: Adult; Carcinoid Tumor; Cell Division; Humans; Male; Ornithine Decarboxylase; Pancreatic Neoplasms; Serotonin; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Cells, Cultured

Pancreatic cancer is an extremely aggressive malignancy. The factors allowing human pancreatic cancer cells to escape normal growth constraints are not known. However, it has been proposed that certain cancer cells may obtain a growth advantage as the result of a lack of responsiveness to negative growth regulators such as transforming growth factor-beta 1 (TGF-beta 1). We now show that two established pancreatic carcinoma cell lines, COLO 357 and PANC-I, are sensitive to growth inhibition by TGF-beta 1. The growth of COLO 357 cells is inhibited by 50% when incubated in the presence of TGF-beta 1 (5 ng/ml) under low serum conditions (0.5%). PANC-I cells are growth inhibited by 25% under the same conditions. In COLO 357 cells, but not PANC-I cells, TGF-beta 1 also causes a marked alteration in cell morphology. In both cell lines, TGF-beta 1 induces TGF-beta 1 mRNA levels in a time and dose-dependent manner. However, TGF-beta 1 does not increase the amount of TGF-beta 2 or TGF-beta 3 mRNA in these cells. In spite of its growth inhibitory effects, TGF-beta 1 fails to suppress c-myc mRNA levels. These findings suggest that TGF-beta 1 inhibits the growth of human pancreatic cancer cells and point to a significant dysfunction in the ability of TGF-beta 1 to suppress c-myc expression in these cells.

Topics: Cell Division; Cell Membrane; Gene Expression; Genes, myc; Humans; Pancreatic Neoplasms; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

We have shown recently that 5-HT is an autocrine growth stimulatory factor for a cell line (BON) that is derived from a human pancreatic carcinoid tumor. This action is mediated by a 5-HT receptor-linked decrease of cyclic adenosine monophosphate (AMP) production, but not mediated by a 5-HT receptor-linked stimulation of phosphatidylinositol hydrolysis. The BON cells also express transforming growth factor betas (TGF beta s) (1, 2, and 3) and release TGF beta into their medium. In this study, we examined the effects of TGF beta on the secretion of 5-HT, on signal transduction pathways involved in 5-HT secretion, and on growth of BON cells. TGF beta 1 inhibited basal and acetylcholine-stimulated release of 5-HT, but did not inhibit isobutylmethylxanthine-stimulated release of 5-HT. TGF beta 1 inhibited both basal and acetylcholine-stimulated hydrolysis of phosphatidylinositol in a dose dependent manner, but did not affect cyclic AMP production. TGF-beta 1 inhibited growth of BON cells in culture; this effect was reversed by exogenously administered 5-HT. Three different specific and saturable TGF beta 1 binding sites were identified; binding assays performed after mild acid wash (0.1% acetic acid, pH 2.5) conditions uncovered TGF beta receptors that were apparently occupied by endogenously produced TGF beta species. Affinity cross-linking assay showed that BON cells had three different TGF beta binding proteins. These results suggest that TGF beta 1 can inhibit growth of BON cells by altering secretory responses of 5-HT by means of receptor-mediated inhibition of phosphatidylinositol hydrolysis. We conclude that growth of BON cells is regulated, at least in part, by the opposing receptor-mediated autocrine actions of 5-HT and TGF beta.

Topics: Affinity Labels; Carcinoid Tumor; Cell Division; Cyclic AMP; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Pancreatic Neoplasms; Phosphatidylinositols; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Serotonin; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Neuroendocrine tumors of the digestive system are slow growing neoplasms which often present with pronounced fibrosis around tumor cells and in the peritoneal cavity. In this report 30 midgut carcinoids and endocrine pancreatic tumors were examined for the expression of peptide growth factors and their receptors, both by immunohistochemistry and in situ hybridization. Our data indicate that multiple peptide growth factors, PDGF, TGF-beta, and bFGF are expressed by these tumors. PDGF was expressed on tumor cells and stroma in 70% of tissues examined. PDGF alpha-receptor was seen on clusters of tumor cells and occasionally on adjacent stroma, whereas PDGF beta-receptor was seen only in the stroma. Our data suggest that PDGF may be involved in the autocrine stimulation of tumor cells and stimulation of stromal cell growth through paracrine and possibly autocrine mechanism. In addition, tumor tissues express all three isoforms of TGF-beta in more than half of the tissues examined. Tumor cells produce small latent complexes causing an escape from potent inhibitory effect of TGF-beta and stimulation of stromal cell growth and matrix deposition through paracrine mechanism. bFGF, a potent stimulant of endothelial cell growth, was expressed by all tumor tissues examined. Our data suggest that multiple peptide growth factors may have an important role in tumor progression and desmoplastic reaction accompanying these tumors.

Topics: Adenoma, Islet Cell; Adolescent; Adult; Aged; Aged, 80 and over; Animals; Carcinoid Tumor; Digestive System Neoplasms; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunohistochemistry; Mice; Middle Aged; Neoplasm Proteins; Pancreatic Neoplasms; Peptide Biosynthesis; Peptides; Platelet-Derived Growth Factor; Rabbits; Receptors, Platelet-Derived Growth Factor; Receptors, Somatotropin; Transforming Growth Factor beta

To ascertain whether the tumor cells can regulate the host immune systems through the production of the cytokines or their receptors, we examined the expressions of tumor necrosis factor alpha (TNF alpha), tumor necrosis factor beta (TNF beta), interleukin 2 (IL-2) and interleukin 2 receptor alpha chain (IL-2R alpha) on the human cancer cell lines by Northern blot analysis. We used K562 (leukemia cell line), MCF-7 (breast cancer cell line), LS180, HT29 (colon cancer cell lines), SH101 (gastric cancer cell line) and PH101 (pancreas cancer cell line). Expressions of TNF alpha, TNF beta and IL-2 mRNA were not detected in any of the tumor cell lines. However, 1.4 and 3.5 kilobases of the IL-2R alpha mRNA were expressed in the PH101 cells, but not in the other five cell lines. Furthermore, IL-2R alpha was detected on the cell surface of the PH101 cells by the flow-cytometric analysis with an anti-IL-2R alpha monoclonal antibody. Interestingly, the soluble IL-2R alpha (sIL-2R alpha) was found in the conditioned media obtained from the PH101 cell culture with a sandwich enzyme immunoassay. Moreover, the sIL-2R alpha secreted from the PH101 cells blocked the IL-2 dependent lymphocyte proliferation. These results indicate that the expression of IL-2R alpha on PH101 might suppress the IL-2 induced lymphocyte proliferation.

Topics: Cell Division; Cell Line; Cell-Free System; Humans; Immunosuppressive Agents; Interleukin-2; Lymphocyte Activation; Lymphotoxin-alpha; Neutralization Tests; Pancreatic Neoplasms; Receptors, Interleukin-2; Transforming Growth Factor beta; Tumor Cells, Cultured

The relationship between cell differentiation and transforming growth factor alpha (TGF-alpha) expression in human pancreatic cancer cells was analyzed in Capan 1 cells. These cells differentiate either spontaneously or after butyrate treatment. During differentiation (spontaneous or butyrate induced), TGF-alpha messenger RNA (mRNA) levels decreased, whereas the TGF-beta 1 mRNA levels remained unchanged. TGF-alpha was present in cells as proTGF-alpha, which decreased after butyrate treatment. Secretion of TGF-alpha was not found. Under the two conditions of differentiation, the membrane-bound protein kinase C activity was also reduced. Conversely, long-term phorbol ester treatment increased both membrane-bound protein kinase C activity (260%) and TGF-alpha mRNA level (500%), a not significant increase of TGF-beta 1 mRNA was observed. However, phorbol 12-myristate-13-acetate did not induce TGF-alpha synthesis or secretion. These data suggest that expression of TGF-alpha can be reduced in cancer cells; they also suggest the existence of a relationship between TGF-alpha expression and cell differentiation. In addition, the protein kinase C-induced TGF-alpha mRNA level was not followed by the increase of TGF-alpha biosynthesis, suggesting a translational control. Finally, the expression of TGF-alpha and -beta 1 messengers appears to be differently regulated.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Gene Expression Regulation; Humans; Pancreatic Neoplasms; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Advanced gastrointestinal endocrine tumors respond poorly to conventional chemotherapy. In this study we examined the effects of two agents that promote cellular differentiation, sodium butyrate and hexamethylene bisacetamide, on the in vitro growth and secretory responses of a human pancreatic carcinoid (BON) and human gastrinoma (PT-2 and PT-SM) cell lines that have been established in our laboratory. We found that both sodium butyrate and hexamethylene bisacetamide strongly inhibited growth of BON, PT-2, and PT-SM cells. With continuous exposure of BON cells to sodium butyrate (2 mmol/L), the doubling time was prolonged, from 60 hours in controls to 156 hours, and saturation density was reduced to 28% that of controls. Hexamethylene bisacetamide (4 mmol/L) reduced saturation density to 37% that of controls in BON cells and prolonged the doubling time, from 60 hours to 103 hours. Antiproliferative effects of similar magnitudes were observed in the gastrinoma cell lines. In contrast, differential effects were produced on amine biosynthesis in BON cells; sodium butyrate stimulated levels of 5-hydroxytryptamine in the cells, whereas hexamethylene bisacetamide caused a profound dose-dependent inhibition of amine biosynthesis. The significant antiproliferative activity of sodium butyrate and hexamethylene bisacetamide and the inhibitory effects of hexamethylene bisacetamide on amine biosynthesis warrant evaluation of these agents or analogues for treatment of metastatic carcinoid and gastrinoma.

Topics: 5-Hydroxytryptophan; Acetamides; Analysis of Variance; Butyrates; Butyric Acid; Carcinoid Tumor; Cell Division; Gastrinoma; Humans; Hydroxyindoleacetic Acid; Pancreatic Neoplasms; Serotonin; Transforming Growth Factor beta; Tumor Cells, Cultured

A serotonin-secreting human pancreatic carcinoid cell line (BON) is demonstrated to express transcripts for all three mammalian types of transforming growth factor beta (TGF beta 1, 2, and 3). Similarly, freshly excised carcinoid tumors from six patients were also found to express mRNA for all three of the type-beta TGFs. Medium conditioned by BON cells had detectable TGF beta activity, although most of the activity was latent as determined by radioreceptor assay with and without prior acid treatment. However, nonactivated BON-conditioned medium stimulated DNA synthesis, soft agar growth, and an increase in TGF beta 1 and fibronectin mRNA expression in AKR-2B fibroblasts. In addition, BON-conditioned medium had a potent endothelial cell growth-stimulatory activity. Since the TGF beta s inhibit growth of endothelial cells, the presence of other growth factors was suspected. TGF alpha, c-sis, and basic fibroblast growth factor transcripts were also found to be expressed by the BON carcinoid cells. These data indicate that multiple peptide growth factors may have a paracrine role in the desmoplastic reaction accompanying carcinoid tumors.

Topics: Amino Acid Isomerases; Blotting, Northern; Carcinoid Tumor; Carrier Proteins; Cell Division; DNA; Dose-Response Relationship, Drug; Endothelium; Fibroblasts; Fibronectins; Growth Substances; Humans; Insulin; Pancreatic Neoplasms; Peptidylprolyl Isomerase; Procollagen; RNA, Messenger; Time Factors; Transcription, Genetic; Transforming Growth Factor alpha; Transforming Growth Factor beta

A murine keratinocyte cell line that is resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF beta 1) was examined for differential gene expression patterns that may be related to the mechanism of the loss of TGF beta 1 responsiveness. Cells that were resistant to the growth-inhibitory effects of TGF beta 1 (KCR cells) were derived from K-ras-transformed BALB/MK keratinocytes (KC cells). Using a subtractive hybridization procedure with KC and KCR mRNAs, we isolated a complementary DNA clone for murine protein disulfide isomerase (PDI). The mRNA for PDI is inhibited by TGF beta 1 treatment in the parental KC cells, but not in the TGF beta 1-resistant KCR cells. Similar PDI down-regulation also occurs in other TGF beta-sensitive cells, but not in a human pancreatic carcinoma cell line which is insensitive to the growth-inhibitory effects of TGF beta 1. The results suggest that misregulation of PDI, an important component of co- and posttranslational modification systems, may be involved in the mechanism by which some cells escape from the growth-inhibitory effects of TGF beta 1.

Topics: Animals; Carcinoma; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; DNA; Drug Resistance; Enzyme Induction; Humans; Isomerases; Keratinocytes; Kirsten murine sarcoma virus; Mice; Neoplasm Proteins; Pancreatic Neoplasms; Protein Disulfide-Isomerases; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-myc; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured