transforming-growth-factor-beta and Colorectal-Neoplasms

Growing evidence supports an important role of the tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC). Resident cells such as fibroblasts or immune cells infiltrating into the TME maintain continuous crosstalk with cancer cells and thereby regulate CRC progression. One of the most important molecules involved is the immunoregulatory cytokine transforming growth factor-β (TGFβ). TGFβ is released by various cells in the TME, including macrophages and fibroblasts, and it modulates cancer cell growth, differentiation, and cell death. Mutations in components of the TGF pathway, including TGFβ receptor type 2 or SMAD4, are among the most frequently detected mutations in CRC and have been associated with the clinical course of disease. Within this review, we will discuss our current understanding about the role of TGFβ in the pathogenesis of CRC. This includes novel data on the molecular mechanisms of TGFβ signaling in TME, as well as possible strategies for CRC therapy targeting the TGFβ pathway, including potential combinations with immune checkpoint inhibitors.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Humans; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The epithelial-mesenchymal transition (EMT) is a cellular reprogramming process that occurs during embryonic development and adult tissue homeostasis. This process involves epithelial cells acquiring a mesenchymal phenotype. Through EMT, cancer cells acquire properties associated with a more aggressive phenotype. EMT and its opposite, mesenchymal-epithelial transition (MET), have been described in more tumors over the past ten years, including colorectal cancer (CRC). When EMT is activated, the expression of the epithelial marker E-cadherin is decreased and the expression of the mesenchymal marker vimentin is raised. As a result, cells temporarily take on a mesenchymal phenotype, becoming motile and promoting the spread of tumor cells. Epithelial-mesenchymal plasticity (EMP) has become a hot issue in CRC because strong inducers of EMT (such as transforming growth factor β, TGF-β) can initiate EMT and regulate metastasis, microenvironment, and immune system resistance in CRC. In this review, we take into account the significance of EMT-MET in CRC and the impact of the epithelial cells' plasticity on the prognosis of CRC. The analysis of connection between EMT and colorectal cancer stem cells (CCSCs) will help to further clarify the current meager understandings of EMT. Recent advances affecting important EMT transcription factors and EMT and CCSCs are highlighted. We come to the conclusion that the regulatory network for EMT in CRC is complicated, with a great deal of crosstalk and alternate paths. More thorough research is required to more effectively connect the clinical management of CRC with biomarkers and targeted treatments associated with EMT.

Topics: Adult; Cadherins; Cell Differentiation; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment

Colorectal cancer (CRC) is a complex malignancy responsible for the second-highest cancer deaths worldwide. TGF-β maintains normal cellular homeostasis by inhibiting the cell cycle and inducing apoptosis, but its elevated level is correlated with colorectal cancer progression, as TGF-β is a master regulator of the epithelial-to-mesenchymal transition, a critical step of metastasis. Tumors, including CRC, use elevated TGF-β levels to avoid immune surveillance by modulating immune cell differentiation, proliferation, and effector function. Presently, the treatment of advanced CRC is mainly based on chemotherapy, with multiple adverse effects. Thus, there is a need to develop alternate tactics because CRC continue to be mostly resistant to the present therapeutic regimen. TGF-β blockade has emerged as a promising therapeutic target in cancer therapy. Blocking TGF-β with phytochemicals and other molecules, such as antisense oligonucleotides, monoclonal antibodies, and bifunctional traps, alone or in combination, may be a safer and more effective way to treat CRC. Furthermore, combination immunotherapy comprising TGF-β blockers and immune checkpoint inhibitors is gaining popularity because both molecules work synergistically to suppress the immune system. Here, we summarize the current understanding of TGF-β as a therapeutic target for managing CRC and its context-dependent tumor-promoting or tumor-suppressing nature.

Topics: Colorectal Neoplasms; Humans; Immunosuppression Therapy; Immunotherapy; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the leading causes of mortality among cancers. Many aspects of this cancer are under investigation to find established markers of diagnosis, prognosis, and also potential drug targets. In this review article, we are going to discuss the possible solution to all these aims by investigating the literature about cancer-associated fibroblasts (CAFs) involved in CRC. Moreover, we are going to review their interaction with the tumor microenvironment (TME) and vitamin D and their role in tumorigenesis and metastasis. Moreover, we are going to expand more on some markers produced by them or related to them including FAP, a-SMA, CXCL12, TGF- β, POSTN, and β1-Integrin. Some signaling pathways related to CAFs are as follows: FAK, AKT, activin A, and YAP/TAZ. Some genes related to the CAFs which are found to be possible therapeutic targets include COL3A1, JAM3, AEBP1 and, CAF-derived TGFB3, WNT2, and WNT54.

Topics: Cancer-Associated Fibroblasts; Carboxypeptidases; Carcinogenesis; Colorectal Neoplasms; Fibroblasts; Humans; Prognosis; Repressor Proteins; Transforming Growth Factor beta; Tumor Microenvironment

According to data from 2020, Slovakia has long been among the top five countries with the highest incidence rate of colorectal cancer (CRC) worldwide, and the rate is continuing to rise every year. In approximately 80% of CRC cases, allelic loss (loss of heterozygosity, LOH) occurs in the long arm of chromosome 18q. The most important genes that can be silenced by 18q LOH or mutations are small mothers against decapentaplegic homolog (SMAD) 2 and SMAD4, which are intracellular mediators of transforming growth factor (TGF)-β superfamily signals. TGF-β plays an important role in the pro-oncogenic processes, including such properties as invasion, epithelial-mesenchymal transition (commonly known as EMT), promotion of angiogenesis, and immunomodulatory effects. Several recent studies have reported that activation of TGF-β signaling is related to drug resistance in CRC. Because the mechanisms of drug resistance are different between patients in different stages of CRC, personalized treatment is more effective. Therefore, knowledge of the activation and inhibition of factors that affect the TGF-β signaling pathway is very important.

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

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, largely due to the development of colorectal liver metastases (CRLM). For the establishment of CRLM, CRC cells must remodel their tumor-microenvironment (TME), avoid the immune system, invade the underlying stroma, survive the hostile environment of the circulation, extravasate into the liver, reprogram the hepatic microenvironment into a permissive pre-metastatic niche, and finally, awake from a dormant state to grow out into clinically detectable CRLM. These steps form part of the invasion-metastasis cascade that relies on reciprocal interactions between the tumor and its ever-changing microenvironment. Such interplay provides a strong rational for therapeutically targeting the TME. In fact, several TME constituents, such as VEGF, TGF-β coreceptor endoglin, and CXCR4, are already targeted in clinical trials. It is, however, of utmost importance to fully understand the complex interactions in the invasion-metastasis cascade to identify novel potential therapeutic targets and prevent the establishment of CRLM, which may ultimately greatly improve patient outcome.

Topics: Colorectal Neoplasms; Humans; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The liver is the most common metastatic site in colorectal cancer (CRC) patients. Indeed, 25-30% of the cases develop colorectal liver metastasis (CLM), showing an extremely poor 5-year survival rate and resistance to conventional anticancer therapies. Tumor-associated macrophages (TAMs) provide a nurturing microenvironment for CRC metastasis, promoting epithelial-to-mesenchymal transition (EMT) through the TGF-β signaling pathway, thus driving tumor cells to acquire mesenchymal properties that allow them to migrate from the primary tumor and invade the new metastatic site. EMT is known to contribute to the disruption of blood vessel integrity and the generation of circulating tumor cells (CTCs), thus being closely related to high metastatic potential in numerous solid cancers. Despite the fact that it is well-recognized that the crosstalk between tumor cells and the inflammatory microenvironment is crucial in the EMT process, the association between the EMT and the role of TAMs is still poorly understood. In this review, we elaborated on the role that TAMs exert in the induction of EMT during CLM development. Since TAMs are the major source of TGF-β in the liver, we also focused on novel insights into their role in TGF-β-induced EMT.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages

Fibroblasts constitute a ubiquitous mesenchymal cell type and produce the extracellular matrix (ECM) of connective tissue, thereby providing the structural basis of various organs. Fibroblasts display differential transcriptional patterns unique to the organ of their origin and they can be activated by common stimuli such as transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) signaling. Cancer-associated fibroblasts (CAFs) reside in the cancer tissue and contribute to cancer progression by influencing cancer cell growth, invasion, angiogenesis and tumor immunity. CAFs impact on the tumor microenvironment by remodeling the ECM and secreting soluble factors such as chemokines and growth factors. Differential expression patterns of molecular markers suggest heterogeneous features of CAFs in terms of their function, pathogenic role and cellular origin. Recent studies elucidated the bimodal action of CAFs on cancer progression and suggest a subgroup of CAFs with tumor-suppressive effects. This review attempts to describe cellular features of colorectal CAFs with an emphasis on their heterogeneity and functional diversity.

Topics: Cancer-Associated Fibroblasts; Colorectal Neoplasms; Extracellular Matrix; Humans; Immune Checkpoint Inhibitors; Organ Specificity; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC) is a heterogeneous disease that includes both hereditary and sporadic types of tumors. Tumor initiation and growth is driven by mutational or epigenetic changes that alter the function or expression of multiple genes. The genes predominantly encode components of various intracellular signaling cascades. In this review, we present mouse intestinal cancer models that include alterations in the Wnt, Hippo, p53, epidermal growth factor (EGF), and transforming growth factor β (TGFβ) pathways; models of impaired DNA mismatch repair and chemically induced tumorigenesis are included. Based on their molecular biology characteristics and mutational and epigenetic status, human colorectal carcinomas were divided into four so-called consensus molecular subtype (CMS) groups. It was shown subsequently that the CMS classification system could be applied to various cell lines derived from intestinal tumors and tumor-derived organoids. Although the CMS system facilitates characterization of human CRC, individual mouse models were not assigned to some of the CMS groups. Thus, we also indicate the possible assignment of described animal models to the CMS group. This might be helpful for selection of a suitable mouse strain to study a particular type of CRC.

Topics: Animals; Carcinogenesis; Cell Transformation, Neoplastic; Colonic Neoplasms; Colorectal Neoplasms; Disease Models, Animal; DNA Mismatch Repair; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, p53; Hippo Signaling Pathway; Humans; Mice; Protein Serine-Threonine Kinases; Transforming Growth Factor beta; Wnt Signaling Pathway

Colorectal cancer (CRC) is one of the most common types of cancer and is associated with an increasing rate of mortality. Transforming Growth Factor-Beta (TGF-β) is often upregulated in CRC, and appears to play an important role in regulating cell proliferation, migration, immune surveillance, apoptosis, cell differentiation, drug-resistance and many cellular processes that may be involved in CRC, and therefore underscores its potential value as a therapeutic target in the treatment of CRC. An increased expression of the TGF- β pathway has been associated with poor prognosis in several cancer types, including CRC.. Here, we describe the critical role of the TGF-β pathway in CRC as well as the preclinical and clinical investigations on TGF-β inhibitors, with particular emphasis on recent findings with small-molecule inhibitors in CRC. Several TGF-β inhibitors (e.g., Trabedersen, Galunisertib, Gradalis, PF-03446962, NIS793) have been generated over the past decade for targeting this pathway.. There is accumulating evidence of the therapeutic potential of this and other TGF-β inhibitors for the treatment of other malignancies. These inhibitors might be used in combination with chemotherapy as well as with other biological agents, in order to overcome different resistance mechanisms. However, further studies are needed to identify determinants of the activity of TGF-β inhibitors, through the analysis of genetic and environmental alterations affecting TGF-β and parallel pro-cancer pathways.. These studies will be critical to improving the efficacy and selectivity of current and future anticancer strategies targeting TGF-β.

Topics: Colorectal Neoplasms; Humans; Molecular Targeted Therapy; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) signaling is one of the important cellular pathways that play key roles for tissue maintenance. In particular, it is important in the context of inflammation and tumorigenesis by modulating cell growth, differentiation, apoptosis, and homeostasis. TGF-β receptor type 2 (

Topics: Animals; Colorectal Neoplasms; Humans; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The transforming growth factor-β (TGF-β) signaling pathway plays an important role in cancer cell proliferation, growth, metastasis, and apoptosis. It has been shown that TGF-β acts as a tumor suppressor in the early stages of the disease, and as a tumor promoter in its late stages. Mutations in the TGF-β signaling components, the TGF-β receptors and cytoplasmic signaling transducers, are frequently observed in colorectal carcinomas. Exploiting specific TGF-β receptor agonist and antagonist with antitumor properties may be a way of controlling cancer progression. This review summarizes the regulatory role of TGF-β signaling in the pathogenesis of colorectal cancer.

Topics: Colorectal Neoplasms; Drug Resistance, Neoplasm; Humans; Mutation; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the most common causes of cancer deaths worldwide and the number of CRC patients is increasing progressively. Despite the improvement of the surgical techniques and chemotherapy, we have not completely overcome this disease yet due to the metastases. Therefore, understanding the mechanisms through which metastasis occurs is important for overcoming CRC. Normal host cells in the tumor microenvironment, such as macrophages and fibroblasts, have been reported to promote the growth of CRCs. Although neutrophils were originally considered to have defensive functions against tumor cells, it has been revealed that some populations of neutrophils, called as tumor-associated neutrophils (TANs), have tumor-supportive functions. The plasticity between tumor-suppressive and -supportive neutrophils are regulated by transforming growth factor (TGF)-β and Interferon-β signaling. Some studies have demonstrated that TANs promote the spread of cancer cells to distant organs. TANs contribute to the tumor invasion and angiogenesis through the production of matrix metalloproteinase-9 (MMP9), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in the primary and metastatic sites. Neutrophils also promotes tumor cell dissemination by capturing circulating tumor cells using neutrophil extracellular traps and promote their migration to distant sites. The neutrophil-to-lymphocyte ratio is a well-defined predictive marker for CRC patients. In this review, we highlight the molecular signaling between TANs and CRC cells and the possibility of TANs as a potential target for cancer therapy.

Topics: Colorectal Neoplasms; Hepatocyte Growth Factor; Humans; Interferon-beta; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neutrophils; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A

Small mothers against decapentaplegic (SMAD) proteins are a family of signal transduction molecules in transforming growth factor β (TGFβ) ligand pathways that have been found to have a key role in the pathogenesis of inflammatory bowel disease (IBD). Long standing IBD predisposes individuals to colitis-associated colorectal cancer (CAC), an entity that possess unique characteristics compared to hereditary and sporadic cancer. The ligands of the TGFβ super family along with SMADs have also been implicated in several aspects of colorectal cancer formation. SMAD proteins are shown to be involved in a number of potentially carcinogenic mechanisms such as altering gene transcription, controlling stem cell differentiation to causing epigenetic changes. Modulation of these proteins has emerged as a novel therapeutic intervention for IBD although its effect on carcinogenesis remains elusive. This account reviews available evidence linking SMAD proteins to CAC and explores the potential areas for future research in this area.

Topics: Animals; Carcinogenesis; Cell Differentiation; Cell Proliferation; Colorectal Neoplasms; Humans; Inflammatory Bowel Diseases; Phosphorylation; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The increasing availability of detailed structural information on many biological systems provides an avenue for manipulation of these structures, either for probing mechanism or for developing novel therapeutic agents for treating disease. This has been accompanied by the advent of several powerful new methods, such as the ability to incorporate non-natural amino acids or perform fragment screening, increasing the capacity to leverage this new structural information to aid in these pursuits. The abundance of structural information also provides new opportunities for protein engineering, which may become more and more relevant as treatment of diseases using gene therapy approaches become increasingly common. This is illustrated by example with the TGF-β family of proteins, for which there is ample structural information, yet no approved inhibitors for treating diseases, such as cancer and fibrosis that are promoted by excessive TGF-β signaling. The results presented demonstrate that through several relatively simple modifications, primarily involving the removal of an α-helix and replacement of it with a flexible loop, it is possible to alter TGF-βs from being potent signaling proteins into inhibitors of TGF-β signaling. The engineered TGF-βs have improved specificity relative to kinase inhibitors and a much smaller size compared to monoclonal antibodies, and thus may prove successful as either as an injected therapeutic or as a gene therapy-based therapeutic, where other classes of inhibitors have failed.

Topics: Colorectal Neoplasms; Drug Design; Humans; Protein Engineering; Protein Structure, Tertiary; Receptors, Transforming Growth Factor beta; Signal Transduction; Structure-Activity Relationship; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the most common cancers globally and is associated with a high mortality rate. The transforming growth factor beta (TGF-β) signaling pathway plays an important role in normal intestinal tissue function, but has also been implicated in the development of CRC. MicroRNAs (miRNAs) have also recently emerged as important regulators of cancer development and progression. They act by targeting multiple signaling pathways including the TGF-β signaling pathway. There is growing evidence demonstrating that miRNAs target various components of the TGF-β signaling pathway, including TGF-β1, TGF-β2, regulatory SMADs (SMAD1, 2, 3, 5 and 9), co-mediator SMAD4, inhibitory SMADs (SMAD6 and 7) and the TGF-β receptors, and thereby alter the proliferation and migration of CRC cells. In this review, we summarize the data concerning the interaction between TGF-β signaling pathway and miRNAs with the aim to better understanding the CRC molecular mechanisms and hence better management of this disease.

Topics: Colorectal Neoplasms; Humans; MicroRNAs; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Colorectal Neoplasms; Disease Models, Animal; Disease Progression; Doxycycline; Gene Expression Regulation, Neoplastic; Genes, ras; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) is associated with a higher incidence of distant metastasis and decreased survival. Whether TGF-β can be used as a prognostic indicator of colorectal cancer (CRC) remains controversial.. The Medline, EMBASE and Cochrane databases were searched from their inception to March 2016. The studies that focused on TGF-β as a prognostic factor in patients with CRC were included in this analysis. Overall survival (OS) and disease-free survival (DFS) were analysed separately. A meta-analysis was performed, and hazard ratios (HR) with 95% confidence intervals (CI) were calculated.. Twelve studies were included in the analysis, of which 8 were used for OS and 7 for DFS. In all, 1622 patients with CRC undergoing surgery were included. Combined HRs suggested that high expression of TGF-β had a favourable impact on OS (HR = 1.68, 95% CI: 1.10-2.59) and DFS (HR = 1.11, 95% CI: 1.03-1.19) in CRC patients. For OS, the combined HRs of Asian studies and Western studies were 1.50 (95% CI: 0.61-3.68) and 1.80 (95% CI: 1.33-2.45), respectively. For DFS, the combined HRs of Asian studies and Western studies were 1.42 (95% CI: 0.61-3.31) and 1.11 (95% CI: 1.03-1.20), respectively.. This meta-analysis demonstrates that TGF-β can be used as a prognostic biomarker for CRC patients undergoing surgery, especially for CRC patients from Western countries.

Topics: Asian People; Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Female; Humans; Male; Prognosis; Proportional Hazards Models; Transforming Growth Factor beta

Colorectal cancer is one of the most common malignancies and is the second-leading cause of cancer-related death world-wide, which is linked to genetic mutations, epigenetic alterations, and oncogenic signaling activation. MicroRNAs, one of the categories of epigenetics, have been demonstrated significant roles in carcinogenesis and progression through regulating of oncogenic signaling pathways, stem cells, epithelial-mesenchymal transition, and metastasis. This review summarizes the roles of microRNAs in the regulating of Wnt, Ras, TGF-β, and inflammatory signaling pathways, stemness, and epithelial-mesenchymal transition, for carcinogenesis and metastasis in colorectal cancer. Improving our understanding of the mechanisms of regulatory interactions of microRNAs with signaling pathways in colorectal cancer formation and progression will aid in determining the genes responsible for colorectal cancer initiation, progression, metastasis, and recurrence and, finally, in developing personalized approaches for cancer prevention and therapy.

Topics: Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Humans; MicroRNAs; Neoplasm Metastasis; Neoplastic Stem Cells; ras Proteins; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Colorectal cancer (CRC) is one of the most frequent tumor types in Western countries. Approximately 20 % of patients show metastasis at the time of diagnosis, with the liver being one of the most affected organs. Transforming growth factor-beta (TGF-β) plays a regulatory role not only in the physiology of the normal colon but also in the development of CRC and its metastatic process. In this review, we analyze the molecular mechanisms leading to TGF-β dysregulation in tumor and stroma cells and the modification of the microenvironment that fosters CRC metastasis. Recent genomic studies have identified a CRC subtype with a mesenchymal and aggressive phenotype having TGF-β as a hub gene of this signature. Consistent with these findings, the inhibition of TGF-β signaling has been shown to impair experimental CRC metastasis to the liver. Based on these and other results conducted in various tumor types, the pharmaceutical industry has developed a variety of strategies to target TGF-β. We provide up-to-date information of these therapies, which are currently in preclinical or clinical trials.

Topics: Animals; Colon; Colorectal Neoplasms; Drug Discovery; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplastic Stem Cells; Rectum; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-β cytokines signal via a complex network of pathways to regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. A high percentage of colorectal tumors contain mutations that disrupt TGF-β family member signaling. We review how TGF-β family member signaling is altered during development of colorectal cancer, models of study, interaction of pathways, and potential therapeutic strategies.

Topics: Activins; Animals; Bone Morphogenetic Proteins; Colorectal Neoplasms; Germ-Line Mutation; Homeostasis; Humans; Mice; Mice, Knockout; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Colorectal cancer (CRC) diagnosis often occurs at late stages when tumor cells have already disseminated. Current therapies are poorly effective for metastatic disease, the main cause of death in CRC. Despite mounting evidence implicating the tumor microenvironment in CRC progression and metastasis, clinical practice remains predominantly focused on targeting the epithelial compartment. Because CRCs remain largely refractory to current therapies, we must devise alternative strategies. Transforming growth factor (TGF)-β has emerged as a key architect of the microenvironment in poor-prognosis cancers. Disseminated tumor cells show a strong dependency on a TGF-β-activated stroma during the establishment and subsequent expansion of metastasis. We review and discuss the development of integrated approaches focused on targeting the ecosystem of poor-prognosis CRCs.

Topics: Animals; Colorectal Neoplasms; Humans; Intestinal Mucosa; Signal Transduction; Transcriptome; Transforming Growth Factor beta; Tumor Microenvironment

The β6 subunit of the αvβ6 integrin heterodimer has long been an enigma in cancer biology though recent research has provided many new insights into its biology. Collectively, these findings include discovery of the transcriptional, translational and cell biological mechanisms by which β6 acts, the identification of the cellular influences β6 exerts upon the cell proteome, the characterisation of multiple β6-centric pro-metastatic signalling systems and the search for pharmacological therapies (industry and academia) targeted against β6. Once expressional restriction is overcome in early colorectal cancer (CRC), epithelial cell surface restricted αvβ6 can physically interact with, and activate, known oncoproteins, and has the potential to enable the cross-talk through non-canonical signal transduction pathways, resulting in the adoption of an invasive/metastatic phenotype. This recent research has identified numerous interconnections and potential feedback loops, highlighting the fact that the expression of the β6 subunit may initiate a cascade of downstream effects on the CRC cell rather than acting through a single mechanism. We here review these recent studies and postulate that the existence of a cell surface uPAR/αvβ6/TGFβ "metastasome" interactome in/on a proportion of colorectal cancer cells, where β6 expression sequesters and activates multiple systems at the invasive front of tumour lesions, promoting cancer metastasis and hence explaining why β6 has been correlated with reduced patient survival in CRC.

Topics: Antigens, Neoplasm; Cell Movement; Chemokine CXCL12; Colorectal Neoplasms; Eukaryotic Initiation Factor-4E; Humans; Integrins; Neoplasm Metastasis; Promoter Regions, Genetic; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

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

Acute inflammation is a response to an alteration induced by a pathogen or a physical or chemical insult, which functions to eliminate the source of the damage and restore homeostasis to the affected tissue. However, chronic inflammation triggers cellular events that can promote malignant transformation of cells and carcinogenesis. Several inflammatory mediators, such as TNF-α, IL-6, TGF-β, and IL-10, have been shown to participate in both the initiation and progression of cancer. In this review, we explore the role of these cytokines in important events of carcinogenesis, such as their capacity to generate reactive oxygen and nitrogen species, their potential mutagenic effect, and their involvement in mechanisms for epithelial mesenchymal transition, angiogenesis, and metastasis. Finally, we will provide an in-depth analysis of the participation of these cytokines in two types of cancer attributable to chronic inflammatory disease: colitis-associated colorectal cancer and cholangiocarcinoma.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinogenesis; Cholangiocarcinoma; Chronic Disease; Colitis; Colorectal Neoplasms; Gene Expression; Humans; Inflammation; Interleukin-10; Interleukin-6; Oxidative Stress; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Dissemination of cancer cells to distant organ sites is the leading cause of death due to treatment failure in different types of cancer. Mehlen and Puisieux have reviewed the importance of the development of inappropriate cell survival signaling for various steps in the metastatic process and have noted the particular importance of aberrant cell survival to successful colonization at the metastatic site. Therefore, the understanding of mechanisms that govern cell survival fate of these metastatic cells could lead to the understanding of a new paradigm for the control of metastatic potential and could provide the basis for developing novel strategies for the treatment of metastases. Numerous studies have documented the widespread role of Akt in cell survival and metastasis in colorectal cancer, as well as many other types of cancer. Akt acts as a key signaling node that bridges the link between oncogenic receptors to many essential pro-survival cellular functions, and is perhaps the most commonly activated signaling pathway in human cancer. In recent years, Akt2 and Akt3 have emerged as significant contributors to malignancy alongside the well-characterized Akt1 isoform, with distinct non-overlapping functions. This review is aimed at gaining a better understanding of the Akt-driven cell survival mechanisms that contribute to cancer progression and metastasis and the pharmacological inhibitors in clinical trials designed to counter the Akt-driven cell survival responses in cancer.

Topics: Antineoplastic Agents; Cell Survival; Colorectal Neoplasms; Humans; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Protein Isoforms; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Insulin-Like Growth Factor I; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

Chronic inflammation is thought to be the leading cause of many human cancers including colorectal cancer (CRC). Accordingly, epidemiologic and clinical studies indicate that patients affected by ulcerative colitis and Crohn's disease, the two major forms of inflammatory bowel disease, have an increased risk of developing CRC. In recent years, the role of immune cells and their products have been shown to be pivotal in initiation and progression of colitis-associated CRC. On the other hand, activation of the immune system has been shown to cause dysplastic cell elimination and cancer suppression in other settings. Clinical and experimental data herein reviewed, while confirming chronic inflammation as a risk factor for colon carcinogenesis, do not completely rule out the possibility that under certain conditions the chronic activation of the mucosal immune system might protect from colonic dysplasia.

Topics: Animals; Cell Transformation, Neoplastic; Chemokines; Colorectal Neoplasms; Humans; Immune System; Inflammation; Inflammatory Bowel Diseases; Interleukin-10; Interleukin-6; Intestines; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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

Recent progress in the field of molecular biology has allowed us to identify at least two different molecular mechanisms implicated in colorectal carcinogenesis (CRC): chromosomal instability (CIN) and genetic instability. Even though the two molecular mechanisms differ, their signalling pathways, implicated in malignant transformation of colonic epithelial cells, appear to be similar. The most frequent group of CRC, which represents 80% of sporadic CRC, is characterized by allelic losses on the short arm of chromosome 17 and 8 and on the long arm of chromosome 5, 18 and 22. These allelic losses are associated with mutations in TP53, APC, SMAD2 and SMAD4 genes. All of these alterations are grouped under the phenotype CIN. A genetic instability termed MSI (microsatellite instability), which results from a mismatch repair (MMR) deficiency, appears in 12-15% of CRC cases. The presence of MMR deficiency leads to the accumulation of mutations in genes controlling cell cycle and apoptosis (TGFBRII, BAX or CASPASE5). More recently, the existence of a third phenotype was suggested. The main alteration associated with this group of tumors is the hypermethylation of the promoter region of numerous genes, leading to their inactivation. An activating mutation of BRAF is frequently associated with this phenotype. As described above, CRC shows genetic heterogeneity, however the consequences in terms of signalling pathway alterations are similar. For example, the activation of Wnt signalling pathways can result from the inactivation of the APC gene in the CIN phenotype or from an activating mutation in the β-catenin gene in MSI tumors. The inactivation of TGFβ pathways is also present in both tumor types and is driven by SMAD4, and more rarely by a SMAD2 inactivating mutation in CIN tumors, or by the existence of a frame-shift mutation occurring in a polyG coding track of the TGFβ (transforming growth factor) receptor type II in MSI tumors. The RAS-MAP kinase pathway is activated by KRAS mutations in CIN tumors or by BRAF mutations in MSI tumors. The p53 pathway is inactivated by TP53 inactivation in CIN tumors or by BAX inactivating mutations in MSI tumors.

Topics: beta Catenin; Chromosomal Instability; Colorectal Neoplasms; DNA Methylation; DNA Mismatch Repair; Elafin; ErbB Receptors; Genes, p53; Genes, Tumor Suppressor; Humans; Microsatellite Instability; Phenotype; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Transforming Growth Factor beta

Topics: Adenocarcinoma; Bone Morphogenetic Proteins; Cadherins; Claudin-1; Colorectal Neoplasms; Epigenesis, Genetic; Humans; Intercellular Junctions; Membrane Proteins; Neoplasm Metastasis; Neoplastic Processes; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Colorectal cancer (CRC) is the third most common malignancy in the United States. Advances in molecular biology have enhanced the understanding of colorectal carcinogenesis. Approximately 75% of CRCs are sporadic; the rest are hereditary or belong to a familial syndrome. Identification of familial forms of CRC have enabled the development of several models of carcinogenesis and made CRC a well-studied malignancy in terms of molecular pathogenesis. Pathways containing multiple mutations and genetic alterations that play a role in hereditary CRC pathogenesis have been elucidated. Many of the molecular changes seen in these pathways also are involved in the development of sporadic cancers.

Topics: Antineoplastic Agents; Cell Proliferation; Chemotherapy, Adjuvant; Chromosomal Instability; Colorectal Neoplasms; Disease Progression; Humans; Mass Screening; Microsatellite Instability; Molecular Biology; Neovascularization, Pathologic; Positron-Emission Tomography; Prognosis; Tomography, X-Ray Computed; Transforming Growth Factor beta

In 2006, more than 55,000 patients died of colorectal cancer in the US, accounting for approximately 10% of all cancer deaths. Despite significant progress in screening combined with the development of novel effective therapies, colorectal cancer ranks second to lung cancer as a cause of cancer death. Twin studies indicate that 35% of all colorectal cancers are inherited, but high-penetrance tumor susceptibility genes only account for approximately 3-6% of all cases. The remainder of the unexplained familial risk is presumably due to other high-penetrance genes, but polygenic mechanisms and low-penetrance tumor susceptibility genes are likely to account for a greater proportion of familial colorectal cancers. In this regard, there is growing evidence that a common hypomorphic variant of the type I TGF-beta receptor, TGFBR1*6A, may account for approximately 3% of all colorectal cancer cases, a fraction higher than that attributable to mismatch repair genes MLH1, MSH2, MSH6 and PMS2. Furthermore, TGFBR1*6A is emerging as a potent modifier of colorectal cancer risk among individuals with a strong family of colorectal cancer. The TGF-beta signaling pathway plays a central but paradoxical role in the predisposition and progression of colorectal cancer. TGF-beta is a potent inhibitor of normal colonic epithelial cells acting as a tumor suppressor. However, TGF-beta promotes the survival, invasion and metastasis of colorectal cancer cells, thereby acting as an oncogene. Understanding how selective alterations of the TGF-beta signaling pathway contribute to colorectal cancer development and progression will likely permit the identification of an additional fraction of inherited colorectal cancer cases and provide novel opportunities for therapeutic intervention.

Topics: Animals; Colorectal Neoplasms; Humans; Models, Biological; Mutation; Oncogenes; Polymorphism, Genetic; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor Cross-Talk; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wnt Proteins

Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective therapy against human colorectal cancer, involving inhibition of JNK/pSmad3L pathway.

Topics: Adenocarcinoma; Adenoma; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Progression; Humans; MAP Kinase Kinase 4; Neoplasm Invasiveness; Phosphorylation; Protein Isoforms; Serine; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

It has been well documented that there are two major pathways in colorectal carcinogenesis. One is the chromosomal instability pathway (adenoma-carcinoma sequence), which is characterized by allelic losses on chromosome 5q (APC), 17p (p53), and 18q (DCC/SMAD4), and the other is a pathway that involves microsatellite instability. Recent progress in molecular biology, however, has shown that colorectal carcinogenesis is not necessarily clearly divided into these two pathways, but is in fact more complicated. Other routes, including the transforming growth factor-beta/SMAD pathway, the serrated pathway, and the epigenetic pathway, have been reported. Cross talk among these pathways has also been reported. In the invasion and metastasis steps of colorectal cancers, many more genes have now been identified as being involved in proteolysis, adhesion, angiogenesis, and cell growth. Recently accumulated evidence indicates that colorectal cancer is a genetically heterogeneous and complicated disease.

Topics: Adenocarcinoma; Carcinoma; Cell Transformation, Neoplastic; Chromosomal Instability; Colorectal Neoplasms; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Microsatellite Instability; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins

Transforming growth factor-beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells, JNK/pSmad3L-mediated signaling is involved in invasion by activated mesenchymal cells. During sporadic human colorectal carcinogenesis, TGF-beta signaling confers a selective advantage on tumor cells by shifting from the TbetaRI/pSmad3C pathway characteristic of mature epithelial cells to the JNK/pSmad3L pathway, which is more characteristic of the state of flux shown by the activated mesenchymal cells. JNK acts as a regulator of TGF-beta signaling by increasing the basal level of pSmad3L available for action in the nuclei of the invasive adenocarcinoma, in the meantime shutting down TGF-beta-dependent nuclear activity of pSmad3C. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. From the viewpoint of TGF-beta signaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal colorectal epithelial cells at the expense of effects promoting aggressive behavior of the adenocarcinoma. Specific inhibitors of the JNK/pSmad3L pathway might prove useful in this respect. In the case of molecularly targeted therapy for human cancer, pSmad3L and pSmad3C could be assessed as biomarkers to evaluate the likely benefit from specific inhibition of the JNK/pSmad3L pathway.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; Epithelium; Humans; Signal Transduction; Transforming Growth Factor beta

Advances in immunology and molecular biology have shown that colorectal cancer is potentially immunogenic and that host immune responses influence survival. However, immune surveillance and activation is frequently ineffective in preventing and/or controlling tumour growth.. To discuss potential ways in which colorectal cancer induces immune suppression, its effect upon prognosis and avenues for therapeutic development.. A literature review was undertaken for evidence of colorectal cancer-induced immune suppression using PubMed and Medline searches. Further studies were identified from the reference lists of identified papers.. Immune suppression occurs at a molecular and cellular level and can result in a shift from cellular to humoral immunity. Several mechanisms for immune suppression have been described affecting innate and adaptive immunity with suppression linked to poorer clinical outcome.. Colorectal cancer causes direct inhibition of the host's immune response with a detrimental effect upon prognosis. Immunotherapy offers a therapeutic strategy to counteract these effects with promising results seen particularly in precancerous conditions and early tumours. This review strongly suggests that immunotherapy should be incorporated into adjuvant therapeutic trials for stage 2 tumours and be considered as adjuvant treatment in conjunction with standard chemotherapy regimes for advanced disease.

Topics: Apoptosis; CD4-Positive T-Lymphocytes; Colorectal Neoplasms; Cyclooxygenase 2; Down-Regulation; Histamine; Histocompatibility Antigens Class I; HLA Antigens; HLA-G Antigens; Humans; Immune Tolerance; Interleukin-10; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-beta (TGF-beta) activates not only TGF-beta type I receptor (Tbeta RI) but also c-Jun N-terminal kinase (JNK), converting the mediator Smad3 to two distinct phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While Tbeta RI/pSmad3C pathway inhibits growth of normal epithelial cells, the activated mesenchymal cells invade via JNK/pSmad3L pathway. During sporadic human colorectal carcinogenesis, TGF-beta signaling confers a selective advantage upon tumor cells by shifting from epithelial Tbeta RI/pSmad3C pathway to mesenchymal JNK/pSmad3L pathway. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. In a future, specific inhibition of the JNK/pSmad3L pathway will become a therapy for human colorectal cancer that restores the lost tumor-suppressive function observed in normal colorectal epithelial cells at the expense of effects promoting the aggressive behavior.

Topics: Animals; Cell Differentiation; Colorectal Neoplasms; Epithelium; Humans; Mesoderm; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta

During embryonic development, epithelial cells must escape the structural constraints imposed by tissue architecture and adopt a phenotype more amenable to cell movement, a process known as the epithelial-mesenchymal transition (EMT). The progression of carcinomas to invasive and metastatic disease may also involve localized occurrences of EMT. However, data that support the actual occurrence of EMT in specific carcinomas and the relevance of this process to the progression of these tumors had been scant. This review highlights recent studies that substantiate the importance of the EMT to colorectal carcinoma. Specifically, a novel model for studying the EMT of colorectal carcinoma has been used to gain insight into the nature of the EMT itself and to identify molecular events that contribute to disease progression. Although loss of E-cadherin function is a primal event for the EMT, the expression of specific integrins such as alpha(v)beta6 as a consequence of the EMT enables invasive cells to interact with interstitial matrices and to sustain activation of TGF-beta. Of note, alpha(v)beta6 expression in tumors is a marker of cells that have undergone an EMT and it is prognostic for tumors that will progress more rapidly to terminal disease. The EMT also induces autocrine signaling involving VEGF and Flt-1 that enable invasive cells to become 'self-sufficient' for survival. Thus, the EMT appears to be an integral component of colorectal cancer progression and its analysis can yield novel targets for prognosis and therapy.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; Epithelium; Humans; Integrins; Mesoderm; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

It is well established that concentration gradients of signaling molecules (the so-called "morphogens") organize and pattern tissues in developing animals. In particular, studies in Drosophila and different vertebrates have shown that gradients of the Wnt, Hedgehog (Hh) and transforming growth factor-beta (TGF-beta) families of morphogens play critical roles in limb patterning. Morphogens are often expressed in organizing centres and can act over a long range to coordinate the patterning of an entire field of cells. These observations imply that exposure to different concentrations of these diffusible factors may trigger differential cellular responses. In order to study these dosage-dependent Wnt/beta-catenin signaling effects, we have generated several hypomorphic mutant alleles at the mouse Apc locus and studied their cellular and phenotypic outcomes in stem cell renewal and differentiation, and in tumorigenesis. The results clearly show that Apc mutations differentially affect the capacity of stem cells to differentiate in a dosage-dependent fashion. Likewise, different Apc mutations (and the corresponding Wnt signaling dosages) confer different degrees of susceptibility to tumorigenesis in the corresponding mouse models. These results have implications for the understanding of the molecular and cellular basis of tumor initiation by defects in the Wnt pathway. We propose a model in which adult somatic stem cell compartments are characterized by tissue-specific beta-catenin threshold levels for cell proliferation, differentiation and apoptosis. Different APC mutations will result in different levels of beta-catenin signaling, thus conferring different degrees of tumor susceptibility in different tissues. Hence, beta-catenin dosage-dependent effects may not only explain how a single pathway is involved in the development and homeostasis of different tissues, but also its pleiotrophic role in tumorigenesis.

Topics: Adenomatous Polyposis Coli Protein; Alleles; Animals; Cell Differentiation; Colorectal Neoplasms; Disease Progression; Dose-Response Relationship, Drug; Genes, APC; Mice; Models, Biological; Models, Genetic; Mutation; Neoplasms; Phenotype; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Colitis, Ulcerative; Colorectal Neoplasms; Cyclooxygenase 2; Cytokines; DNA Repair; DNA-Binding Proteins; Genes, APC; Genes, DCC; Genes, p53; Genes, ras; Humans; Inflammation Mediators; Isoenzymes; Membrane Proteins; Mutation; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nitric Oxide; Nuclear Proteins; Precancerous Conditions; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Colorectal Neoplasms; Genes, APC; Genes, p53; Humans; Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

Molecular biology studies have led to the identification of two different types of colorectal carcinomas. The first group, called LOH (for loss of heterozygosity), represents 80% of colorectal cancers and is characterised by aneuploidy, allelic losses and a location in the distal colon. The second group displays phenotypic microsatellite instability (MSI-positive tumours), has a near-diploid karyotype and a relatively low frequency of allelic losses. It accounts for 15% of all colorectal cancers and for about 30% of right-sided cancers. Four different pathways have been identified as responsible for tumour progression: the WNT/Wingless, the K-ras, the Transforming growth factor (TGF) and the P53 pathways. The involvement of these pathways depends on the tumour type. In LOH-positive tumours, the WNT/Wingless pathway is activated through an APC mutation, whereas MSI+ tumours do so through a catenin stabilising mutation. The TGFb growth inhibitory pathway is altered either by mutations in the signal transduction molecules SMAD2 and SMAD4 in LOH positive tumours or by mutations of TGFbRII in MSI+ tumours. In the p53 pathway, mutations in BAX may contribute to the adenoma-carcinoma transition just as p53 mutations may do in LOH positive tumours. Until now, cancer phenotype determination has had no clinical implications. However, the predictive value of the MSI status was recently stressed as a predictive factor for response to chemotherapy. Immunohistochemistry could represent a complementary strategy to molecular biology in assessing MSI status. This simple test would allow to screen all colorectal carcinomas for MSI status, which would provide valuable management information in addition to the histological assessment for tumour stage and grade.

Topics: Aneuploidy; Colonic Neoplasms; Colorectal Neoplasms; Disease Progression; Genes, ras; Humans; Loss of Heterozygosity; Molecular Biology; Precancerous Conditions; Proto-Oncogene Proteins; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

Colorectal carcinogenesis is a multistep process during which the specialised epithelial cells of intestinal mucosa surface (e.g. colonocytes) accumulate a series of genetic and epigenetic events which lead to a perturbation of their normal cellular functions and turnover. This review will address the mechanisms and biological effects of these abnormalities on the growth control, differentiation, adhesion and survival of the colonocytes.

Topics: Animals; beta Catenin; Cadherins; Cell Adhesion; Cell Differentiation; Colorectal Neoplasms; Cytoskeletal Proteins; Genes, APC; Humans; Intestinal Mucosa; Mutation; Proto-Oncogene Proteins; Signal Transduction; Trans-Activators; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

Topics: Adenomatous Polyposis Coli; Biomarkers, Tumor; Colorectal Neoplasms; Genes, APC; Genes, p53; Genes, ras; Germ-Line Mutation; Humans; Loss of Heterozygosity; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

There is substantial evidence to support the contention that the Smad portion of the TGF-beta signal transduction pathway provides an important tumor-suppressor function. Mutational loss of function of Smad pathway members have been associated with the development of human cancers and appear to be causative in selected rodent carcinogenesis models. TGF-beta also has multiple other actions that appear to be independent of the growth-inhibitory/tumor suppressor effects. The predominant effect of TGF-beta appears to be dependent on the context of the responding cell. Once transformation has occurred, TGF-beta effects may be detrimental and may actually promote tumor cell survival, invasion, and metastasis. Recent work suggests that these effects may involve TGF-beta regulation of COX-2 and other pathways that may contribute to tumor cell aggressiveness. In gaining a better understanding of the mechanisms by which TGF-beta may promote tumor progression, it is likely that new therapeutic strategies may be developed that preserve tumor-suppressor function of TGF-beta while inhibiting the tumor-promoting effects.

Topics: Adenoma; Animals; Colorectal Neoplasms; Humans; Mice; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The growth hormone (GH)-insulin-like growth factor (IGF)-I axis is an important modulator of growth and development, but in addition to their classical role as endocrine hormones, its components also regulate a wide range of biological functions through paracrine and autocrine mechanisms. Their potent mitogenic and anti-apoptotic effects play a critical role in the regulation of rapidly renewing epithelial cell populations such as those found in the colon. Recent evidence suggests an association between inappropriate regulation of the GH-IGF-I axis and the development of colorectal cancer. However, the molecular mechanisms and signalling pathways responsible are only beginning to be unravelled, as are the relative contributions of the endocrine and autocrine or paracrine effects.

Topics: Acromegaly; Animals; Apoptosis; Colorectal Neoplasms; Cyclooxygenase 2; Endothelial Growth Factors; Growth Hormone; Humans; Immune Tolerance; Insulin-Like Growth Factor Binding Proteins; Isoenzymes; Lymphokines; Membrane Proteins; Neovascularization, Pathologic; Prostaglandin-Endoperoxide Synthases; Receptors, Somatomedin; Signal Transduction; Somatomedins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Colorectal carcinogenesis is a multi-step process during which genetic and epigenetic events determine the transition from a normal cell to a malignant cell. In the past decade, intensive research led to the identification of two types of colorectal cancers that are distinct by their carcinogenesis process. The first group, named LOH (Loss of Heterozygosity) type, is characterized by allelic losses of tumor suppressor genes. The second group, called MSI (Microsatellite Instability) type, is characterized by genetic instability at microsatellite loci. In addition, recent studies suggest the four different signaling pathways, Wnt/Wingless pathway, K-ras pathway, TGF-beta pathway and p53 pathway, could be implicated in tumor progression. In this review we discuss the development of colorectal cancer on the basis of molecular biology and recent advances in research of the colorectal cancer.

Topics: Colorectal Neoplasms; Humans; Loss of Heterozygosity; Microsatellite Repeats; ras Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Stem cell research provides a foundation for therapeutic advancement in oncology, clinical genetics and a diverse array of degenerative disorders. For example, the elucidation of pathways governing proliferative regulation and differentiation within cellular systems will result in medical strategies aimed at the root cause of cancer. At present the characterization of reliable stem cell markers is the immediate aim in this particular field. Over the past 30 years investigators have determined many of the physical and functional properties of stem cells through careful and imaginative experimentation. Intestinal stem cells reside at the crypt base and give rise to all cell types found within the crypt. They readily undergo altruistic apoptosis in response to toxic stimuli although their progeny are hardier and will regain stem cell function to repopulate the tissue compartment, giving rise to the concept of a proliferative hierarchy. Contention exists when deciding whether the full complement of cells within a crypt is derived from either a single or multiple stems. Evidence has also arisen to challenge the long held view that colorectal tumours arise from a single mutated stem cell, as early adenomas from a human XO/XY mosaic contained distinct clones. Mechanisms governing the stem cell cycle and subsequent proliferative activity largely remain obscure. The adenomatous polyposis coli gene product has, however, been shown to promote the degradation of beta-catenin, an enhancer of cell proliferation, thereby downregulating this activity in healthy individuals.

Topics: Adenomatous Polyposis Coli; Animals; Cell Differentiation; Cell Division; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cyclooxygenase 2; Glucagon-Like Peptides; Humans; Intestinal Mucosa; Intestines; Isoenzymes; Membrane Proteins; Neoplasms; Peptides; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stem Cells; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Topics: Adenomatous Polyposis Coli; Base Pair Mismatch; beta Catenin; Colorectal Neoplasms; Cytoskeletal Proteins; DNA Repair; DNA-Binding Proteins; Humans; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Topics: Adenoma; Animals; Cell Division; Cell Transformation, Neoplastic; Colorectal Neoplasms; DNA-Binding Proteins; Gene Expression Regulation; Humans; Intestinal Polyps; Mice; Mutation; Signal Transduction; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Recent work has shown that: 1) loss of transforming growth factor-beta response is associated with malignant progression, 2) maintenance of autocrine negative transforming growth factor-beta activity is a key impediment to malignant progression, and 3) the major mechanism for loss of RII expression in replication error-positive colorectal cancer patients is mutation of the poly A tract of the transforming growth factor-beta receptor type II (RII) gene resulting in the generation of a premature STOP codon. Major issues for the role of RII in cancer are identified as the determination of the penetrance of mechanisms of RII loss in non-replication error tumors and other types of malignancies in addition to colon cancer. Analysis of mechanism of RII loss may prove to have clinical use in defining the clinical course of subset of different types of malignancies and, in addition, it may result in the identification of new therapeutic targets and approaches for some subsets of cancers.

Topics: Colorectal Neoplasms; Genes, Tumor Suppressor; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

There are a number of lines of evidence suggesting that transforming growth factor beta (TGF beta) has an important role in the control of intestinal growth and differentiation. In vivo localization studies show that TGF beta expression occurs predominantly in the differentiated non proliferating cells of the intestinal epithelium. The use of an antisense expression vector for TGF beta resulted in an increased tumorigenicity in an antisense-transfected cancer cell line. In vitro proliferation studies showed colorectal premalignant adenoma cells to be more sensitive to the growth inhibitory effects of TGF beta than colorectal cancer cells. Furthermore the conversion of an adenoma to a carcinoma was accompanied by a reduced response to the inhibitory effects of TGF beta. The acquisition of partial or complete resistance to the inhibitory effects of TGF beta may be an important late event in colorectal carcinogenesis. Of further interest is the possibility that clonal selection could occur even more rapidly in colorectal tumour cells which not only had lost response to TGF beta inhibition but produced TGF beta and were growth stimulated by it. This could have the advantage of not only inhibiting the growth of surrounding less malignantly advanced cells but of also escaping from their potential growth suppressive influence. Carcinogenesis is not, however, simply losing response to negative regulators of growth; the fully malignant cell has to acquire new characteristics of invasiveness and metastatic potential. Growth factors including TGF beta may have a role in the complex cascade of events leading to the activation of proteolytic enzymes which are involved in progression to an invasive phenotype. Cell proliferation in the large bowel, as well as being under the control of endogenous growth factors, is also under the influence of dietary components in the lumen such as the naturally occurring fatty acid sodium butyrate. Sodium butyrate at physiological concentrations induces apoptosis (programmed cell death) in colonic tumour cell lines. Since sodium butyrate occurs naturally in the colorectum, being produced by bacterial fermentation of dietary fibre, it may be involved in the control of cell death in human colorectal epithelium. This could, in part, explain the apparent protective effects of dietary fibre. Clonal evolution and tumour progression in colorectal carcinogenesis could therefore involve loss of response to endogenous growth factors such as TGF

Topics: Apoptosis; Butyrates; Butyric Acid; Cell Division; Colorectal Neoplasms; Dietary Fiber; Growth Substances; Humans; Neoplasm Invasiveness; Transforming Growth Factor beta

Topics: Animals; Cell Division; Colorectal Neoplasms; Drug Resistance; Gene Expression Regulation; Humans; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

The gene for familial adenomatous polyposis coli (APC or FAP), which has previously been linked to chromosome 5q21 has been identified. The APC gene has been found to be altered by point mutations in the germ line of both adenomatous polyposis coli and Gardner's syndrome patients and somatically in tumors from sporadic colorectal cancer patients. During the hunt for the APC gene, the closely linked MCC (mutated in colorectal cancer) gene was identified and found to be altered somatically in tumors from sporadic cancer patients. These data suggest that more than one gene on chromosome 5q21 may contribute to colorectal carcinogenesis and that mutations at the APC gene can cause both adenomatous polyposis coli and Gardner's syndrome. The identification of these genes should aid in the counseling of patients with genetic predispositions to colorectal cancer. Progress has also been made in identifying specific genetic changes that occur in other gastrointestinal cancers. A mutational "hotspot" in the p53 gene in human hepatocellular carcinomas has been identified that could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1.

Topics: Adenomatous Polyposis Coli; Animals; Biomarkers, Tumor; Chromosome Aberrations; Chromosome Deletion; Colorectal Neoplasms; Diet; Gardner Syndrome; Gastrointestinal Neoplasms; Genes, p53; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Oncogenes; Rats; Transforming Growth Factor beta

Colorectal cancer (CRC) is a heterogeneous and complex disease with limited treatment options. Targeting transforming growth factor β (TGF-β) and programmed death ligand 1 pathways may enhance antitumor efficacy. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-β receptor II (a TGF-β "trap") fused to a human IgG1 monoclonal antibody blocking programmed cell death ligand 1. We report results from an expansion cohort of a phase I study (NCT02517398) in patients with heavily pretreated advanced CRC treated with bintrafusp alfa. As of May 15, 2020, 32 patients with advanced CRC had received bintrafusp alfa for a median duration of 7.1 weeks. The objective response rate was 3.1% and the disease control rate was 6.3% (1 partial response, 1 stable disease); 2 patients were not evaluable. The safety profile was consistent with previously reported data.

Topics: Antibodies, Monoclonal; B7-H1 Antigen; Colorectal Neoplasms; Humans; Immunologic Factors; Transforming Growth Factor beta

CALGB/SWOG 80405 was a randomized phase III trial in first-line patients with metastatic colorectal cancer treated with bevacizumab, cetuximab, or both, plus chemotherapy. We tested the effect of tumor immune features on overall survival (OS).. Primary tumors (N = 554) were profiled by RNA sequencing. Immune signatures of macrophages, lymphocytes, TGFβ, IFNγ, wound healing, and cytotoxicity were measured. CIBERSORTx scores of naive and memory B cells, plasma cells, CD8+ T cells, resting and activated memory CD4+ T cells, M0 and M2 macrophages, and activated mast cells were measured.. Increased M2 macrophage score [HR, 6.30; 95% confidence interval (CI), 3.0-12.15] and TGFβ signature expression (HR, 1.35; 95% CI, 1.05-1.77) were associated with shorter OS. Increased scores of plasma cells (HR, 0.55; 95% CI, 0.38-0.87) and activated memory CD4+ T cells (HR, 0.34; 95% CI, 0.16-0.65) were associated with longer OS. Using optimal cutoffs from these four features, patients were categorized as having either 4, 3, 2, or 0-1 beneficial features associated with longer OS, and the median (95% CI) OS decreased from 42.5 (35.8-47.8) to 31.0 (28.8-34.4), 25.2 (20.6-27.9), and 17.7 (13.5-20.4) months respectively (P = 3.48e-11).. New immune features can be further evaluated to improve patient response. They provide the rationale for more effective immunotherapy strategies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Cetuximab; Colorectal Neoplasms; Humans; Transforming Growth Factor beta; Treatment Outcome

Inflammation, hormones and energy-related factors have been associated with colorectal cancer (CRC) and it has been proposed that convergence and interactions of these factors importantly influence CRC risk. We have previously hypothesized that genetic variation in the CHIEF (convergence of hormones, inflammation and energy-related factors) pathway would influence risk of CRC. In this paper, we utilize an Adaptive Rank Truncation Product (ARTP) statistical method to determine the overall pathway significance and then use that method to identify the key elements within the pathway associated with disease risk. Data from two population-based case-control studies of colon (n = 1555 cases and 1956 controls) and rectal (n = 754 cases and 959 controls) cancer were used. We use ARTP to estimate pathway and gene significance and polygenic scores based on ARTP findings to further estimate the risk associated with the pathway. Associations were further assessed based on tumor molecular phenotype. The CHIEF pathway was statistically significant for colon cancer (P(ARTP)= 0.03) with the most significant interferons (P(ARTP) = 0.0253), JAK/STAT/SOCS (P(ARTP) = 0.0111), telomere (P(ARTP) = 0.0399) and transforming growth factor β (P(ARTP) = 0.0043) being the most significant subpathways for colon cancer. For rectal cancer, interleukins (P(ARTP) = 0.0235) and selenoproteins (P ARTP = 0.0047) were statistically significant although the pathway overall was of borderline significance (P(ARTP) = 0.06). Interleukins (P(ARTP) = 0.0456) and mitogen-activated protein kinase (P(ARTP) = 0.0392) subpathways were uniquely significant for CpG island methylator phenotype-positive colon tumors. Increasing number of at-risk alleles was significantly associated with both colon [odds ratio (OR) = 6.21, 95% confidence interval (CI): 4.72, 8.16] and rectal (OR = 7.82, 95% CI: 5.26, 11.62) cancer. We conclude that elements of the CHIEF pathway are important for CRC risk.

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Colorectal Neoplasms; Female; Follow-Up Studies; Genetic Predisposition to Disease; Genotype; Humans; Interferons; Janus Kinase 1; Male; Middle Aged; Phenotype; Polymorphism, Single Nucleotide; Prognosis; Risk Factors; Signal Transduction; STAT1 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Telomere; Transforming Growth Factor beta; United States

The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFβ, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by

Topics: Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Humans; Multiomics; Smad4 Protein; Transforming Growth Factor beta

Adenosine N6-methylation (m6A) and N6,2'-O-dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalyzed by the methyl transferase phosphorylated CTD-interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9-mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti-PD-1 antibody treatment by decreasing intratumoral TGF-β levels and increasing intratumoral IFN-γ, TNF-α levels, and tumor-infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti-PD-1 in a context-dependent mechanism with PCIF1 directly targeting FOS, IFITM3, and STAT1 via m6Am modifications. PCIF1 stabilizes FOS mRNA, which in turn leads to FOS-dependent TGF-β regulation and tumor growth. While during immunotherapy, Pcif1-Fos-TGF-β, as well as Pcif1-Stat1/Ifitm3-IFN-γ axes, contributes to the resistance of anti-PD-1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti-PD-1 therapy, supporting that the combination of PCIF1 inhibition with anti-PD-1 treatment is a potential therapeutic strategy to enhance CRC response to immunotherapy. Finally, we developed a lipid nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)-based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof-of-concept for tumor growth suppression using LNP-CMsiRNA to silence target genes in cancer.

Topics: Adaptor Proteins, Signal Transducing; Animals; Colorectal Neoplasms; Humans; Immunotherapy; Membrane Proteins; Methylation; Mice; Nuclear Proteins; RNA-Binding Proteins; RNA, Messenger; Transforming Growth Factor beta

Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor β (TGFβ) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFβ signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFβ receptor 1 (TGFβR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFβR1 mRNA and protein expression in these cells. Effects on TGFβR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFβR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFβR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCα→ERK→Runx2→TGFβR1 signaling axis. In further support of a link between PKCα and TGFβ signaling, PKCα knockdown reduced the ability of TGFβ to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFβR1 decreased PKCα-induced upregulation of p21

Topics: Colorectal Neoplasms; Core Binding Factor Alpha 1 Subunit; Epithelial Cells; Humans; Intestines; Protein Kinase C-alpha; Receptor, Transforming Growth Factor-beta Type I; RNA, Messenger; Transforming Growth Factor beta

Perturbations in transforming growth factor-β (TGF-β) signaling can lead to a plethora of diseases, including cancer. Mutations and posttranslational modifications (PTMs) of the partner of SMAD complexes contribute to the dysregulation of TGF-β signaling. Here, we reported a PTM of SMAD4, R361 methylation, that was critical for SMAD complexes formation and TGF-β signaling activation. Through mass spectrometric, co-immunoprecipitation (Co-IP) and immunofluorescent (IF) assays, we found that oncogene protein arginine methyltransferase 5 (PRMT5) interacted with SMAD4 under TGF-β1 treatment. Mechanically, PRMT5 triggered SMAD4 methylation at R361 and induced SMAD complexes formation and nuclear import. Furthermore, we emphasized that PRMT5 interacting and methylating SMAD4 was required for TGF-β1-induced epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis, and SMAD4 R361 mutation diminished PRMT5 and TGF-β1-induced metastasis. In addition, highly expressed PRMT5 or high level of SMAD4 R361 methylation indicated worse outcomes in clinical specimens analysis. Collectively, our study highlights the critical interaction of PRMT5 and SMAD4 and the roles of SMAD4 R361 methylation for controlling TGF-β signaling during metastasis. We provided a new insight for SMAD4 activation. And this study indicated that blocking PRMT5-SMAD4 signaling might be an effective targeting strategy in SMAD4 wild-type CRC.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Humans; Neoplasm Metastasis; Protein-Arginine N-Methyltransferases; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hsa-miR-495 (miR-495) has been extensively investigated in cancer initiation and progression. On the other hand, our bioinformatics analysis suggested that miR-495 exerts its effects through targeting of TGFβ signaling components.. In order to investigate such an effect, miR-495 precursor was overexpressed in HEK293T, SW480, and HCT116 cells, which was followed by downregulation of TGFβR1, TGFβR2, SMAD4, and BUB1 putative target genes, detected by RT-qPCR. Also, luciferase assay supported the direct interaction of miR-495 with 3'UTR sequences of TGFβR1, TGFβR2, SMAD4, and BUB1 genes. Furthermore, a negative correlation of expression between miR-495-3p and some of these target genes was deduced in a set of colorectal and breast cancer cell lines. Then, flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase. Consistently, western blotting analysis showed a significant reduction of the Cyclin D1 protein in the cells overexpressing miR-495, pointing to downregulation of the TGFβ signaling pathway and cell cycle arrest. Finally, microarray data analysis showed that miR-495-3p is significantly downregulated in colorectal tumors, compared to the normal pairs.. Overall, the results of the current study introduced miR-495-3p as a cell cycle progression suppressor, which may negatively regulate TGFβR1, TGFβR2, SMAD4, and BUB1 genes. This finding suggests miR-495-3p as a tumor suppressor candidate for further evaluation.

Topics: Cell Cycle Checkpoints; Colorectal Neoplasms; HEK293 Cells; Humans; MicroRNAs; Protein Serine-Threonine Kinases; Smad4 Protein; Transforming Growth Factor beta

Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. Transwell

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Proto-Oncogene Proteins c-myc; RNA-Binding Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Immunosuppressive factors within the tumor microenvironment (TME), such as Transforming growth factor beta (TGF-β), constitute a crucial hindrance to immunotherapeutic approaches in colorectal cancer (CRC). Furthermore, immune checkpoint factors (e.g., programmed death-ligand 1 [PD-L1]) inhibit T-cell proliferation and activation. To cope with the inhibitory effect of immune checkpoints, the therapeutic value of dual targeting PD-L1 and TGF-β pathways via M7824 plus 5-FU in CRC has been evaluated. Integrative-systems biology approaches and RNAseq were used to assess the differential level of genes associated with 88 metastatic-CRC patients. The level of PD-L1 and TGF-β was evaluated in a validation cohort. The anti-proliferative, migratory, and apoptotic effects of PD-L1/TGF-β inhibitor, M7824, were assessed by MTT, wound-healing assay, and flow cytometry. Anti-tumor activity was assessed in a xenograft model, followed by biochemical studies and histological staining, and gene/protein expression analyses by RT-PCR and ELISA/IHC. The result of differentially expressed genes (DEGs) analysis showed 1268 upregulated and 1074 downregulated genes in CRC patients. Among the highest scoring genes and dysregulated pathways associated with CRC, PD-L1, and TGF-β were identified and further validated in 92 CRC patients. Targeting of PD-L1-TGF-β inhibited cell growth and migration, associated with modulation of CyclinD1 and MMP9. Furthermore, M7824 inhibited tumor growth via targeting TGF-β and PD-L1 pathways, resulting in modulation of inflammatory response and fibrosis via TNF-α/IL6/CD4-8 and COL1A1/1A2, respectively. In conclusion, our data illustrated that co-targeting PD-L1 and TGF-β pathways increased the effect of Fluorouracil (5-FU) and reduced the tumor growth in PD-L1/TGF-β expressing tumors, providing a new therapeutic option in the treatment of CRC.

Topics: B7-H1 Antigen; Colorectal Neoplasms; Fluorouracil; Humans; Transforming Growth Factor beta; Tumor Microenvironment

The Makorin ring finger protein 1 (MKRN1) gene, also called RNF61, is located on the long arm of chromosome 7 and is a member of the RING finger protein family. The E3 ubiquitin ligase MKRN1 is closely linked to tumour development, but the exact mechanism needs to be elucidated. In this study, we aimed to investigate the specific mechanism and role of MKRN1 in colorectal cancer (CRC) development.. MKRN1 expression in CRC was analysed using the Cancer Cell Line Encyclopaedia and the Cancer Genome Atlas (TCGA) databases. Rectal tumour tissues were frozen to explore the MKRN1 expression in CRC and its clinical significance. The impact of MKRN1 on CRC cell proliferation and migration was observed using CCK8, colony formation, wound healing, and transwell assays. A combination of MKRN1 quantitative proteomics, ubiquitination modification omics analysis, and a string of in vitro and in vivo experiments revealed the potential mechanisms by which MKRN1 regulates CRC metastasis.. MKRN1 expression was significantly elevated in CRC tissues compared to paracancerous tissues and was positively linked with prognosis (P < 0.01). MKRN1 downregulation inhibits CRC cell proliferation, migration, and invasion. Conversely, MKRN1 overexpression promotes the proliferation, migration, and invasion of CRC cells. Mechanistically, MKRN1 induces epithelial-mesenchymal transition (EMT) in CRC cells via ubiquitination and degradation of Smad nuclear-interacting protein 1 (SNIP1). Furthermore, SNIP1 inhibits transforming growth factor-β (TGF-β) signalling, and MKRN1 promotes TGF-β signalling by degrading SNIP1 to induce EMT in CRC cells. Finally, using conditional knockout mice, intestinal lesions and metastatic liver microlesions were greatly reduced in the intestinal knockout MKRN1 group compared to that in the control group.. High MKRN1 levels promote TGF-β signalling through ubiquitination and degradation of SNIP1, thereby facilitating CRC metastasis, and supporting MKRN1 as a CRC pro-cancer factor. The MKRN1/SNIP1/TGF-β axis may be a potential therapeutic target in CRC.

Topics: Animals; Cell Line; Cell Proliferation; Colorectal Neoplasms; Humans; Mice; Proteolysis; Ribonucleoproteins; RNA-Binding Proteins; Signal Transduction; Transforming Growth Factor beta

Several studies have shown that the TGF-β signaling pathway plays a critical role in colorectal cancer (CRC) pathogenesis. The aim of the current study is to investigate the therapeutic potential of Vactosertib (EW-7197), a selective inhibitor of TGF-β receptor type I, either alone or in combination with the standard first-line chemotherapeutic treatment, 5-Fluorouracil (5-FU), in CRC progression in both cellular and animal models.. Real-Time PCR, Zymography, enzyme-linked immunosorbent assay (ELISA), Hematoxylin and Eosin (H&E) tissue staining, and Flow cytometry techniques were applied to determine the anti-tumor properties of this novel TGF-β inhibitor in in vitro (CT-26 cell line) and in vivo (inbred BALB/C mice) samples.. Our findings showed that Vactosertib decreased cell proliferation and induced spheroid shrinkage. Moreover, this inhibitor suppressed the cell cycle and its administration either alone or in combination with 5-FU induced apoptosis by regulating the expression of p53 and BAX proteins. It also improved 5-FU anti-cancer effects by decreasing the tumor volume and weight, increasing tumor necrosis, and regulating tumor fibrosis and inflammation in an animal model. Vactosertib also enhanced the inhibitory effect of 5-FU on invasive behavior of CRC cells by upregulating the expression of E-cadherin and inhibiting MMP-9 enzymatic activity.. This study demonstrating the potent anti-tumor effects of Vactosertib against CRC progression. Our results clearly suggest that this inhibitor could be a promising agent reducing CRC tumor progression when administered either alone or in combination with standard treatment in CRC patients.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Colorectal Neoplasms; Fluorouracil; Humans; Mice; Mice, Inbred BALB C; Transforming Growth Factor beta

Metastasis is the leading cause of cancer-related death, where TGFβ-induced epithelial-mesenchymal transition (EMT) process confers on cancer cells increased metastatic potential. However, the involvement of circRNAs in this process is still obscure. Here, we identify a TGFβ-induced circRNA called circITGB6 as an indispensable factor during the TGFβ-mediated EMT process. circITGB6 is significantly upregulated in metastatic cancer samples and its higher abundance is closely correlated to worse prognosis of colorectal cancer (CRC) patients. Through gain- and loss-of-function assays, circITGB6 is found to potently promote EMT process and tumor metastasis in various models in vitro and in vivo. Mechanistically, circITGB6 enhances the mRNA stability of PDPN, an EMT-promoting gene, by directly interacting with IGF2BP3. Notably, interfering circITGB6 with PEI-coated specific siRNA effectively represses liver metastasis. Therefore, our study reveals the function of a TGFβ-regulated circRNA in tumor metastasis and suggests that targeting circITGB6 is a promising strategy for cancer therapy.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Neoplasm Metastasis; RNA, Circular; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the most prevalent and deadly cancers worldwide. It is still necessary to further define the mechanisms and explore the therapeutic targets of CRC. Long non-coding RNA taurine upregulated gene 1 (LncRNA TUG1) was initially discovered as a transcript upregulated by taurine and is observed to be expressed in numerous human cancers. The Study Aim: This article was to explore the correlation between transforming growth factor-beta (TGF-β)/tumor protein 53 (P53) signaling mechanisms as regulators for LncRNA TUG1 in Egyptian patients with CRC.. Immunohistochemical (IHC) staining was achieved to study TGF-β and P53 expression in CRC specimens vs. normal colonic specimens and quantitative real-time PCR (qRT-PCR) was used to analyze LncRNA TUG1, TGF-β, and P53 relative gene expression in 96 tissue specimens (neoplastic specimens and the corresponding adjacent non-neoplastic specimens).. The expressions of LncRNA TUG1, TGF-β, and P53 were overexpressed significantly in CRC specimens as opposed to the matched neighboring non-neoplastic specimens (P<0.001), also LncRNA TUG1 was significantly positively correlated to the expression of TGF-β and P53 (r=0.89, 0.91 respectively, P<0.001).. These findings reveal that LncRNA TUG1 may be a molecular component in the TGF-β/P53 signaling pathway, and LncRNA TUG1 could function as a CRC possible oncogene. LncRNA TUG1 may serve as a potential oncogene for CRC. The TGF-β/P53/LncRNA TUG1 interactions may be employed as potential targets for CRC diagnosis, prognostic evaluation, and cure.

Topics: Colorectal Neoplasms; Humans; RNA, Long Noncoding; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Monocytic myeloid-derived suppressor cells (M-MDSCs) are significantly expanded in the blood of colorectal cancer (CRC) patients. However, their presence and underlying mechanisms in the tumour microenvironment of CRC have not been examined in detail.. Tumour tissues and peripheral blood from CRC patients were analysed for the presence of M-MDSCs. The mechanisms of suppression were analysed by blocking pathways by which MDSCs abrogate T cell proliferation. Co-culture of CRC cells with monocytes were performed with and without cytokine blocking antibodies to determine the mechanism by which CRC cells polarise monocytes. Multi-spectral IHC was used to demonstrate the intra-tumoral location of M-MDSCs.. Tumour tissues and blood of CRC patients contain M-MDSCs which inhibit T cell proliferation. Whilst inhibition of arginase and nitric oxide synthase 2 fail to rescue T cell proliferation, blockade of IL-10 released by these HLA-DR. Our study demonstrates the cross-talk between CRC cells, M-MDSCs and T cells. Characterisation of CRC M-MDSCs point to therapeutic avenues to target these cells in addition to TGF-β blockade.

Topics: Colorectal Neoplasms; HLA-DR Antigens; Humans; Interleukin-10; Monocytes; Myeloid-Derived Suppressor Cells; Phenotype; Transforming Growth Factor beta; Tumor Microenvironment

Fuzheng Xiaojijinzhan (FZXJJZF) decoction is an effective prescription for treating colorectal cancer liver metastasis (LMCRC).. To elucidate the pharmacological mechanism of the FZXJJZF decoction therapy on LMCRC.. Firstly, a network pharmacological approach was used to characterize the underlying targets of FZXJJZF on LMCRC. Secondly, LMCRC-related genes are obtained from the public database TCGA, and those genes are further screened and clustered through Mfuzz, an R package tool. Then, targets of FZXJJZF predicted by network pharmacology were overlapped with LMCRC related genes screened by Mfuzz. Meanwhile, FZJZXJF intervened in LMCRC model,epithelial-to-mesenchymal transition (EMT), and migration and invasion of HCT-116 cells. Thirdly, the transcriptomics data of FZJZXJF inhibited HCT-116 cells of EMT cells were overlapped with EMT database data to narrow the possible range of targets. Based on this, the potential targets and signal pathways of FZJZXJF were speculated by combining the transcriptomics data with the targets from network pharmacology-TCGA. Finally, the anti-cancer mechanism of FZXJJZF on LMCRC was verified in vitro by Real-Time PCR and Western Blot in vitro.. By network pharmacological analysis, 282 ingredients and 429 potential targets of FZXJJZF were predicted. The 9268 LMCRC-related genes in the TCGA database were classified into 10 clusters by the Mfuzz. The two clustering genes with the most similar clustering trends were overlapped with 429 potential targets, and 32 genes were found, such as CD34, TRPV3, PGR, VDR, etc. In vivo experiments, FZJZXJF inhibited the tumor size in LMCRC models, and the EMT, migration, and invasion of HCT-116 also be inhibited. Intersecting transcriptomics dates with 32 target genes, it is speculated that the VDR-TGF-β signaling pathway may be an effective mechanism of FZXJJZF. Additionally, it is shown that FZXJJZF up-regulated the expression levels of VDR and E-cadherin and down-regulated the expression levels of TGF-β and Snail1 in vitro. These results confirmed that FZXJJZF plays an effective role in LMCRC mainly by inhibiting EMT phenotype via the VDR-TGF-β signaling pathway.. Collectively, this study reveals the anti-LMCRC effect of FZXJJZF and its potential therapeutic mechanism from the perspective of potential targets and potential pathways.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Movement; Colorectal Neoplasms; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; HCT116 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Network Pharmacology; Receptors, Calcitriol; Signal Transduction; Snail Family Transcription Factors; Transcriptome; Transforming Growth Factor beta

As conventional therapeutics failed to provide satisfied outcomes against one of the most prevalent cancers, colorectal cancer (CRC), we purposed to implicate MicroRNA (miR)-34a, as a major tumor suppressor, to be delivered by tumor-derived exosomes (TEXs) and investigated its anti-tumor functions in-vivo.. TEXs were isolated from CT-26 cell line and loaded with miR-34a mimic. Then, mice bearing CRC were treated with miR-34a-enriched TEX (TEX-miR-34a) and then examined for the relative tumor-suppressive impacts of the TEX as well as its potential in promoting an anti-tumor immune response.. TEX-miR-34a significantly reduced tumor size and prolonged survival of mice bearing CRC. TEX-miR-34a was able to diminish gene expressions related to invasion, angiogenesis and immune evasion. It was also capable of inducing T cell polarization toward CD8+ T subsets among tumor-infiltrating lymphocytes, draining lymph nodes (DLNs) and spleen cells. Moreover, cytotoxic T cells were professionally induced in mice receiving TEX-miR-34a and the secretion of interleukin (IL)-6, IL-17A and tumor necrosis factor (TGF)-β was reduced in DLNs. However, the enhanced levels of interferon-γ were evaluated in DLN and spleen displaying the polarization of anti-tumor immune responses. Interestingly, mice receiving TEX alone showed a noticeable reduction in certain oncogenic gene expressions as well as IL-17A secretion in DLNs.. TEX-miR-34a demonstrated the potential to induce beneficial anti-tumor immune responses and TEXs, aside from the delivery function of miRNA, revealed certain anti-tumor beneficial characteristics which could introduce TEX-miR-34a as a promising approach in CRC combination therapies.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Drug Delivery Systems; Exosomes; Gene Expression; Gene Expression Regulation, Neoplastic; Interleukin-17; Lymphocytes, Tumor-Infiltrating; Mice; MicroRNAs; T-Lymphocytes, Cytotoxic; Tomography, X-Ray Computed; Transforming Growth Factor beta

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic Kras

Topics: Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Snail Family Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment; Zinc Finger E-box-Binding Homeobox 1

Different molecular signaling pathways have been involved in the incidence and progression of CRC. We aimed to examine the correlation between eight candidate genes, including TFGβ, SMAD2, SMAD4, RhoA, EGFR, MAP2K1, MTA1, and LEF1 in the progression of colorectal cancer (CRC) and their association with clinicopathological variables and CRC patients prognosis. Immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) analysis 2

Topics: Adult; Aged; Colorectal Neoplasms; Disease Progression; ErbB Receptors; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Transforming Growth Factor beta; Wnt Signaling Pathway; Young Adult

Metastasis is the primary cause of cancer mortality, and colorectal cancer (CRC) frequently metastasizes to the liver. Our previous studies demonstrated the critical role of KIAA1199 in tumor invasion and metastasis in CRC. In the present study, we described an immune regulatory effect of KIAA1199 that creates a permissive environment for metastasis.. Flow cytometry was used to examine the effects of KIAA1199 on the infiltration of tumor immune cells. Neutrophils and T cells were isolated, stimulated, and/or cultured for in vitro function assays. In the patients with CRC, high expression levels of KIAA1199 were associated with an increased neutrophil infiltration into the liver. This result was further validated in mouse metastasis models. The increased influx of neutrophils contributed to the KIAA1199-driven CRC liver metastasis. Mechanistically, KIAA1199 activated the TGFβ signaling pathway by interacting with the TGFBR1/2 to stimulate CXCL1 and CXCL3 production, thereby driving the aggregation of immunosuppressive neutrophils. Genetic blockade or pharmacologic inhibition of KIAA1199 restored tumor immune infiltration, impeded tumor progression, and potentiated response to immune checkpoint blockade (ICB).. These findings indicated that KIAA1199 could facilitate the liver infiltration of immunosuppressive neutrophils via the TGFβ-chemokine (C-X-C motif) ligand (CXCL)3/1-CXCR2 axis, which might be clinically targeted for the treatment of hepatic metastasis.

Topics: Animals; Colorectal Neoplasms; Immune Checkpoint Inhibitors; Ligands; Liver Neoplasms; Mice; Neutrophil Infiltration; Receptor, Transforming Growth Factor-beta Type I; Transforming Growth Factor beta

PTEN is one of the most frequently mutated genes in human cancer. Although the roles of canonical PTEN protein and PTEN isoforms have been extensively explored, the current understanding of PTEN family members cannot fully illustrate the diversity of their roles in biological processes and tumor development. Notably, the function of noncoding RNAs arising from PTEN has been less elucidated.. We searched circBase and circInteractome to analyze the potential of PTEN for generating circRNAs. Then, Sanger sequencing, RNase R and Actinomycin D assays were used to verify the ring structure of circPTEN1. In situ hybridization and qRT-PCR were used to determine the level of circPTEN1 in peritumor and tumor tissues of colorectal cancer (CRC). Furthermore, functional experiments, including Transwell assay, 3D multicellular tumor spheroid invasion assay and metastasis models, were performed using circPTEN1 knockdown and overexpression cell lines in vitro and in vivo to investigate the effects of circPTEN1 on tumor metastasis in CRC. Mechanistically, luciferase reporter assay, fluorescence in situ hybridization, electrophoretic mobility shift assay, RNA immunoprecipitation, RNA pull-down and mass spectrometry were executed.. We identified a circular RNA generated from the PTEN gene, designated circPTEN1, that is frequently downregulated in colorectal cancer, and decreased expression of circPTEN1 predicts poor survival. Low expression of circPTEN1 promotes metastasis in PDX models in vivo and accelerates cancer cell invasion in vitro, whereas overexpression of circPTEN1 reveals opposite roles. Mechanically, we found that circPTEN1 is capable of binding the MH2 domain of Smad4 to disrupt its physical interaction with Smad2/3, which reduces the formation and subsequent nucleus translocation of Smad complexes and consequently suppresses the expression of its downstream genes associated with epithelial-mesenchymal transition upon TGF-β stimulation. Furthermore, we found that eIF4A3 suppresses the cyclization of circPTEN1 by directly binding to the circPTEN1 flanking region.. Our study uncovered a novel PTEN gene-generated circRNA with a tumor suppression function, and further revealed the mechanism of circPTEN1 in CRC metastasis mediated by TGF-β. The identification of circPTEN1 provides a new direction for PTEN investigation, and elucidation of circPTEN1/TGF-β/Smad signaling may pave the way for the development of a potential therapeutic strategy for the suppression of cancer progression.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; MicroRNAs; PTEN Phosphohydrolase; RNA, Circular; Transforming Growth Factor beta

Multiple evidence shows that metformin serves as a potential agent for Colorectal Cancer (CRC) treatment, while its molecular mechanisms still require detailed investigation. Here, we revealed that metformin specifically suppressed the proliferation of CRC cells by causing G1/S arrest, and INHBA is a potential target for metformin to play an anti-proliferation effect in CRC. We verified the oncogene role of INHBA by knocking down and overexpressing INHBA in CRC cells. Silencing INHBA abrogated the cell growth, while overexpression INHBA promotes the proliferation of CRC cells. As an oncogene, INHBA was aberrant overexpression in CRC tissues and closely related to the poor prognosis of CRC patients. In mechanism, INHBA is an important ligand of TGF-β signaling and metformin blocked the activation of TGF-β signaling by targeting INHBA, and then down-regulated the activity of PI3K/Akt pathway, leading to the reduction of cyclinD1 and cell cycle arrest. Together, these findings indicate that metformin down-regulates the expression of INHBA, then attenuating TGF-β/PI3K/Akt signaling transduction, thus inhibiting the proliferation of CRC. Our study elucidated a novel molecular mechanism for the anti-proliferation effect of metformin, providing a theoretical basis for the application of metformin in CRC therapy.

Topics: Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Metformin; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-β to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-β reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-β. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL7; Chemokine CXCL5; Colorectal Neoplasms; Humans; Kruppel-Like Transcription Factors; Mesenchymal Stem Cells; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta; Tumor Microenvironment

TGF-β signaling pathways promote tumour development and control several downstream genes such as CTGF and MMPs. This study aimed to investigate the association between CTGF and MMP-1 mRNA expressions with clinicopathological status and survival rate in colorectal cancer patients. We investigated expression levels of CTGF and MMP-1 genes in paraffin-embedded tumours and adjacent normal tissue blocks (ADJ) by Real Time-PCR. Then, the expression of Smad2 and Smad4 proteins in the TGF-β canonical pathway was evaluated by immunohistochemistry. Finally, the correlation between CTGF, MMP-1, and the canonical TGF-β-signalling pathway with the clinicopathological features was investigated. Expression levels of MMP-1and CTGF were higher in tumours compared with adjacent normal tissues. Overexpression levels of MMP-1 and CTGF were associated with lymph node metastasis, distant metastasis, tumour histopathological grading, advanced stage, and poor survival (p < 0.05). Additionally, a significant association between the upregulation of MMP-1 and tumour location was noted. Upregulation of Smad2 and Smad4 proteins were also significantly correlated with lymph node metastasis, distant metastasis, advanced stage, and poor survival (p < 0.0001). This study showed that canonical TGF-β signalling regulates both CTGF and MMP-1 expression and CRC progression. Moreover, TGF-β signalling and its downstream genes could be used as novel biomarkers and novel approaches for targeted therapy in CRC.

Topics: Colorectal Neoplasms; Connective Tissue Growth Factor; Humans; Lymphatic Metastasis; Matrix Metalloproteinase 1; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. The effects of CAFs on the progression of colorectal cancer remain controversial. In this study, we found the ectopic overexpression of Fibronectin leucine-rich transmembrane protein 3 (FLRT3) inhibited the process of epithelial-mesenchymal transition (EMT), as well as the proliferation, migration, invasion, and promote apoptosis of colorectal cancer cells, whereas silencing FLRT3 expression resulted in the opposite phenomenon. FLRT3 downregulation was associated with a poor prognosis in colorectal cancer. Also, FLRT3 expression was significantly related to some clinicopathologic factors, including T stage (P = 0.037), N stage (P = 0.042), and E-cadherin (P = 0.002) level. Via univariate and multivariate analyses, M stage (P < 0.0001), FLRT3 (P = 0.044), and E-cadherin (P = 0.003) were associated with overall survival and were independent prognostic factors for it. Mechanistically, CAFs secreted TGF-β, which downregulated FLRT3 expression by activating SMAD4 to promote aggressive phenotypes in colorectal cancer cells. Moreover, FLRT3 repressed tumorigenesis and lung metastasis, which could be reversed by LY2109761, a dual inhibitor of TGF-β receptor type I and II. Treatment with LY2109761 increased IFN-γ expression in CD8+ T cells and reduced the number of regulatory T cells in the tumor microenvironment. Taken together, we revealed the metastasis-suppressive function of FLRT3, which was attenuated during the CAFs-mediated activation of the TGF-β/SMAD4 signaling pathway to promote EMT in colorectal cancer. LY2109761 that significantly inhibited metastasis could be a new treatment option for advanced colorectal cancer.. CAFs enhance colorectal cancer aggressiveness by reducing FLRT3 expression through activating TGF-β/SMAD4 signaling pathway. CAF-targeted therapy and/or LY2109761 were promising treatments for colorectal cancer.

Topics: Antigens, CD; Cadherins; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Pyrazoles; Pyrroles; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Colorectal Neoplasms; Glycosylation; Humans; Nerve Tissue Proteins; Potassium Channels; Transforming Growth Factor beta

The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies - MUC CRCs either display a conventional 'apical-in' polarity or, more frequently, harbor an inverted 'apical-out' topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFβ and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.

Topics: Cell Adhesion; Colorectal Neoplasms; Humans; Organoids; Signal Transduction; Transforming Growth Factor beta

TGFβ contributes to chemoresistance in advanced colorectal cancer (CRC) via diverse immune-microenvironment mechanisms. Here, we found that cancer cell autonomous TGFβ directly triggered tumor programmed cell death 1 ligand 1 (PD-L1) upregulation, resulting in resistance to chemotherapy. Inhibition of tumor PD-L1 expression sensitized cancer cells to chemotherapy, reduced lung metastasis and increased the influx of CD8

Topics: B7-H1 Antigen; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Humans; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Macrophages; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor beta; Tumor Microenvironment

Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFβ1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFβ signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFβ signaling, while KIT deletion sensitized PDOs to TGFβ-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFβ signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFβ expression, and protects the tumor cells from tumor-suppressive TGFβ signaling by inhibiting SMAD2 expression.

Topics: Colorectal Neoplasms; Humans; Proto-Oncogene Proteins c-kit; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Colorectal cancer (CRC) is a heterogeneous disease and activation of WNT and TGFβ mediated oncogenic pathways is frequently observed in this pathology. However, to date, limited reports have been published addressing the association of circadian clock with CRC pathogenesis and stratification. The current study aims at assessing the expression of important circadian markers, PER2, PER3 and NR1D1, in independent CRC cohorts and their associations with CRC-related pathways.. Gene expression analysis was performed using available GEO (GSE39582) and TCGA datasets. Quantitative real time polymerase chain reaction was used to quantify the expression levels of PER2, PER3 and NRID1 in FFPE (formalin fixed paraffin embedded) CRC tissue samples. Furthermore, enrichment of circadian markers in WNT and TGFβ pathways-activated tumors was assessed.. Statistically significant downregulation of PER3 was found in tumor versus control samples in GEO (P<0.0001) and TCGA colon and rectal adenocarcinoma datasets (P<0.05). Analysis of GEO dataset revealed a statistically significant upregulation of PER2 (P<0.01), and NR1D1 in colon adenocarcinoma, which was confirmed by qRT-PCR in CRC tumor samples versus controls in FFPE validation cohort. Higher expression of NR1D1 was associated with poor prognosis in colon adenocarcinoma. Contrastingly, PER3 was significantly downregulated in tumors (P<0.001) compared to controls and was associated with high-grade CRC tumors versus low-grade tumors. Tumors with WNT pathway activation had significantly low PER3 and slightly upregulated PER2 (<0.0001) expression. Interestingly, differential expression of PER3 and NR1D1 was significantly correlated with TGFβ1-expressing tumors (P<0.0001). Moreover, MYC- amplified tumors exhibited decreased PER3 levels.. Thus, low PER3 expression in CRC and poor survival of patients with NR1D1-high tumors reveal that genes in the suppressor loop of circadian rhythm are dysregulated in CRC, hence pointing out to the importance of dissecting the circadian pathway in cancer.

Topics: Adenocarcinoma; Circadian Clocks; Circadian Rhythm; Colonic Neoplasms; Colorectal Neoplasms; Humans; Transforming Growth Factor beta

Colorectal cancer (CRC) is a major cause of cancer-related deaths. Metastasis is enhanced through epithelial-mesenchymal transition (EMT), a process primarily induced by the transforming growth factor beta (TGF-β)-mediated canonical Smad pathway. This study focused on plexin D1 (PLXND1), a chemoreceptor for the ligand SEMA3E to mechanosensory, showing that PLXND1 induces EMT via activation of the PI3K/AKT pathway in CRC cells. The findings showed that PLXND1-knockdown decreases cell migration and invasion significantly, and that the binding of p61-SEMA3E to the PLXND1 enhances the invasiveness and migration through EMT. Furin inhibitor suppresses EMT, decreasing cell migration and invasion. Furin cleaves full-length SEMA3E and converts it to p61-SEMA3E, suggesting that furin inhibitors block PLXND1 and p61-SEMA3E binding. Furin is a potential therapeutic target for the purpose of suppressing EMT by inhibiting the binding of p61-SEMA3E to PLXND1. In vivo experiments have shown that PLXND1-knockdown suppresses EMT. Mesenchymal cells labeled with ZEB1 showed heterogeneity depending on PLXND1 expression status. The high-expression group of PLXND1 in 182 CRC samples was significantly associated with poor overall survival compared with the low-expression group (P = 0.0352, median follow-up period of 60.7 months) using quantitative real-time polymerase chain reaction analysis. Further research is needed to determine whether cell fractions with a different expression of PLXND1 have different functions.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Furin; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Membrane Glycoproteins; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Semaphorins; Signal Transduction; Transforming Growth Factor beta

Anastomotic leakage is the most feared complication after colonic anastomosis. The purpose of the study is to determine the effects of phenytoin applied by different application routes, on the healing process of colorectal anastomoses.. Wistar Albino rats were divided into Intraperitoneal Phenytoin Group, Oral Phenytoin Group (OAP), Rectal Phenytoin Group (RAP), and control groups. The molecular effect of phenytoin on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), fibroblast growth factor 2 (FGF2), and p53 genes was evaluated at mRNA and protein level. The effects of phenytoin on anastomotic bursting pressure analysis measured as well as pathohistological examinations.. There are statistically significant increase in anastomotic bursting pressure values between control and application groups. Inflammatory cell infiltration of all groups increased in the intestinal anastomosis region compared to control. Collagen scores were found to be significantly higher in the OAP and RAP groups compared to the control group. mRNA of TGF-ß and FGF2 expression increased in all routes of phenytoin applications.. Three different administration routes show considerably increase on the bursting pressure. Regarding the results of the expression of FGF2, TGF-β, p53, and VEGF genes, there is a significant increase FGF2 and TGF-β at mRNA and protein level in most administration routes.

Topics: Anastomosis, Surgical; Animals; Colon; Colorectal Neoplasms; Fibroblast Growth Factor 2; Phenytoin; Rats; Rats, Wistar; Rectum; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.

Topics: Cell Line; Colorectal Neoplasms; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Humans; Neoplasm Metastasis; Neoplastic Stem Cells; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Onco-immunogenic molecule CD155 is overexpressed in various tumor microenvironments (TME) including in colorectal cancer (CRC). Tumor-associated macrophages (TAMs) are the most abundant immune cells in CRC TME and play a vital role in CRC progression and metastasis. Most studies have focused on investigating the role of CRC cell-specific CD155 on CRC progression, while the contribution of TAMs-specific CD155 is still unknown. Here, we sought to investigate the expression pattern of CD155 in CRC TAMs and its role in tumor immunity and progression.. CD155 expression patterns in CRC TAMs and macrophages in paratumor or adjacent normal tissue were analyzed in 50 patients with CRC using flow cytometry and in 141 patients with CRC using immunohistochemistry. The correlation of CD155 expression level in TAMs with M1 and M2 phenotypic transition was analyzed. The role of macrophage-specific CD155 in CRC progression and tumor immune response was investigated in vitro and in vivo. We further analyzed the effect of CRC cells on the regulation of CD155 expression in macrophages.. CRC TAMs from clinical samples showed robustly higher expression of CD155 than macrophages from paratumor and adjacent normal tissues. The CD155 expression level was higher in TAMs of CRC at III/IV stages compared with the I/II stages and was negatively associated with the survival of patients with CRC. CD155. These findings indicated that CD155

Topics: Animals; Cell Movement; Colorectal Neoplasms; Immunosuppression Therapy; Mice; Phenotype; Receptors, Virus; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages

In clinical practice, colorectal cancer (CRC) is difficult to distinguish from ulcerative colitis and colon polyps. Practical markers are useful for diagnosing and treating patients with CRC. Carcinoembryonic antigen (CEA) is a biomarker for diagnosing patients with CRC. However, the diagnostic sensitivity and specificity of CEA are not high. Interleukin (IL)-10, IL-17A, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and transforming growth factor beta (TGF-β) are assumed to be closely related to the occurrence and development of human cancer. Some have been used as diagnostic markers in CRC. It remains unclear whether cytokines in combination with CEA could be used as biomarkers for the diagnosis of CRC. Serum levels of IL-10, IL-17, TNF-α, IFN-γ, and TGF-β in patients with CRC, ulcerative colitis, colonic polyps, stomach cancer, and healthy controls were measured by enzyme-linked immunosorbent assay. The serum level of CEA was detected using electrochemiluminescence. The value of the cytokines combined with CEA as a biomarker panel for the diagnosis of CRC was assessed. CEA, IL-10, IL-17A, TNF-α, and TGF-β levels were significantly increased in CRC. CEA displayed a higher specificity than the other cytokines. IL-17A, TNF-α, and TGF-β displayed higher sensitivities than CEA, IL-10, and IFN-γ in the diagnosis of CRC. The combination of serum CEA, IL-17A, and TNF-α achieved higher diagnostic efficacy for CRC (area under the curve = 0.935). The combination of CEA, IL-17, and TNF-α has better diagnostic efficacy than CEA alone in CRC. A panel containing IL-17A, TNF-α, and CEA could be a promising molecular biomarker panel to diagnostically differentiate CRC from ulcerative colitis, colon polyps, and stomach cancer.

Topics: Biomarkers, Tumor; Carcinoembryonic Antigen; Colitis, Ulcerative; Colonic Polyps; Colorectal Neoplasms; Cytokines; Humans; Interferon-gamma; Interleukin-10; Interleukin-17; Stomach Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The purpose of this study was to evaluate recombinant human endostatin (rHE)-induced normalization of the tumor vasculature in colorectal cancer (CRC) and to evaluate the therapeutic effects of combined treatment with rHE and a programmed death ligand-1 (PD-L1) inhibitor.. A mouse subcutaneous tumorigenesis model was established to evaluate the antitumor effects of endostatin combined with a PD-L1 inhibitor on CRC. Intravoxel incoherent motion diffusion-weighted magnetic resonance imaging (IVIM-DW MRI) was used to evaluate changes in the intratumor microcirculation in response to combined treatment with endostatin and a PD-L1 inhibitor. The infiltration density and function of CD8+ T cells in tumors were evaluated using flow cytometry. Finally, clinical specimens were used to evaluate the expression area of tumor vascular pericytes and CD8+ T cells in tumor tissues.. The antitumor effects of endostatin combined with a PD-L1 inhibitor were significantly greater than those of endostatin or a PD-L1 inhibitor alone. On the ninth day of intervention, the endostatin group showed significantly higher pseudo diffusion parameter (D*) and microvascular volume fraction (F) values in tumors than those in the control group or PD-L1 group. After 27 days of intervention, the endostatin groups showed significantly lower levels of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β than those in the control group. Treatment of CD8+ T cells with endostatin for 24 h did not alter the expression levels of markers of reduced T-cell activity. However, endostatin reversed the VEGF-mediated inhibition of the secretion of interferon (IFN)-γ from T cells. The results in CRC clinical samples showed that treatment with endostatin induced significantly higher infiltration of CD8+ T cells compared with treatment that did not include endostatin. Furthermore, the expression area of pericytes was significantly positively related to the infiltration density of CD8+ T cells and overall survival time.. Endostatin improved the antitumor effects of PD-L1 inhibitors on CRC, significantly increased the activity of CD8+ T cells, and synergistically improved the tumor treatment effect of the two inhibitors.

Topics: Animals; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Endostatins; Humans; Immune Checkpoint Inhibitors; Immunologic Factors; Immunotherapy; Matrix Metalloproteinase Inhibitors; Mice; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Despite the medico-surgical progress that has been made in the management of patients with colorectal cancer (CRC), the prognosis at five years remains poor. This resistance of cancer cells partly results from their phenotypic characteristics in connection with the epithelial-mesenchymal transition (EMT). In the present study, we have explored the ability of a polyphenol, tannic acid (TA), to counteract CRC cell proliferation and invasion through an action on the EMT. We highlight that TA decreases human SW480 and SW620 CRC cell and murine CT26 CRC cell viability, and TA inhibits their adhesion in the presence of important factors comprising the extracellular matrix, particularly in the presence of collagen type I and IV, and fibronectin. Moreover, these properties were associated with TA's ability to disrupt CRC cell migration and invasion, which are induced by transforming growth factor-β (TGF-β), as evidence in the video microscopy experiments showing that TA blocks the TGF-β1-induced migration of SW480 and CT26 cells. At the molecular level, TA promotes a reversal of the epithelial-mesenchymal transition by repressing the mesenchymal markers (i.e., Slug, Snail, ZEB1, and N-cadherin) and re-expressing the epithelial markers (i.e., E-cadherin and β-catenin). These effects could result from a disruption of the non-canonical signaling pathway that is induced by TGF-β1, where TA strongly decreases the phosphorylation of extracellular-signal regulated kinase ERK1/2, P38 and the AKT proteins that are well known to contribute to the EMT, the cell motility, and the acquisition of invasive properties by tumor cells. Very interestingly, a preclinical study of mice with subcutaneous murine tumor colon CT26 cells has shown that TA was able to significantly delay the growth of tumors without hepato- and nephrotoxicities.

Topics: Animals; Cadherins; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Hydrolyzable Tannins; Mice; Tannins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; RNA, Long Noncoding; Transforming Growth Factor beta; Tumor Escape

Transforming growth factor-beta (TGF-β) signalling pathway has been reported to be involved in metastasis and at the same time has been considered compellingly an important mediator of epithelial-to-mesenchymal transition (EMT). Besides, EMT process is maintained by zinc-finger E-box-binding homeobox 1 (ZEB1) gene which is induced by TGF-β pathway. TGF-β has been shown to be associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) phenomenon, which is one of the prognostic biomarkers of colorectal cancer (CRC). This study was conducted to determine the link among ZEB1-induced TGF-β, EMAST status and metastasis.. The expression level of ZEB1 was evaluated using quantitative reverse transcription (qRT) real-time PCR in 122 formalin fixed paraffin-embedded tissues of CRC sample with known EMAST status and TGF-β/Smad-dependent pathways. The association among ZEB1 expression, TGF-β signalling pathway, EMAST status and metastatic behaviour was examined.. Our results validated significant association between activated TGF-β signalling pathway and EMAST

Topics: Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Microsatellite Repeats; Neoplasm Metastasis; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Colorectal cancers (CRCs) continue to be the leading cause of cancer-related deaths worldwide. The exact landscape of the molecular features of TGF-β pathway-inducing CRCs remains uncharacterized.. Unsupervised hierarchical clustering was performed to stratify samples into two clusters based on the differences in TGF-β pathways. Weighted gene co-expression network analysis was applied to identify the key gene modules mediating the different characteristics between two subtypes. An algorithm integrating the least absolute shrinkage and selection operator (LASSO), XGBoost, and random forest regression was performed to narrow down the candidate genes. Further bioinformatic analyses were performed focusing on COMP-related immune infiltration and functions.. The integrated machine learning algorithm identified COMP as the hub gene, which exhibited a significant predictive value for two subtypes with an area under the curve (AUC) value equaling 0.91. Further bioinformatic analysis revealed that COMP was significantly upregulated in various cancers, especially in advanced CRCs, and regulated the immune infiltration, especially M2 macrophages and cancer-associated fibroblasts in CRCs.. Comprehensive immune analysis and experimental validation demonstrate that COMP is a reliable signature for subtype prediction. Our results could provide a new point for TGFβ-targeted anticancer drugs and contribute to guiding clinical decision making for CRC patients.

Topics: Algorithms; Area Under Curve; Cancer-Associated Fibroblasts; Cartilage Oligomeric Matrix Protein; Colorectal Neoplasms; Humans; Transforming Growth Factor beta

Topics: Apoptosis Regulatory Proteins; Colorectal Neoplasms; Down-Regulation; Humans; MicroRNAs; Neoplasm Proteins; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal carcinogenesis is frequently induced by hypoxia to trigger the reprogramming of cellular metabolism and gain of malignant phenotypes. Previously, hyperbaric oxygen (HBO) therapy and melatonin have been reported to alter the hypoxic microenvironment, resulting in inhibiting cancer cell survival. Accordingly, this study tested the hypothesis whether HBO and melatonin effectively inhibited CRC carcinogenesis. In vitro results indicated that melatonin therapy significantly suppressed the malignant phenotypes, including colony formation, growth, invasion, migration and cancer stemness with dose-dependent manners in CRC cell lines through multifaceted mechanisms. Similar to in vitro study, in vivo findings further demonstrated the melatonin, HBO and combined treatments effectively promoted apoptosis (cleaved-caspase 3/ cleaved-PARP) and arrested tumor proliferation, followed by inhibiting colorectal tumorigenesis in CRC xenograft tumor model. Moreover, melatonin, HBO and combined treatments modulated multifaceted mechanisms, including decreasing HIF-1α expression, alleviating AKT activation, repressing glycolytic metabolism (HK-2/PFK1/PKM2/LDH), restraining cancer stemness pathway (TGF-β/p-Smad3/Oct4/Nanog), reducing inflammation (p-NFκB/ COX-2), diminishing immune escape (PD-L1), and reversing expression of epithelial mesenchymal transition (E-cadherin/N-cadherin/MMP9). In conclusion, melatonin and HBO therapies suppressed colorectal carcinogenesis through the pleiotropic effects and multifaceted mechanisms, suggesting melatonin and HBO treatments could be novel therapeutic strategies for CRC treatment.

Topics: Animals; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Combined Modality Therapy; Humans; Hyperbaric Oxygenation; Male; Melatonin; Mice; Mice, Inbred BALB C; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Inhibitory SMAD7 and common mediator SMAD4 play crucial roles in SMAD-dependent TGF-β signaling that is often disrupted in colorectal cancer (CRC). This study aimed to profile the expression of SMAD7 and SMAD4 in primary and metastatic CRC and to evaluate their significance in disease progression and therapy response. The expression of SMAD7 and SMAD4 genes was analyzed by quantitative real-time PCR in tissues from 35 primary and metastatic CRC patients and in vitro in 7 human cell lines originating from colon tissue. Expression levels of SMAD7 and SMAD4, as well as their ratio, were determined and their association with tumor characteristics and response to therapy were evaluated. SMAD4 level was significantly lower in tumors compared to non-tumor tissues in both primary (p = 0.001) and metastatic (p = 0.001) CRC patients, while tumor expression of SMAD7 was significantly lower from non-tumor tissue only in metastatic patients (p = 0.017). SMAD7/SMAD4 ratio was elevated in CRC primary tumor tissues and cell lines compared to corresponding non-tumor tissues and cell line, respectively (p = 0.003). SMAD7 expression was significantly elevated in primary tumor tissues obtained from responders to neoadjuvant chemoradiotherapy (nCRT) compared to non-responders (p = 0.014). Alterations of expression and ratio of SMAD7 and SMAD4 in CRC cell lines, primary rectal cancer, and liver metastasis emphasize the importance of these genes in different stages of disease progression. Differential expression of SMAD7 in responders versus non-responders to nCRT should be further investigated for its potential predictive value.

Topics: Colorectal Neoplasms; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Signal Transduction; Smad4 Protein; Smad7 Protein; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the most common cancer types around the world. The prognosis of patients with advanced diseases is still poor in spite of currently available therapeutic options. Regorafenib is an oral tyrosine kinase inhibitor (TKI) approved to treat refractory metastatic colorectal cancer (mCRC). We investigated Somatic mutations in several genes involved in immunological response and cancer progression in both long/short responder mCRC patients who underwent third-line therapy with regorafenib to identify predictive biomarkers of response using Ion Torrent PGM sequencing and bioinformatic tools. We found Somatic mutations in TGFBR1, TGFBR2, and TGFBR3 genes in primary tumor and metastases samples of long-responder patients. Furthermore, our bioinformatic results show that they were mainly enriched in immune response, cell junction, and cell adhesion in long responder patients, particularly in primary tumor and metastatic sites. These data suggest that the TGF-b pattern could be the leading actor of a prolonged response to this drug.

Topics: Colorectal Neoplasms; Humans; Phenylurea Compounds; Pyridines; Transforming Growth Factor beta

Inhibin, beta A (INHBA) is a member of the transforming growth factor (TGF-β) family. The carcinogenic mechanisms of INHBA during the development of colorectal cancer (CRC) remain unclear. In the present study, we further elucidated the role of INHBA in CRC. We analysed the expression of INHBA in CRC and its relationship with patient prognosis using data from public databases. INHBA expression was evaluated in CRC tissues and cell lines using immunohistochemistry and western blotting. After inhibiting the expression of INHBA, the effect of INHBA on the function of CRC cells was evaluated in vitro. We found that INHBA was upregulated in CRC. High INHBA expression is closely related to poor prognosis in patients with CRC. Knockdown of INHBA in vitro can inhibit the proliferation, migration, and invasion of CRC cells. In terms of mechanism, we found that high INHBA expression activates the TGF-β pathway. SIGNIFICANCE OF THE STUDY: INHBA acts as an oncogene in the progression of CRC and may, therefore, be a potential therapeutic target for CRC.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibin-beta Subunits; Male; Middle Aged; Prognosis; Progression-Free Survival; Proportional Hazards Models; RNA Interference; RNA, Small Interfering; Signal Transduction; Survival Rate; Transforming Growth Factor beta

LINC00941 is a novel lncRNA that has been found to exhibit protumorigenic and prometastatic behaviors during tumorigenesis. However, its role in metastatic CRC remains unknown. We aimed to investigate the functions and mechanisms of LINC00941 in CRC metastasis. LINC00941 was shown to be upregulated in CRC, and upregulated LINC00941 was associated with poor prognosis. Functionally, LINC00941 promoted migratory and invasive capacities and accelerated lung metastasis in nude mice. Mechanistically, LINC00941 activated EMT in CRC cells, as indicated by the increased expression of key molecular markers of cell invasion and metastasis (Vimentin, Fibronectin, and Twist1) and simultaneous decreased expression of the main invasion suppressors E-cadherin and ZO-1. LINC00941 was found to activate EMT by directly binding the SMAD4 protein MH2 domain and competing with β-TrCP to prevent SMAD4 protein degradation, thus activating the TGF-β/SMAD2/3 signaling pathway. Our data reveal the essential role of LINC00941 in metastatic CRC via activation of the TGF-β/SMAD2/3 axis, which provides new insight into the mechanism of metastatic CRC and a novel potential therapeutic target for advanced CRC.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; RNA, Long Noncoding; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Vimentin

The tumor microenvironment significantly affects tumor progression, and tumor cells can also remodel the tumor microenvironment through complex interaction. Inflammasomes are innate immune system receptors/sensors that regulate an inflammatory response mainly mediated by the nucleotide-binding oligomerization domain-like receptors in macrophages, which can also influence the formation, progression and therapeutic response of cancer. However, the effects of tumor-derived factors in the microenvironment on inflammasomes have rarely been reported. In this study, we found that lactate, as the main metabolite of tumor cells could specifically activate the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain-containing protein 3 inflammasome through increasing the level of reactive oxygen species (ROS) in THP-1-derived macrophages. Furthermore, we showed that transforming growth factor-β (TGF-β), a cytokine accumulated in the tumor microenvironment, could be induced by lactate treatment in tumor cells, and in turn inhibit inflammasome activation induced by lactate and other canonical ligands in macrophages. In addition, TGF-β might induce autophagy of macrophages in a SMAD-dependent manner, leading to ROS clearance and eventually inhibiting the activation of inflammasomes. Collectively, these results indicated that in the tumor microenvironment, tumor-derived lactate could act as a danger signal alerting innate immunity, but nevertheless tumor cells produced more TGF-β to avoid immune surveillance.

Topics: Colorectal Neoplasms; Culture Media, Conditioned; HCT116 Cells; Humans; Immunity, Innate; Inflammasomes; Interleukin-1beta; Lactic Acid; Macrophages; NLR Family, Pyrin Domain-Containing 3 Protein; Paracrine Communication; Reactive Oxygen Species; Signal Transduction; THP-1 Cells; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment

The transforming growth factor-β (TGF-β) pathway plays a pivotal role in inducing epithelial-mesenchymal transition (EMT), which is a key step in cancer invasion and metastasis. However, the regulatory mechanism of TGF-β in inducing EMT in colorectal cancer (CRC) has not been fully elucidated. In previous studies, it was found that S100A8 may regulate EMT. This study aimed to clarify the role of S100A8 in TGF-β-induced EMT and explore the underlying mechanism in CRC.. S100A8 and upstream transcription factor 2 (USF2) expression was detected by immunohistochemistry in 412 CRC tissues. Kaplan-Meier survival analysis was performed. In vitro, Western blot, and migration and invasion assays were performed to investigate the effects of S100A8 and USF2 on TGF-β-induced EMT. Mouse metastasis models were used to determine in vivo metastasis ability. Luciferase reporter and chromatin immunoprecipitation assay were used to explore the role of USF2 on S100A8 transcription.. During TGF-β-induced EMT in CRC cells, S100A8 and the transcription factor USF2 were upregulated. S100A8 promoted cell migration and invasion and EMT. USF2 transcriptionally regulated S100A8 expression by directly binding to its promoter region. Furthermore, TGF-β enhanced the USF2/S100A8 signaling axis of CRC cells whereas extracellular S100A8 inhibited the USF2/S100A8 axis of CRC cells. S100A8 expression in tumor cells was associated with poor overall survival in CRC. USF2 expression was positively related to S100A8 expression in tumor cells but negatively related to S100A8-positive stromal cells.. TGF-β was found to promote EMT and metastasis through the USF2/S100A8 axis in CRC while extracellular S100A8 suppressed the USF2/S100A8 axis. USF2 was identified as an important switch on the intracellular and extracellular S100A8 feedback loop.

Topics: Animals; Calgranulin A; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Transforming Growth Factor beta; Upstream Stimulatory Factors

Cross-talk among inflammation and colorectal cancer cells is chiefly reported through a complex of cytokines, chemokines, and growth factors. MicroRNA performs strategic roles in controlling a variety of signaling cascades. miR-34a is known as a master regulator of tumor suppression. Combined application of different miRNA-based agents and chemotherapeutic drugs has been used to augment drug sensitivity and may reinforce the antitumor effect. A lot of studies specify a substantial increase in the effectiveness of combination therapies. The anti-inflammatory activity of Zerumbone (ZER) was investigated in many cancers. In this study the level of the inflammatory cytokines including CXCL-12 (SDF-1), CCL-2 (MCP-1), TGF-β and IL-33 has been measured in pmiR-34a-5p transfected and pmiR-34a-5p +ZER treated CRC cell lines (HCT-116 and SW48) by QRT-PCR and ELISA methods, respectively. The results showed that miR-34a could significantly inhibit cytokine expression in both cell lines for 48 and 72 h except SDF-1 which no inhibition was observed in SW48 cells. ZER suppressed SDF-1 for all three time points in both cell lines, while in SW48 cells IL-33 and TGF-β were inhibited in 72 h and in HCT-116 cells MCP-1 diminished for only 24 h and TGF-β diminished for all three times. Combination of both miR-34a and ZER suppressed TGF-β, SDF-1 and MCP-1 in HCT-116 cells in all time points while in SW48 cells, suppression of most cytokines was observed in 48 and 72 h. Furthermore Colony formation assay and scratch test were employed to detect changes of proliferation and migration in CRC transfected and treated cells. Generally, we found that miR-34a could considerably decrease the expression of inflammatory cytokines and the combination of ZER+ miR-34 boosted this effect. Moreover the migration and proliferation decreased in treated and transfected cells and this reduction was more severe in miR-34a +ZER treatment. It is important to note that in the case of cell resistance to each of these therapeutic agents, inhibition of cytokines can be compensated by another one.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL2; Chemokine CXCL12; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Inflammation; Interleukin-33; MicroRNAs; Sesquiterpenes; Transforming Growth Factor beta

Increased integrin β5 (ITGB5) expression is associated with the progression and metastasis of several types of cancers. However, whether upregulated ITGB5 expression can act as a prognostic factor for colorectal cancer (CRC) remains controversial. In this study, we aimed to identify the role ITGB5 plays during the pathogenesis of human CRC and explore the underlying molecular mechanism. Here, we show that ITGB5 expression is upregulated in CRC and is significantly associated with exacerbated CRC malignancy and an unfavourable overall survival rate among CRC patients. ITGB5 silencing significantly inhibited the proliferation and invasion of human CRC cell lines (HCT116 and HT29) in vitro and suppressed the growth and metastasis of implanted CRC tumours in vivo. Mechanistically, upregulated ITGB5 expression enhanced transforming growth factor β/Smad signalling and facilitated the epithelial-mesenchymal transition in CRC cells. Together, such findings indicate that ITGB5 acts as an oncogenic factor to enhance the malignancy of CRC and suggest that ITGB5 may be a therapeutic target.

Topics: Animals; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Integrin beta Chains; Mice; Mice, Inbred BALB C; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; Up-Regulation; Xenograft Model Antitumor Assays

To reveal the effects and related mechanism of cisatracurium on colorectal cancer (CRC) development.. HCT116 and SW480 cells were treated with various concentrations of cisatracurium or transforming growth factor-β (TGF-β). Chemokine C-X-C-Motif Receptor 4 (CXCR4) was overexpressed and let-7a-5p was silenced in cells by transfection with pcDNA3.1-CXCR4 or let-7a-5p inhibitor. Cell Counting Kit-8 (CCK-8) assay measured cell viability, and transwell and wound healing assays evaluated cell invasion and migration, respectively. The expression levels of let-7a-5p and CXCR4 were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blotting was conducted to test the levels of CXCR4, TGF-β/SMAD2/3 signalling and metastasis-related proteins. A tumour xenograft assay was performed to assess tumour growth.. Cisatracurium treatment suppressed the viability and metastasis of HCT116 and SW480 cells in a concentration-dependent manner, whereas activating TGF-β/SMAD2/3 signalling significantly reversed these effects. Cisatracurium treatment markedly reduced CXCR4 expression by inhibiting TGF-β/SMAD2/3 signalling. Besides, let-7a-5p was identified as a target of CXCR4 and could be upregulated by cisatracurium. Both CXCR4 overexpression and let-7a-5p knockdown alleviated the biological roles of cisatracurium in CRC cells. Moreover, a tumour xenograft assay further confirmed that cisatracurium inhibited tumour growth and metastasis by increasing let-7a-5p expression.. Cisatracurium suppressed the viability, metastasis and tumour growth of CRC by regulating the CXCR4/let-7a-5p axis via inhibiting TGF-β/SMAD2/3 signalling. These findings provide a theoretical basis for the role of cisatracurium in the prognosis of CRC patients.

Topics: Animals; Atracurium; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; HCT116 Cells; Heterografts; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Receptors, CXCR4; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation; Xenograft Model Antitumor Assays

The mechanisms and key factors involved in tumor environments for lung metastasis of CRC are still unclear. Here, using clinical samples from lung metastases of CRC patients, we found that intestinal immune network for IgA production was significantly dysregulated in lung metastases of CRC. Single-cell RNA sequencing discovered a subtype of B cells positive for Erbin, one member of the leucine-rich repeat and PDZ domain (LAP) family, was involved in the lung metastases. Erbin deletion in B cells suppressed lung metastasis of CRC in vivo. And, deletion of Erbin in B cells enhanced the killing effects of CD8

Topics: Adaptor Proteins, Signal Transducing; Aged; Animals; Antibodies, Neutralizing; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Immunoglobulin A; Intestines; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Metastasis; Programmed Cell Death 1 Receptor; Receptors, CXCR5; RNA-Seq; Single-Cell Analysis; STAT6 Transcription Factor; Transforming Growth Factor beta

Poorly differentiated colorectal cancer (CRC) is characterized by aggressive invasion and stromal fibroblast activation, which results in rapid progression and poor therapeutic consequences. However, the regulatory mechanism involved remains unclear. Here, we showed that ZNF37A, a member of KRAB-ZFP family, was upregulated in poorly differentiated CRCs and associated with tumor metastasis. ZNF37A enhanced the metastatic potential of multiple CRC cell lines and promoted distant metastasis in an orthotopic CRC model. Further investigation attributed the ZNF37A-exacerbated metastasis to increased extracellular TGF-β and the consequent activation of cancer-associated fibroblasts (CAFs) in tumor microenvironment (TME). Mechanistically, ZNF37A formed a complex with KAP1 and bound to the promoter of THSD4, a TME modulator, to suppress its transcription, which is required for ZNF37A-mediated TGF-β activation and CRC metastasis. Collectively, our study indicates that ZNF37A promotes TGF-β signaling in CRC cells and activates CAFs by transcriptionally repressing THSD4 to drive CRC metastasis, implicating ZNF37A as a potential biomarker for CRC differentiation and progression.

Topics: ADAM Proteins; Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Progression; Heterografts; Humans; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Serine Endopeptidases; Survival Rate; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Computational Biology; Humans; MicroRNAs; Signal Transduction; Smad6 Protein; Smad7 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Emerging data show a rise in colorectal cancer (CRC) incidence in young men and women that is often chemoresistant. One potential risk factor is an alteration in the microbiome. Here, we investigated the role of TGF-β signaling on the intestinal microbiome and the efficacy of chemotherapy for CRC induced by azoxymethane and dextran sodium sulfate in mice. We used two genotypes of TGF-β-signaling-deficient mice (Smad4

Topics: Animals; Antineoplastic Agents; Azoxymethane; Colon; Colorectal Neoplasms; Dextran Sulfate; Female; Fluorouracil; Gastrointestinal Microbiome; Male; Mice; Mice, Inbred C57BL; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a drastic phenotypic change during cancer metastasis and is one of the most important hallmarks of aggressive cancer. Although the overexpression of some specific transcription factors explains the functional alteration of EMT-induced cells, a complete picture of this biological process is yet to be elucidated. To comprehensively profile EMT-related genes in colorectal cancer, we quantified the EMT induction ability of each gene according to its similarity to the cancer stromal gene signature and termed it "mesenchymal score." This bioinformatic approach successfully identified 90 candidate EMT mediators, which are strongly predictive of survival in clinical samples. Among these candidates, we discovered that the neuronal gene ARC, possibly originating from the retrotransposon, unexpectedly plays a crucial role in EMT induction. Profiling of novel EMT mediators we demonstrated here may help understand the complexity of the EMT program and open up new avenues for therapeutic intervention in colorectal cancer.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Computational Biology; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Male; Middle Aged; Molecular Sequence Annotation; Reproducibility of Results; Transcriptome; Transforming Growth Factor beta

Tumor-associated macrophages (TAMs), one of the most common cell components in the tumor microenvironment, have been reported as key contributors to cancer-related inflammation and enhanced metastatic progression of tumors. To explore the underlying mechanism of TAM-induced tumor progression, TAMs were isolated from colorectal cancer patients, and the functional interaction with colorectal cancer cells was analyzed. Our study found that coculture of TAMs contributed to a glycolytic state in colorectal cancer, which promoted the stem-like phenotypes and invasion of tumor cells. TAMs produced the cytokine transforming growth factor-β to support hypoxia-inducible factor 1α (HIF1α) expression, thereby upregulating Tribbles pseudokinase 3 (TRIB3) in tumor cells. Elevated expression of TRIB3 resulted in activation of the β-catenin/Wnt signaling pathway, which eventually enhanced the stem-like phenotypes and cell invasion in colorectal cancer. Our findings provided evidence that TAMs promoted colorectal cancer progression in a HIF1α/TRIB3-dependent manner, and blockade of HIF1α signals efficiently improved the outcome of chemotherapy, describing an innovative approach for colorectal cancer treatment.

Topics: Animals; Cell Cycle Proteins; Cell Proliferation; Coculture Techniques; Colorectal Neoplasms; Disease Progression; Female; HCT116 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplastic Stem Cells; Phenotype; Protein Serine-Threonine Kinases; Repressor Proteins; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages; Up-Regulation; Wnt Signaling Pathway

The primary cause of colorectal cancer (CRC) recurrence is increased distant metastasis after radiotherapy, so there is a need for targeted therapeutic approaches to reduce the metastatic-relapse risk. Dysregulation of the cell-surface glycoprotein podocalyxin-like protein (PODXL) plays an important role in promoting cancer-cell motility and is associated with poor prognoses for many malignancy types. We found that CRC cells exposed to radiation demonstrated increased TGFβ and PODXL expressions, resulting in increased migration and invasiveness due to increased extracellular matrix deposition. In addition, both TGFβ and PODXL were highly expressed in tissue samples from radiotherapy-treated CRC patients compared to those from patients without this treatment. However, it is unclear whether TGFβ and PODXL interactions are involved in cancer-progression resistance after radiation exposure in CRC. Here, using CRC cells, we showed that silencing PODXL blocked radiation-induced cell migration and invasiveness. Cell treatment with galunisertib (a TGFβ-pathway inhibitor) also led to reduced viability and migration, suggesting that its clinical use may enhance the cytotoxic effects of radiation and lead to the effective inhibition of CRC progression. Overall, the results demonstrate that downregulation of TGFβ and its-mediated PODXL may provide potential therapeutic targets for patients with radiotherapy-resistant CRC.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Cell Survival; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Prognosis; Pyrazoles; Quinolines; Radiation, Ionizing; RNA Interference; RNA, Small Interfering; Sialoglycoproteins; Transforming Growth Factor beta; Up-Regulation; Vimentin

GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) are sequentially responsible for de novo synthesis of tetrahydrobiopterin (BH4), a known co-factor for nitric oxide synthase (NOS). The implication of BH4-biosynthesis process in tumorigenesis remains to be investigated. Here, we show that PTPS, which is highly expressed in early-stage colorectal cancer, is phosphorylated at Thr 58 by AMPK under hypoxia; this phosphorylation promotes PTPS binding to LTBP1 and subsequently drives iNOS-mediated LTBP1 S-nitrosylation through proximal-coupling BH4 production within the PTPS/iNOS/LTBP1 complex. In turn, LTBP1 S-nitrosylation results in proteasome-dependent LTBP1 protein degradation, revealing an inverse relationship between PTPS pT58 and LTBP1 stability. Physiologically, the repressive effect of PTPS on LTBP1 leads to impaired transforming growth factor β (TGF-β) secretion and thereby maintains tumor cell growth under hypoxia. Our findings illustrate a molecular mechanism underlying the regulation of LTBP1-TGF-β signaling by the BH4-biosynthesis pathway and highlight the specific requirement of PTPS for tumor growth.

Topics: Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; Hypoxia; Latent TGF-beta Binding Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Nitric Oxide Synthase; Phosphorus-Oxygen Lyases; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC), a leading cause of cancer death, has recently been known as the most prevalent malignancy worldwide. Although chemotherapy is an important therapeutic option for CRC patients, multidrug resistance (MDR) still remains a major cause of chemotherapy failure. Transmembrane protein 45A (TMEM45A) has been found highly expressed in various cancers, and is also proposed as an interesting biomarker for chemoresistance. However, the association between TMEM45A and MDR in CRC remains unclear. This study aimed to investigate the key role of TMEM45A in CRC by knockdown of its expression in 5-FU-resistant CRC cells (HCT-8/5-FU and SW480/5-FU) and their parental cells (HCT-8 and SW480). Data showed that TMEM45A was significantly up-regulated in HCT-8/5-FU and SW480/5-FU cells in comparison with their parental HCT-8 and SW480 cells. Knockdown of TMEM45A enhanced 5-FU sensitivity and 5-FU-induced apoptosis in HCT-8/5-FU and SW480/5-FU cells. It was also found that inhibition of TMEM45A increased the intracellular accumulation of Rhodamine-123 and down-regulated the expression of MDR1 in HCT-8/5-FU and SW480/5-FU cells. In addition, knockdown of TMEM45A suppressed migration and invasion of HCT-8/5-FU and SW480/5-FU cells. Furthermore, knockdown of TMEM45A not only attenuated MDR-enhanced epithelial-mesenchymal transition (EMT), but also suppressed MDR-enhanced activation of the TGF-β signalling pathway in HCT-8/5-FU and SW480/5-FU cells. Taken together, our study suggests that knockdown of TMEM45A can effectively overcome MDR and inhibit EMT via suppression of the TGF-β signalling pathway in human CRC cells, and that targeting TMEM45A will be a potential strategy in the treatment of MDR in CRC.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Fluorouracil; Gene Knockdown Techniques; Humans; Membrane Proteins; Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC) is the second deadliest cancer in the US due to its propensity to metastasize. Stromal cells and especially cancer-associated fibroblasts (CAF) play a critical biophysical role in cancer progression, but the precise pro-metastatic mechanisms are not clear. Activin A, a TGF-β family member, is a strong pro-metastatic cytokine in the context of CRC. Here, we assessed the link between biophysical forces and pro-metastatic signaling by testing the hypothesis that CAF-generated mechanical forces lead to activin A release and associated downstream effects. Consistent with our hypothesis, we first determined that stromal activin A secretion increased with increasing substrate stiffness. Then we found that stromally-secreted activin A induced ligand-dependent CRC epithelial cell migration and epithelial to mesenchymal transition (EMT). In addition, serum activin A levels are significantly increased in metastatic (stage IV) CRC patients (1.558 ng/ml versus 0.4179 ng/ml, p < 0.05). We propose that increased tumor microenvironment stiffness leads to stromal cell-mediated TGF-β family signaling relying on the induction and utilization of activin A signaling.

Topics: Activins; Aged; Aged, 80 and over; Cadherins; Cancer-Associated Fibroblasts; Case-Control Studies; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment

The serrated neoplasia pathway gives rise to a distinct subgroup of colorectal cancers distinguished by the presence of mutant BRAF

Topics: Animals; beta Catenin; Colorectal Neoplasms; CpG Islands; Disease Models, Animal; DNA Methylation; Exome Sequencing; Humans; Mice; Microsatellite Instability; Microsatellite Repeats; Mutation; Proto-Oncogene Proteins B-raf; Transforming Growth Factor beta; Wnt Signaling Pathway

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, and metastasis is the major cause of CRC-related mortality. Transforming growth factor-beta (TGF-β) has a central role not only in the regulation of the normal colon but also in the development and metastasis of CRC. However, TGF-β is not considered an ideal therapeutic target because it shows both pro-tumorigenic and anti-tumorigenic activity, depending on the tumor stage. Therefore, it is important to find a downstream signaling component of TGF-β that can be targeted to impair CRC metastasis. Here, we show that TGF-β promotes CRC migration and upregulates the expression of long-noncoding RNA Taurine Upregulated Gene 1 (TUG1). TUG1 knockdown inhibited migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells in vitro, and reduced CRC lung metastasis in vivo. TGF-β induced metastasis, and TUG1 knockdown inhibited these effects. In addition, TGF-β could not reverse the anti-metastasis effects of TUG1 knockdown. These data demonstrate that TUG1 is a downstream molecular of TGF-β. Moreover, TWIST1 expression was increased with TGF-β treatment, and TUG1 knockdown decreased TWIST1 expression in CRC cells. TWIST1 knockdown inhibited invasion and EMT in CRC cells; these effects were not changed by simultaneous TUG1 knockdown, indicating that TWIST1 is a downstream mediator of TUG1. Moreover, TUG1 was significantly overexpressed in CRC patients. In conclusion, TGF-β promotes metastasis of CRC via a TUG1/TWIST1/EMT signaling pathway. TUG1 may be a promising drug target to inhibit TGF-β pathway activation in the treatment of CRC.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nuclear Proteins; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Twist-Related Protein 1

Non-coding RNAs (ncRNAs) are reported to be regulators of signaling pathways that are involved in colorectal cancer (CRC) progression. Aiming at finding ncRNAs (miRNAs) that are differentially expressed in tumor versus normal colorectal tissue samples, online RNA-seq data were analyzed. Of between 18 candidate miRNAs, hsa-miR-29b-1 (miR-29b-1) represented the highest fold change of expression level. Hsa-miR-29b-1 is encoded from the third intron of LOC646329 long ncRNA gene. Surprisingly, two miR-29b sponging sites were predicted within exons of LOC646329 gene. Then, dual luciferase assay supported the interaction of miR-29b-1 with LOC646329-variant D transcript. Also, a direct indication of miR-29b-1 with 3'UTR sequence of SMAD3 gene was verified through dual luciferase assay and RT-qPCR analysis. Furthermore, a reverse pattern of expression was detected between miR-29b-1 and LOC646329-variant D transcript in about 25 pairs of CRC tumor samples, detected by RTqPCR. Consistently, overexpression of LOC646329-variant D transcript was followed by increased SMAD3 and p21 genes expression level and downregulation of CyclinD1 genes in HCT116 cells, detected by RT-qPCR, and western analysis. Also, overexpression of it was followed by increased G1 cell population of HCT-116 cells. All of these data suggested a tumor suppressor effect for LOC646329-variant D in CRC tumor tissue samples, consistent to its reduced expression level at late stages of CRC progression. Data also indicated that LOC646329-variant D exerts its suppression effect on CRC progression through sponging miR-29b, which in turn regulates Wnt and TGFB signaling pathways. This makes LOC646329-variant D transcript as a novel potential therapy target.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Computational Biology; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; MicroRNAs; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Collagen and calcium-binding EGF domain-1 (CCBE1) is essential for lymphatic vascular development as it promotes vascular endothelial growth factor C (VEGFC) proteolysis. A recent study reported that CCBE1 was overexpressed in epithelial colorectal cancer (CRC) cells; however, the role of CCBE1 in tumor lymphangiogenesis and the mechanism underlying dysregulated CCBE1 expression in CRC remain undefined.

Topics: Aged; Animals; Calcium-Binding Proteins; Cancer-Associated Fibroblasts; Colorectal Neoplasms; Endothelial Cells; Female; HCT116 Cells; Humans; Lymphangiogenesis; Lymphatic Metastasis; Male; Mice; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor C

Characterising context-dependent gene functions is crucial for understanding the genetic bases of health and disease. To date, inference of gene functions from large-scale genetic perturbation screens is based on ad hoc analysis pipelines involving unsupervised clustering and functional enrichment. We present Knowledge- and Context-driven Machine Learning (KCML), a framework that systematically predicts multiple context-specific functions for a given gene based on the similarity of its perturbation phenotype to those with known function. As a proof of concept, we test KCML on three datasets describing phenotypes at the molecular, cellular and population levels and show that it outperforms traditional analysis pipelines. In particular, KCML identified an abnormal multicellular organisation phenotype associated with the depletion of olfactory receptors, and TGFβ and WNT signalling genes in colorectal cancer cells. We validate these predictions in colorectal cancer patients and show that olfactory receptors expression is predictive of worse patient outcomes. These results highlight KCML as a systematic framework for discovering novel scale-crossing and context-dependent gene functions. KCML is highly generalisable and applicable to various large-scale genetic perturbation screens.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; HCT116 Cells; Humans; MCF-7 Cells; Neoplasm Grading; Phenotype; Prognosis; Receptors, Odorant; Signal Transduction; Support Vector Machine; Systems Biology; Transforming Growth Factor beta; Wnt Signaling Pathway

Radioresistance observed in patients with colorectal cancer (CRC) may be related to the presence of cancer stem cells (CSCs), but the underlying mechanism(s) remain unclear. Cancer-associated fibroblasts (CAFs) can regulate the stemness of cancer cells and tumor radiosensitivity. In addition, exosomes have been reported to modify treatment response by mediating cell-cell communication. In this study, we aimed to investigate whether exosomes derived from CAFs (CAF-exosomes) are involved in mediating resistance to radiotherapy in colorectal cancer and to explore the underlying mechanism. We found that CSCs were inherently resistant to cell death induced by radiotherapy. CAF-derived CM promoted clonogenicity and radioresistance of CRC cells. Further investigations revealed that exosomes isolated from CM induced the above effects whereas exosome-depleted CM (solution) was not able to induce clonogenicity and radioresistance. Finally, exosomes could activate transforming growth factor-β (TGF-β) signaling pathway and TGFβ1-neutralizing antibody inhibit this effect and decrease clonogenicity and expression levels of stemness genes. In conclusion,our findings suggest CAFs promote stemness of CRC cells and thus increase radiation resistance. Exosomes derived from CAFs play a crucial role through activating TGF-β signaling pathway in this process.

Topics: Animals; Apoptosis; Cancer-Associated Fibroblasts; Cell Proliferation; Colorectal Neoplasms; Exosomes; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Phenotype; Radiation Tolerance; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Colorectal Cancer (CRC) therapy confronts challenges as chemoresistance and side effects. Therefore, drugs with antitumor properties that downmodulate aggressiveness mediators are required. Studies have shown the relevance of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP), Protein Tyrosine Phosphatase 1B (PTP1B), and Transforming Growth Factor β (TGFβ) in mediating proliferation, chemoresistance, and metastasis. In this study, we aimed to investigate the responsiveness of colorectal cancer lines (HT29 and HCT116) towards Vemurafenib and whether this treatment could modulate these aggressiveness mediators. Cytotoxicity Assays (MTT and Trypan Exclusion Test) were performed to evaluate the viability of HT29 and HCT116 cells treated with Vemurafenib. Western blotting was performed to analyze the amount and/or the activity of mediators (LMWPTP, PTP1B, TGFβ, SMAD3), and the immunoprecipitation was performed to evaluate LMWPTP activity. This study brought up novel aspects of Vemurafenib action in colorectal cancer, which can decrease the activity of protein tyrosine phosphatases (LMWPTP and PTP1B) and the TGFβ pathway, making them important in the CRC aggressiveness. By downmodulating colorectal cancer hallmarks, Vemurafenib appears as an interesting candidate for CRC therapeutic protocols.

Topics: Antineoplastic Agents; Colorectal Neoplasms; HCT116 Cells; HT29 Cells; Humans; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Smad3 Protein; Transforming Growth Factor beta; Vemurafenib

Topics: Carcinogenesis; Colorectal Neoplasms; Gastrointestinal Microbiome; Humans; Transforming Growth Factor beta; Transforming Growth Factors

To date, no systematic analyses are available assessing concordance of molecular classifications between primary tumors (PT) and matched liver metastases (LM) of metastatic colorectal cancer (mCRC). We investigated concordance between PT and LM for four clinically relevant CRC gene signatures. Twenty-seven fresh and 55 formalin-fixed paraffin-embedded pairs of PT and synchronous LM of untreated mCRC patients were retrospectively collected and classified according to the MSI-like, BRAF-like, TGFB activated-like and the Consensus Molecular Subtypes (CMS) classification. We investigated classification concordance between PT and LM and association of TGFBa-like and CMS classification with overall survival. Fifty-one successfully profiled matched pairs were used for analyses. PT and matched LM were highly concordant in terms of BRAF-like and MSI-like signatures, (90.2% and 98% concordance, respectively). In contrast, 40% to 70% of PT that were classified as mesenchymal-like, based on the CMS and the TGFBa-like signature, respectively, lost this phenotype in their matched LM (60.8% and 76.5% concordance, respectively). This molecular switch was independent of the microenvironment composition. In addition, the significant change in subtypes was observed also by using methods developed to detect cancer cell-intrinsic subtypes. More importantly, the molecular switch did not influence the survival. PT classified as mesenchymal had worse survival as compared to nonmesenchymal PT (CMS4 vs CMS2, hazard ratio [HR] = 5.2, 95% CI = 1.5-18.5, P = .0048; TGFBa-like vs TGFBi-like, HR = 2.5, 95% CI = 1.1-5.6, P = .028). The same was not true for LM. Our study highlights that the origin of the tissue may have major consequences for precision medicine in mCRC.

Topics: Aged; Colorectal Neoplasms; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Retrospective Studies; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Carcinogenesis; Colorectal Neoplasms; Gastrointestinal Microbiome; Humans; Transforming Growth Factor beta

Innate lymphoid cells (ILCs) reside in mucosal surfaces to potentiate immune responses, sustain mucosal integrity and maintain tissue homeostasis. However, how tumor infiltrating ILCs modulate tumor development and progression is unclear. Here we profiled tumor infiltrating ILCs during colorectal cancer (CRC) progression by single-cell RNA sequencing. We identified six clusters of tumor infiltrating ILCs with unique features. ILC1s expressed inhibitory receptors and underwent inhibitory functional conversion at the late stage of CRC. ILC2s were classified into three subsets (called ILC2-A, -B, -C), of which ILC2-C subset could facilitate tumor progression. HS3ST1 and PD1 were highly expressed in ILC2s of late stage CRC tumors and deficiency of HS3ST1 or PD1 in ILC2s suppressed tumor growth. Moreover, ILC3s transdifferentiated into ILCregs during CRC progression and ILCregs promoted tumor growth. Of note, TGF-β signaling initiated the conversion of ILC3s to ILCregs and blockade of TGF-β signaling could disrupt the ILCreg transdifferentiation and inhibited tumor growth. Thus, intervention of ILC conversions might be a potential strategy for CRC immunotherapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Transdifferentiation; Colorectal Neoplasms; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunity, Innate; Lymphocytes; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasm Staging; Transforming Growth Factor beta

IL-22 has dual functions during tumorigenesis. Short term IL-22 production protects against genotoxic stress, whereas uncontrolled IL-22 activity promotes tumor growth; therefore, tight regulation of IL-22 is essential. TGF-β1 promotes the differentiation of Th17 cells, which are known to be a major source of IL-22, but the effect of TGF-β signaling on the production of IL-22 in CD4+ T cells is controversial. Here we show an increased presence of IL-17+IL-22+ cells and TGF-β1 in colorectal cancer compared to normal adjacent tissue, whereas the frequency of IL-22 single producing cells is not changed. Accordingly, TGF-β signaling in CD4+ T cells (specifically Th17 cells) promotes the emergence of IL-22-producing Th17 cells and thereby tumorigenesis in mice. IL-22 single producing T cells, however, are not dependent on TGF-β signaling. We show that TGF-β, via AhR induction, and PI3K signaling promotes IL-22 production in Th17 cells.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinogenesis; Cell Differentiation; Colitis; Colonic Neoplasms; Colorectal Neoplasms; Disease Models, Animal; Female; Humans; Interleukin-17; Interleukin-22; Interleukins; Lymphocytes, Tumor-Infiltrating; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Receptors, Aryl Hydrocarbon; Signal Transduction; Th17 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Colorectal Neoplasms; DNA Mismatch Repair; Female; Gene Expression Profiling; Humans; Immune Checkpoint Inhibitors; Male; Microsatellite Instability; Proteomics; Transforming Growth Factor beta

MicroRNAs (miRNAs) regulate the development of colorectal cancer (CRC). We aimed to investigate miRNAs and their relation to cancer-related signaling pathways in site-specific CRC.. We used a total of 24 left- and right-sided Finnish CRC samples (discovery cohort) and The Cancer Genome Atlas public mature miRSeq dataset of 201 CRC samples (validation cohort). MiRNA differential expression and biological pathway analyses were performed using DESeq2 and the DIANA/mirPath tool, respectively.. We found 17 significantly differentially up-regulated [false discovery rate (FDR) <0.05] miRNAs in left-sided CRC ("left miRNAs"), and 15 in right-sided CRC ("right miRNAs"). The left miRNAs participate in the mTor, Wnt, PI3K-Akt signaling pathways (FDR<0.05). The right miRNAs participate in the TGF-β signaling pathway. We also observed that both cohorts share six miRNAs. One of these (hsa-miR-196b-5p) was significantly (FDR<0.05) up-regulated in left-sided CRC. The rest of them (hsa-miR-625-3p, hsa-miR-155-5p, hsa-miR-625-5p, hsa-miR-31-5p and hsa-miR-330-5p) showed significant (FDR<0.05) up-regulation in right-sided CRC.. Left and right miRNAs are associated with predominant biological pathways of left- and right-sided CRC, respectively. Our results may be beneficial for classifying CRC and for future biomarker studies of site-specific CRC.

Topics: Aged; Cohort Studies; Colorectal Neoplasms; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF-β receptor activation. The prognostic value of a TGF-β response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF-β1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP-3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Humans; Insulin-Like Growth Factor Binding Protein 3; Mice, Nude; Neoplasm Invasiveness; Neoplastic Stem Cells; Paracrine Communication; Pericytes; Phenotype; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC) was one of the most malignant tumors worldwide due to its metastasis. Epithelial-to-mesenchymal transition (EMT) plays an important role in CRC migration, and transforming growth factor-β (TGF-β) works as a dominating cytokine in CRC EMT process. Here, we originally identified RUNX1 as an important factor among TGF-β induced EMT in CRC. We found that RUNX1 was overexpressed with the treatment of TGF-β, accompanied with enhanced cancer cell migration and EMT which was characterized by up-graded N-Cadherin levels. Vice versa, knockdown of RUNX1 attenuated the migration ability of TGF-β induced CRC cells. In addition, decreased expression of N-Cadherin suggested that EMT was also attenuated after knocking down RUNX1. Similar decrease was observed in EMT regulator snail family transcriptional repressor 1 (SNAI1). And the knockdown effect of RUNX1 cannot be reversed by the addition of TGF-β. Moreover, we observed that RUNX1 expression was higher in CRC tumor tissues than in normal epithelial tissues. The enhanced expression was detected in cancer cell nucleus. These results revealed RUNX1 could regulate colorectal cancer migration via TGF-β signaling pathway, and RUNX1 might serve as a potential target for preventing CRC metastasis.

Topics: Cell Movement; Colorectal Neoplasms; Core Binding Factor Alpha 2 Subunit; Epithelial-Mesenchymal Transition; Humans; Transforming Growth Factor beta

Clinical and molecular characteristics differ between right-sided and left-sided colorectal cancer (CRC). This study aimed to clarify the correlation between CRC sidedness and tumor immunity.. A total of 102 patients who underwent curative colectomy for stage II/III CRC were included in this study. The expression of programmed cell death (PD)-1, PD1-ligand 1 (PD-L1), forkhead box P3 (Foxp3), transforming growth factor (TGF)-β, and indoleamine-pyrrole 2,3-dioxygenase (IDO) were examined using immunohistochemistry and the relationships between sidedness and several prognostic factors were examined.. Clinicopathological factors were not significantly different between right- and left-sided CRC. The tumor immunity-related molecule PD-L1 was more highly expressed in right-sided than in left-sided CRC (62.9% vs. 30.6%, p<0.01). No significant difference was found in overall survival (OS) and disease-free survival (DFS) by sidedness. PD-1 and Foxp3 expression were significant prognostic factors for OS. Lymph node metastasis (N), lymphatic invasion (ly), and PD-L1 expression were significant prognostic factors for DFS. In right-sided CRC, IDO-positive patients had a poor OS (p<0.05), and IDO was the only independent prognostic indicator for OS. N and venous invasion were identified as independent prognostic indicators for DFS. In left-sided CRC, univariate analysis identified PD-1, PD-L1, and Foxp3 expression as significant predictors of poor OS. Multivariate analysis confirmed PD-L1 expression as an independent prognostic indicator. N, ly, and PD-L1 expression levels were identified as significant predictors of poor DFS.. The prognostic factors were IDO in right-sided CRC and PD-L1 and Foxp3 in left-sided CRC. These findings indicated that tumor immunity might play different roles depending upon sidedness. Tumor location may be an important factor to consider when assessing immune response and therapeutic decisions in CRC patients.

Topics: Adult; Aged; Aged, 80 and over; B7-H1 Antigen; Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Male; Middle Aged; Neoplasm Staging; Prognosis; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta; Up-Regulation

Inhibin subunit beta B (INHBB) is a protein-coding gene that participated in the synthesis of the transforming growth factor-. Real-time PCR and analyses of Oncomine, Gene Expression Omnibus (GEO), and The Cancer Genome Atlas (TCGA) databases were utilized to evaluate the INHBB gene transcription level of colorectal cancer (CRC) tissue. We evaluated the INHBB methylation level and the relationship between expression and methylation levels of CpG islands in CRC tissue. The corresponding clinical data were obtained to further explore the association of INHBB with clinical and survival features. In addition, Gene Set Enrichment Analysis (GSEA) was performed to explore the gene ontology and signaling pathways of INHBB involved.. INHBB expression was elevated in CRC tissue. Although the promoter of INHBB was hypermethylated in CRC, methylation did not ultimately correlate with the expression of INHBB. Overexpression of INHBB was significantly and positively associated with invasion depth, distant metastasis, and TNM stage. Cox regression analyses and Kaplan-Meier survival analysis indicated that high expression of INHBB was correlated with worse overall survival (OS) and disease-free survival (DFS). GSEA showed that INHBB was closely correlated with 5 cancer-promoting signaling pathways including the Hedgehog signaling pathway, ECM receptor interaction, TGF-. INHBB might be a potential prognostic biomarker and a novel therapeutic target for CRC.

Topics: Aged; Biomarkers, Tumor; Colorectal Neoplasms; CpG Islands; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Gene Ontology; Hedgehog Proteins; Humans; Inhibin-beta Subunits; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Promoter Regions, Genetic; Proportional Hazards Models; Signal Transduction; Transforming Growth Factor beta

Tumor metastasis is the leading cause of death in patients with colorectal cancer (CRC), in which epithelial-mesenchymal transition(EMT) plays a vital role. However, the exact mechanisms of this process remain largely unknown. The aim of the present study was to determine the role of phenethyl isothiocyanate (PEITC) in CRC metastasis by regulating EMT.. Wound healing assays and Transwell matrix assays were used to evaluate the potential of PEITC to inhibit CRC cells invasion and migration in vitro. Western blotting, light microscopy and immunofluorescence assays were used to detect the occurrence of EMT. Luciferase activity assay, real time-PCR and western blotting were used to investigate TGF-β1/Smad signaling activity.. We observed that PEITC, an isothiocyanate compound from crucifer with chemopreventive potential, inhibited the invasion and migration of CRC cells. Moreover, we showed that PEITC regulated the EMT of CRC cells. Additionally, we demonstrated that PEITC blocked the activation of the TGF-β1/Smad pathway and significantly suppressed TGF-β1-induced EMT.. Our results suggested that PEITC plays a crucial role in inhibiting the invasion and migration of CRC cells by regulating TGF-β1-induced EMT. The results of the present study provide a theoretical basis for the use of PEITC to treat CRC.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Isothiocyanates; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Higher circulating 25-hydroxyvitamin D levels (25(OH)D) have been found to be associated with lower risk for colorectal cancer (CRC) in prospective studies. Whether this association is modified by genetic variation in genes related to vitamin D metabolism and action has not been well studied in humans. We investigated 1307 functional and tagging single-nucleotide polymorphisms (SNPs; individually, and by gene/pathway) in 86 vitamin D-related genes in 1420 incident CRC cases matched to controls from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. We also evaluated the association between these SNPs and circulating 25(OH)D in a subset of controls. We confirmed previously reported CRC risk associations between SNPs in the

Topics: Aged; Case-Control Studies; Colorectal Neoplasms; Europe; Female; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Nutritional Physiological Phenomena; Polymorphism, Single Nucleotide; Prospective Studies; Risk Factors; Signal Transduction; Transforming Growth Factor beta; Vitamin D

The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in Kras

Topics: Animals; Colorectal Neoplasms; Datasets as Topic; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Liver Neoplasms; Male; Mice; Mutation; Neutrophil Activation; Neutrophils; Prognosis; Proto-Oncogene Proteins p21(ras); Receptor, Notch1; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; Tumor Microenvironment

Solid tumour growth is the consequence of a complex interplay between cancer cells and their microenvironment. Recently, a new global transcriptomic immune classification of solid tumours has identified six immune subtypes (ISs) (C1-C6). Our aim was to specifically characterise ISs in colorectal cancer (CRC) and assess their interplay with the consensus molecular subtypes (CMSs).. Clinical and molecular information, including CMSs and ISs, were obtained from The Cancer Genome Atlas (TCGA) (N = 625). Immune cell populations, differential gene expression and gene set enrichment analysis were performed to characterise ISs in the global CRC population by using CMSs.. Only 5 ISs were identified in CRC, predominantly C1 wound healing (77%) and C2 IFN-γ dominant (17%). CMS1 showed the highest proportion of C2 (53%), whereas C1 was particularly dominant in CMS2 (91%). CMS3 had the highest representation of C3 inflammatory (7%) and C4 lymphocyte depleted ISs (4%), whereas all C6 TGF-β dominant cases belonged to CMS4 (2.3%). Prognostic relevance of ISs in CRC substantially differed from that reported for the global TCGA, and ISs had a greater ability to stratify the prognosis of CRC patients than CMS classification. C2 had higher densities of CD8, CD4 activated, follicular helper T cells, regulatory T cells and neutrophils and the highest M1/M2 polarisation. C2 had a heightened activation of pathways related to the immune system, apoptosis and DNA repair, mTOR signalling and oxidative phosphorylation, whereas C1 was more dependent of metabolic pathways.. The correlation of IS and CMS allows a more precise categorisation of patients with relevant clinical and biological implications, which may be valuable tools to improve tailored therapeutic interventions in CRC patients.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Aged; CD8-Positive T-Lymphocytes; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Genes, MHC Class I; Humans; Inflammation; Interferon-gamma; Lymphocytes; Macrophages; Male; Microsatellite Instability; Monocytes; Neovascularization, Pathologic; Prognosis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Receptors, Antigen, T-Cell; Signal Transduction; Th1 Cells; Th17 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway; Wound Healing

Long non-coding RNA (lncRNA) plays a key regulatory role in the pathogenesis of colorectal cancer (CRC). In the present study, the specific regulatory role of lncRNA ezrin antisense RNA 1 (EZR-AS1) on CRC was investigated. The expression of lncRNA EZR-AS1 was significantly up-regulated in CRC cell lines (HCT8, HCT116, HT29, and SW620 cells), which was significantly different from that of normal human fetal colonic mucosa cells (FHC cells) (P<0.01). HCT116 and HT29 cells were then transfected with EZR-AS1 shRNA (sh-EZR-AS1) to silence lncRNA EZR-AS1 (P<0.01). When compared with the Control, after transfection of SH-EZR-AS1, E-cadherin was up-regulated, Vimentin was down-regulated, the apoptosis rate was increased, the cell viability, wound healing rate, and the number of invasive cells were decreased in HCT116 and HT29 cells (P<0.05). Silencing of lncRNA EZR-AS also significantly reduced the tumor volume and weight in mice injected with sh-EZR-AS1-transfected HCT116 and HT29 cells (P<0.05). The regulatory relationship between lncRNA EZR-AS1 and transforming growth factor β (TGF-β) signaling was further identified in CRC cells. Silencing of lncRNA EZR-AS1 significantly down-regulated TGF-β, Smad2, and α-SMA expression in HCT116 and HT29 cells at the protein level (P<0.05). The intervention of SB431542 (a TGF-β receptor blocker) and silencing of Smad2 both significantly down-regulated lncRNA EZR-AS1 expression in HCT116 and HT29 cells (P<0.01). In conclusion, silencing of lncRNA EZR-AS1 inhibited the proliferation, invasion, migration, and epithelial-mesenchymal transition, and promoted the apoptosis of CRC cells through blocking TGF-β signaling.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice; Neoplasm Invasiveness; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Alcohol intake is a well-known lifestyle risk factor for CRC, and an increasing number of studies have revealed that alcohol intake is also tightly associated with CRC metastasis. However, the effect of alcohol on CRC metastasis and its underlying mechanism remain unclear.. A retrospective cohort study was performed to investigate the characteristics of patients with alcohol-related CRC. The effects of ethanol on the biological behaviours of CRC cells were assessed through in vivo and in vitro assays using the Lieber-DeCarli ethanol liquid diet and ethanol, respectively. The ethanol-mediated signalling pathway and downstream factors were screened through ELISA, western blot, immunofluorescence and co-immunoprecipitation.. Most patients with alcohol-related CRC, particularly those with tumour metastasis, were characterized by a notably higher circulating ethanol level and a lower systemic acetaldehyde level. Moreover, CRC cells accumulated in ethanol, but not acetaldehyde, to notably higher levels compared with adjacent normal cells. Alcohol intake significantly promoted CRC metastasis via the ethanol-mediated TGF-β/Smad/Snail axis, and ethanol induced the cytoplasmic mislocalization of RUNX3 and further promoted the aggressiveness of CRC by targeting Snail. Pirfenidone (PFD) significantly eliminated the effects of ethanol on CRC metastasis by specifically blocking TGF-β signalling.. Alcohol intake plays a vital role in CRC metastasis via the ethanol-mediated TGF-β/RUNX3/Snail axis, and PFD might be a novel therapeutic management strategy for CRC.

Topics: Alcohol Drinking; Animals; Biomarkers; Cell Line, Tumor; Colorectal Neoplasms; Core Binding Factor Alpha 3 Subunit; Disease Models, Animal; Epithelial-Mesenchymal Transition; Humans; Incidence; Mice; Neoplasm Metastasis; Neurophysins; Protein Binding; Protein Precursors; Protein Transport; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vasopressins; Xenograft Model Antitumor Assays

The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers.. Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material.. Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39. We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-β pathway to reinforce T cell reactivity in this patient group.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; DNA Mismatch Repair; DNA-Binding Proteins; Epitopes; Humans; Immunotherapy; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; Microfilament Proteins; Mutation; Peptides; T-Lymphocyte Subsets; Transcription Factors; Transforming Growth Factor beta

We investigated the role of tumor-associated macrophages (TAMs) on the epithelial to mesenchymal transition (EMT) of colorectal cancer cells and determined the potential mechanism involved in the metastatic process.. In this study, flow cytometry was used to detect the expression of target proteins. We used transwell assay to evaluate the migration of cancer cells under specific conditions. Using real-time polymerase chain reaction, we examined the expressions of cytokines and EMT-related markers in mRNA level. Animal assay was performed for analysis in vivo and hematoxylin and eosin was used to visualize the effect of TAMs on tumor metastasis. We also used immunohistochemistry and Western blotting to detect the expression of target proteins.. Here, we observed enrichment of TAMs in colorectal tumor tissues, resulting in high metastasis in clinical therapy. Moreover, those TAMs could facilitate the EMT progression of colorectal cancer cells, which is induced by the transforming growth factor-β (TGF-β) derived from TAMs, leading to the invasion and migration of cancer cells.. Our results demonstrated that TAMs contributed the EMT progression through a TGF-β/Smad2,3-4/Snail signaling pathway, and disrupting this pathway with TGF-β receptor inhibitor could suppress metastasis, readjusting our focus to the connection of TAMs and cancer metastasis.

Topics: Animals; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; HCT116 Cells; Humans; Lung Neoplasms; Macrophages; Male; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

Cancer stem cells (CSCs) possess the capacity for self-renewal and the potential to differentiate into non-CSCs. The recent discoveries of dynamic equilibrium between CSCs and non-CSCs revealed the significance of acquiring CSC-like properties in non-CSCs as an important process in progression of cancer. The mechanism underlying acquisition of CSC-like properties has mainly been investigated in the context of epithelial-mesenchymal transition. Here, we demonstrate the dedifferentiation process may be an alternative mechanism in acquisition of CSC-like properties in human colorectal cancer cells. By exploring the single-cell gene expression analysis of organoids developed from CD44

Topics: Cell Dedifferentiation; Colorectal Neoplasms; Female; Humans; Male; Neoplasm Proteins; Neoplastic Stem Cells; Nuclear Proteins; Signal Transduction; Transforming Growth Factor beta; Twist-Related Protein 1

Conjugated linoleic acid (CLA) has been shown to activate the nuclear receptor PPAR-γ and modulate metabolic and immune functions. Despite the worldwide use of CLA dietary supplementation, strong scientific evidence for its proposed beneficial actions are missing. We found that CLA-supplemented diet reduced mucosal damage and inflammatory infiltrate in the dextran sodium sulfate (DSS)-induced colitis model. Conditional deletion of PPAR-γ in macrophages from mice supplemented with CLA diet resulted in loss of this protective effect of CLA, suggesting a PPAR-γ-dependent mechanism mediated by macrophages. However, CLA supplementation significantly worsened colorectal tumor formation induced by azoxymethane and DSS by inducing macrophage and T-cell-producing TGF-β via PPAR-γ activation. Accordingly, either macrophage-specific deletion of PPAR-γ or in vivo neutralization of latency-associated peptide (LAP, a membrane-bound TGF-β)-expressing cells abrogated the protumorigenic effect of CLA. Thus, the anti-inflammatory properties of CLA are associated with prevention of colitis but also with development of colorectal cancer.

Topics: Aminosalicylic Acid; Animals; Carcinogenesis; Cells, Cultured; Colitis; Colorectal Neoplasms; Dextran Sulfate; Dietary Supplements; Disease Models, Animal; Female; Humans; Inflammatory Bowel Diseases; Linoleic Acids, Conjugated; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; PPAR gamma; T-Lymphocytes; Transforming Growth Factor beta

The epithelial mesenchymal transition (EMT) is one step in the process through which carcinoma cells metastasize by gaining the cellular mobility associated with mesenchymal cells. This work examines the dual influence of the TGF-β pathway and intercellular contact on the activation of EMT in colon (SW480) and breast (MCF7) carcinoma cells. While the SW480 population revealed an intermediate state between the epithelial and mesenchymal states, the MC7 cells exhibited highly adhesive behavior. However, for both cell lines, an exogenous TGF-β signal and a reduction in cellular confluence can push a subgroup of the population towards the mesenchymal phenotype. Together, these results highlight that, while EMT is induced by the synergy of multiple signals, this activation varies across cell types.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Adhesion; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Colorectal cancer (CRC) is frequently a lethal disease because of metastasis. Actin cytoskeletal rearrangement is an essential step in cell migration during activation of the epithelial-mesenchymal transition (EMT) program, which is associated with metastatic properties of cancer cells. Cofilin-1 protein modulates actin dynamics by promoting actin treadmilling, thereby driving membrane protrusion and cell migration and invasion. However, the role of cofilin-1 during EMT in CRC is unknown. Here, we show that cofilin-1 and p-cofilin-1 have distinct subcellular distribution in EMT cells, as determined by super-resolution microscopy images, indicating distinct roles in different areas of cells. Silenced cofilin-1 cells treated with TGF-β (siCofilin-1/TGF-β) evaded p-LIMK2-p-cofilin-1 status, leading to recovery of E-cadherin and claudin-3 at the cell-cell contact and their respective protein levels, actin reorganization, and decreased mesenchymal protein level. Furthermore, siCofilin-1/TGF-β cells exhibited decreased migration and invasion rates as well as MMP-2 and -9 activity and augmented focal adhesion size. The expression of an inactive phospho-cofilin-1 mimetic (S3E) reduced E-cadherin and claudin-3 in cell-cell contacts, reduced their protein levels, and increased vimentin protein. Based on our findings, we suggest that cofilin-1 is crucial to switching from epithelial to mesenchymal-like morphology and cell migration and invasion by regulating actin cytoskeleton organization through activation of RhoA-LIMK2-cofilin-1 signaling, impacting the cell-cell adhesion organization of colon cancer cells in EMT.

Topics: Actin Cytoskeleton; Actins; Antigens, CD; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Claudin-3; Cofilin 1; Colorectal Neoplasms; Cytoskeleton; Epithelial-Mesenchymal Transition; Humans; Lim Kinases; Neoplasm Invasiveness; rhoA GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta; Vimentin

Colorectal cancer (CRC) is characterized by DNA methylation, which is associated with genomic instability and tumor initiation. As an important epigenetic regulation, DNA methylation can be used as a potential therapeutic target for CRC. In our study, we downloaded DNA methylation profiles (GSE17648 and GSE29490) and RNA sequencing microarray data (GSE25070 and GSE32323) from the Gene Expression Omnibus (GEO) database. As a result, 14 aberrantly methylated differentially expressed genes (DEGs) were screened according to the different criteria. We further validated these DEGs in The Cancer Genome Atlas (TCGA) database and obtained Pearson's correlation coefficient (COR) for the relationship between gene expression and DNA methylation. Three candidate genes (SOX9, TCN1, and TGFBI) with COR greater than 0.3 were screened out as Hub genes. The receiver operating characteristic result indicated that SOX9 and TGFBI effectively serve as biomarkers for the early diagnosis of CRC. Furthermore, the potential prognosis of the Hub genes for CRC patients was evaluated. Only TGFBI, which is regulated by methylation, can predict patient disease-free survival. Additionally, we examined the methylation level of the Hub genes in CRC cells in the Cancer Cell Line Encyclopedia database. Considering that methylation status tends to be highly modified on CpG islands in tumorigenesis, we screened the CpG island methylation of TGFBI based on the TCGA database and verified its diagnostic value in the GEO database. Our result revealed two Hub genes (TCN1 and TGFBI) whose aberrant expressions were regulated by DNA methylation. Additionally, we uncovered the hypermethylation of TGFBI on CpG islands and its clinical value in the diagnosis of CRC.

Topics: Atlases as Topic; Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; CpG Islands; Databases, Genetic; DNA Methylation; Early Detection of Cancer; Epigenesis, Genetic; Extracellular Matrix Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Staging; RNA, Messenger; Signal Transduction; SOX9 Transcription Factor; Survival Analysis; Transcobalamins; Transforming Growth Factor beta

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti-PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti-PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGFβ blockade in combination with radioimmunotherapy results in greater efficacy.

Topics: Animals; Antibodies, Monoclonal; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Colorectal Neoplasms; Combined Modality Therapy; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Granzymes; Humans; Mice; Programmed Cell Death 1 Receptor; Radioimmunotherapy; Transforming Growth Factor beta; Tumor Necrosis Factor Receptor Superfamily, Member 9

The aim of this study was to investigate the underlying mechanisms that transforming growth factor-β (TGF-β)-mediated epithelial-to-mesenchymal transition (EMT) in tumor cells contributes to 5-FU resistance. A series of experiments involving cell viability and caspase activity analyses, siRNA transfection, RNA isolation, and quantitative-PCR (qPCR) assay, cell migration analysis, Western blotting analysis of total protein and membrane protein were performed in this study. Mouse xenograft model was used to determine the effect of the PAR2 inhibitor in vivo. In this study, we found that protease-activated receptor 2 (PAR2) induction in 5-FU therapy is correlated with TGF-β-mediated EMT and apoptosis resistance. PAR2 and TGF-β were both activated in response to 5-FU treatment in vivo and in vitro, and whereas TGF-β inhibition sensitized CRC cells to 5-FU and suppressed cell migration, PAR2 activation eliminated the effect of TGF-β inhibition. Conversely, siRNA-mediated PAR2 depletion or PAR2 inhibition with a specific inhibitor produced a similar phenotype as TGF-β signal inhibition: 5-FU sensitization and cell migration suppression. Moreover, the results of xenograft experiments indicated that the PAR2 inhibitor can enhance cell killing by 5-FU in vivo and suppress EMT signaling. Our results reveal that the TGF-β effects require the coordinating action of PAR2, suggesting that PAR2 inhibition could be a new therapeutic strategy to combat 5-FU resistance in CRC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Fluorouracil; HCT116 Cells; HT29 Cells; Humans; Mice; Piperazines; Receptor, PAR-2; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transfection; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Topics: Animals; Colorectal Neoplasms; Disease Models, Animal; Immunotherapy; Neoplasm Metastasis; Neoplasms, Experimental; Organoids; Prognosis; Transforming Growth Factor beta

Prostate transmembrane protein androgen induced 1 (PMEPA1) has been reported to promote cancer progression. Metastasis is the main factor leading to cancer progression and poor prognosis, and at the beginning of metastasis, epithelial-to-mesenchymal transition (EMT) is a crucial activation. However, the relationship between PMEPA1 and EMT in colorectal cancer metastasis is still poorly understood. In this study, we first testified that PMEPA1 expresses higher in tumour than normal tissue in Gene Expression Omnibus database, in the Cancer Genome Atlas (TCGA) as well as in the clinical data we collected. Moreover, the higher expression was associated with poor prognosis. We furthermore demonstrated PMEPA1 promotes colorectal cancer metastasis and EMT in vivo and in vitro. We found that PMEPA1 activates the bone morphogenetic proteins (BMP) signalling of TGF-β signalling resulting in promoting EMT and accelerating the proliferation and metastasis of colorectal cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; Male; Membrane Proteins; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Middle Aged; RNAi Therapeutics; Signal Transduction; Transforming Growth Factor beta; Tumor Burden; Xenograft Model Antitumor Assays

Epithelial to mesenchymal transition (EMT) is compulsory for metastatic dissemination and is stimulated by TGF-β. Although targeting EMT has significant therapeutic potential, very few pharmacological agents have been shown to exert anti-metastatic effects. BI-69A11, a competitive Akt inhibitor, displays anti-tumor activity toward melanoma and colon carcinoma. This study provides molecular and biochemical insights into the effects of BI-69A11 on EMT in colon carcinoma cells in vitro and in vivo. BI-69A11 inhibited metastasis-associated cellular migration, invasion and adhesion by inhibiting the Akt-β-catenin pathway. The underlying mechanism of BI-69A11-mediated inhibition of EMT included suppression of nuclear transport of β-catenin and diminished phosphorylation of β-catenin, which was accompanied by enhanced E-cadherin-β-catenin complex formation at the plasma membrane. Additionally, BI-69A11 caused increased accumulation of vinculin in the plasma membrane, which fortified focal adhesion junctions leading to inhibition of metastasis. BI-69A11 downregulated activation of the TGF-β-induced non-canonical Akt/NF-κB pathway and blocked TGF-β-induced enhanced expression of Snail causing restoration of E-cadherin. Overall, this study enhances our understanding of the molecular mechanism of BI-69A11-induced reversal of EMT in colorectal carcinoma cells in vitro, in vivo and in TGF-β-induced model systems.

Topics: Antigens, CD; Antineoplastic Agents; Benzimidazoles; beta Catenin; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Quinolones; Transforming Growth Factor beta; Vinculin

Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR.. Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-β, and PI3K).. Mutation of genes within the WNT, P53, RTK-RAS, TGF-β, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-β pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-β pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p <  0.001). Mutations in genes within other critical pathways were not associated with SAR. When MAC was excluded as a covariate, multivariate analysis revealed TGF-β pathway mutation and curative resection after distant recurrence as an independent prognostic factor for SAR. The impact of TGF-β pathway mutations were predicted using the PROVEAN, SIFT, and PolyPhen-2. Among 25 mutations, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation.. Mutation in genes within TGF-β pathway may have negative prognostic role for SAR in CRC. Other pathway mutations were not associated with SAR.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Capecitabine; Chemotherapy, Adjuvant; Colon; Colorectal Neoplasms; Deoxycytidine; Female; Fluorouracil; Follow-Up Studies; Humans; Leucovorin; Male; Microsatellite Instability; Middle Aged; Neoplasm Recurrence, Local; Organoplatinum Compounds; Oxaloacetates; Palliative Care; Prognosis; Rectum; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

Hyaluronic acid synthase 2 (HAS2) is suggested to play a critical role in malignancy and is abnormally expressed in many carcinomas. However, its role in colorectal cancer (CRC) malignancy and specific signaling mechanisms remain obscure. Here, we report that HAS2 was markedly increased in both CRC tissue and malignant CRC cell lines. Depletion of HAS2 in HCT116 and DLD1 cells, which express high levels of HAS2, critically increased sensitivity of radiation/oxaliplatin-mediated apoptotic cell death. Moreover, downregulation of HAS2 suppressed migration, invasion and metastasis in nude mice. Conversely, ectopic overexpression of HAS2 in SW480 cells, which express low levels of HAS2, showed the opposite effect. Notably, HAS2 loss- and gain-of-function experiments revealed that it regulates CRC malignancy through TGF-β expression and SMAD2/Snail downstream components. Collectively, our findings suggest that HAS2 contributes to malignant phenotypes of CRC, at least partly, through activation of the TGF-β signaling pathway, and shed light on the novel mechanisms behind the constitutive activation of HAS2 signaling in CRC, thereby highlighting its potential as a therapeutic target.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HCT116 Cells; Humans; Hyaluronan Synthases; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Radiation Tolerance; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta; Up-Regulation

Fibroblasts become cancer-associated fibroblasts (CAFs) in the tumor microenvironment after activation by transforming growth factor-β (TGF-β) and are critically involved in cancer progression. However, it is unknown whether the TGF superfamily member Nodal, which is expressed in various tumors but not expressed in normal adult tissue, influences the fibroblast to CAF conversion. Here, we report that Nodal has a positive correlation with α-smooth muscle actin (α-SMA) in clinical melanoma and colorectal cancer (CRC) tissues. We show the Nodal converts normal fibroblasts to CAFs, together with Snail and TGF-β signaling pathway activation in fibroblasts. Activated CAFs promote cancer growth in vitro and tumor-bearing mouse models in vivo. These results demonstrate that intercellular crosstalk between cancer cells and fibroblasts is mediated by Nodal, which controls tumor growth, providing potential targets for the prevention and treatment of tumors.

Topics: Actins; Animals; Cancer-Associated Fibroblasts; Cell Differentiation; Cell Line; Colorectal Neoplasms; Female; Humans; Melanoma; Mice; Mice, Nude; Nodal Protein; Proliferating Cell Nuclear Antigen; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

We developed a molecular beacon targeting miR-141-3p, aberrantly increased in 5-fluorouracil-resistant colorectal cancer cells (R-CRCCs). It consists of a fluorophore-labeled oligonucleotide, antisense to miR-141-3p, and a quencher. It detected R-CRCCs and recovered the chemosensitivity of them to 5-fluorouracil by hybridization with miR-141-3p, which is applicable to cancer treatment.

Topics: Antagomirs; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; DNA-Binding Proteins; Drug Resistance, Neoplasm; Fluorescence Recovery After Photobleaching; Fluorouracil; Humans; MicroRNAs; Microscopy, Confocal; Nucleic Acid Hybridization; Transforming Growth Factor beta; Tumor Suppressor Proteins

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer-related death worldwide. Increasing evidence indicates that the deregulation of long noncoding RNAs (lncRNAs) contributes to tumor initiation and progression; however, little is known about the biological role of cancer susceptibility candidate 9 (CASC9) in CRC.. Novel lncRNAs potentially involved in CRC tumorigenesis were identified from datasets downloaded from The Cancer LncRNome Atlas and The Atlas of Noncoding RNAs in Cancer. The CRC cell lines HCT-116, HCT-116 p53. CASC9 was frequently upregulated in CRC, which was correlated with advanced TNM stage, and higher CASC9 levels were associated with poor patient outcomes. Knockdown of CASC9 inhibited growth and promoted apoptosis in CRC cells, whereas ectopic CASC9 expression promoted cell growth in vitro and in vivo. We demonstrated that CPSF3 is a CASC9-interacting protein, and knockdown of CPSF3 mimicked the effects of CASC9 knockdown in CRC cells. Furthermore, we found that CASC9 exerts its oncogenic activity by modulating TGFβ2 mRNA stability and upregulating the levels of TGFβ2 and TERT, resulting in an increase in phosphorylated SMAD3 and activation of TGF-β signaling, and enhanced TERT complex function in CRC cells. Finally, CPSF3 was significantly upregulated in CRC tissues as compared with adjacent or non-adjacent normal colon tissues, and CASC9, CPSF3, and TGFβ2 levels in human CRC tissues were positively correlated.. CASC9 is a promising prognostic predictor for patients with CRC and the CASC9-CPSF3-TGFβ2 axis is a potential therapeutic target for CRC treatment.

Topics: Aged; Aged, 80 and over; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cleavage And Polyadenylation Specificity Factor; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; RNA Interference; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Tumor Burden

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Nerve Tissue Proteins; Rats; Receptors, Immunologic; Roundabout Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Comprehensive transcriptomic analyses have shown that colorectal cancer (CRC) is heterogeneous and have led to the definition of molecular subtypes among which the stem-cell, mesenchymal-like group is associated with poor prognosis. The molecular pathways orchestrating the emergence of this subtype are incompletely understood. In line with the contribution of the cellular prion protein PrP. We assessed the distribution of the PrP. We found that PRNP gene expression is enriched in tumours of the mesenchymal subtype and is associated with poor survival. Our in vitro analyses revealed that PrP. Our findings define PrP. Grant support was provided by the following: Cancéropôle Ile de France (grant number 2016-1-EMERG-36-UP 5-1), Association pour la Recherche sur le Cancer (grant number PJA 20171206220), SATT Ile de France Innov (grant number 415) as well as INSERM.

Topics: Adult; Aged; Aged, 80 and over; Animals; Biomarkers; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression; Hippo Signaling Pathway; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Prion Proteins; Prognosis; Protein Serine-Threonine Kinases; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

This study aims to investigate the effects of glucose transport l (Glut1) gene on proliferation, differentiation, and apoptosis of colorectal cancer (CRC) cells by regulating the TGF-β/PI3K-AKT-mTOR signaling pathway. Immunohistochemistry was conducted to detect the positive Glut1 expression. Normal human CRC epithelial cells (CCD-18Co) and CRC cell line HCT116 were grouped into the control, blank, negative control (NC), and shGlut1-1 groups. RT-qPCR and Western blotting were performed to detect the expressions of Glut1, TGF-β1, PI3K, AKT, PTEN, mTOR, Bcl-2, and Bax. Protein expression of phosphorylated-PI3K (p-PI3K), p-S473-AKT, p-S389-S6K1, p-T70-4EBP1, Cleaved caspase-3 and Cleaved-PARP were detected. MTT assay, flow cytometry, and colony formation assay were performed in order to detect cell viability, cell cycle, and apoptosis, respectively. The positive expression rate of Glut1 in CRC tissues was 75% ± 8%, while in the adjacent normal tissues it was 0%. In comparison to adjacent normal tissues, CRC tissues had increased Glut1, TGF-β1, PI3K, AKT, mTOR, and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1, and p-T70-4EBP1 expressions; and decreased PTEN, Bax, Cleaved caspase-3, and Cleaved-PARP expressions. In comparison with the blank and NC groups, cells in the shGlut1-1 group showed decreased Glut1, TGF-β1, PI3K, AKT, mTOR, and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1, and p-T70-4EBP1 expressions; and increased PTEN, Bax, Cleaved caspase-3, and Cleaved-PARP expressions, along with more arrested cells in C0/C1 phase than in S phase and slower cell growth. These results suggested that silencing the Glut1 gene inhibited proliferation and promoted apoptosis of CRC cells by inactivating TGF-β/PI3K-AKT-mTOR signaling pathway.

Topics: Apoptosis; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Gene Silencing; Glucose Transporter Type 1; HCT116 Cells; Humans; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) activated by cancer cells has a central role in development and malignant biological behavior in colorectal cancer (CRC). Adult fibroblasts do not express Snail, but Snail-positive fibroblasts are discovered in the stroma of malignant CRC and reported to be the key role to chemoresistance. However, the reciprocal effect of CAFs expressed Snail to chemoresistance on CRC cells and the underlying molecular mechanisms are not fully characterized.. Snail-overexpressed 3T3 stable cell lines were generated by lipidosome and CT26 mixed with 3T3-Snail subcutaneous transplanted CRC models were established by subcutaneous injection. Cell Counting Kit-8, flow cytometry and western blotting assays were performed, and immunohistochemistry staining was studied. The cytokines participated in chemoresistance was validated with reverse transcriptase-polymerase chain reaction and heatmap.. Snail-expression fibroblasts are discovered in human and mouse spontaneous CRCs. Overexpression of Snail induces 3T3 fibroblasts transdifferentiation to CAFs. CT26 co-cultured with 3T3-Snail resisted the impairment from 5-fluorouracil and paclitaxel in vitro. The subcutaneous transplanted tumor models included 3T3-Snail cells develop without restrictions even after treating with 5-fluorouracil or paclitaxel. Moreover, these chemoresistant processes may be mediated by CCL1 secreted by Snail-expression fibroblasts via transforming growth factor β/nuclear factor-κB signaling pathways.. Taken together, Snail-expressing 3T3 fibroblasts display CAFs properties that support 5-fluorouracil and paclitaxel chemoresistance in CRC via participation of CCL1 and suggest that inhibition of the Snail-expression fibroblasts in tumor may be a useful strategy to limit chemoresistance.

Topics: 3T3 Cells; Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Chemokine CCL1; Coculture Techniques; Colorectal Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; Humans; Mice; Neoplasm Transplantation; NF-kappa B; Paclitaxel; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

Only mismatch repair (MMR)-deficient colorectal cancer (CRC) appears to respond well to programmed death (PD)-1 inhibition at the present time. Emerging evidence suggests a role for micro-environmental factors such as CD25

Topics: Animals; Biomarkers; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclooxygenase 2; Cytokines; Disease Models, Animal; Forkhead Transcription Factors; Genes, APC; Immunohistochemistry; Immunophenotyping; Interleukin-2 Receptor alpha Subunit; Intestinal Mucosa; Mice; Mice, Knockout; Mucosal-Associated Invariant T Cells; T-Lymphocytes, Regulatory; Thymic Stromal Lymphopoietin; Transforming Growth Factor beta

Ectopic production of free β human chorionic gonadotropin (hCGβ) has been associated with aggressive behavior in non-trophoblastic tumors. hCGβ shares common evolutionary sequences with transforming growth factor-β (TGF-β), which represents a major driving force of epithelial-to-mesenchymal transition (EMT). In this study, we examined the biological roles of hCGβ during EMT and its clinical significance in colorectal cancer (CRC) progression. Eighty CRC specimens and 54 preoperative serum samples were analyzed. hCGβ-overexpressing human CRC cell lines were examined for invasiveness and tumorigenicity, and the expression of EMT-associated genes was investigated. In human CRC, histologic hCGβ positivity [13/80 (16.3%)] was lower than serologic hCGβ positivity [13/54 (24.1%)]. However, it was significantly correlated with several clinicopathological features and unfavorable outcome (P < 0.05). hCGβ-overexpressing cell lines had increased invasiveness, migratory ability, and metastatic potential in mice (P < 0.01). Western blot, PCR, and microarray analyses showed hCGβ altered expression of EMT-related genes, including E-cadherin, phosphorylated SMAD2, SNAIL, and TWIST. hCGβ-induced SNAIL and TWIST overexpression levels were reversible by type I and type II TGF-β receptor inhibitors (P < 0.05). hCGβ thus induces EMT via the TGF-β signaling pathway, and it may represent a molecular target in CRC treatment.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chorionic Gonadotropin, beta Subunit, Human; Colorectal Neoplasms; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Mice; Neoplasm Invasiveness; Phosphorylation; Prognosis; Signal Transduction; Transforming Growth Factor beta

Advances in multiplex immunohistochemistry (IHC) techniques and digital pathology platforms allow quantification of multiple proteins at same tissue section and produce continuous data. TGF-β signaling plays crucial and complex roles in colorectal cancer (CRC). We here aimed to investigate clinical pathological relevant of proteins involved in TGF-β signaling at CRC tissues. Multiplex fluorescent IHC was used to quantitative analysis. The levels of eight proteins (TGF-β1, TGFBRI, TGFBRII, SMAD4, SMAD2/3, p-SMAD2/3, SMAD1/5/9, and p-SMAD1/5/9) were determined in TMA sections. Quantitative analysis was carried out by a scoring system by InForm software. It revealed that TGF-β signaling was hyper active. The levels of TGF-β1, TGFBRI, TGFBRII, SMAD4, SMAD1/5/9 and p-SMAD2/3 were significantly increased in cancer tissues when compared their levels in normal tissues. Furthermore, the levels of eight proteins in stroma were significantly lower than the levels that in cancer tissues. Clinical pathological relevant analysis exhibited that TGF-β signaling inclined to suppress the progression of tumor. SMAD1/5/9, TGFBRII, SMAD2/3 were confirmed as significant predictors for overall survival. In conclusion, we established a method based on multispectral imaging to extensively explore the clinical relevant of TGF-β signaling proteins. These results provided an opportunity to consider the novel application for proteins involving TGF-β signaling that used as diagnostic or prognostic biomarkers to conduct tumor therapy.

Topics: Aged; Biomarkers, Tumor; Colorectal Neoplasms; Datasets as Topic; Disease Progression; Female; Fluoroimmunoassay; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Phosphorylation; Prognosis; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta

The aim of the present study was to further develop our previous study on c-Met expression in colorectal cancer and epithelial-mesenchymal transition (EMT) induced by hepatocyte growth factor (HGF), to investigate EMT in the process of liver metastases, and evaluate the effects of chemotherapy on EMT cells as a therapeutic strategy for colorectal liver metastasis.. CT26 colon cancer cells were treated with 5-FU and oxaliplatin with or without HGF. The signaling pathway was evaluated by western blotting analysis, and drug resistance was evaluated by the MTT (3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide) assay.. Under pretreatment with HGF for 96 h, 5 μM and 10 μM of 5-FU mediated significant growth inhibition by 72.5±3.9% and 76.2±2.4%, respectively, compared to HGF alone, and by 105.1±2.8% and 103.5±2.9%, respectively, without HGF. The expression of E2F1 was decreased significantly to 50.5±3.8% after 24 hours by HGF with a reduction of both cyclin D1 to 52.1±7.0% and E to 73.7±3.8%. Thymidylate synthase was also decreased in a time-dependent manner to 80.6±2.0% after 24 h and to 52.7±1.5% after 96 h.. The presence of HGF was found to increase the 5-FU-induced death signal, JNK pathway, and inhibition of cell growth. As its mechanism, HGF was shown to decrease E2F-1 by reducing cyclin D or E by cell-cycle activation, resulting in inactivation of thymidylate synthase. The chemotherapeutic effect of 5-FU was increased in HGF- but not TGF-β-induced EMT.

Topics: Animals; Antimetabolites, Antineoplastic; Cadherins; Cell Line, Tumor; Colorectal Neoplasms; E2F1 Transcription Factor; Epithelial-Mesenchymal Transition; Fluorouracil; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Mice; Organoplatinum Compounds; Oxaliplatin; Signal Transduction; Thymidylate Synthase; Time Factors; Transforming Growth Factor beta

Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-β signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

Topics: Animals; Biomarkers, Tumor; Caco-2 Cells; Cell Movement; Cell Polarity; Colorectal Neoplasms; DNA Methylation; Epithelial Cells; Genetic Predisposition to Disease; Humans; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Peritoneal Neoplasms; Phenotype; Prospective Studies; Signal Transduction; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

BACKGROUND F-box protein 32 (FBXO32) (also known as atrogin-1), a member of the F-box protein family, was recently shown to be a transforming growth factor beta (TGF-β)/Smad4 target gene involved in regulating cell survival. It can be transcriptionally silenced by epigenetic mechanisms in some cancers, but its role in colorectal carcinoma (CRC) is unclear. We investigated the role of FBXO32 in CRC and determined its prognostic significance. MATERIAL AND METHODS We used real-time quantitative PCR, Western blot, and immunohistochemistry to elucidate the role of FBXO32 in clinical specimens and primary CRC cell lines. Differences in patient survival were determined by the Kaplan-Meier method and log-rank test. RESULTS We found that the FBXO32 and SMAD4 levels were higher in normal tissues than in CRC tissues, but PAI-1 and VEGF levels showed the opposite pattern. The expressions of FBXO32 and SMAD4 were related to clinicopathological parameters in CRC. Kaplan-Meier analyses showed that the 5-year overall survival of the low-FBXO32 expression group was significantly shorter than that of the high-FBXO32 expression group (p=0.010). CONCLUSIONS The fbxo32 gene is a novel tumor suppressor that inhibits CRC progression by inducing differentiation. Elevated expression of FBXO32 predicts longer survival in CRC patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; Epigenesis, Genetic; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Muscle Proteins; Prognosis; Signal Transduction; SKP Cullin F-Box Protein Ligases; Smad4 Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. During EMT, tumor cells acquire the capacity to migrate and invade the stroma. Activation of the transforming growth factor-b (TGF-b) signaling pathway is of major importance for the initiation of EMT. Smad4, an essential protein of this pathway, is known to complex with multiple transcription factors (e.g. Snail-1, Slug, Twist-1), in various types of cancer, promoting the repression or activation of target genes. The role of Smad4 in colorectal cancer (CRC) is not straightforward so far. In the present study forty eight resected CRC tumor specimens were immunohistochemically examined in order to assess the expression of Smad4 and its association with E-cadherin, Snail-1, Slug, Twist-1 protein expression and with various pathological parameters. Smad4 was found to be positively correlated with Snail-1, Slug and Twist-1 expression (p < 0.001). On the other hand it was negatively correlated with the expression of E-cadherin (p < 0.001). Furthermore, lymphatic invasion could be clearly associated with Smad4 expression, a finding complying with the metastatic ability of EMT cells. In conclusion, Smad4 could be considered as a central component of EMT transition in human colorectal cancer that combines with transcriptional factors to reduce E-cadherin and alter the expression of the epithelial phenotype.

Topics: Biomarkers, Tumor; Cadherins; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Immunohistochemistry; Signal Transduction; Smad4 Protein; Transcription Factors; Transforming Growth Factor beta

Topics: Colonic Neoplasms; Colorectal Neoplasms; Humans; Immune Evasion; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs.. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo.. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo.. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs.

Topics: Actins; Animals; Cancer-Associated Fibroblasts; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Disease Progression; Exosomes; Genes, Reporter; HCT116 Cells; Heterografts; Humans; Luciferases; Male; Mice; Mice, SCID; MicroRNAs; Transforming Growth Factor beta

Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR), is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA) derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread.

Topics: Antigens, CD; Blood Platelets; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Dimerization; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Lysophospholipids; Platelet Activation; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; RNA Interference; RNA, Small Interfering; Signal Transduction; Tight Junctions; Transforming Growth Factor beta

Novel immune therapeutic tools are rapidly expanding the anticancer arsenal. Despite this progress, patients with colorectal cancer (CRC) that spreads to vital parts of the body still have a dismal outcome. Transforming growth factor-β (TGF-β) plays a pivotal role in the development of CRC and metastasis. Important new work by Tauriello and colleagues has revealed that inhibition of TGF-β prevents tumor metastasis by enhancing a cytotoxic T cell response, suggesting that TGF-β inhibition is a promising pro-immunogenic therapy.

Topics: Colonic Neoplasms; Colorectal Neoplasms; Humans; Immune Evasion; Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC) is a malignancy that has high morbidity and mortality and is initiated from accumulative genetic events. Although much effort has been made to elucidate the genetic mechanism underlying this disease, it still remains unknown. Here, we discovered a novel role for multiple epidermal growth factor-like domains protein 6 (MEGF6) in CRC, namely, that it induces the epithelial-to-mesenchymal transition (EMT) to promote CRC metastasis via the transforming growth factor beta (TGFβ)/SMAD signaling pathway.. RNA sequencing data from the Gene Expression Omnibus database were analyzed using R software. Based on The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) cohort, the clinical significance of MEGF6 was investigated. HCT8R, HCT116, and LoVo CRC cells were transfected with small interfering RNA against MEGF6, and their proliferation and sensitivity to fluorouracil were evaluated with the MTT cell proliferation and colony formation assays. Proteins associated with cell growth were detected by western blot analysis. The apoptosis of cells was evaluated by Annexin V/propidium iodide staining, and transwell assays were performed to assess the involvement of MEGF6 in cell migration. Markers of EMT and TGFβ/SMAD signaling were evaluated by quantitative PCR and western blotting, and the correlation between MEGF6 and these markers was assessed in the TCGA colon and renal adenocarcinoma cohort.. The results showed that MEGF6 was upregulated in HCT8R cells. In addition, MEGF6 was significantly overexpressed in tumor tissue and predicted a poor survival in the TCGA-COAD cohort. Moreover, MEGF6 accelerated CRC cell growth and inhibited apoptosis, and promoted CRC metastasis by inducing the EMT. Finally, we found that TGFβ/SMAD signaling triggered the expression of Slug, which regulates the MEGF6-mediated EMT.. MEGF6 may serve as an oncogene to promote cell proliferation and inhibit apoptosis. MEGF6 can also accelerate cell migration via TGFβ/SMAD signaling-mediated EMT.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colon; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Intercellular Signaling Peptides and Proteins; Liver; Liver Neoplasms; Mice; Neoplasm Metastasis; Rectum; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Programmed cell death protein 1 (PD-1)/ programmed cell death ligand 1(PD-L1) axis is associated with immune tolerance via inhibition of T cell activation. The aim of this study was to clarify the significance of PD-1 and PD-L1 expressions and analyze the relationships between PD-1, PD-L1, transforming growth factor-β (TGF-β) and Forkhead box P3 (Foxp3) expressions in colorectal cancer (CRC).. A total of 116 patients who underwent curative colectomy for stage II/III CRC were included in the study. PD-1, PD-L1, TGF-β, and Foxp3 expressions were examined by immunohistochemistry and related to prognostic factors by Kaplan-Meier.. PD-1 expression was correlated with PD-L1, TGF-β, and Foxp3 expressions. Overall survival rates were significantly poorer in the PD-1 and PD-L1-positive groups. Multivariate analysis showed that PD-L1-positive is an independent risk factor. Disease-free survival (DFS) was tended in the PD-L1-positive group. The group with double-positive expression had significantly poorer prognosis.. PD-1 and PD-L1 expressions were associated with a poor prognosis and correlated with TGF-β and Foxp3 expressions in patients with CRC.

Topics: Adult; Aged; Aged, 80 and over; B7-H1 Antigen; Colectomy; Colorectal Neoplasms; Female; Forkhead Transcription Factors; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Prognosis; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis.. CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed. CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival.. Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations.

Topics: Animals; Biomarkers; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Cell Survival; Colorectal Neoplasms; Disease Models, Animal; Endoglin; Fibroblasts; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ligands; Male; Mice; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Zebrafish

It remains unclear if the vascular and connective tissue structures of primary and metastatic tumors are intrinsically determined or whether these characteristics are defined by the host tissue. Therefore we examined the microanatomical steps of vasculature and connective tissue development of C38 colon carcinoma in different tissues.. Tumors produced in mice at five different locations (the cecal wall, skin, liver, lung, and brain) were analyzed using fluorescent immunohistochemistry, electron microscopy and quantitative real-time polymerase chain reaction.. We found that in the cecal wall, skin, liver, and lung, resident fibroblasts differentiate into collagenous matrix-producing myofibroblasts at the tumor periphery. These activated fibroblasts together with the produced matrix were incorporated by the tumor. The connective tissue development culminated in the appearance of intratumoral tissue columns (centrally located single microvessels embedded in connective tissue and smooth muscle actin-expressing myofibroblasts surrounded by basement membrane). Conversely, in the brain (which lacks fibroblasts), C38 metastases only induced the development of vascularized desmoplastic tissue columns when the growing tumor reached the fibroblast-containing meninges.. Our data suggest that the desmoplastic host tissue response is induced by tumor-derived fibrogenic molecules acting on host tissue fibroblasts. We concluded that not only the host tissue characteristics but also the tumor-derived fibrogenic signals determine the vascular and connective tissue structure of tumors.

Topics: Actins; Animals; Cell Differentiation; Cell Line, Tumor; Colorectal Neoplasms; Connective Tissue; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation, Neoplastic; Male; Melanoma, Experimental; Mice, Inbred C57BL; Muscle, Smooth; Myofibroblasts; Neovascularization, Pathologic; Transforming Growth Factor beta

Common variants have explained less than the amount of heritability expected for complex diseases, which has led to interest in less-common variants and more powerful approaches to the analysis of whole-genome scans. Because of low frequency (low statistical power), less-common variants are best analyzed using SNP-set methods such as gene-set or pathway-based analyses. However, there is as yet no clear consensus regarding how to focus in on potential risk variants following set-based analyses. We used a stepwise, telescoping approach to analyze common- and rare-variant data from the Illumina Metabochip array to assess genomic association with colorectal cancer (CRC) in the Japanese sub-population of the Multiethnic Cohort (676 cases, 7180 controls). We started with pathway analysis of SNPs that are in genes and pathways having known mechanistic roles in colorectal cancer, then focused on genes within the pathways that evidenced association with CRC, and finally assessed individual SNPs within the genes that evidenced association. Pathway SNPs downloaded from the dbSNP database were cross-matched with Metabochip SNPs and analyzed using the logistic kernel machine regression approach (logistic SNP-set kernel-machine association test, or sequence kernel association test; SKAT) and related methods.. The TGF-β and WNT pathways were associated with all CRC, and the WNT pathway was associated with colon cancer. Individual genes demonstrating the strongest associations were TGFBR2 in the TGF-β pathway and SMAD7 (which is involved in both the TGF-β and WNT pathways). As partial validation of our approach, a known CRC risk variant in SMAD7 (in both the TGF-β and WNT pathways: rs11874392) was associated with CRC risk in our data. We also detected two novel candidate CRC risk variants (rs13075948 and rs17025857) in TGFBR2, a gene known to be associated with CRC risk.. A stepwise, telescoping approach identified some potentially novel risk variants associated with colorectal cancer, so it may be a useful method for following up on results of set-based SNP analyses. Further work is required to assess the statistical characteristics of the approach, and additional applications should aid in better clarifying its utility.

Topics: Asian; Cohort Studies; Colorectal Neoplasms; Genome-Wide Association Study; Genotype; Humans; Japan; Polymorphism, Single Nucleotide; Receptor, Transforming Growth Factor-beta Type II; Risk; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

The TGFβ-signaling pathway plays an important role in the pathogenesis of colorectal cancer (CRC). Loss of function of several genes within this pathway, such as bone morphogenetic proteins (BMPs) have been seen as key events in CRC progression.. In this study we comprehensively evaluate differential gene expression (RNASeq) of 81 genes in the TGFβ-signaling pathway and evaluate how dysregulated genes are associated with miRNA expression (Agilent Human miRNA Microarray V19.0). We utilize paired carcinoma and normal tissue from 217 CRC cases. We evaluate the associations between differentially expressed genes and miRNAs and sex, age, disease stage, and survival months.. Thirteen genes were significantly downregulated and 14 were significantly upregulated after considering fold change (FC) of > 1.50 or < 0.67 and multiple comparison adjustment. Bone morphogenetic protein genes BMP5, BMP6, and BMP2 and growth differentiation factor GDF7 were downregulated. BMP4, BMP7, INHBA (Inhibin beta A), TGFBR1, TGFB2, TGIF1, TGIF2, and TFDP1 were upregulated. In general, genes with the greatest dysregulation, such as BMP5 (FC 0.17, BMP6 (FC 0.25), BMP2 (FC 0.32), CDKN2B (FC 0.32), MYC (FC 3.70), BMP7 (FC 4.17), and INHBA (FC 9.34) showed dysregulation in the majority of the population (84.3, 77.4, 81.1, 80.2, 82.0, 51.2, and 75.1% respectively). Four genes, TGFBR2, ID4, ID1, and PITX2, were un-associated or slightly upregulated in microsatellite-stable (MSS) tumors while downregulated in microsatellite-unstable (MSI) tumors. Eight dysregulated genes were associated with miRNA differential expression. E2F5 and THBS1 were associated with one or two miRNAs; RBL1, TGFBR1, TGIF2, and INHBA were associated with seven or more miRNAs with multiple seed-region matches. Evaluation of the joint effects of mRNA:miRNA identified interactions that were stronger in more advanced disease stages and varied by survival months.. These data support an interaction between miRNAs and genes in the TGFβ-signaling pathway in association with CRC risk. These interactions are associated with unique clinical characteristics that may provide targets for further investigations.

Topics: Adult; Aged; Cell Line, Tumor; Colorectal Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; RNA, Messenger; Signal Transduction; Statistics, Nonparametric; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression, metastasis and drug resistance. The transcription factor(TF) and microRNA (miR) chimeric [SNAIL/miR-34]:[ZEB/miR-200] unit is the core regulatory system for the EMT process. Here, we proposed to assess the anti-EMT abilities and explore the inherent pharmacological mechanisms of the classic hypoglycaemic agent metformin for colorectal cancer(CRC). For the EMT model, the TGF-β-induced CRC cell lines SW480 and HCT116 were treated with metformin. The viability, migration and invasion abilities of the cells were evaluated with the Cell Counting Kit-8, wound-healing and trans-well assay. The alterations of the [SNAIL/miR-34]:[ZEB/miR-200] system and the EMT markers E-cadherin and vimentin were detected by western blot, qPCR and immunofluorescent staining. Metformin exhibited inhibitory effects on the proliferation, migration and invasion of the CRC SW480 cells. The up-regulation of E-cadherin and the down-regulation of vimentin for both SW480 and HCT116 cells revealed the anti-EMT abilities of metformin. For the [SNAIL/miR-34]:[ZEB/miR-200] system, metformin increased miR-200a, miR-200c and miR-429 levels and decreased miR-34a, SNAIL1 and ZEB1 levels in the TGF-β-induced EMT. From immunofluorescence, we observed increased E-cadherin and ZEB1 co-expression in metformin-treated cells. Metformin may perform bidirectional regulations of the [SNAIL/miR-34]:[ZEB/miR-200] system in the EMT process for colorectal cancer. Such regulation is expressed as the inhibition of EMT in general as well as an increased higher proportion of E/M hybrid cells in the total population.

Topics: Cell Movement; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; HCT116 Cells; Humans; Metformin; MicroRNAs; Neoplasm Invasiveness; Snail Family Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Annexin A2 (ANXA2) is upregulated in several malignancies, including colorectal cancer (CRC). However, there is little knowledge on the molecular mechanisms involved to its upregulation. The aim of this study was to identify the mechanism through which ANXA2 overexpression leads to CRC progression and evaluate its potential prognostic value. We used human CRC samples to analyse the correlation between ANXA2 levels and tumour staging. ANXA2 expression was increased in CRC tissues compared to normal colon tissues. In addition, we observe increased ANXA2 levels in stage IV tumours and metastasis, when compared to stage I-III. Whereas E-cadherin, an epithelial marker, decreased in stage II-IV and increased in metastasis. We've also shown that TGF-β, a classic EMT inductor, caused upregulation of ANXA2, and internalization of both E-cadherin and ANXA2 in CRC cells. ANXA2 silencing hindered TGF-β-induced invasiveness, and inhibitors of the Src/ANXA2/STAT3 pathway reversed the EMT. In silico analysis confirmed overexpression of ANXA2 and association to the consensus moleculars subtypes (CMS) with the worst prognosis. Therefore, ANXA2 overexpression play a pivotal role in CRC invasiveness through Src/ANXA2/STAT3 pathway activation. The association of ANXA2 to distinct CMSs suggests the possible use of ANXA2 as a prognostic marker or directed target therapy.

Topics: Annexin A2; Colon; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Staging; src-Family Kinases; STAT3 Transcription Factor; Transforming Growth Factor beta

Collagen triple helix repeat containing (CTHRC1), which was identified as a cancer-related factor, is a promigratory protein involved in multiple processes, including vascular remodeling, antifibrosis, metabolism, bone formation, and cancer. In this study, we aimed to investigate the clinical significance and possible role of CTHRC1 in the process of epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC). Here, we revealed that CTHRC1 mRNA and protein levels are both upregulated in CRC tissues compared with those of paired noncancerous tissues. Moreover, the overexpression of CTHRC1 correlated with poor prognosis in patients with CRC (especially colon cancer). Furthermore, we showed that CTHRC1 induced EMT and promoted cell motility in CRC cells. Importantly, we demonstrated that CTHRC1 promoted EMT by activating transforming growth factor-β (TGF-β) signaling, revealing a possible effective therapeutic treatment for patients with CRC.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Helicase‑like transcription factor (HLTF) has been identified as a tumor suppressor gene. The hypermethylation of HTLF is frequently observed in various types of cancer, including colorectal cancer (CRC). However, the mechanisms through which HLTF suppresses CRC progression remain unclear. Thus, the aim of the present study was to explore the biological function of HLTF in CRC cells and the underlying mechanisms. CRC tissues and cells were used to detect the expression of HLTF. Wound‑healing and Transwell assays were performed to assess the motility of CRC cells. The results revealed that HLTF expression was significantly associated with the differentiation status, invasion depth, lymph node metastasis and distant metastasis. A low HLTF expression was significantly associated with a poor survival. Furthermore, HTLF knockdown or ectopic overexpression significantly promoted or suppressed the motility of CRC cells, respectively. With regard to the underlying molecular mechanisms, the protein expression of HTLF was upregulated when the CRC cells were stimulated with transforming growth factor (TGF)‑β, and HLTF upregulation induced an increase in SMAD4 and p‑SMAD2/3 expression and a decrease in levels of the TGF‑β/SMAD pathway downstream genes, Vimentin and zinc finger e‑box binding homeobox 1 (ZEB1). On the whole, the findings of this study suggest that HLTF is negatively associated with the progression of CRC, and its overexpression suppresses the migration and invasion of CRC cells by targeting the TGF‑β/SMAD pathway.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Progression; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HCT116 Cells; HT29 Cells; Humans; In Vitro Techniques; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Phosphorylation; Prognosis; Signal Transduction; Smad Proteins; Survival Analysis; Transforming Growth Factor beta

Tumor-associated macrophages (TAMs) were reported to be involved in colorectal cancer (CRC) progression. However, its biological role and underlying mechanism in CRC remained to be elucidated. In this study, the expressions of the macrophage marker CD68 and transforming growth factor β1 (TGF-β1) in CRC tumor tissues and adjacent tissues were detected by immunohistochemistry. The expression levels of miR-34a, TGF-β1 and vascular endothelial growth factor (VEGF) in CRC tumor tissues and peripheral blood macrophages were measured by quantitative real-time PCR (qRT-PCR) and western blot. TGF-β1 levels in culture supernatant were detected by ELISA. The cell proliferation and invasion of human CRC cell lines CL187 and HCT116 were determined by MTT assay and Transwell assay, respectively. The results showed that the expression of miR-34a was downregulated whereas TGF-β1 and VEGF were upregulated in CRC tumor tissues and peripheral blood macrophages. TGF-β1 secreted by TAMs promoted the proliferation and invasion of CRC cells. TGF-β1-mediated miR-34a downregulation contributed to the proliferation and invasion of CRC cells via upregulating VEGF. MiR-34a in vivo exerted anti-tumor effect in CRC via inhibiting VEGF expression. In conclusion, TGF-β1 secreted by TAMs promoted CRC proliferation and invasion through regulating miR-34a/VEGF axis.

Topics: Animals; Antagomirs; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Colorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC remains incomplete. Recognising the potential of studying different human populations to reveal novel insights in disease pathogenesis, we conducted genomic analysis of CRC in Saudi patients.. In the discovery phase of the study, we conducted whole genome sequencing of tumour and corresponding germline DNA in 27 patients with CRC. In addition to known driver mutations, we identified three. In order to gain insight into a plausible biological mechanism for the potential role of. Our data expand the recently described role of

Topics: Aged; Biomarkers, Tumor; Cohort Studies; Colorectal Neoplasms; Exome; Female; Humans; Male; Mediator Complex; Middle Aged; Middle East; Mutation; Predictive Value of Tests; Prognosis; Sensitivity and Specificity; Transforming Growth Factor beta

miR-34a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.. Expression of miR-34a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34a in CRC patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in CRC cells.

Topics: Aged; Antineoplastic Agents; Apoptosis; Autophagy; Beclin-1; Colorectal Neoplasms; Drug Resistance, Neoplasm; Flow Cytometry; HT29 Cells; Humans; MicroRNAs; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Middle Aged; Organoplatinum Compounds; Oxaliplatin; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Inflammation is frequently associated with initiation, progression, and metastasis of colorectal cancer (CRC). Here, we unveil a CRC-specific metastatic programme that is triggered via the transcriptional repressor, GFI1. Using data from a large cohort of clinical samples including inflammatory bowel disease and CRC, and a cellular model of CRC progression mediated by cross-talk between the cancer cell and the inflammatory microenvironment, we identified GFI1 as a gating regulator responsible for a constitutively activated signalling circuit that renders CRC cells competent for metastatic spread. Further analysis of mouse models with metastatic CRC and human clinical specimens reinforced the influence of GFI1 downregulation in promoting CRC metastatic spread. The novel role of GFI1 is uncovered for the first time in a human solid tumour such as CRC. Our results imply that GFI1 is a potential therapeutic target for interfering with inflammation-induced CRC progression and spread.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Progression; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Inflammation; Inflammatory Bowel Diseases; Liver Neoplasms; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Receptors, Prostaglandin E, EP2 Subtype; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment

Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-β pathway activity, epithelial-mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances.

Topics: Animals; Antineoplastic Agents, Immunological; Cell Lineage; Cetuximab; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Profiling; Genes, p53; Glycolysis; Heterografts; Humans; Insulin-Like Growth Factor II; Male; Mice; Microsatellite Instability; Mutation; Prognosis; Signal Transduction; Stromal Cells; Transcriptome; Transforming Growth Factor beta

Nonylphenol (NP) is a well-known endocrine-disrupting chemical (EDC), which can enhance the progression of cancer by functioning as an estrogen‑like factor. In the present study, the effects of different concentrations of NP on COLO205 colorectal cancer (CRC) cells were examined. The results of flow cytometric analysis revealed that NP significantly decreased the proportion of cells in the G0/G1 phase in a dose‑dependent manner, which was accompanied by a marginal increase in the proportions of cells in S and G2/M phases. NP did not induce apoptosis, whereas estradiol (E2) did induce apoptosis. To elucidate the mechanisms underlying the action of NP on COLO205 cells, the transcriptional levels of extracellular signal‑regulated kinase (ERK)1, ERK2 and phosphoinositide 3‑kinase (PI3K) were assessed using reverse transcription‑quantitative polymerase chain reaction analysis. The expressions levels of ERK1, ERK2 and PI3K were increased by treatment with NP in a dose‑dependent manner. On examining protein levels, the expression of PI3K p38 was increased by NP and E2, and the expression of ERK1/2 was increased by NP. The phosphorylation of the ERK protein was significantly increased by treatment with NP at a high concentration (10‑4 M; P<0.01), but significantly decreased by E2 (P<0.01). Two key proteins in the transforming growth factor (TGF)β pathway (c‑Fos and SnoN) were selected for analysis using western blot analysis in the COLO205 cells treated with NP and E2. The expression levels of c‑Fos and SnoN were significantly increased by treatment with E2 (10‑7 M; P<0.01) and NP (10‑7‑10‑4 M; P<0.01). Taken together, these results indicated that NP affected the development of CRC via the ERK signaling pathway and TGFβ pathway.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Endocrine Disruptors; Estradiol; G1 Phase Cell Cycle Checkpoints; Humans; Intracellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phenols; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Advanced colorectal cancer (CRC) remains a critical health care challenge worldwide. Various TGF-β superfamily members are important in colorectal cancer metastasis, but their signaling effects and predictive value have only been assessed in isolation. Here, we examine cross-regulation and combined functions of the two most prominent TGF-β superfamily members activin and TGF-β in advanced colorectal cancer. In two clinical cohorts we observed by immune-based assay that combined serum and tissue activin and TGF-β ligand levels predicts outcome in CRC patients and is superior to single ligand assessment. While TGF-β growth suppression is independent of activin, TGF-β treatment leads to increased activin secretion in colon cancer cells and TGF-β induced cellular migration is dependent on activin, indicating pathway cross-regulation and functional interaction in vitro. mRNA expression of activin and TGF-β pathway members were queried in silico using the TCGA data set. Coordinated ligand and receptor expression is common in solid tumors for activin and TGF-β pathway members. In conclusion, activin and TGF-β are strongly connected signaling pathways that are important in advanced CRC. Assessing activin and TGF-β signaling as a unit yields important insights applicable to future diagnostic and therapeutic interventions.

Topics: Activins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Male; Neoplasm Staging; Prognosis; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; Up-Regulation

Topics: Animals; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression; Heterografts; Humans; Immunohistochemistry; Integrin alphaV; Mice; Neoplastic Stem Cells; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor β (TGF-β) pathway. Importantly, the effects of TGF-β signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-β signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-β signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-β signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-β receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/β-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.

Topics: Adenocarcinoma; Adenoma; Animals; Colorectal Neoplasms; DNA Transposable Elements; Genes, Neoplasm; Genes, Tumor Suppressor; Genetic Association Studies; Humans; Mice; Mice, Knockout; Mice, Transgenic; Mutagenesis, Insertional; Neoplasm Proteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Analysis, DNA; Signal Transduction; Species Specificity; Transforming Growth Factor beta

The Cancer Genome Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor's genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two hundred and seven colorectal carcinomas that had undergone whole-exome sequencing as part of The Cancer Genome Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics constituted our study population. Upon analysis, a tight association between 'microsatellite instability-high histology' and microsatellite instability-high (P<0.001) was readily detected and helped validate our image-based histology evaluation. Further, we showed, (1) among all histologies, the not otherwise specified type had the lowest overall mutation count (P<0.001 for entire cohort, P<0.03 for the microsatellite-instable group), and among the microsatellite-instable tumors, this type also correlated with fewer frameshift mutations in coding mononucleotide repeats of a defined set of relevant genes (P<0.01); (2) cytosine phosphate guanine island methylator phenotype-high colorectal cancers with or without microsatellite instability tended to have different histological patterns: the former more often mucinous and the latter more often not otherwise specified; (3) mucinous histology was associated with more frequent alterations in BRAF, PIK3CA, and the transforming growth factor-β pathway when compared with non-mucinous histologies (P<0.001, P=0.01, and P<0.001, respectively); and (4) few colorectal cancers (<9%) exhibited upregulation of immune-inhibitory genes including major immune checkpoints; these tumors were primarily microsatellite-instable (up to 43%, vs <3% in microsatellite-stable group) and had distinctly non-mucinous histologies with a solid growth. These morphology-molecular associations are interesting and propose important clinical implications. The morphological patterns associated with alterations of immune checkpoint genes bear the potential to guide patient selection for clinical trials that target immune checkpoints in colorectal cancer, and provide directions for future studies.

Topics: Carcinoma; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; DNA Methylation; Humans; Microsatellite Instability; Proto-Oncogene Proteins B-raf; Transforming Growth Factor beta

The TGF-β signaling pathway is involved in regulation of cell growth, angiogenesis, and metastasis. We test the hypothesis that genetic variation in the TGF-β signaling pathway alters miRNA expression.We use data from 1188 colorectal cancer cases to evaluate associations between 80 SNPs in 21 genes.Seven variants eIF4E rs12498533, NFκB1 rs230510, TGFB1 rs4803455, TGFBR1 rs1571590 and rs6478974, SMAD3 rs3743343, and RUNX1 rs8134179 were associated with expression level of miRNAs in normal colorectal mucosa. RUNX2 rs12333172 and BMPR1B rs13134042 were associated with miRNAs in normal colon mucosa; eIF4EBP3 rs250425, SMAD3 rs12904944, SMAD7 rs3736242, and PTEN rs532678 were associated with miRNA expression in normal rectal mucosa. Evaluation of the differential expression between carcinoma and normal mucosa showed that SMAD3 rs12708491 and rs2414937, NFκB1 rs230510 and rs3821958, and RUNX3 rs6672420 were associated with several miRNAs for colorectal carcinoma. Evaluation of site-specific differential miRNA expression showed that BMPR1B rs2120834, BMPR2 rs2228545, and eIF4EBP3 rs250425 were associated with differential miRNA expression in colon tissue and SMAD3 rs12901071, rs1498506, and rs2414937, BMPR2 rs2228545, and RUNX2 rs2819854, altered differential miRNA expression in rectal tissue.These data support the importance of the TGF-β signaling pathway to the carcinogenic process, possibly through their influence on miRNA expression levels.

Topics: Aged; Colon; Colorectal Neoplasms; Female; Genetic Variation; Humans; Intestinal Mucosa; Male; MicroRNAs; Middle Aged; Polymorphism, Single Nucleotide; Rectum; Transforming Growth Factor beta

Despite advancements in the diagnosis and treatment of colorectal cancer (CRC), many patients die because of tumor metastasis or recurrence. Therefore, identifying new prognostic markers and elucidating the mechanisms of CRC metastasis and recurrence will help to improve the prognosis of the disease. As dysregulation of microRNAs is strongly related to cancer progression, the aim of this study was to identify the role of miR-4775 in the prognosis of CRC patients and the underling mechanisms involved in CRC progression.. qPCR and in situ hybridization were used to evaluate the expression of miR-4775 in 544 pairs of paraffin-embedded normal and CRC tissues. Kaplan-Meier analysis with the log-rank test was used for survival analyses. Immunohistochemical staining was applied to investigate the expression of miR-4775-regulated Smad7/TGFβ pathway-associated markers. In vitro and in vivo invasion and metastasis assays were used to explore the function of miR-4775 in the progression of CRC.. miR-4775 was identified as a high-risk factor for CRC metastasis and recurrence, with high levels predicting poor survival among the 544 studied CRC patients. Furthermore, high miR-4775 expression promoted the invasion of CRC cells as well as metastasis and the epithelial to mesenchymal transition (EMT) via Smad7-mediated activation of TGFβ signaling both in vitro and in vivo. Downregulating miR-4775 or overexpressing Smad7 reversed the tumor-promoting roles of miR-4775/Smad7/TGFβ in vitro and in vivo.. miR-4775 promotes CRC metastasis and recurrence in a Smad7/TGFβ signaling-dependent manner, providing a new therapeutic target for inhibiting the metastasis or recurrence of the disease.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Models, Animal; Epithelial-Mesenchymal Transition; Heterografts; Humans; Kaplan-Meier Estimate; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Metastasis is one of the most decisive factors influencing CRC patient prognosis and current studies suggest that a molecular mechanism known as EMT broadly regulates cancer metastasis. NR2F2 is a key molecule in the development of CRC, but the roles and underlying mechanisms of NR2F2 in TGF-β induced EMT in CRC remain largely unknown. In the current study, we were interested to examine the role of NR2F2 in the TGF-β-induced EMT in CRC. Here, we found NR2F2 was upregulated in CRC cells and promotes TGF-β-induced EMT in CRC. Using comparative miRNA profiling TGF-β pre-treated CRC cells in which NR2F2 had been knocked down with that of control cells, we identified miR-21 as a commonly downregulated miRNA in HT29 cells treated with TGF-β and NR2F2 siRNA, and its downregulation inhibiting migration and invasion of CRC cells. Moreover, we found NR2F2 could transcriptional activated miR-21 expression by binding to miR-21 promoter in HT29 by ChIP and luciferase assay. In the last, our data demonstrated that Smad7 was the direct target of miR-21 in CRC cells. Thus, NR2F2 could promote TGF-β-induced EMT and inhibit Smad7 expression via transactivation of miR-21, and NR2F2 may be a new common therapeutic target for CRC.

Topics: Cell Line; Cell Line, Tumor; Colon; Colorectal Neoplasms; COUP Transcription Factor II; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Rectum; Smad7 Protein; Transcriptional Activation; Transforming Growth Factor beta

The TGF-β pathway plays an essential role in regulating cell proliferation and differentiation. GWASs and candidate approaches have identified a battery of genetic variants in the TGF-β pathway contributing to colorectal cancer (CRC). However, most of the significant variants are common variants and their functions remain ambiguous. To identify causal variants with low-frequency in the TGF-β pathway contributing to CRC susceptibility in Chinese population, we performed targeted sequencing of 12 key genes in TGF-β signaling in CRC patients followed by a two-stage case-control study with a total of 5109 cases and 5169 controls. Bioinformatic annotations and biochemical experiments were applied to reveal the potential functions of significant variants. Seven low-frequency genetic variants were captured through targeted sequencing. The two stage association studies showed that missense variant rs3764482 (c. 83C>T; p. S28F) in the gene SMAD7 was consistently and significantly associated with CRC risk. Compared with the wild type, the ORs for variant allele were 1.37 (95%CI: 1.10-1.70, P = 0.005), 1.55 (95%CI: 1.30-1.86, P = 1.15 × 10

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; China; Colorectal Neoplasms; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Genome-Wide Association Study; Genotype; Humans; Male; Middle Aged; Neoplasm Staging; Phosphorylation; Polymorphism, Single Nucleotide; Prognosis; Risk Factors; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-β signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize-i.e., to colonize distant sites-is the direct consequence of the loss of dependency on specific niche signals.

Topics: Adult; Animals; Cell Movement; Colorectal Neoplasms; Disease Models, Animal; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genetic Engineering; Humans; Male; Mice; Mice, Inbred NOD; Middle Aged; Mutation; Neoplasm Metastasis; Neoplastic Processes; Organoids; Signal Transduction; Transforming Growth Factor beta

Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Genotype; Humans; Mice; Mice, Inbred C57BL; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins p21(ras); Transcriptome; Transforming Growth Factor beta

Topics: Adult; Aged; Angiopoietin-Like Protein 2; Angiopoietin-like Proteins; Animals; Bevacizumab; Cell Line, Tumor; Chemokine CXCL1; Colorectal Neoplasms; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Interleukin-8; Male; Mice; Middle Aged; Neoplasm Metastasis; Prognosis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) α is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAPα, we investigate how FAPα shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAPα activity has a strong impact on the secreted CAF proteome ("secretome"), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) β, and an impact on matrix processing enzymes. Functionally, FAPα mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAPα leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGFβ. In collagen contraction assays, FAPα induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAPα, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By "terminal amine labeling of substrates" (TAILS) we explored FAPα-dependent cleavage events. Although FAPα acts predominantly as an amino-dipeptidase, putative FAPα cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAPα cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAPα with distinct positional profiles. Generally, our findings link FAPα to key aspects of CAF biology and attribute an important role in tumor-stroma interaction to FAPα.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; Endopeptidases; Fibroblasts; Gelatinases; Humans; Membrane Proteins; Neoplasm Proteins; Proteolysis; Proteome; Serine Endopeptidases; Stromal Cells; Transforming Growth Factor beta

Constitutive overactivation of TGFβ signaling is a common event in human cancer progression and acts as a major inducer of epithelial-mesenchymal transition (EMT). In pre-metastatic colorectal cancer (CRC) cells, however, this cascade is tightly controlled and the underlying mechanism in TGFβ stimulated hyperactivation of downstream Smad pathway remains elusive. In this study, expression of miR-187 was downregulated in colorectal cancer (CRC) compared with adjacent normal tissues. miR-187 could suppress the formation of aggressive phenotype in CRC and inactivate Smad pathway, thus preventing EMT. TGFβ stimulation significantly suppressed the expression of miR-187, and overexpressed miR-187 counteracted the influence of TGFβ on cell phenotype and downstream pathway. Furthermore, we found that miR-187 directly suppressed the expression of SOX4, NT5E and PTK6, which were identified as essential upstream effectors of Smad pathway. Together with the fact that high SOX4 or NT5E levels were associated with poor prognosis, we also demonstrated that downregulation of miR-187 was closely related to tumor metastasis and poor prognosis in CRC. These findings revealed a plausible mechanism for sustained TGFβ activation in cancer progression and might have suggested a novel miR-187-based clinical intervention target for patients with advanced CRC.

Topics: 5'-Nucleotidase; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Profiling; GPI-Linked Proteins; Humans; MicroRNAs; Neoplasm Proteins; Protein-Tyrosine Kinases; Signal Transduction; Smad Proteins; SOXC Transcription Factors; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer death. However, the molecular mechanisms underlying CRC initiation, growth and metastasis are poorly understood. In this study, based on our previous work for comprehensively analyzing miRNA sequencing data, we examined a series of colorectal cancer microRNAs expression profiles data. Results show that all these CRC samples share the same four pathways including TGF-beta signaling pathway, which is important in colorectal carcinogenesis. Twenty-one microRNAs that evolved in the four overlapped pathways were then discovered. Further analysis selected miR-21 as an important regulator for CRC through TGF-beta pathways. This study develops methods for discovering tumor specific miRNA cluster as biomarker and for screening new cancer therapy targets based on miRNA sequencing.

Topics: Biomarkers, Tumor; Case-Control Studies; Colorectal Neoplasms; Humans; MicroRNAs; Oligonucleotides, Antisense; Real-Time Polymerase Chain Reaction; Sequence Analysis, RNA; Signal Transduction; Smad6 Protein; Transcriptome; Transforming Growth Factor beta

TGF-β pathway is generally associated with the processes of metastasis, angiogenesis and EMT in cancer. Very little is known, however, about the role of TGF-β in cancer drug resistance. In this work, we show a specific activation of the TGF-β pathway in consequence of chemotherapeutic treatment in in vivo and in vitro models of colorectal carcinoma. 5-Fluorouracil (5FU) was able to stimulate the activation of SMAD3 and the transcription of specific genes such as ACVRL1, FN1 and TGFB1. On the other hand, the specific inhibition of TGF-βRI was able to repress the 5FU-induced genes transcription and to restore the sensitivity of chemoresistant cells to the toxic action of the drug, by decreasing the expression of BCL2L1 and ID1 genes. The role of the TGF-β molecule in the chemoresistant colon carcinoma cells' response to 5FU was further demonstrated by conditioned medium (CM) experiments: CM from 5FU-treated chemoresistant cells was able to protect chemosensitive cells against the toxic action of 5FU. In conclusion, these findings showed the pivotal role of TGF-β pathway in colon cancer mechanisms of drug resistance suggesting new possible approaches in diagnosis and treatment of colon cancer patients.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Colorectal Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; HCT116 Cells; Humans; Mice; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actin (αSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts.

Topics: Amine Oxidase (Copper-Containing); Cell Adhesion Molecules; Cell Differentiation; Cell Line; Cell Line, Tumor; Cells, Cultured; Colon; Colorectal Neoplasms; Fibroblasts; Gene Expression; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Oligonucleotide Array Sequence Analysis; Proteins; RNA, Messenger; Skin; T-Box Domain Proteins; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

UGT1A1*28/*6 as predictors of severe irinotecan-related diarrhea (SIRD) were duplicated by many studies. However, some patients of lower risk genotype (UGT1A1*1/*1) still suffered SIRD and the extremely low frequency of UGT1A1*6/*6 limited its clinical usage. Previous studies proved that the transforming growth factor (TGFB) family may have some effect on MTX-induced mucositis. However, the associations between TGFB gene variants and SIRD have never been reported so far. Our aim was to improve the predictive value of UGT1A1 gene variants on SIRD.. Six SNPs (TGFB1 rs1800469; TGFBR1 rs10733710, rs334354 and rs6478974; TGFBR2 rs3087465; UGT1A1*6) and UGT1A1*28 were selected for genotyping in 160 metastatic colorectal cancer patients treated with irinotecan in a prospective multicenter trial (NCT01282658).. UGT1A1*6, UGT1A1*28, rs1800469 and rs3087465 were all associated with SIRD (p = 0.026, 0.014, 0.047 and 0.045 respectively). A novel genetic score model (with a cut off value of 1.5) based on them was created to predict SIRD (OR = 11.718; 95 % CI 2.489-55.157, p = 0.002). In patients of gene score > 1.5, the risk of SIRD was much higher (23.5 vs. 2.8 %, p = 2.24E-04) and continued in the first 6 cycles of chemotherapy, while in patients with gene score ≤1.5, the risk was much lower and none of them suffered SIRD after the first cycle of chemotherapy (p = 0.0003).. The novel genetic score model improved the predictive value of UGT1A1 on SIRD. If validated, it will provide valuable information for clinical use of irinotecan.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents, Phytogenic; Camptothecin; Colorectal Neoplasms; Diarrhea; Female; Glucuronosyltransferase; Humans; Irinotecan; Male; Middle Aged; Neoplasm Metastasis; Prospective Studies; Transforming Growth Factor beta; Young Adult

LIM and SH3 protein 1(LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the mechanism remains unclear. Here, we show that LASP1 interacts with S100 calcium binding protein A11(S100A11) and enhances its expression in CRC. LASP1-S100A11 axis is essential for TGFβ-mediated epithelial-mesenchymal transition (EMT) and cell aggressive phenotype. Clinically, S100A11 is overexpressed in CRC tissues and localized in both the cytoplasm and the nucleus of CRC cells. Overexpression of S100A11 in cytoplasmic and nuclear subcellular compartments is associated with tumor metastasis and poor prognosis of CRC patients. Introduction of cytoplasmic and nuclear S100A11 promotes aggressive phenotypes of CRC cells in vitro as well as growth and metastasis of CRC xenografts, whereas suppressing S100A11 abrogates these effects. Furthermore, we identify flotillin-1 (FLOT1) and histone H1 as downstream factors for cytoplasmic and nuclear pathway of S100A11, which are required for LASP1-S100A11 axis-mediated EMT and CRC progression. These findings indicate S100A11, combined with LASP1, plays a critical role in promoting CRC metastasis via its subcellular effectors, FLOT1 and histone H1.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line, Tumor; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; Cytoskeletal Proteins; Epithelial-Mesenchymal Transition; Heterografts; Humans; LIM Domain Proteins; Membrane Proteins; Mice; Neoplasm Transplantation; S100 Proteins; Signal Transduction; Transforming Growth Factor beta

Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development.

Topics: Animals; Carcinogenesis; Colorectal Neoplasms; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Genome, Human; Humans; Male; Mice, Inbred NOD; Mice, SCID; Mutation; Organoids; Stem Cell Niche; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Wnt Proteins

The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFβ plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFβ is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFβ treatment prevails in a genetically engineered organoid culture carrying a BRAF(V) (600E) mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFβ signaling is already active in SSA precursor lesions and that TGFβ is a critical cue for directing SSAs to the mesenchymal, poor-prognosis CMS4 of CRC.

Topics: Adenoma; Carcinogenesis; Colorectal Neoplasms; Humans; Organoids; Signal Transduction; Transforming Growth Factor beta

A member of homeodomain protein namely TGIF2LX has been implicated as a tumor suppressor gene in human malignancy as well as in spermatogenesis. However, to our knowledge, dynamic functional evidence of the TGIF2LX has not yet been provided. The aim of the present study was to investigate the human TGIF2LX target gene(s) using a cDNA-AFLP as a differential display method. A pEGFP-TGIF2LX construct containing the wild-type TGIF2LX cDNA was stably transfected into SW48 cells. UV microscopic analysis and Real-time RT-PCR were used to confirm TGIF2LX expression. The mRNA expressions of TGIF2LX in transfected SW48 cells, the cells containing empty vector (pEGFP-N), and untransfected cells were compared. Also, a Real-time PCR technique was applied to validate cDNA-AFLP results. The results revealed a significant down-regulation and up-regulationby TGIF2LX of Nir1 and Nir2 genes, respectively. The genes are engaged in the cell morphogenesis process. Our findings may provide new insight into the complex molecular pathways underlying colorectal cancer development.

Topics: Amplified Fragment Length Polymorphism Analysis; Cell Line, Tumor; Colorectal Neoplasms; Down-Regulation; Homeodomain Proteins; Humans; Morphogenesis; Real-Time Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta

Drug resistance is one of the main causes of colon cancer recurrence. However, our understanding of the underlying mechanisms and availability of therapeutic options remains limited. Here we show that expression of pyruvate dehydrogenase kinase 4 (PDK4) is positively correlated with drug resistance of colon cancer cells and induced by 5-fluorouracil (5-FU) treatment in drug-resistant but not drug-sensitive cells. Knockdown of PDK4 expression sensitizes colon cancer cells to 5-FU or oxaliplatin-induced apoptosis in vitro and increases the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo In addition, we demonstrate for the first time that TGFβ mediates drug resistance by regulating PDK4 expression and that 5-FU induces PDK4 expression in a TGFβ signaling-dependent manner. Mechanistically, knockdown or inhibition of PDK4 significantly increases the inhibitory effect of 5-FU on expression of the anti-apoptotic factors Bcl-2 and survivin. Importantly, studies of patient samples indicate that expression of PDK4 and phosphorylation of Smad2, an indicator of TGFβ pathway activation, show a strong correlation and that both positively associate with chemoresistance in colorectal cancer. These findings indicate that the TGFβ/PDK4 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of PDK4 may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, which warrants the development of PDK4-specific inhibitors.

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Organoplatinum Compounds; Oxaliplatin; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Colorectal cancer (CRC) is one of the most common cancers worldwide, and the identification of new biomarkers for CRC is valuable for its diagnosis and treatment. We aimed to screen differentially expressed glycoproteins (especially O-glycoproteins) and to identify diagnostic or therapeutic candidates for colorectal cancer (CRC) based on different Tn antigen expression levels. Fresh cancer tissues and adjacent healthy tissues were obtained from CRC patients and classified into three groups based on their Tn antigen expression: CRC with negative Tn expression (CRC Tn‑), CRC with positive Tn expression (CRC Tn+) and normal control without Tn expression (NC). Protein extractions were separated and identified by iTRAQ technology. Glycoproteins and O-glycoproteins were selected using UniProt and DAVID. Deep bioinformatic analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KO), was used to annotate this O-glycoprotein interaction network. Subsequently, two O‑glycoproteins were verified by western blotting and immunohistochemistry in either LS174T cells or CRC tissues. We found that 330 differentially expressed proteins were identified by iTRAQ between CRC Tn‑ and NC tissues, 317 between CRC Tn+ and NC tissues, and 316 between CRC Tn‑ and Tn+ tissues. Of the 316 proteins, 55 glycoproteins and 19 O‑glycoproteins were identified and analyzed via deep informatics. Namely, different Tn antigen expression levels in CRC led to differential protein expression patterns, especially for glycoproteins and O‑glycoproteins. Decorin and SORBS1, two representative functional O-glycoproteins, were significantly downregulated in the CRC Tn+ tissues compared with the level in the CRC Tn‑ or NC tissues. Based on this deep bioinformatic analysis, Decorin and SORBS1 are hypothesized to be involved in the TGF‑β and PPAR‑γ signaling pathways, respectively.

Topics: Aged; Aged, 80 and over; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Colorectal Neoplasms; Down-Regulation; Female; Glycoproteins; Humans; Male; Microfilament Proteins; Middle Aged; PPAR gamma; Transforming Growth Factor beta

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-β receptor expression and signaling, and blocking TGF-β receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-β signaling with BET bromodomain inhibition may offer new therapeutic benefits.

Topics: Azepines; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Drug Resistance; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Molecular Structure; Proteins; Proto-Oncogene Proteins c-myc; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Triazoles

T lymphocyte-mediated immune responses are critical for antitumour immunity; however, T cell function is impaired in the tumour environment. MicroRNAs are involved in regulation of the immune system. While little is known about the function of intrinsic microRNAs in CD8(+) T cells in the tumour microenvironment. Here, we found that miR-491 was upregulated in CD8(+) T cells from mice with colorectal cancer. Retroviral overexpression of miR-491 in CD8(+) and CD4(+) T cells inhibited cell proliferation and promoted cell apoptosis and decreased the production of interferon-γ in CD8(+) T cells. We found that miR-491 directly targeted cyclin-dependent kinase 4, the transcription factor T cell factor 1 and the anti-apoptotic protein B-cell lymphoma 2-like 1 in CD8(+) T cells. Furthermore, tumour-derived TGF-β induced miR-491 expression in CD8(+) T cells. Taken together, our results suggest that miR-491 can act as a negative regulator of T lymphocytes, especially CD8(+) T cells, in the tumour environment; thus, this study provides a novel insight on dysfunctional CD8(+) T cells during tumourigenesis and cancer progression. In conclusion, miR-491 may be a new target for antitumour immunotherapy.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; CD8-Positive T-Lymphocytes; Cells, Cultured; Colorectal Neoplasms; Female; Interferon-gamma; Lymphocyte Activation; Mice; Mice, Inbred C57BL; MicroRNAs; Transforming Growth Factor beta; Tumor Microenvironment

PSG9 is a member of the pregnancy-specific glycoprotein (PSG) family and has been shown to contribute to the progression of colorectal cancer (CRC) and cancer-related angiogenesis. Here, we aim to investigate abnormal PSG9 levels in patients with CRC and to emphasize the role of PSG9 in driving tumorigenesis. Serum from 140 patients with CRC and 125 healthy controls as well as 74 paired tumors and adjacent normal tissue were used to determine PSG9 levels. We discovered that PSG9 was significantly increased in serum (P<0.001) and in tumor tissues (P<0.001) from patients with CRC. Interestingly, the increased PSG9 levels correlated with poor survival (P=0.009) and microvessel density (MVD) (P=0.034). The overexpression of PSG9 strongly promoted the proliferation and migration of HCT-116 and HT-29 cells. However, PSG9 depletion inhibited the proliferation of SW-480 cells. Using a human umbilical vein endothelial cell tube-forming assay, we found that PSG9 promoted angiogenesis. The overexpression of PSG9 also increased the growth of tumor xenografts in nude mice. Co-immunoprecipitation experiments revealed that PSG9 was bound to SMAD4. The PSG9/SMAD4 complex recruited cytoplasmic SMAD2/3 to form a complex, which enhanced SMAD4 nuclear retention. The PSG9 and SMAD4 complex activated the expression of multiple angiogenesis-related genes (included IGFBP-3, PDGF-AA, GM-CSF, and VEGFA). Together, our findings illustrate the innovative mechanism by which PSG9 drives the progression of CRC and tumor angiogenesis. This occurs via nuclear translocation of PSG9/SMAD4, which activates angiogenic cytokines. Therefore, our study may provide evidence for novel treatment strategies by targeting PSG9 in antiangiogenic cancer therapy.

Topics: Animals; Carcinogenesis; Cell Movement; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Granulocyte-Macrophage Colony-Stimulating Factor; HCT116 Cells; HT29 Cells; Humans; Immunoprecipitation; Insulin-Like Growth Factor Binding Protein 3; Kaplan-Meier Estimate; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Pregnancy-Specific beta 1-Glycoproteins; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Kindlin-1, an integrin-interacting protein, has been implicated in TGF-β/Smad3 signaling. However, the molecular mechanism underlying Kindlin-1 regulation of TGF-β/Smad3 signaling remains elusive. Here, we reported that Kindlin-1 is an important mediator of TGF-β/Smad3 signaling by showing that Kindlin-1 physically interacts with TGF-β receptor I (TβRI), Smad anchor for receptor activation (SARA) and Smad3. Kindlin-1 is required for the interaction of Smad3 with TβRI, Smad3 phosphorylation, nuclear translocation, and finally the activation of TGF-β/Smad3 signaling pathway. Functionally, Kindlin-1 promoted colorectal cancer (CRC) cell proliferation in vitro and tumor growth in vivo, and was also required for CRC cell migration and invasion via an epithelial to mesenchymal transition. Kindlin-1 was found to be increased with the CRC progression from stages I to IV. Importantly, raised expression level of Kindlin-1 correlates with poor outcome in CRC patients. Taken together, we demonstrated that Kindlin-1 promotes CRC progression by recruiting SARA and Smad3 to TβRI and thereby activates TGF-β/Smad3 signaling. Thus, Kindlin-1 is a novel regulator of TGF-β/Smad3 signaling and may also be a potential target for CRC therapeutics.

Topics: Cell Proliferation; Colorectal Neoplasms; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Neoplasm Proteins; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Serine Endopeptidases; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Mutations in TGFBR2, a component of the transforming growth factor (TGF)-β signaling pathway, occur in high-frequency microsatellite instability (MSI-H) colorectal cancer (CRC). In mouse models, Tgfbr2 inactivation in the intestinal epithelium accelerates the development of malignant intestinal tumors in combination with disruption of the Wnt-β-catenin pathway. However, no studies have further identified the genes influenced by TGFBR2 inactivation following disruption of the Wnt-β-catenin pathway. We previously described CDX2P-G19Cre;Apcflox/flox mice, which is stochastically null for Apc in the colon epithelium. In this study, we generated CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice, with simultaneous loss of Apc and Tgfbr2. These mice developed tumors, including adenocarcinoma in the proximal colon. We compared gene expression profiles between tumors of the two types of mice using microarray analysis. Our results showed that the expression of the murine homolog of GSDMC was significantly upregulated by 9.25-fold in tumors of CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice compared with those of CDX2P-G19Cre;Apcflox/flox mice. We then investigated the role of GSDMC in regulating CRC tumorigenesis. The silencing of GSDMC led to a significant reduction in the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GSDMC enhanced cell proliferation. These results suggested that GSDMC functioned as an oncogene, promoting cell proliferation in colorectal carcinogenesis. In conclusion, combined inactivation of both Apc and Tgfbr2 in the colon epithelium of a CRC mouse model promoted development of adenocarcinoma in the proximal colon. Moreover, GSDMC was upregulated by TGFBR2 mutation in CRC and promoted tumor cell proliferation in CRC carcinogenesis, suggesting that GSDMC may be a promising therapeutic target.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli Protein; Animals; Biomarkers, Tumor; CDX2 Transcription Factor; Cell Line, Tumor; Cell Proliferation; Colon; Colorectal Neoplasms; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Transgenic; Microarray Analysis; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-β) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-β-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-β-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-β receptor II (TβRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF-β1 stimulation.. Our study demonstrates a requirement of VASP for TGF-β-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver.

Topics: Animals; Cell Adhesion Molecules; Cell Membrane; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hepatic Stellate Cells; HT29 Cells; Humans; Liver Neoplasms, Experimental; Mice; Microfilament Proteins; Myofibroblasts; Paracrine Communication; Phosphoproteins; Protein Serine-Threonine Kinases; rab GTP-Binding Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Smad3 and Smad4 are signaling mediators in the transforming growth factor β (TGFβ) pathway and play a major role in the progression and migration of many types of cancers. The TGFβ pathway is correlated with resistance against both targeted and conventional chemotherapeutic drugs. The aim of this study was to determine the effect of Smad3/4 on drug sensitivity in chemotherapy-resistant colorectal cancer (CRC) cells. We isolated the TGFβ-mediated chemoresistant CRC cell line DLD1-5FU-C10, which showed high expression of Smad3/4 and p21. In order to analyze the influence of Smad3/4 on drug sensitivity in DLD1-5FU-C10 cells, we knocked down Smad3/4 using small interfering RNAs (siRNA). The results showed similar drug sensitivity between the DLD1‑5FU-C10 and the DLD1 control cells and reduced p21 expression. In addition, we found a significant increase in the levels of 3 TGFβ downstream factors: interleukin 6 (IL6), plasminogen activator (PLAU) and prostaglandin-endoperoxide synthase 2 (PTGS2). Furthermore, we showed that Smad3/4 regulated the JAK1/STAT3 pathway via IL6 in the chemoresistant CRC cell line. In conclusion, we identified Smad3/4 as a novel drug sensitivity regulator in TGFβ-mediated chemotherapy-resistant CRC cells. Our results suggest that Smad3/4 regulate p-STAT3 signaling by IL6 and p21 and highlight an important role for STAT3 signaling in Smad3/4 regulated drug sensitivity in chemoresistant CRC cells.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Cell Movement; Cell Survival; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Signal Transduction; Smad3 Protein; Smad4 Protein; STAT3 Transcription Factor; Transcriptional Activation; Transforming Growth Factor beta

The cytotoxic activity and count of natural killer (NK) cells appear to be reduced after surgery; however, it is unknown whether the magnitude of this immune suppression is similar among different types of oncological surgery. In this study, we compared the innate immune function of patients undergoing three different oncological surgeries.. We compared the number and function of NK cells obtained from patients who had undergone mastectomies (n = 17), thoracotomies (n = 21), or liver resections for cancer (n = 22). Cytotoxicity assays were performed to measure the function of NK cells. We also determined the plasma concentrations of interleukins (IL) 2 and 4, interferon-γ, granzyme B, perforin, soluble major histocompatibility complex class I-related chain A, and epinephrine, both before and 24 h after surgery. Differences in immunologic parameters were compared preoperatively and postoperatively and by type of surgery. P values <0.05 were considered statistically significant.. The preoperative NK cell count differed statistically (P < 0.006) among all three types of surgeries; however, within surgery postoperative counts and changes compared with baseline did not. The postoperative function of NK cells was similar among types of surgeries, but was significantly reduced compared with preoperative levels (mastectomy P < 0.0001, thoracotomy P = 0.001, and liver resections P = 0.002). We observed a significant increase in the postoperative plasma concentrations of epinephrine, whereas the concentrations of major histocompatibility class I polypeptide-related sequence A and the IL-2 and/or IL-4 ratio remained unchanged before and after surgery.. The magnitude of innate immune suppression is similar among different oncological procedures. More studies are needed to better understand this complex phenomenon.

Topics: Adult; Aged; Breast Neoplasms; Colorectal Neoplasms; Cytokines; Epinephrine; Female; Humans; Immunity, Innate; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

The interconnected network of pathways downstream of the TGFβ, WNT and EGF-families of receptor ligands play an important role in colorectal cancer pathogenesis.We studied and implemented dynamic simulations of multiple downstream pathways and described the section of the signaling network considered as a Molecular Interaction Map (MIM). Our simulations used Ordinary Differential Equations (ODEs), which involved 447 reactants and their interactions.Starting from an initial "physiologic condition", the model can be adapted to simulate individual pathologic cancer conditions implementing alterations/mutations in relevant onco-proteins. We verified some salient model predictions using the mutated colorectal cancer lines HCT116 and HT29. We measured the amount of MYC and CCND1 mRNAs and AKT and ERK phosphorylated proteins, in response to individual or combination onco-protein inhibitor treatments. Experimental and simulation results were well correlated. Recent independently published results were also predicted by our model.Even in the presence of an approximate and incomplete signaling network information, a predictive dynamic modeling seems already possible. An important long term road seems to be open and can be pursued further, by incremental steps, toward even larger and better parameterized MIMs. Personalized treatment strategies with rational associations of signaling-proteins inhibitors, could become a realistic goal.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Epidermal Growth Factor; G1 Phase; HCT116 Cells; HT29 Cells; Humans; Models, Biological; Molecular Targeted Therapy; Neoplasm Proteins; Resting Phase, Cell Cycle; Transforming Growth Factor beta; Wnt Signaling Pathway

Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor β receptor II (TGFBR2). TGFβ signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFβ. We investigated how TGFβ signaling might continue in MSI-H CRC cells.. We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGFβ signaling was detected by SMAD2 phosphorylation and through use of a TGFβ-responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGFβ, and their activities were measured.. SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2—even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein.. TGFβ signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGFβ signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated.

Topics: Colorectal Neoplasms; Frameshift Mutation; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; HCT116 Cells; HEK293 Cells; Humans; Microsatellite Instability; Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad2 Protein; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Transforming growth factor-beta-induced (TGFBI) serves as a linker protein and plays a role in the activation of morphogenesis, cell proliferation, adhesion, migration, differentiation and inflammation. High expression levels of the human TGFBI gene are correlated with numerous human malignancies. In order to explore the roles of TGFBI in the tumor progression of colorectal cancer, colorectal cancer specimens from 115 patients with strict follow-up were selected for the analysis of TGFBI by immunohistochemistry. The correlations between TGFBI expression and the clinicopathological features of colorectal cancers were evaluated. In the colorectal cancer tissues, TGFBI was mainly localized in the cytoplasm and stroma and scarcely in the nucleus. TGFBI expression in the cytoplasm and stroma was not found to be associated with age, gender, tumor histopathological grading, PT category and tumor location (P > 0.05 for each). However, high TGFBI expression in the cytoplasm and stroma correlated with lymph node metastasis, distant metastasis and Dukes stage (P < 0.05 for each). The survival rate was significantly lower in patients with high TGFBI expression than in those with low TGFBI expression. Furthermore, we found that tumor node metastasis (TNM) staging (HR: 2.963; 95% CI: 1.573-1.664; P = 0.000), differentiation (HR: 1.574; 95% CI: 1.001-2.476; P = 0.049) and high TGFBI cytoplasmic expression (HR: 3.332; 95% CI: 1.410-7.873; P = 0.000) proved to be independent prognostic factors for survival in colorectal cancer. In conclusion, TGFBI plays an important role in the progression of colorectal cancers and it is an independent poor prognostic factor for colorectal cancer patients.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; Extracellular Matrix Proteins; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Prognosis; Proportional Hazards Models; Transforming Growth Factor beta

DACH1 has been found down-regulated in a variety of human cancers, but its clinical significance and functional roles in colorectal cancer (CRC) remain unknown. In this study, we identified DACH1 as a tumor suppressor in CRC. Suppression of DACH1 strikingly increased cell growth, migration and invasion potential of CRC cell line SW480. Expression analysis of a set of epithelial-mesenchymal transition (EMT) markers by RT-qPCR and western blot showed an increase in the expression of mesenchymal markers (vimentin and N-cadherin) and a reduction in the expression of epithelial marker (E-cadherin and γ-catenin). Furthermore, EMT characteristics in DACH1-downregulated CRC cells were abrogated by TGF-β inhibitor SB431542. DACH1 overexpression reduced TGF-β-induced EMT and inhibited SW480 cell invasion which can be reversed in the presence of TGF-β. Thus, our results suggest that DACH1 loss of function results in increased cell growth, motility and invasiveness through TGF-β-mediated EMT, and DACH1 loss of function has important therapeutic implications for targeted therapies of CRC.

Topics: Base Sequence; Biomarkers, Tumor; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; DNA Primers; Down-Regulation; Epithelial-Mesenchymal Transition; Eye Proteins; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

Topics: Aged; Animals; Carcinogenesis; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Insulin-Like Growth Factor Binding Proteins; Male; Mice; Middle Aged; Neoplasm Metastasis; Smad Proteins; Transforming Growth Factor beta; Wnt Signaling Pathway

As patient survival drops precipitously from early-stage cancers to late-stage and metastatic cancers, microRNAs that promote relapse and metastasis can serve as prognostic and predictive markers as well as therapeutic targets for chemoprevention. Here we show that miR-1269a promotes colorectal cancer (CRC) metastasis and forms a positive feedback loop with TGF-β signalling. miR-1269a is upregulated in late-stage CRCs, and long-term monitoring of 100 stage II CRC patients revealed that miR-1269a expression in their surgically removed primary tumours is strongly associated with risk of CRC relapse and metastasis. Consistent with clinical observations, miR-1269a significantly increases the ability of CRC cells to invade and metastasize in vivo. TGF-β activates miR-1269 via Sox4, while miR-1269a enhances TGF-β signalling by targeting Smad7 and HOXD10, hence forming a positive feedback loop. Our findings suggest that miR-1269a is a potential marker to inform adjuvant chemotherapy decisions for CRC patients and a potential therapeutic target to deter metastasis.

Topics: Adult; Aged; Aged, 80 and over; Animals; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin Immunoprecipitation; Colorectal Neoplasms; Feedback, Physiological; Female; Fluorescent Antibody Technique; HCT116 Cells; Homeodomain Proteins; HT29 Cells; Humans; Male; Mice; MicroRNAs; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Prognosis; Real-Time Polymerase Chain Reaction; Smad7 Protein; SOXC Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Young Adult

Topics: Colorectal Neoplasms; Frameshift Mutation; Humans; Microsatellite Instability; Protein Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Stepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC). These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC.. We analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β) growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC) markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5%) but not in tumors with downregulated but non-alternating PTEN expression (14.3%). There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases.. In conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt/β-catenin pathways. This bottleneck-like zone is usually followed by the emergence of invasive tumors with intact PTEN expression but dysregulated TP53 and uniformly high proliferation rates.

Topics: Adenocarcinoma; Adenoma; beta Catenin; Biomarkers, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Progression; Haploinsufficiency; Humans; Mutation; Neoplasm Invasiveness; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Signaling Pathway

In colorectal cancer (CRC), genetic alterations are involved in disease progression from adenoma to carcinoma and metastatic disease. Three different carcinogenesis mechanisms exist: chromosomal instability phenotype (CIN), microsatellite instability phenotype (MSI) and the hypermetylator phenotype (CIMP for CpG Island methylation phenotype). These molecular alterations lead to signal transduction dysfunction, and signaling pathways alterations are involved in cancer mechanisms, such as EGFR pathway, WNT/APC pathway, TGFB pathway and p53 pathway. Recently, molecular signatures have been established, allowing distinction of 4 different types of CCR. Finally, circulating biomarkers are investigated for molecular characterization.

Topics: Chromosomal Instability; Colorectal Neoplasms; CpG Islands; DNA Methylation; ErbB Receptors; Humans; Microsatellite Instability; Transforming Growth Factor beta

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-β activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

Topics: Carcinoma; Colorectal Neoplasms; Consensus; CpG Islands; DNA Copy Number Variations; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Information Dissemination; Microsatellite Instability; Mutation; Neovascularization, Pathologic; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Transforming Growth Factor beta; Wnt Signaling Pathway

Topics: Carcinoma; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Neovascularization, Pathologic; Transforming Growth Factor beta

Tumours frequently activate genes whose expression is otherwise biased to the testis, collectively known as cancer-testis antigens (CTAs). The extent to which CTA expression represents epiphenomena or confers tumorigenic traits is unknown. In this study, to address this, we implemented a multidimensional functional genomics approach that incorporates 7 different phenotypic assays in 11 distinct disease settings. We identify 26 CTAs that are essential for tumor cell viability and/or are pathological drivers of HIF, WNT or TGFβ signalling. In particular, we discover that Foetal and Adult Testis Expressed 1 (FATE1) is a key survival factor in multiple oncogenic backgrounds. FATE1 prevents the accumulation of the stress-sensing BH3-only protein, BCL-2-Interacting Killer (BIK), thereby permitting viability in the presence of toxic stimuli. Furthermore, ZNF165 promotes TGFβ signalling by directly suppressing the expression of negative feedback regulatory pathways. This action is essential for the survival of triple negative breast cancer cells in vitro and in vivo. Thus, CTAs make significant direct contributions to tumour biology.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antigens, Neoplasm; Apoptosis Regulatory Proteins; Carcinogenesis; Cell Line; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; DNA-Binding Proteins; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Immunoblotting; In Vitro Techniques; Lung Neoplasms; Membrane Proteins; Mice, Inbred NOD; Mitochondrial Proteins; Neoplasm Transplantation; Neoplasms; Prognosis; Proportional Hazards Models; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad7 Protein; Transcription Factors; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Ubiquitin-Protein Ligases; Wnt Signaling Pathway

Although elevated CD4⁺Foxp3⁺ regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4⁺Foxp3⁺ T cells (Tregs) were Helios⁺ and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ∼30% of intratumoral CD4⁺Foxp3⁻ T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and was ∼50-fold more suppressive than Foxp3⁺ Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Colorectal Neoplasms; Female; Forkhead Transcription Factors; Humans; Immunophenotyping; Inducible T-Cell Co-Stimulator Protein; Integrin alpha Chains; Interleukin-10; Lymphocyte Activation Gene 3 Protein; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Neoplasm Staging; Phenotype; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Adenomatous polyposis coli (APC)-regulated Wnt and transforming growth factor-β (TGFβ) signaling cooperate in the intestine to maintain normal enterocyte functions. Human clinical trials showed that estrogen [17β-estradiol (E2)], the ligand of nuclear receptors estrogen receptor α (ERα) and ERβ, inhibited colorectal cancer (CRC) in women. Consistent with this finding, we reported that E2, ERα and ERβ suppressed intestinal tumorigenesis in the C57BL/6J-Min/+ (Min/+) mouse, a CRC model. Here, we extended our results with further comparisons of colon and small intestine from intact female Apc (+/+) (WT), Min/+ and ER-deficient Min/+ mice. In the colon of ER-deficient Min/+ mice, ER loss reduced TGFβ signaling in crypt base cells as evidenced by minimal expression of the effectors Smad 2, 3 and 4 in these strains. We also found reduced expression of Indian hedgehog (Ihh), bone morphogenetic protein 4 and hepatocyte nuclear factor 3β or FoxA2, factors needed for paracrine signaling between enterocytes and mesenchyme. In proximal colon, ER loss produced a >10-fold increased incidence of crypt fission, a marker for wound healing and tumor promotion. These data, combined with our previous work detailing the specific roles of E2, ERα and ERβ in the colon, suggest that ER activity helps to maintain the intestinal stem cell (ISC) microenvironment by modulating epithelial-stromal crosstalk in ways that regulate cytokine, Wnt and Ihh availability in the extracellular matrix (ECM).

Topics: Aberrant Crypt Foci; Animals; Cellular Microenvironment; Colon; Colorectal Neoplasms; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Hedgehog Proteins; Hepatocyte Nuclear Factor 3-beta; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Paneth Cells; Signal Transduction; Smad4 Protein; Stem Cells; Transforming Growth Factor beta

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

Topics: Animals; Apoptosis; Blotting, Western; Bone Marrow; Cadherins; Cell Adhesion; Cell Communication; Cell Membrane; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokines; Colorectal Neoplasms; Cytokines; Epithelial-Mesenchymal Transition; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Humans; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a major inducer of epithelial-mesenchymal transition (EMT) in different cell types. TGF-β-mediated EMT is thought to contribute to tumour cell spread and metastasis. Sialyl Lewis antigens synthesised by fucosyltransferase (FUT) 3 and FUT6 are highly expressed in patients with metastatic colorectal cancer (CRC) and are utilised as tumour markers for cancer detection and evaluation of treatment efficacy. However, the role of FUT3 and FUT6 in augmenting the malignant potential of CRC induced by TGF-β is unclear.. Colorectal cancer cell lines were transfected with siRNAs for FUT3/6 and were examined by cell proliferation, invasion and migration assays. The expression and phosphorylation status of TGF-β downstream molecules were analysed by western blot. Fucosylation of TGF-β receptor (TβR) was examined by lectin blot analysis.. Inhibition of FUT3/6 expression by siRNAs suppressed the fucosylation of type I TβR and phosphorylation of the downstream molecules, thereby inhibiting the invasion and migration of CRC cells by EMT.. Fucosyltransferase 3/6 has an essential role in cancer cell adhesion to endothelial cells by upregulation of sialyl Lewis antigens and also by enhancement of cancer cell migration through TGF-β-mediated EMT.

Topics: Cell Adhesion; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Fucosyltransferases; HT29 Cells; Humans; Phosphorylation; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transfection; Transforming Growth Factor beta; Up-Regulation

Evidence shows that single-nucleotide polymorphisms in microRNA (miRNA) target sites can create, destroy, or modify the miRNA/mRNA binding, therefore modulating gene expression and affecting cancer susceptibility. The transforming growth factor-β (TGF-β) signaling pathway plays a pivotal role in tumor initiation and progression. Intriguingly, recent advances of genome-wide association studies have identified multiple risk loci in this pathway to be associated with risk of colorectal cancer (CRC). To test the hypothesis that genetic variants in miRNA target sites in genes of the TGF-β signaling pathway may also be associated with CRC risk, we first systematically scanned the single-nucleotide polymorphisms (SNPs) in genes of TGF-β signaling pathway which potentially affect the miRNA/mRNA bindings. Through a series of filters, we narrowed down these candidates to four SNPs. Then, we conducted a case-control study with 600 CRC patients and 638 controls in Han Chinese population. We observed that compared with A carriers (AA + AG), the GG genotype of rs12997:ACVR1 is associated with a significantly higher risk of CRC (OR = 1.52, 95% confidence interval (95% CI) = 1.04-2.21, P = 0.031), particularly in nonsmokers with a higher OR of 1.63 (95% CI = 1.04-2.55, P = 0.032). Our study suggested that SNPs in miRNA target sites could contribute to the likelihood of CRC susceptibility and emphasized the important role of polymorphisms at miRNA-regulatory elements in carcinogenesis.

Topics: Activin Receptors, Type I; Adult; Aged; Case-Control Studies; Colorectal Neoplasms; Female; Humans; Male; MicroRNAs; Middle Aged; Polymorphism, Single Nucleotide; Signal Transduction; Transforming Growth Factor beta

Fibroblasts in the tumor microenvironment are a key determinant in cancer progression and may be a promising target for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is known as a tumor suppressor in colorectal cancer (CRC). The present study investigated the inductive mechanism of IGFBP7 expression in fibroblasts by supernatant from the CRC cell line, SW620. The results showed that the expression of IGFBP7 was up-regulated in the fibroblasts when treated with SW620 supernatant and exogenous TGF-β1. The IGFBP7 induced by SW620 supernatant or TGF-β1 was partially inhibited by the TGF-β1 specific antibody AF and TGF-β1 receptor antagonist SB431542. The Wnt signaling-targeted genes, c-Myc, CCND1 and the proteins Dvl2/3, were all up-regulated in fibroblasts expressing high levels of IGFBP7, and the up-regulation could be inhibited both by the Wnt signaling antagonist Dickkopf-1 (DKK1) and by the TGF-β1 receptor antagonist SB431542. In conclusion, CRC cells promote the high expression of IGFBP7 in fibroblasts, most likely through the co-regulation of TGF-β and Wnt signaling in a Smad2/3-Dvl2/3 dependent manner. Taken together, these data suggest that the fibroblasts could be a novel therapeutic target in tumor therapy.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Colorectal Neoplasms; Culture Media, Conditioned; Dishevelled Proteins; Fibroblasts; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Proteins; Phosphoproteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation; Wnt Signaling Pathway

Transforming growth factor-beta (TGF-β) is deeply involved in colorectal cancer development and the disruption of the TGF-β signaling in dysplastic cells is required for tumor to grow. Nevertheless, tumor cells express TGF-β to escape the immune surveillance mediated by T cells. T-cell expression of Smad7, an intracellular inhibitor of the TGF-β signaling, protects against colitis-associated colorectal cancer. However, whether Smad7 in T cells might influence colorectal cancer growth independently of chronic inflammation and which T-cell subset is involved in this process is unknown. To address this issue, T-cell-specific Smad7 transgenic mice and wild-type (WT) littermates were subcutaneously transplanted with syngenic MC38 colon carcinoma cells. Smad7Tg mice were resistant to tumor development compared with WT mice and protection was dependent on CD4(+) T cells. Smad7 expression in T cells increased the number of tumor-infiltrating Tbet/ROR-γ-t double-positive CD4 T cells characterized by the expression of tumor necrosis factor-alpha (TNF-α) and interferon-gamma but lower IL17A. The low expression of IL17A caused by the Smad7 expression in tumor-infiltrating CD4(+) T cells enabled the TNF-α-mediated killing of cancer cells both in vitro and in vivo, thus indicating that the Smad7-mediated plastic effect on T-cell phenotype induces protection against colorectal cancer.

Topics: Animals; Apoptosis; Cell Proliferation; Colorectal Neoplasms; Flow Cytometry; Humans; Immunoenzyme Techniques; Interferon-gamma; Interleukin-17; Lymphocytes, Tumor-Infiltrating; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad7 Protein; Th17 Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The c-Myc oncoprotein is activated in the majority of colorectal cancers (CRCs), whereas the TGF-β pathway is frequently affected by loss-of-function mutations, for example in SMAD2/3/4 genes. The canonical model places Myc downstream of inhibitory TGF-β signaling. However, we previously demonstrated that Myc also inhibits TGF-β signaling through the miR-17~92 microRNA cluster, raising the question about functional relationships between these two pathways.. We engineered a series of genetically complex murine and human CRC cell lines in which Myc and TGF-β activities could be manipulated simultaneously. This was achieved through retroviral expression of the Myc-estrogen receptor fusion protein and through Smad4 short hairpin RNA knockdown. Cell lines thus modified were injected subcutaneously in immunocompromised mice, and the resultant tumors (n = 5-10 per treatment group) were analyzed for overall growth and neovascularization. Additionally, the distribution of MYC and TGF-β pathway mutations was analyzed in previously profiled human CRC samples.. In kras-mutated/trp53-deleted murine colonocytes, either Myc activation or TGF-β inactivation increased tumor sizes and microvascular densities approximately 1.5- to 2.5-fold, chiefly through downregulation of thrombospondin-1 and related type I repeat-containing proteins. Combining Myc activation with TGF-β inactivation did not further accelerate tumorigenesis. This redundancy and the negative effect of TGF-β signaling on angiogenesis were also demonstrated using xenografts of human CRC cell lines. Furthermore, the analysis of the Cancer Genome Atlas data revealed that in CRC without microsatellite instability, overexpression of Myc and inactivation of Smads (including acquired mutations in SMAD2) are mutually exclusive, with odds ratio less than 0.1.. In human CRC, gain-of-function alterations in Myc and loss-of-function alterations in TGF-β exhibit a masking epistatic interaction and are functionally redundant.

Topics: Angiogenesis Inhibitors; Animals; Blotting, Western; Cell Line, Tumor; Colorectal Neoplasms; Epistasis, Genetic; Heterografts; Humans; Immunohistochemistry; Mice; Mutation; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs), and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT)-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins c-fos; Signal Transduction; Transforming Growth Factor beta

WJ1376-1 and WJ1398-1 are new synthetic compounds developed based on the structure of the Chinese herbal medicine osthole. Previously, we reported that WJ1376-1 and WJ1398-1 can induce cell-cycle arrest by activating ATR kinase (ataxia telangiectasia and rad3 related kinase) and inhibiting the phosphorylation of Aurora A kinase. In this study, we determined that WJ1376-1 and WJ1398-1 strongly inhibited the migration and invasion in human colorectal cancer cells at concentrations as low as 1μM. In the transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition model, WJ1376-1 and WJ1398-1 potently downregulated the transcription factor Snail1, the mesenchymal protein vimentin, and matrix metalloprotease-9, but upregulated the epithelial protein E-cadherin. WJ1376-1 and WJ1398-1 also inhibited the TGF-β-induced phosphorylation of Smad2 and of Akt at Ser 473, and the nuclear translocation of Smad2 was substantially lower in WJ1376-1- and WJ1398-1-treated cells than it was in control cells. In transient transfection experiments, we observed that WJ1376-1 and WJ1398-1 strongly inhibited TGF-β-stimulated activity of a Smad reporter. Finally, WJ1376-1 and WJ1398-1 blocked TGF-β-induced phosphorylation of the TGF-β Type I receptor (TGF-βRI). These results suggest that WJ1376-1 and WJ1398-1 inhibit cell migration and invasion by suppressing TGF-βRI phosphorylation and subsequently hindering both Smad2 and phosphatidylinositol 3-kinase/Akt signaling pathways.

Topics: Adenocarcinoma; Cell Movement; Cell Survival; Colorectal Neoplasms; Coumarins; Epithelial-Mesenchymal Transition; HCT116 Cells; Humans; Hydroxamic Acids; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA; Smad2 Protein; Transforming Growth Factor beta

Conventional cancer chemotherapy preferentially destroys non-stem cancer cells within a tumor, and a subpopulation of cancer stem cells (CSCs) is more resistant and survives, leading to relapses and metastasis. Howeve, recent studies suggest that CD24 and susceptibility to epithelial-mesenchymal transition (EMT) can serve as markers of CSCs. We report that CD24(+) cells are susceptible to induction of EMT, a phenotype important for cancer metastasis. We studied the responsiveness of CSC markers to TGF-β , an effective EMT inducer. The data on CD24 demonstrated that CD24(+) cells are susceptible to EMT, a phenotype important for cancer metastasis in two colorectal cancer cell lines, the CaR-1 and CCK81. CD24(+) cells expressed Notch 1 in response to exposure to TGF-β in culture and showed higher tumorigenic activity compared to controls. This evidence shows that CD24(+) cells are susceptible to EMT induction and to cancer progression and is indicative of the candidacy of CD24 as a therapeutic target in CSC.

Topics: Animals; CD24 Antigen; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Heterografts; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Effects of transforming growth factor-beta (TGF-β) were investigated in human colorectal cancer, and the influence of cantharidinate in inhibiting TGF-β1 expression was explored. Relationships among TGF-β1 and sex, age, tumor size, tumor location, tumor stage were also analyzed. H and E and immunohistochemistry staining were employed to assess colorectal cancer and TGF-β1 expression, respectively. Then, HCT-116 CRC cells were randomly divided into four groups, controls, no serum-treated, chemotherapy and cantharidinate-treated. Immunohistochemistry and real-time PCR were employed to assess the expression of TGF-β1 in CRC cells. Our data showed that the expression of TGF-β1 might be associated with tumor size and tumor location (P<0.05). The expression of TGF-β1 in CRC groups was higher than in adjacent groups (P<0.05). In addition, the expression of TGF-β1 in cantharidinate-treated group was much lower than in CRC group (P<0.05). Taken together, these results suggest that TGF-β1 plays an important role in CRC development. Cantharidinate might inhibit the expression of TGF-β1 and control the development of colorectal cancer.

Topics: Cantharidin; Cell Line, Tumor; Colorectal Neoplasms; Enzyme Inhibitors; HCT116 Cells; Humans; Mutation; Random Allocation; Transforming Growth Factor beta

Drug-induced gene-expression profiles that invert disease profiles have recently been illustrated to be a starting point for drug repositioning. In this study, we validate this approach and focus on prediction of novel drugs for colorectal cancer, for which there is a pressing need to find novel antimetastatic compounds. We computationally predicted three novel and still unknown compounds against colorectal cancer: citalopram (an antidepressant), troglitazone (an antidiabetic), and enilconazole (a fungicide). We verified the compounds by in vitro assays of clonogenic survival, proliferation, and migration and in a subcutaneous mouse model. We found evidence that the mode of action of these compounds may be through inhibition of TGFβ signaling. Furthermore, one compound, citalopram, reduced tumor size as well as the number of circulating tumor cells and metastases in an orthotopic mouse model of colorectal cancer. This study proposes citalopram as a potential therapeutic option for patients with colorectal cancer, illustrating the potential of systems pharmacology.

Topics: Animals; Antineoplastic Agents; Chromans; Citalopram; Colorectal Neoplasms; Gene Expression Profiling; HCT116 Cells; HT29 Cells; Humans; Imidazoles; Mice, Inbred NOD; Neoplastic Cells, Circulating; Signal Transduction; Thiazolidinediones; Transcriptome; Transforming Growth Factor beta; Troglitazone; Xenograft Model Antitumor Assays

The immune infiltrate in colorectal cancer has been correlated with outcome, such that individuals with higher infiltrations of T cells have increased survival independent of the disease stage. For patients with lower immune infiltrates, overall survival is limited. Because the patients with colorectal cancer studied have received conventional cancer therapies, these data may indicate that the pretreatment tumor environment increases the efficacy of treatments such as chemotherapy, surgery, and radiotherapy. This study was designed to test the hypothesis that an improved immune environment in the tumor at the time of treatment will increase the efficacy of radiotherapy. We demonstrate that inhibition of TGFβ using the orally available small-molecule inhibitor SM16 improved the immune environment of tumors in mice and significantly improved the efficacy of subsequent radiotherapy. This effect was not due to changes in radiosensitivity, epithelial-mesenchymal transition, or changes in vascular function in the tumor; rather, this effect was dependent on adaptive immunity and resulted in long-term protective immunity in cured mice. These data demonstrate that immunotherapy is an option to improve the immune status of patients with poor tumor infiltrates and that pretreatment improves the efficacy of radiotherapy.

Topics: Adaptive Immunity; Animals; Antineoplastic Agents; Azabicyclo Compounds; Chemotherapy, Adjuvant; Colorectal Neoplasms; Drug Evaluation, Preclinical; Female; Kaplan-Meier Estimate; Lymphocytes, Tumor-Infiltrating; Mammary Neoplasms, Experimental; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoadjuvant Therapy; Neoplasm Transplantation; Radiation Tolerance; Transforming Growth Factor beta; Tumor Cells, Cultured

Colon carcinoma is a malignant tumor showing a marked preference to metastasize to distant organs. The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. However, whether circulating cytokines are linked to the circulation of tumor cells, as individual cells or clusters, remain unclear. In this study, we investigated the circulating levels of TGF-beta, CXCL1, VEGF and PAI-1 as potential bioindicators of the presence of CTCs in patients with metastatic colon cancer.. Circulating tumor cells (CTCs) were isolated from peripheral blood by immunomagnetic separation and phenotypically characterized in a cohort of 103 patients with metastatic colon cancer. TGF-beta, CXCL1, VEGF and PAI-1 concentrations were determined by immunoassay in plasma samples from the same patients.. We detected two different populations of CTCs, single cells or clusters in patients with metastatic colon cancer. Importantly, we found that the presence of clustered CTCs is significantly associated with elevated circulating levels of TGF-beta and CXCL1 and with reduced overall survival. Finally, we observed that circulating levels of cytokines are differently associated with the two populations of CTCs.. Taken together, these findings show that detection of clustered CTCs represents a negative prognostic factor in patients with metastatic colon cancer. The presence of clustered CTCs is associated with elevated circulating levels of cytokines such as TGF-beta and CXCL1. This suggests an additional role for circulating cytokines as predictive tool for cancer prognosis and diagnosis of minimal residual disease as well as assessment of tumor sensitivity to anticancer therapy.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Chemokine CXCL1; Colorectal Neoplasms; Cytokines; Female; Genetic Predisposition to Disease; Humans; Italy; Male; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Phenotype; Prognosis; Transforming Growth Factor beta

Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) signaling are required for hepatic stellate cell (HSC) activation under pathological conditions such as liver metastatic tumor growth. These two signaling pathways are functionally divergent; PDGF signaling promotes proliferation and migration of HSCs, and TGF-β induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-β-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-β activation of HSCs has not been investigated. Here we report that PDGF receptor-α (PDGFR-α) is required for TGF-β signaling of cultured human HSCs although HSCs express both PDGF-α and -β receptors. PDGFR-α knockdown inhibits TGF-β-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-β signaling. PDGFR-α knockdown suppresses TGF-β receptor I (TβRI) but increases TβRII gene transcription. At the protein level, PDGFR-α is recruited to TβRI/TβRII complexes by TGF-β stimulation. PDGFR-α knockdown blocks TGF-β-mediated internalization of TβRII and induces accumulation of TβRII at the plasma membrane, thereby inhibiting TGF-β phosphorylation of SMAD2. Functionally, knockdown of PDGFR-α reduces paracrine effects of HSCs on colorectal cancer cell proliferation and migration in vitro. In mice and patients, colorectal cancer cell invasion of the liver induces upregulation of PDGFR-α of HSCs. In summary, our finding that PDGFR-α knockdown inhibits SMAD-dependent TGF-β signaling by repressing TβRI transcriptionally and blocking endocytosis of TGF-β receptors highlights a convergence of PDGF and TGF-β signaling for HSC activation and PDGFR-α as a therapeutic target for liver metastasis and other settings of HSC activation.

Topics: Active Transport, Cell Nucleus; Animals; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation; HEK293 Cells; Hepatic Stellate Cells; HT29 Cells; Humans; Liver Neoplasms; Mice, SCID; Paracrine Communication; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad2 Protein; Time Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta

The expression of LIM and SH3 protein 1 (LASP1) was upregulated in colorectal cancer cases, thereby contributing to the aggressive phenotypes of colorectal cancer cells. However, we still cannot decipher the underlying molecular mechanism associated with colorectal cancer metastasis.. In this study, IHC was performed to investigate the expression of proteins in human colorectal cancer tissues. Western blot analysis was used to assess the LASP1-induced signal pathway. Two-dimensional difference gel electrophoresis was performed to screen LASP1-modulated proteins and uncover the molecular mechanism of LASP1. TGFβ was used to induce an epithelial-mesenchymal transition (EMT).. LASP1 expression was correlated with the mesenchymal marker vimentin and was inversely correlated with epithelial markers, namely, E-cadherin and β-catenin, in clinical colorectal cancer samples. The gain- and loss-of-function assay showed that LASP1 induces EMT-like phenotypes in vitro and in vivo. S100A4, identified as a LASP1-modulated protein, was upregulated by LASP1. Moreover, it is frequently coexpressed with LASP1 in colorectal cancer. S100A4 was required for EMT, and an increased cell invasiveness of colorectal cancer cell is induced by LASP1. Furthermore, the stimulation of TGFβ resulted in an activated Smad pathway that increased the expression of LASP1 and S100A4. The depletion of LASP1 or S100A4 expression inhibited the TGFβ signaling pathway. Moreover, it significantly weakened the proinvasive effects of TGFβ on colorectal cancer cells.. These findings elucidate the central role of LASP1 in the TGFβ-mediated EMT process and suggest a potential target for the clinical intervention in patients with advanced colorectal cancer.

Topics: Adaptor Proteins, Signal Transducing; beta Catenin; Cadherins; Cell Line, Tumor; Colorectal Neoplasms; Cytoskeletal Proteins; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; LIM Domain Proteins; Neoplasm Invasiveness; S100 Calcium-Binding Protein A4; S100 Proteins; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Vimentin

Latency-associated peptide (LAP) - expressing regulatory T cells (Tregs) are important for immunological self-tolerance and immune homeostasis. In order to investigate the role of LAP in human CD4⁺Foxp3⁺ Tregs, we designed a cross-sectional study that involved 42 colorectal cancer (CRC) patients. The phenotypes, cytokine-release patterns, and suppressive ability of Tregs isolated from peripheral blood and tumor tissues were analyzed. We found that the population of LAP-positive CD4⁺Foxp3⁺ Tregs significantly increased in peripheral blood and cancer tissues of CRC patients as compared to that in the peripheral blood and tissues of healthy subjects. Both LAP⁺ and LAP⁻ Tregs had a similar effector/memory phenotype. However, LAP⁺ Tregs expressed more effector molecules, including tumor necrosis factor receptor II, granzyme B, perforin, Ki67, and CCR5, than their LAP⁻ negative counterparts. The in vitro immunosuppressive activity of LAP⁺ Tregs, exerted via a transforming growth factor-β-mediated mechanism, was more potent than that of LAP⁻ Tregs. Furthermore, the enrichment of LAP⁺ Treg population in peripheral blood mononuclear cells (PBMCs) of CRC patients correlated with cancer metastases. In conclusion, we found that LAP⁺ Foxp3⁺ CD4⁺ Treg cells represented an activated subgroup of Tregs having more potent regulatory activity in CRC patients. The increased frequency of LAP⁺ Tregs in PBMCs of CRC patients suggests their potential role in controlling immune response to cancer and presents LAP as a marker of tumor-specific Tregs in CRC patients.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Case-Control Studies; Colorectal Neoplasms; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Granzymes; Humans; Immune Tolerance; Immunologic Memory; Ki-67 Antigen; Lymphatic Metastasis; Male; Middle Aged; Peptides; Perforin; Protein Precursors; Receptors, CCR5; Receptors, Tumor Necrosis Factor, Type II; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-β acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-β. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-β. In addition, Irx5 expression confers a growth advantage in the presence of TGF-β, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-β activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-β, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-β pathway components.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Cell Line, Tumor; Cells, Cultured; Colorectal Neoplasms; Drosophila; Drosophila Proteins; Eye Proteins; Homeodomain Proteins; Humans; Intestinal Mucosa; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have led to the identification of a number of common variants associated with modest risk. Several risk variants map within the vicinity of TGFβ/BMP signaling pathway genes, including rs4939827 within an intron of SMAD7 at 18q21.1. A previous study implicated a novel SNP (novel 1 or rs58920878) as a functional variant within an enhancer element in SMAD7 intron 4. In this study, we show that four SNPs including novel 1 (rs6507874, rs6507875, rs8085824, and rs58920878) in linkage disequilibrium (LD) with the index SNP rs4939827 demonstrate allele-specific enhancer effects in a large, multi-component enhancer of SMAD7. All four SNPs demonstrate allele-specific protein binding to nuclear extracts of CRC cell lines. Furthermore, some of the risk-associated alleles correlate with increased expression of SMAD7 in normal colon tissues. Finally, we show that the enhancer is responsive to BMP4 stimulation. Taken together, we propose that the associated CRC risk at 18q21.1 is due to four functional variants that regulate SMAD7 expression and potentially perturb a BMP negative feedback loop in TGFβ/BMP signaling pathways.

Topics: Bone Morphogenetic Proteins; Cell Line, Tumor; Chromosomes, Human, Pair 18; Colorectal Neoplasms; Enhancer Elements, Genetic; Genetic Variation; HCT116 Cells; HEK293 Cells; Humans; Linkage Disequilibrium; Polymorphism, Single Nucleotide; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mesenchymal Stem Cells; Mice; Platelet Activation; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Transcriptome; Transforming Growth Factor beta

Prostaglandin (PG) E2 plays a critical role in colorectal cancer (CRC) progression, including epithelial-mesenchymal transition (EMT). Activity of the rate-limiting enzyme for PGE2 catabolism (15-hydroxyprostaglandin dehydrogenase [15-PGDH]) is dependent on availability of NAD+. We tested the hypothesis that there is intra-tumoral variability in PGE2 content, as well as in levels and activity of 15-PGDH, in human CRC liver metastases (CRCLM). To understand possible underlying mechanisms, we investigated the relationship between hypoxia, 15-PGDH and PGE2 in human CRC cells in vitro.. Tissue from the periphery and centre of 20 human CRCLM was analysed for PGE2 levels, 15-PGDH and cyclooxygenase (COX)-2 expression, 15-PGDH activity, and NAD+/NADH levels. EMT of LIM1863 human CRC cells was induced by transforming growth factor (TGF) β.. PGE2 levels were significantly higher in the centre of CRCLM compared with peripheral tissue (P = 0.04). There were increased levels of 15-PGDH protein in the centre of CRCLM associated with reduced 15-PGDH activity and low NAD+/NADH levels. There was no significant heterogeneity in COX-2 protein expression. NAD+ availability controlled 15-PGDH activity in human CRC cells in vitro. Hypoxia induced 15-PGDH expression in human CRC cells and promoted EMT, in a similar manner to PGE2. Combined 15-PGDH expression and loss of membranous E-cadherin (EMT biomarker) were present in the centre of human CRCLM in vivo.. There is significant intra-tumoral heterogeneity in PGE2 content, 15-PGDH activity and NAD+ availability in human CRCLM. Tumour micro-environment (including hypoxia)-driven differences in PGE2 metabolism should be targeted for novel treatment of advanced CRC.

Topics: Cell Hypoxia; Colorectal Neoplasms; Dinoprostone; Epithelial-Mesenchymal Transition; Humans; Hydroxyprostaglandin Dehydrogenases; Immunohistochemistry; Liver Neoplasms; Microarray Analysis; Neoplasm Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Patients with ulcerative colitis have increased risk of colorectal carcinoma, but little is known about how peritoneal macrophages are involved in ulcerative colitis-associated carcinogenesis. We investigated the alteration of peritoneal macrophages and M1/M2 subpopulations during ulcerative colitis-associated carcinogenesis.. Expression and functional changes in peritoneal macrophages and M1/M2 subpopulations were investigated by histopathology, flow cytometry, immunofluorescence, cytokines expression by ELISA and QRT-PCR in an azoxymethane (AOM)- and dextran sodium sulfate (DSS)-induced chemical colitis-associated carcinoma mouse model using male Crj:CD-1 (ICR) mice.. Striking evidence observed in histopathology, flow cytometry, cytokine detection, and gene expression analysis all revealed that inflammation-associated cytokines (IL-1β, IL-10, IL-12, IL-6, TNF-α) and migration/invasion-associated factors (G-CSF, GM-CSF, CXCR4, VEGF, TGF-β, ICAM-1) induced by peritoneal M2 macrophages increased significantly during the progression from inflammatory hyperplasia to carcinoma and metastasis. Similar functional changes occurred during peritoneal metastasis in M1 macrophages without changed polarization.. These results suggested that peritoneal M2 macrophages played a critical role in ulcerative colitis-associated carcinogenesis, including unbalanced pro-inflammatory and anti-inflammatory axis and enhanced expression of migration/invasion-associated factors. Furthermore, functional changes of M1 macrophages occurred without changed polarization during carcinogenesis and metastasis.

Topics: Animals; Colitis, Ulcerative; Colorectal Neoplasms; Cytokines; Disease Models, Animal; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Intercellular Adhesion Molecule-1; Macrophages, Peritoneal; Male; Mice; Mice, Inbred ICR; Peritoneal Neoplasms; Receptors, CXCR4; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The transforming growth factor β (TGF-β) pathway acts as a double-edged sword in tumorigenesis. By constraining epithelial cell growth, TGF-β is a potent tumor suppressor. However, TGF-β also acts as a key player in the induction of epithelial-to-mesenchymal transition (EMT), thereby enhancing invasiveness and metastasis. Furthermore, TGF-β signaling has recently been correlated with resistance against both targeted and conventional anticancer agents. Here, we present data demonstrating a role for TGF-β in chemotherapy resistance in colorectal cancer (CRC). We discuss these results in the context of recent findings indicating TGF-β signaling as an emerging player in cancer drug resistance.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Mediator Complex; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor β (TGF-β)/Smad signaling is involved in colorectal carcinoma (CRC) development and progression. The frequent loss of SMAD4 is associated with liver metastasis and poor prognosis of CRC, but the underlying mechanism remains elusive. This study aimed to elucidate the role of Smad-independent TGF-β signaling in CRC metastasis. Immunohistochemistry showed that Smad4 level was negatively correlated with TNM stage and phospho-ERK level in human CRCs and liver metastasis samples. Knockdown of Smad4 in CT26 and HCT116 cells activated ERK pathway, altered the expression of MMP2 and COX-2, promoted cell motility, migration, and invasion in vitro, enhanced metastasis, and shortened the survival of metastatic tumor-bearing mice. MEK inhibitor U0126 and GSK1120212 inhibited the motility, migration, and invasion of Smad4 knockdown cells, inhibited metastasis, and prolonged the survival of metastatic tumor-bearing mice. Furthermore, MEK inhibitor could reverse the changes of phospho-ERK, MMP2, and COX-2 levels. In conclusion, our results indicate that ERK pathway plays a key oncogenic role in CRC with SMAD4 inactivation mutations, and implicate ERK as a potential therapeutic target for CRC liver metastasis.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Phosphorylation; Smad4 Protein; Transforming Growth Factor beta

Tumorigenesis is a multistep process that reflects intimate reciprocal interactions between epithelia and underlying stroma. However, tumor-initiating mechanisms coordinating transformation of both epithelial and stromal components are not defined. In humans and mice, initiation of colorectal cancer is universally associated with loss of guanylin and uroguanylin, the endogenous ligands for the tumor suppressor guanylyl cyclase C (GUCY2C), disrupting a network of homeostatic mechanisms along the crypt-surface axis. Here, we reveal that silencing GUCY2C in human colon cancer cells increases Akt-dependent TGF-β secretion, activating fibroblasts through TGF-β type I receptors and Smad3 phosphorylation. In turn, activating TGF-β signaling induces fibroblasts to secrete hepatocyte growth factor (HGF), reciprocally driving colon cancer cell proliferation through cMET-dependent signaling. Elimination of GUCY2C signaling in mice (Gucy2c(-/-)) produces intestinal desmoplasia, with increased reactive myofibroblasts, which is suppressed by anti-TGF-β antibodies or genetic silencing of Akt. Thus, GUCY2C coordinates intestinal epithelial-mesenchymal homeostasis through reciprocal paracrine circuits mediated by TGF-β and HGF. In that context, GUCY2C signaling constitutes a direct link between the initiation of colorectal cancer and the induction of its associated desmoplastic stromal niche. The recent regulatory approval of oral GUCY2C ligands to treat chronic gastrointestinal disorders underscores the potential therapeutic opportunity for oral GUCY2C hormone replacement to prevent remodeling of the microenvironment essential for colorectal tumorigenesis.

Topics: Animals; Caco-2 Cells; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Down-Regulation; Epithelial-Mesenchymal Transition; Fibrosis; HCT116 Cells; Humans; Intestinal Mucosa; Intestines; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Stem Cell Niche; Transforming Growth Factor beta

Chemotherapy-associated liver injury (CALI) has been linked to increased morbidity and poorer disease-specific outcomes in patients undergoing resection of colorectal liver metastases (CRLM). The aim of this study was to assess the contribution of tumour-related factors to the development of FOLFOX-induced liver injury.. We assessed the effect of FOLFOX treatment on the murine liver either in the presence or absence of CRLM to evaluate the contribution of both chemotherapy and tumour death to the development of CALI.. In the presence of liver metastases, there was increased hepatic expression of plasminogen activator inhibitor-1 (146-fold; P<0.01) and vWF (2.4-fold; P<0.01) transcript as compared with sham-operated controls. In addition, we detected large clusters of megakaryocytes in the spleen of FOLFOX-treated tumour-bearing animals. The livers of FOLFOX-treated animals also showed changes in matrix remodelling genes such as TGFβ (P<0.01), MMP2 (P<0.001), TIMP1 (P<0.001) and Pro-Collagen I (P<0.05) which was exacerbated in the presence of tumour. These genes have previously been demonstrated to have a key role in FOLFOX-induced liver injury.. It appears that the toxicity of FOLFOX chemotherapy is enhanced by tumour-related factors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Collagen Type I; Colorectal Neoplasms; Fluorouracil; Hepatic Veno-Occlusive Disease; Inflammation; Leucovorin; Liver; Liver Neoplasms; Matrix Metalloproteinase 2; Megakaryocytes; Mice; Mice, Inbred C57BL; Organoplatinum Compounds; Plasminogen Activator Inhibitor 1; Spleen; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the common malignant tumors worldwide. Both genetic and epigenetic changes are regarded as important factors of colorectal carcinogenesis. Loss of DACH1 expression was found in breast, prostate, and endometrial cancer. To analyze the regulation and function of DACH1 in CRC, 5 colorectal cancer cell lines, 8 cases of normal mucosa, 15 cases of polyps and 100 cases of primary CRC were employed in this study. In CRC cell lines, loss of DACH1 expression was correlated with promoter region hypermethylation, and re-expression of DACH1 was induced by 5-Aza-2'-deoxyazacytidine treatment. We found that DACH1 was frequently methylated in primary CRC and this methylation was associated with reduction in DACH1 expression. These results suggest that DACH1 expression is regulated by promoter region hypermethylation in CRC. DACH1 methylation was associated with late tumor stage, poor differentiation, and lymph node metastasis. Re-expression of DACH1 reduced TCF/LEF luciferase reporter activity and inhibited the expression of Wnt signaling downstream targets (c-Myc and cyclinD1). In xenografts of HCT116 cells in which DACH1 was re-expressed, tumor size was smaller than in controls. In addition, restoration of DACH1 expression induced G2/M phase arrest and sensitized HCT116 cells to docetaxel. DACH1 suppresses CRC growth by inhibiting Wnt signaling both in vitro and in vivo. Silencing of DACH1 expression caused resistance of CRC cells to docetaxel. In conclusion, DACH1 is frequently methylated in human CRC and methylation of DACH1 may serve as detective and prognostic marker in CRC.

Topics: Adenomatous Polyposis Coli; Animals; Cell Line, Tumor; Colorectal Neoplasms; DNA Methylation; Docetaxel; Drug Resistance, Neoplasm; Epigenesis, Genetic; Eye Proteins; Female; Gene Silencing; HCT116 Cells; Heterografts; Humans; Male; Mice; Neoplasm Transplantation; Signal Transduction; Taxoids; Transcription Factors; Transforming Growth Factor beta; Wnt Proteins

There are few mouse models that adequately mimic large bowel cancer in humans or the gastrointestinal inflammation which frequently precedes it. Dextran sodium sulphate (DSS)-induces colitis in many animal models and has been used in combination with the carcinogen azoxymethane (AOM) to induce cancer in mice. Smad3(-/-) mice are deficient in the transforming growth factor beta (TGFβ) signaling molecule, SMAD3, resulting in dysregulation of the cellular pathway most commonly affected in human colorectal cancer, and develop inflammation-associated colon cancer. Previous studies have shown a requirement for a bacterial trigger for the colitis and colon cancer phenotype in Smad3(-/-) mice. Studies presented here in Smad3(-/-) mice detail disease induction with DSS, without the use of AOM, and show a) Smad3(-/-) mice develop a spectrum of lesions ranging from acute and chronic colitis, crypt herniation, repair, dysplasia, adenomatous polyps, disseminated peritoneal adenomucinosis, adenocarcinoma, mucinous adenocarcinoma (MAC) and squamous metaplasia; b) the colon lesions have variable galactin-3 (Mac2) staining c) increased DSS concentration and duration of exposure leads to increased severity of colonic lesions; d) heterozygosity of SMAD3 does not confer increased susceptibility to DSS-induced disease and e) disease is partially controlled by the presence of T and B cells as Smad3(-/-) Rag2(-/-) double knock out (DKO) mice develop a more severe disease phenotype. DSS-induced disease in Smad3(-/-) mice may be a useful animal model to study not only inflammation-driven MAC but other human diseases such as colitis cystica profunda (CCP) and pseudomyxomatous peritonei (PMP).

Topics: Animals; Colitis; Colorectal Neoplasms; Dextran Sulfate; DNA-Binding Proteins; Humans; Inflammation; Mice; Mice, Knockout; Smad3 Protein; Transforming Growth Factor beta

Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5' RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-β-induced transcriptional activity and reversed the growth inhibitory effect of TGF-β in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-β regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.

Topics: Animals; Base Sequence; Cell Line, Tumor; Colon; Colorectal Neoplasms; DNA-Binding Proteins; Exons; Gene Expression Regulation, Neoplastic; Humans; MDS1 and EVI1 Complex Locus Protein; Mice; Promoter Regions, Genetic; Proto-Oncogenes; Rectum; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Activation of the canonical TGF-β signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP) microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-X-Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway.

Topics: Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Colorectal Neoplasms; Female; Humans; Loss of Heterozygosity; Male; Middle Aged; Mutation; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

TGF-β has dual functions as a tumor promoter or a tumor suppressor. Recent studies suggest the roles of TGF-β might change from a tumor suppressor to a tumor promoter, when lacking SMAD4 expression in CRC (colorectal cancer). However, the precise role of TGF-β as tumor promoter in CRC is still unclear.. We evaluated the role of TGF-β in SMAD4-null CRC SW620 cells. In vitro, we measured cell growth and cell cycle distribution by flow cytometry. In vivo, we implanted SW620 cells into mice and measured tumor weight after TGF-β treatment. We also determined cell proliferation ability in tumor by immunohistochemistry of proliferating cell nuclear antigen (PCNA).. Cell growth and DNA synthesis in S phase was remarkably enhanced by TGF-β in vitro. Besides, TGF-β-activated ERK1/2 MAPK signal was also observed. In vivo, TGF-β enhanced tumor growth and cell proliferation in tumor.. TGF-β serves as a tumor promoter, which promotes CRC cell growth in vitro and in vivo. These suggest that TGF-β might be developed as an effective therapeutic target for CRC patients.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Flow Cytometry; Immunohistochemistry; In Vitro Techniques; Mice; Mitogen-Activated Protein Kinase 3; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) has recently been studied to elucidate mechanisms of the liver metastatic process. We investigated EMT in the process of liver metastasis and the effects of chemotherapy on EMT cells as therapeutic strategy for colorectal liver metastasis. We used the CT26 murine colorectal carcinoma cell line to create an in vivo mouse liver metastasis model. Liver tumors were stained immuno-histochemically. Expression of proteins associated with TGF-β/Smad and hepatocyte growth factor (HGF)/c-Met pathways were investigated by western blotting. Cells with c-Met mRNA knockdown by siRNA techniques showed clearly reduced liver metastases compared with regular cells at 21 days. TGF-β and HGF induced EMT expression, but signal transduction was quite different. TGF-β induced ERK, but not Akt phosphory-lation. HGF mediated both ERK and Akt phosphorylation. Akt inhibitor blocked Akt phosphorylation but did not affect TGF-β-induced activation of ERK, Snail and Slug. U-0126 did not reduce Snail activity by TGF-β at a concentration to block ERK phosphorylation. However, Akt inhibitor and U-0126 completely inhibited HGF-induced Slug activation. 5-FU mediated cell death in the EMT process induced by TGF-β more effectively than HGF. ERK/Akt signaling, but not the Smad pathway, may be one of the main processes in HGF-induced EMT, despite the Smad pathway, but not ERK/Akt, being critical for TGF-β-induced EMT. The MAPK/Akt pathway is indispensable in HGF/c-Met signaling. The ERK/Akt pathway particularly may be critical in the HGF-induced EMT process. However, long-term use of chemotherapeutic agents may induce drug resistance and distant metastases through EMT-related signaling pathway activation.

Topics: Animals; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Hepatocyte Growth Factor; Liver Neoplasms; Mice; Mitogen-Activated Protein Kinase 3; Neoplasms, Experimental; Oncogene Protein v-akt; Proto-Oncogene Proteins c-met; Signal Transduction; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta

Recent genome-wide association studies (GWAS) have reported multiple risk loci associated with risk of colorectal cancer (CRC), some of which are involved in the transforming growth factor beta (TGFβ) signaling pathway. We systematically examined associations of common genetic variations in the TGFβ signaling pathway and environmental factors with CRC risk using a two-staged case-control study in a Chinese population. A set of 77 single-nucleotide polymorphisms (SNPs) in 10 candidate genes involved in the TGFβ signaling pathway and several environmental factors including sex, age, smoking and drinking were examined by random forest (RF) to capture the potential gene-gene and gene-environment interactions in stage 1 of the study with 443 CRC patients and 480 controls. Three promising SNPs (SMAD7 rs11874392, TGFBR1 rs10988706 and rs6478972) selected by the RF method were genotyped in stage 2 comprising 351 cases and 360 controls for validation. SMAD7 rs11874392 presented consistently significant associations with a risk of CRC at both stages, with odds ratio = 1.41 (95% confidence interval = 1.21-1.63) using additive modes in combined analyses. Moreover, the potential interactions between SMAD7 rs11874392, TGFBR1 rs10988706 and rs6478972 were indicated consistently in both stages of the study by using pair-wise interaction and multilocus genotype pattern analysis. Additionally, gene-smoking interactions for rs11874392, rs10988706 and rs6478972 were also found to enhance the risk of CRC at both stages, with P for multiplicative interaction equal to 1.162×10(-6), 8.574×10(-8) and 9.410×10(-8) in combined analyses, respectively. This study emphasized the substantial role of the TGFβ signaling pathway in CRC, especially in interaction with smoking.

Topics: Case-Control Studies; China; Colorectal Neoplasms; Female; Gene-Environment Interaction; Genetic Predisposition to Disease; Genetic Variation; Genome-Wide Association Study; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Risk; Signal Transduction; Smad7 Protein; Smoking; Transforming Growth Factor beta

The human suppressive T cells that stably express transcription factor FOXP3, or regulatory T cells (Tregs), are thought to suppress antitumor immune responses. The most specific marker for human Tregs is the demethylation of CpG dinucleotides located in the first intron of FOXP3 (FOXP3i1). FOXP3i1 is completely methylated in other hematopoietic cells, including nonsuppressive T cells that transiently express FOXP3 after activation. Previously, we and others reported estimations of the frequency of Tregs in the blood of melanoma patients using a FOXP3i1 methylation-specific qPCR assay. Here, we attempted to quantify Tregs inside tumor samples using this assay. However, we found demethylated FOXP3i1 sequences in the melanoma cells themselves. This demethylation was not associated with substantial FOXP3 mRNA or protein expression, even though the demethylation extended to the promoter and terminal regions of the gene in some melanoma cells. Our results imply that analyzing Treg frequencies by quantification of demethylated FOXP3i1 will require that tumor-infiltrating T cells be separated from melanoma cells.

Topics: Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Colorectal Neoplasms; CpG Islands; DNA Methylation; Epigenesis, Genetic; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Introns; Lung Neoplasms; Male; Melanoma; Phosphorylation; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Smad2 Protein; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The transforming growth factor beta (TGF-β) signaling pathway has been considered both a tumor suppressor and a cancer promoter. Additionally, downregulation of cell adhesion molecules such as E-cadherin plays an important role in the metastatic potential of colorectal cancer (CRC). The aim of the present study was to evaluate TGF-β, TGF-β type I receptor (TGF-βR1), TGF-β type II receptor (TGF-βR2), Smad4, pSmad2/3, and E-cadherin expression in colorectal carcinoma and to correlate the obtained data with other standard prognostic parameters, such as disease stage, metastases, and patient survival. TGF-β, TGF-βR1, TGF-βR2, Smad4, pSmad2/3, and E-cadherin expression was evaluated immunohistochemically in 195 unrelated CRC specimens and the results subjected to various statistical analyses. TGF-β was expressed in 71.28%, TGF-βR1 in 61.0%, TGF-βR2 in 54.4%, Smad4 in 61.5%, pSmad2/3 in 71.3%, and E-cadherin in 50.26% of the colorectal carcinoma samples tested. The correlation of immunoexpression with the clinicopathological parameters of CRC revealed that the high expression of TGF-β and low expression of TGF-βR1, TGF-βR2, Smad4, pSmad2/3, and E-cadherin were correlated with tumor-node-metastasis (TNM) stage of disease. High TGF-β expression and low TGF-βR1, TGF-βR2, Smad4, and pSmad2/3 expression were also correlated with lymph node metastasis. The Kaplan-Meier survival curves demonstrated a clear association of cancer-specific overall survival with high TGF-β, as well as low TGF-βR1, TGF-βR2, Smad4, pSmad2/3, and E-cadherin expression. Our results suggest that TGF-β, TGF-βR1, TGF-βR2, Smad4, pSmad2/3, and E-cadherin are closely related to TNM stage of CRC. Moreover, TGF-β, TGF-βR2, Smad4, pSmad2/3, and E-cadherin emerge as valuable independent biomarkers of prognosis in CRC patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cadherins; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Phosphorylation; Prognosis; Proportional Hazards Models; 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; Transforming Growth Factor beta

Colorectal cancer (CRC) is driven by genetic alterations causing its progression. Besides accepted tumor suppressor- and onco- genes, a series of genes have been identified, which contribute to transformation into a more malignant stage. We investigated whether the expression level of such genes, alone or in combination, could add to predict the prognosis of CRC patients.. Tumor samples from 118 CRC patients were screened in a retrospective analysis by qRT-PCR for expression of the four tumor progression-associated genes osteopontin (Opn), transforming growth factor β (Tgf-β), matrix metalloproteinase-2 (Mmp-2) and cyclooxigenase-2 (Cox-2). The resulting qRT-PCR values were related to those of housekeeping genes. All patients were clustered for similar expression levels between the four genes with R statistical software using the package pvclust, which provides bootstrap agglomerative hierarchical clustering. Clusters with similar expression of the four genes were analyzed for correlation with UICC stages and survival time.. Expression of the four genes varied considerably within the cohort of patients. Cluster analysis of patients revealed a subgroup (n = 33) who in comparison with the other patients showed tenfold higher expression levels of all four genes (p < 0.001, respectively). However, there was no correlation between patients expressing high or low levels of these four genes and known parameters of clinical prognosis (UICC stages, survival time).. In conclusion, tenfold increased expression levels of Opn, Tgf-β, Mmp-2 and Cox-2 in a subset of CRC patients did not predict for a clinical outcome that is different from that of the remaining patients.

Topics: Aged; Colorectal Neoplasms; Cyclooxygenase 2; Disease Progression; Female; Follow-Up Studies; Gene Expression; Humans; Male; Matrix Metalloproteinase 2; Middle Aged; Osteopontin; Prognosis; Retrospective Studies; Transforming Growth Factor beta

Suppressor T cells are one of the determinants of colorectal cancer (CRC) clinical outcome. LAP(+)CD4(+) T cell is a recently identified subset of suppressor T cells. This study was designed to investigate their clinical relevance in patients with CRC.. Sixty patients with CRC and 24 healthy donors (HD) were enrolled in this study. The percentages of LAP(+)CD4(+) T cells in peripheral blood and tumor tissue were measured. The phenotype and functional relevance of LAP(+)CD4(+) T cells were analyzed subsequently.. The percentages of LAP(+)CD4(+) T cells in peripheral blood of patients with CRC were significantly higher than HD (HD vs. CRC: 3.1% ± 0.78% vs. 8.8% ± 5.8%, P < 0.0001) and in tumor tissue when compared with nontumor tissue (nontumor vs. tumor: 3.2% ± 1.1% vs. 9.5% ± 5.5%, P = 0.0002). In addition, LAP(+)CD4(+) T cells with effector memory (EM) phenotype were more likely to accumulate in the tumor sites than in peripheral blood. These LAP(+)CD4(+) T cells produced significantly higher levels of IFN-γ, IL-17 and comparatively lower IL-2 and very few IL-10. LAP(+)CD4(+) T cells could suppress the proliferation of LAP(-)CD4(+) T cells that were partially mediated by TGF-β. Furthermore, these LAP(+)CD4(+) T cells accumulated in tumor site and increased further in the peripheral blood in patients with metastasis.. LAP(+)CD4(+) T cells as a suppressor subset could accumulate in the tumor microenvironment and circulated more in the peripheral blood with tumor progression in patients with CRC.

Topics: Adult; Aged; CD4-Positive T-Lymphocytes; Colorectal Neoplasms; Disease Progression; Female; Humans; Lymphocyte Activation; Male; Middle Aged; Neoplasm Staging; Peptides; Protein Precursors; Transforming Growth Factor beta; Tumor Microenvironment

Interleukin (IL)-17A is an important pro-inflammatory cytokine and involved in the colorectal carcinogenesis. In this study, the authors evaluated the dynamic change of IL-17A expression in the tumor microenvironment throughout the colorectal adenoma-carcinoma sequence.. Using quantitative real-time PCR (polymerase chain reaction) and semi-quantitative immunohistochemistry, the authors examined the expression level of IL-17A in 50 of human colorectal adenoma tissues, 50 of colorectal cancer (CRC) tissues and 15 controls. The relationship between IL-17A expression and clinicopathological parameters throughout the sequence was also evaluated.. The results revealed a step-up increased IL-17A mRNA level throughout the colorectal adenoma-carcinoma sequence, which began to increase in the adenomas and became even higher in the CRCs; notably, the increase of IL-17A mRNA level in the adenomatous tissues was associated with the severity of dysplasia. Immunohistochemical analysis confirmed the real-time PCR results and revealed gradually increasing IL-17A cells in both the stroma and adenomatous/cancerous epithelium. In addition, the quantitative real-time PCR result has also revealed an increased expression of TH17-stimulating factors throughout the sequence.. IL-17A and TH17 are highly activated throughout the colorectal adenoma-carcinoma sequence.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Analysis of Variance; Carcinoma; Case-Control Studies; Cell Transformation, Neoplastic; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Interleukin-17; Interleukin-1beta; Interleukin-23; Interleukin-6; Male; Middle Aged; Real-Time Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Th17 Cells; Transforming Growth Factor beta

Treatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor destruction. However, a thin rim of viable cells persists in the tumor periphery following treatment, contributing to subsequent recurrence. This study investigates inherent differences in the microenvironment of the tumor periphery that contribute to treatment resistance.. Using a murine colorectal liver metastases model, spatial morphological and molecular differences within the periphery and the center of the tumor that may account for differences in resistance to OXi4503 treatment were investigated. H&E staining and immunostaining were used to examine vessel maturity and stability, hypoxia and HIF1α levels, accumulation of immune cells, expression of proangiogenic factors/receptors (VEGF, TGF-β, b-FGF, and AT1R) and expression of EMT markers (ZEB1, vimentin, E-cadherin and β-catenin) in the periphery and center of established tumors. The effects of OXi4503 on tumor vessels and cell kinetics were also investigated.. Significant differences were found between tumor periphery and central regions, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased evidence of EMT. OXi4503 treatment resulted in collapse of vessels in the tumor center; however vasculature in the periphery remained patent. Similarly, tumor apoptosis and proliferation were differentially modulated between centre and periphery after treatment.. The molecular and morphological differences between tumor periphery and center may account for the observed differential resistance to OXi4503 treatment and could provide targets for drug development to totally eliminate metastases.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cadherins; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Diphosphates; Drug Resistance, Neoplasm; Fibroblast Growth Factors; Homeodomain Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Kruppel-Like Transcription Factors; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Stilbenes; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A; Vimentin; Zinc Finger E-box-Binding Homeobox 1

A large proportion of colorectal cancers (CRCs) display mutational inactivation of the TGF-β pathway, yet, paradoxically, they are characterized by elevated TGF-β production. Here, we unveil a prometastatic program induced by TGF-β in the microenvironment that associates with a high risk of CRC relapse upon treatment. The activity of TGF-β on stromal cells increases the efficiency of organ colonization by CRC cells, whereas mice treated with a pharmacological inhibitor of TGFBR1 are resilient to metastasis formation. Secretion of IL11 by TGF-β-stimulated cancer-associated fibroblasts (CAFs) triggers GP130/STAT3 signaling in tumor cells. This crosstalk confers a survival advantage to metastatic cells. The dependency on the TGF-β stromal program for metastasis initiation could be exploited to improve the diagnosis and treatment of CRC.

Topics: Animals; Colorectal Neoplasms; Cytokine Receptor gp130; HT29 Cells; Humans; Interleukin-11; Mice; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recurrence; Signal Transduction; STAT3 Transcription Factor; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Topics: Colorectal Neoplasms; Humans; Models, Biological; Neoplasm Metastasis; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Both chronic inflammation and somatic mutations likely contribute to the pathogenesis of ulcerative colitis (UC)-associated dysplasia and cancer. On the other hand, both tumor suppression and oncogenesis can result from transforming growth factor (TGF)-β signaling. TGF-β type I receptor (TβRI) and Ras-associated kinases differentially phosphorylate a mediator, Smad3, to become C-terminally phosphorylated Smad3 (pSmad3C), linker phosphorylated Smad3 (pSmad3L), and both C-terminally and linker phosphorylated Smad3 (pSmad3L/C). The pSmad3C/p21(WAF1) pathway transmits a cytostatic TGF-β signal, while pSmad3L and pSmad3L/C promote cell proliferation by upregulating c-Myc oncoprotein. The purpose of this study was to clarify the alteration of Smad3 signaling during UC-associated carcinogenesis.. By immunostaining and immunofluorescence, we compared pSmad3C-, pSmad3L-, and pSmad3L/C-mediated signaling in colorectal specimens representing colitis, dysplasia, or cancer from eight UC patients with signaling in normal colonic crypts. We also investigated p53 expression and mutations of p53 and K-ras genes. We further sought functional meaning of the phosphorylated Smad3-mediated signaling in vitro.. As enterocytes in normal crypts migrated upward toward the lumen, cytostatic pSmad3C/p21(WAF1) tended to increase, while pSmad3L/c-Myc shown by progenitor cells gradually decreased. Colitis specimens showed prominence of pSmad3L/C/c-Myc, mediated by TGF-β and tumor necrosis factor (TNF)-α, in all enterocyte nuclei throughout entire crypts. In proportion with increases in frequency of p53 and K-ras mutations during progression from dysplasia to cancer, the oncogenic pSmad3L/c-Myc pathway came to be dominant with suppression of the pSmad3C/p21(WAF1) pathway.. Oncogenic Smad3 signaling, altered by chronic inflammation and eventually somatic mutations, promotes UC-associated neoplastic progression by upregulating growth-related protein.

Topics: Adenocarcinoma; Adult; Blotting, Western; Cell Transformation, Neoplastic; Chronic Disease; Colitis, Ulcerative; Colorectal Neoplasms; Disease Progression; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Immunoprecipitation; Male; Middle Aged; Phosphorylation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) signalling plays a key role in colorectal cancer (CRC). Bone morphogenetic protein-4 (BMP4) is a member of the TGF-β family of signal transduction molecules. To examine if germline mutation in BMP4 causes CRC we analysed 504 genetically enriched CRC cases (by virtue of early-onset disease, family history of CRC) for mutations in the coding sequence of BMP4. We identified three pathogenic mutations, p.R286X (g.8330C>T), p.W325C (g.8449G>T) and p.C373S (g.8592G>C), amongst the CRC cases which were not observed in 524 healthy controls. p.R286X localizes to the N-terminal of the TGF-β1 prodomain truncating the protein prior to the active domain. p.W325C and p.C373S mutations are predicted from protein homology modelling with BMP2 to impact deleteriously on BMP4 function. Segregation of p.C373S with adenoma and hyperplastic polyp in first-degree relatives of the case suggests germline mutations may confer a juvenile polyposis-type phenotype. These findings suggest mutation of BMP4is a cause of CRC and the value of protein-based modelling in the elucidation of rare disease-causing variants.

Topics: Adult; Aged; Amino Acid Sequence; Bone Morphogenetic Protein 4; Colorectal Neoplasms; Female; Germ-Line Mutation; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Protein Structure, Tertiary; Sequence Alignment; Transforming Growth Factor beta

This study examines changes in the expression of growth factors following thermal ablation (TA) of selected colorectal cancer (CRC) liver metastases.. Using mice with established CRC liver metastases, two tumours in each animal were thermally ablated. Liver and tumour tissues were collected at various time-points (days 0, 1, 2, 3, 5 and 7) following TA treatment from the ablation site and from sites distant from ablated tumour. Changes in growth factor expression (epidermal growth factor [EGF], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF] and transforming growth factor-β[TGF-β]) in comparison with baseline levels (non-ablated) were assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.. Baseline TGF-β and VEGF levels in the liver parenchyma of tumour-bearing mice were significantly higher than levels in naive liver parenchyma. Levels of VEGF and HGF decreased after TA treatment in all tissues. Levels of EGF decreased in ablated and distant tumour tissues, but displayed a tendency to increase in liver tissue. Levels of TGF-β also decreased during the first 2 days following TA, but later increased in liver and tumour tissues distant from the ablation site to a level that reached significance in tumour tissue at day 7 (P < 0.001). Decreases in growth factor levels were also observed in animals that underwent laparotomy without TA treatment, which indicates that these decreases were caused by the experimental procedure.. Tumour induces upregulation of TGF-β and VEGF in liver parenchyma. Growth factors decreased after TA, but this appears to be the result of the experimental procedure rather than the TA itself. However, TA resulted in increased levels of TGF-β, which may contribute to tumour recurrence.

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Hepatocyte Growth Factor; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Laser Therapy; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Time Factors; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The Cox-2 inhibitor, celecoxib (Pfizer Inc., N.Y., USA), is a promising chemopreventive agent [Arber et al.: N Engl J Med 2006;355:885-895; Bertagnolli et al.: N Engl J Med 2006;355:873-884]. This study aims to explore its mechanism by defining changes in gene expression between neoplastic and normal tissue samples before and after treatment.. Patients with documented colorectal neoplasia in screening colonoscopy, destined to undergo surgical colectomy, were randomized for treatment with celecoxib (n = 11; 400 mg/day) or placebo (n = 3) for 30 days. Tissue samples were taken from the tumor and from normal adjacent mucosa during both colonoscopy and surgery. RNA was extracted and analyzed using Affymetrix Genechip®.. 687 genes differentiated tumor samples before and after treatment, among which 310 genes did not show the same differential expression in the placebo group or normal samples. These genes were significantly related to pathways of cell cycle regulation and inflammation, and of note was the TGF-β pathway, which held a strong association with the list of genes formerly found to be associated with the colorectal cancer expression profile in microarray analyses, as summarized in a meta-analysis by Cardoso et al. [Biochim Biophys Acta 2007;1775:103-137].. Celecoxib selectively affects genes and pathways involved in inflammation and malignant transformation in tumor but not normal tissues, this may assist in the development of safer and more effective chemopreventive agents.

Topics: Adenocarcinoma; Celecoxib; Cell Cycle; Cell Transformation, Neoplastic; Colonic Polyps; Colorectal Neoplasms; Cyclooxygenase 2 Inhibitors; Gene Expression; Humans; Inflammation; Intestinal Mucosa; Microarray Analysis; Pyrazoles; Random Allocation; RNA, Neoplasm; Signal Transduction; Sulfonamides; Transforming Growth Factor beta

Vascular endothelial growth factor (VEGF) signaling is a target for antiangiogenic cancer therapy. The authors have previously observed that up to 40% of vessels in colorectal carcinoma (CRC) tumors are negative for VEGF receptor 2 (VEGFR2) expression. Differential activity of transforming growth factor beta (TGF-β) is a potential contributor to this receptor heterogeneity because TGF-β contributes to both angiogenesis and CRC tumor progression.. The authors analyzed VEGFR2 expression by Western blotting, and TGF-β expression in endothelial and CRC cell lines, respectively. In addition, they immunostained endothelial cells in CRC xenografts to find an association between VEGFR2 and TGF-β levels or activity.. In bovine aortic endothelial cells (BAECs), TGF-β1 significantly repressed VEGFR2 protein in a time-dependent and dose-dependent fashion (P < .05). Serum-free conditioned media from various malignant human CRC cell lines (HCT116, 379.2, Dks8, and DLD1) induced down-regulation of VEGFR2 in BAECs. This effect was proportional to the total levels of TGF-β1 and TGF-β2 and was blocked by SB-431542 and SD-208, TGF-β receptor I inhibitors. Immunofluorescence staining of subcutaneous mouse xenografts of HCT116, 379.2, Dks8, and SW480 cells revealed vessels with an inverse relationship between TGF-β activity and VEGFR2 expression. Oxygen and bone morphogenetic protein 9 levels were shown to modulate TGF-β-induced VEGFR2 down-regulation.. In combination with other factors, TGF-β may contribute to the vascular heterogeneity in human colorectal tumors.

Topics: Animals; Caco-2 Cells; Cattle; Cell Hypoxia; Cell Line, Tumor; Colorectal Neoplasms; Endothelial Cells; Female; Growth Differentiation Factor 2; Growth Differentiation Factors; HCT116 Cells; Humans; Male; Mice; Neovascularization, Pathologic; Phosphorylation; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous; Vascular Endothelial Growth Factor Receptor-2

TGF-β signaling provides tumor protection against colorectal cancer (CRC). Mechanisms that support its tumor-suppressive properties remain unclear. The ubiquitin ligase Arkadia/RNF111 enhances TGF-β signaling responses by targeting repressors of the pathway for degradation. The corepressors SnoN/Ski, critical substrates of Arkadia, complex with the activated TGF-β signaling effectors Smad2/3 (pSmad2/3) on the promoters of target genes and block their transcription. Arkadia degrades this complex including pSmad2/3 and unblocks the promoter. Here, we report that Arkadia is expressed highly in the mouse colonic epithelium. Heterozygous Akd(+/-) mice are normal but express less Arkadia. This leads to reduced expression of several TGF-β target genes, suggesting that normal levels of Arkadia are required for efficient signaling responses. Critically, Akd(+/-) mice exhibit increased susceptibility to azoxymethane/dextran sodium sulfate carcinogen-induced CRC, as they develop four-fold more tumors than wild-type mice. Akd(+/-) tumors also exhibit a more aggressive pathology, higher proliferation index, and reduced cytostasis. Therefore, Arkadia functions as a tumor suppressor whose peak expression is required to suppress CRC development and progression. The accumulation of nuclear SnoN and pSmad2, along with the downregulation of TGF-β target genes observed in Akd(+/-) colon and tumors, suggest that tumor-suppressing properties of Arkadia are mediated by its ability to derepress TGF-β signaling. Consistent with this likelihood, we identified mutations in primary colorectal tumors from human patients that reduce Arkadia function and are associated with the accumulation of nuclear SNON. Collectively, our findings reveal that Arkadia enhances TGF-β signaling responses and supports its tumor-suppressing properties in CRC.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Azoxymethane; Base Sequence; Colon; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice; Molecular Sequence Data; Mutation; Nuclear Proteins; Proto-Oncogene Proteins; Severity of Illness Index; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

The TGFβ signalling pathway is a growth inhibitor system that operates in both normal and tumour cells. Alterations to components of this pathway, including SMAD4, result in resistance to growth inhibition and uncontrolled proliferation. The aim of this study was to analyse the relationships between SMAD4, a key protein in the growth-inhibiting TGFβ pathway; cell proliferation proteins Ki67, p27 and S-phase kinase-associated protein 2 (SKP2); and mismatch repair (MMR) proteins as well as prognostic indicators in colorectal adenocarcinomas. A series of 230 sporadic colorectal adenocarcinomas were studied using tissue microarrays by immunohistochemistry for SMAD4, Ki67, p27, SKP2 and MMR protein (hMLH1, hMSH2 and hMSH6) expression. Protein expression was analysed with respect to pathological prognostic criteria. Loss of SMAD4 nuclear expression (27/230, 12%) correlated with the presence of lymph node metastases, MMR protein expression and the absence of p27 in tumour cells (p = 0.04, p = 0.08 and p = 0.03, respectively). A high Ki67 index did not correlate with SMAD4 expression; however, it did correlate with moderate or poor histological differentiation, SKP2 expression and aberrant or absent MMR protein expression (p = 0.02, p < 0.01 and p < 0.01, respectively). In conclusion, the results of our study suggest that the loss of SMAD4, occurring in 12% of colorectal adenocarcinomas, correlated with the presence of lymph node metastases and absence of p27 expression but not with high cellular proliferation. However, high proliferation correlated with SKP2 and aberrant MMR protein expression. Although the advantage of immunohistochemistry is high throughput, our results allow only an initial evaluation, and subsequent studies, including genetic analyses, are required.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Proliferation; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; MutL Protein Homolog 1; Nuclear Proteins; S-Phase Kinase-Associated Proteins; Smad4 Protein; Transforming Growth Factor beta

The conditions Juvenile Polyposis Syndrome (JPS) and Hereditary Mixed Polyposis Syndrome (HMPS) are associated with an increased risk of colorectal carcinoma. The genetic mechanisms which explain these conditions have until recently been poorly understood. Recent interest has focused on the transforming growth factor (TGF)-beta signalling pathway and, in particular, on mutations in the SMAD4 gene. However, not all cases of JPS and HMPS have mutations in SMAD4 and focus has now shifted to other components of the TGF-beta pathway to clarify the genetic mechanisms involved in these conditions. In this report, we describe the significance of a bone morphogenetic protein receptor type 1A gene mutation in an Irish family.

Topics: Adenomatous Polyposis Coli; Adult; Bone Morphogenetic Protein Receptors, Type I; Colonic Polyps; Colorectal Neoplasms; Female; Humans; Male; Mutation; Pedigree; Signal Transduction; Smad4 Protein; Syndrome; Transforming Growth Factor beta

TGF-beta plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id-1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF-beta in regulating Id-1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF-beta on Id-1 expression were analyzed using a luciferase reporter assay, RT-PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF-beta1 downregulated Id-1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF-beta1-induced 4xSBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF-beta1-induced 3xAP-1 luciferase reporter activity. However, the suppression of Id-1 by TGF-beta1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF-beta1 stimulated cellular proliferation and p21(Waf1) protein expression, which might be mediated by suppressing Id-1 expression. In conclusion, this study demonstrated that TGF-beta1 suppressed Id-1 expression in a smad3-dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF-beta function in colorectal cancer cells.

Topics: Blotting, Western; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Gene Silencing; Humans; Immunoblotting; Inhibitor of Differentiation Protein 1; Luciferases; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

The human immune system consists of a balance between immune surveillance against non-self antigens and tolerance of self-antigens. CD8(+) T cells and CD4(+) regulatory T cells (Tregs) are the main players for immune surveillance and tolerance, respectively. We examined immunohistochemically the immunological balance at the tumor site using 94 surgically resected colorectal cancer tissues. Forkhead box P3 (FOXP3)(+) cells were considered to be Tregs in the present study. The number of intratumoral FOXP3(+) cells (itFOXP3(+) cells) was positively correlated with lymph node metastases (P = 0.030). itCD8(+) T/itFOXP3(+) cell ratio negatively correlated with pathological stages (P = 0.048). Next, relationship between the number of itCD8(+) T cells or itFOXP3(+) cells and survival prognosis in 94 patients who underwent a curative resection was analyzed. Only itCD8(+) T/itFOXP3(+) cell ratio positively correlated with disease-free survival (0.023) and overall survival (P = 0.010). Multivariate analysis indicated that itCD8(+) T/itFOXP3(+) cell ratio is an independent prognostic factor (P = 0.035) of overall survival. The number of itFOXP3(+) cells positively correlated with transforming growth factor-beta TGF-beta production at the tumor site (P = 0.020). In conclusion, itCD8(+) T/itFOXP3(+) cell ratio is a predictive marker for both disease-free survival time and overall survival time in patients with colorectal cancer. Importantly, itCD8(+) T/itFOXP3(+) cell ratio may be an independent prognostic factor. And, tumor-producing TGF-beta may contribute to the increased number of itFOXP3(+) cells.

Topics: Aged; Biomarkers, Tumor; CD8-Positive T-Lymphocytes; Cell Separation; Colorectal Neoplasms; Disease-Free Survival; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Prognosis; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-gamma2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFalpha on tumor cells.. Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFalpha with an increased expression of all three chains encoding laminin-332; coincubation with TGFbeta and TNFalpha leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFalpha can induce expression of the gamma2 and beta3 but not the alpha3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of gamma2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble gamma2 is associated with increased cell migration.. Loss of Smad4 may lead to uncoupled induction of laminin-gamma2 in response to TNFalpha and may therefore represent one of the mechanisms which underlie accumulation of laminin-gamma2 at the invasive margin of a tumor. The finding, that gamma2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.

Topics: Adenoma; Amino Acid Sequence; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Drug Synergism; Gene Knockdown Techniques; Humans; Kalinin; Laminin; Mass Spectrometry; Molecular Sequence Data; NF-kappa B; Promoter Regions, Genetic; Protein Conformation; Smad4 Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional cytokines regulating a broad spectrum of biological functions. Recent studies show the presence of BMP receptor 1a mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer, suggesting that aberrations in BMP signaling play an important role in intestinal cancer pathogenesis. However, the exact molecular mechanisms remain poorly understood. The Runt domain transcription factor RUNX3 is an integral component of signaling pathways mediated by TGF-beta and BMPs. RUNX3 is a gastric and colon tumor suppressor, functioning downstream of TGF-beta. Recently, we showed the tumor-suppressive effects of RUNX3 by its ability to attenuate beta-catenin/T-cell factors (TCFs) transactivation in intestinal tumorigenesis. Here, we explore the molecular basis of the tumor-suppressive function of the BMP pathway through RUNX3 in colorectal carcinogenesis. BMP exerted a growth-suppressive effect in HT-29, a human colorectal cancer cell line. c-Myc oncogene was found to be downregulated by BMP and/or RUNX3. We show that upregulation of RUNX3 by BMP reduces c-Myc expression. Evidence is presented suggesting that RUNX3 downregulates c-Myc expression by two parallel pathways-directly at the transcriptional level and through attenuation of beta-catenin/TCFs, downstream of BMPs in colorectal cancer cells.

Topics: Apoptosis; beta Catenin; Blotting, Western; Bone Morphogenetic Proteins; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Colorectal Neoplasms; Core Binding Factor Alpha 3 Subunit; Humans; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; TCF Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

To assess the expression of a cancer-associated fibroblasts (CAFs) marker as an indicator of prognosis, we raised anti-protein gene product 9.5 (PGP9.5) monoclonal antibody against cultured fibroblasts. PGP9.5 expression in cultured normal fibroblasts was increased by transforming growth factor beta stimulation, indicating the phenotypic alteration to activated fibroblast. We immunohistochemically evaluated PGP9.5 expression with the CAFs of 110 colorectal cancer cases under T3 stage. PGP9.5 immunoreactivity in 30% or more of CAFs was defined as high PGP9.5 expression, and the other cases were considered as having low PGP9.5 expression. Patients with high PGP9.5 expression (42.7%) had significantly shorter survival and a higher incidence of recurrence than the low PGP9.5 expression group (P = .002 and P < .001, respectively). Multivariate analysis indicated PGP9.5 expression as an independent prognostic factor for overall and recurrence-free survival partly as well as lymph node metastasis. These results indicate that PGP9.5 expression in CAFs is a helpful finding to represent the overall biologic behavior of advanced colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Biomarkers, Tumor; Colorectal Neoplasms; Electrophoresis, Polyacrylamide Gel; Female; Fibroblasts; Humans; Immunoenzyme Techniques; Japan; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Staging; Prognosis; Proportional Hazards Models; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin Thiolesterase

The development and progression of colorectal cancer has been extensively studied and the genes responsible have been well characterized. However the correlation between the SMAD4 gene mutations with KRAS mutant status has not been explored by many studies so far. Here, in this study we aimed to investigate the role of SMAD4 gene aberrations in the pathogenesis of CRC in Kashmir valley and to correlate it with various clinicopathological variables and KRAS mutant genotype.. We examined the paired tumor and normal tissue specimens of 86 CRC patients for the occurrence of aberrations in MCR region of SMAD4 and exon 1 of KRAS by PCR-SSCP and/or PCR-Direct sequencing.. The overall mutation rate of mutation cluster region (MCR) region of SMAD4 gene among 86 patients was 18.6% (16 of 86). 68.75% (11/16) of the SMAD4 gene mutants were found to have mutations in KRAS gene as well. The association between the KRAS mutant genotype with SMAD4 mutants was found to be significant (P = or < 0.05). Further more, we found a significant association of tumor location, tumor grade, node status, occupational exposure to pesticides and bleeding PR/Constipation with the mutation status of the SMAD4 gene (P = or < 0.05).. Our study suggests that SMAD4 gene aberrations are the common event in CRC development but play a differential role in the progression of CRC in higher tumor grade (C+D) and its association with the KRAS mutant status suggest that these two molecules together are responsible for the progression of the tumor to higher/advanced stage.

Topics: Adenocarcinoma; Adult; Aged; Base Sequence; Carcinoma, Squamous Cell; Chi-Square Distribution; Colorectal Neoplasms; DNA Mutational Analysis; Exons; Female; Gene Expression Regulation, Neoplastic; Genotype; Humans; India; Male; Middle Aged; Molecular Sequence Data; Mutation; Neoplasm Staging; Phenotype; Polymerase Chain Reaction; Prognosis; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

The objective of this study was to elucidate molecular mechanisms behind the antitumor activities of the isothiocyanate sulforaphane (SFN) in colorectal cancer cells.. Cell growth was determined by BrdU incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Ornithine decarboxylase (ODC) activity was assayed radiometrically. Reverse transcriptase-PCR was used for measuring mRNA expression. For reporter gene assays plasmids were transfected into cells via lipofection and luciferase activity was measured luminometrically. Acetyl-histone H3 and H4 chromatin immunoprecipitation (ChIP) assays were performed followed by PCR with TGF-β-receptor II promoter specific primers. We could show that SFN-mediated cell growth inhibition closely correlates with a dose-dependent reduction of protein expression and enzymatic activity of ODC. This effect seems to be due to reduced protein levels and transactivation activity of transcription factor c-myc, a direct regulator of ODC expression, as a consequence of SFN-induced TGF-β/Smad signaling. The coherency of these results was further confirmed by using TGF-β receptor kinase inhibitor SB431542, which largely abolishes inhibitory effects of SFN on both, ODC activity and cell growth.. Since elevated ODC enzyme activity is associated with enhanced tumor development, SFN may be a dietary phytochemical with potential to prevent carcinogenesis.

Topics: Anticarcinogenic Agents; Caco-2 Cells; Cell Proliferation; Colorectal Neoplasms; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Isothiocyanates; Ornithine Decarboxylase; 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; Signal Transduction; Smad Proteins; Smad3 Protein; Smad4 Protein; Sulfoxides; Thiocyanates; Transforming Growth Factor beta

As a tumor marker for colorectal cancers, carcinoembryonic antigen (CEA) enhances the metastatic potential of cancer cells. CEA functions as an intercellular adhesion molecule and is upregulated in a wide variety of human cancers. However, the molecular mechanisms by which CEA mediates metastasis remain to be understood. Transforming growth factor-β (TGF-β) signaling regulates both tumor suppression and metastasis, and also contributes to the stimulation of CEA transcription and secretion in colorectal cancer cells. However, it remains unknown whether CEA, in turn, influences TGF-β functions and if a regulatory cross-talk exists between CEA and the TGF-β signaling pathway. Here, we report that CEA directly interacts with TGF-β receptor and inhibits TGF-β signaling. Targeting CEA with either CEA-specific antibody or siRNA rescues TGF-β response in colorectal cancer cell lines with elevated CEA, thereby restoring the inhibitory effects of TGF-β signaling on proliferation. CEA also enhances the survival of colorectal cancer cells in both local colonization and liver metastasis in animal study. Our study provides novel insights into the interaction between CEA and TGF-β signaling pathway and establishes a negative feedback loop in amplifying the progression of colon cancer cells to more invasive phenotypes. These findings offer new therapeutic opportunities to inhibit colorectal cancer cell proliferation by cotargeting CEA in promoting tumor-inhibitory action of the TGF-β pathway.

Topics: Carcinoembryonic Antigen; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; DNA Primers; Genes, myc; Humans; Microsatellite Repeats; Microscopy, Confocal; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

FMNL2 is a member of diaphanous-related formins that control actin-dependent processes such as cell motility and invasion. Its overexpression in metastatic cell lines and tissues of colorectal carcinoma has been associated with aggressive tumor development in our previous study. But its specific role in cancer is largely unknown. Here we report that FMNL2 is involved in epithelial-mesenchymal transition (EMT) maintenance in human colorectal carcinoma cells. A positive correlation between FMNL2 and vimentin expression and an inverse correlation between FMNL2 and E-cadherin expression were found in colorectal carcinoma cell lines and cancer tissues. Specific knockdown of FMNL2 led to an epithelial-state transition, confirmed by the cobblestone-like phenotype, upregulation of E-cadherin, α-catenin, and γ-catenin, and downregulation of vimentin, snail, slug. Loss of FMNL2 expression lowered the ability of TGF-β to induce cell invasion and EMT, as shown by morphology and the expression levels. Upregulation of vimentin, slug, snail, downregulation of E-cadherin and activation of receptor-Smad3 phosphorylation were observed in M5 and MDCK cells induced by TGF-β, whereas altered expression of these markers was not obvious in FMNL2-depleting M5 cells. High levels of activation of p-MAPK and p-MEK, but not p-PI3K and p-AKT, were observed in SW480/FMNL2+ cells compared with control cells. Treatment with U0126 could abrogate the activation of p-MAPK and p-MEK, whereas LY294002 treatment had no effect on the PI3K/AKT pathway. In conclusion, these findings identify a novel EMT and tumor promoting function for FMNL2, which is involved in TGF-β-induced EMT and colorectal carcinoma cell invasion via Smad3 effectors, or in collaboration with MAPK/MEK pathway.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Down-Regulation; Epithelial-Mesenchymal Transition; Formins; Gene Knockdown Techniques; HT29 Cells; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Proteins; Transforming Growth Factor beta; Up-Regulation; Vimentin

TGFbeta is an important cell growth regulator which may have a role in metastasis formation. Microsatellite unstable (MSI-H) colon cancer serves as a unique model to demonstrate this as most MSI-H colon cancers have a mutation in the transforming growth factor beta receptor II (TGFbetaRII) gene and a low metastatic rate.. To demonstrate an increase in invasion and metastasis in a MSI-H colorectal cancer cell line with a known mutation in TGFbetaRII.. By restoring the wild-type TGFbetaRII gene in the KM12C MSI-H colorectal carcinoma cell line with a known mutation in TGFbetaRII, we have demonstrated that both invasion and metastasis in this cell line was significantly increased. A mouse metastatic model have shown that liver metastases were increased in mice inoculated with cells containing a wild-type TGFbetaRII gene (42% for the transfected group compared with 15% for the control group; p = 0.0379), despite a reduction in the size of primary tumours.. This study highlights an important mechanism which may contribute to the low metastatic rate of MSI-H colon cancers and demonstrates the importance of TGFbeta signalling in metastasis formation. Previous studies involving breast cancer cell lines have shown that blocking TGFbeta signalling results in a reduction in metastasis formation. This study is the first study to use a cell line with a low metastatic rate and TGFbetaRII mutations to demonstrate that restoring TGFbeta signalling increases the metastatic rate.

Topics: Animals; Cell Line, Tumor; Collagen; Colorectal Neoplasms; Drug Combinations; Humans; Laminin; Mice; Microsatellite Instability; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta

Increased transforming growth factor-beta (TGF-beta) levels are associated with fibrosis, affected cell proliferation, and postsurgical adhesion development, but the knowledge regarding TGF-beta response to the surgical trauma is limited. This study investigated TGF-beta(1-3) isoforms and fibrinolytical factors in peritoneal serosal fluid during abdominal surgery, together with the in vitro effect of TGF-beta(1-3) on human mesothelial cell proliferation.. Total as well as biologically active TGF-beta(1-3) and fibrinolytic factors: t-PA, uPA, and PAI-1 were measured in serosal fluid and plasma from 23 patients undergoing colorectal cancer surgery. In vitro proliferation of human primary mesothelial cell cultures upon TGF-beta(1-3) stimulation was also investigated.. Total TGF-beta1 and TGF-beta2 levels were similar in serosal fluid and plasma while active fractions were increased in serosal fluid. In contrast, total fraction of TGF-beta3 was higher in serosal fluid compared with plasma, while levels of active fractions did not differ. Plasminogen activators (t-PA, uPA) were elevated while the inhibitor (PAI-1) was decreased in serosal fluid compared with plasma. The in vitro mesothelial cell proliferation studies revealed that high TGF-beta(1-3) concentrations decreased cell proliferation, while lower concentrations of TGF-beta1 increased mesothelial cell proliferation.. This human study shows increased active TGF-beta levels in peritoneal serosal fluid, compared with plasma, during abdominal surgery and that TGF-beta1 at physiological concentrations increased human mesothelial cell proliferation in vitro. TGF-beta cytokines may be involved in postsurgical adhesion formation.

Topics: Abdomen; Aged; Body Fluids; Cell Division; Cells, Cultured; Colorectal Neoplasms; Epithelial Cells; Epithelium; Female; Fibrinolysis; Humans; In Vitro Techniques; Male; Peritoneal Cavity; Tissue Adhesions; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3

The TGF-beta signaling repressors SnoN and Ski have been critically implicated in human cancer.. To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR.. SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with beta-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels.. Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease.

Topics: Colorectal Neoplasms; Disease Progression; DNA-Binding Proteins; Down-Regulation; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) signaling is frequently altered in colorectal cancer. Using a novel model of mice heterozygous for a targeted null mutation of Tgfbr1 crossed with Apc(Min/+) mice, we show that Apc(Min/+);Tgfbr1(+/-) mice develop twice as many intestinal tumors as Apc(Min/+);Tgfbr1(+/+) mice, as well as adenocarcinoma of the colon, without loss of heterozygosity at the Tgfbr1 locus. Decreased Smad2 and Smad3 phosphorylation and increased cellular proliferation are observed in the colonic epithelium crypts of Apc(Min/+); Tgfbr1(+/-) mice. Smad-mediated TGF-beta signaling is preserved in both Apc(Min/+);Tgfbr1(+/+) and Apc(Min/+);Tgfbr1(+/-) intestinal tumors, but cyclin D1 expression and cellular proliferation are significantly higher in Apc(Min/+);Tgfbr1(+/-) tumors. These results show that constitutively reduced Tgfbr1-mediated TGF-beta signaling significantly enhances colorectal cancer development and results in increased tumor cell proliferation. These findings provide a plausible molecular mechanism for colorectal cancer development in individuals with constitutively altered TGFBR1 expression, a recently identified common form of human colorectal cancer.

Topics: Animals; Cell Growth Processes; Colorectal Neoplasms; Disease Models, Animal; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Hematopoiesis; Inbreeding; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Much is known about the genes and mutations that cause colorectal cancer (CRC), yet only a few have been associated with CRC metastasis. We performed expression-profiling experiments to identify genetic markers of risk and to elucidate the molecular mechanisms of CRC metastasis.. We compared gene expression patterns between metastatic and nonmetastatic stage-matched human colorectal carcinomas by microarray analysis. Correlations between BAMBI and metastasis-free survival were examined by quantitative real-time polymerase chain reaction (PCR) using an independent set of human colon carcinomas. Human colon cancer cell lines were analyzed for BAMBI regulation, cell motility, and experimental metastasis.. We established a signature of 115 genes that differentiated metastatic from nonmetastatic primary tumors. Among these, the transforming growth factor (TGF) beta inhibitor BAMBI was highly expressed in approximately half of metastatic primary tumors and metastases but not in nonmetastatic tumors. BAMBI is a target of canonical Wnt signaling that involves the beta-catenin coactivator BCL9-2. We observed an inverse correlation between level of BAMBI expression and metastasis-free survival time of patients. BAMBI inhibits TGF-beta signaling and increases migration in colon cancer cells. In mice, overexpression of BAMBI caused colon cancer cells to form tumors that metastasized more frequently to liver and lymph nodes than control cancer cells.. BAMBI regulates CRC metastasis by connecting the Wnt/beta-catenin and TGF-beta-signaling pathways. The metastatic expression signature we describe, along with BAMBI levels, can be used in prognosis. Developmental signaling pathways appear to act in hierarchies and cooperate in tumor cell migration, invasion, and metastasis.

Topics: Adenocarcinoma; Animals; beta Catenin; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Shape; Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; HCT116 Cells; Humans; Kaplan-Meier Estimate; Membrane Proteins; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Signal Transduction; Transforming Growth Factor beta; Treatment Outcome; Wnt Proteins

Topics: Adenocarcinoma; Animals; Cell Movement; Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

SMAD7 and GREM1 are signaling components on the transforming growth factor-beta pathway, which regulates normal mammary gland development and has been implicated in breast tumor invasion and metastasis. Three variants within SMAD7 and two variants in CRAC1 (a colorectal cancer-associated region on chromosome 15 in which GREM1 is located) have been associated with colorectal cancer risks [odds ratios (OR), 0.85-1.26; all P < 10(-7)]. We genotyped these five variants in a series of 1,267 bilateral breast cancer cases and 900 controls to determine whether they are associated with breast as well as colorectal cancer risk. None of these single nucleotide polymorphisms were associated with breast cancer risk in our study and the 95% confidence limits of our data, pooled with data from the Cancer Genetic Markers of Susceptibility study, exclude per allele ORs of <0.94 or >1.08. One or more of these variants may be associated with a very small OR for breast cancer, but our data suggest that the effects of these alleles are cancer site-specific.

Topics: Adult; Aged; Breast Neoplasms; Calcium Channels; Colorectal Neoplasms; Female; Genetic Predisposition to Disease; Genotype; Humans; Intercellular Signaling Peptides and Proteins; Middle Aged; ORAI1 Protein; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Risk Factors; Smad7 Protein; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

Topics: Animals; Cell Division; Colorectal Neoplasms; Disease Progression; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Liver; Mice; Mice, Knockout; Neoplasm Staging; Phosphorylation; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Recent large-scale tumor resequencing studies have identified a number of mutations that might be involved in tumorigenesis. Analysis of the frequency of specific mutations across different tumors has been able to identify some, but not all of the mutated genes that contribute to tumor initiation and progression. One reason for this is that other functionally important genes are likely to be mutated more rarely and only in specific contexts. Thus, for example, mutation in one member of a collection of functionally related genes may result in the same net effect, and/or mutations in certain genes may be observed less frequently if they play functional roles in later stages of tumor development, such as metastasis. We modified and applied a network reconstruction and coexpression module identification-based approach to identify functionally related gene modules targeted by somatic mutations in cancer. This method was applied to available breast cancer, colorectal cancer, and glioblastoma sequence data, and identified Wnt/TGF-beta cross-talk, Wnt/VEGF signaling, and MAPK/focal adhesion kinase pathways as targets of rare driver mutations in breast, colorectal cancer, and glioblastoma, respectively. These mutations do not appear to alter genes that play a central role in these pathways, but rather contribute to a more refined shaping or "tuning" of the functioning of these pathways in such a way as to result in the inhibition of their tumor-suppressive signaling arms, and thereby conserve or enhance tumor-promoting processes.

Topics: Algorithms; Breast Neoplasms; Colorectal Neoplasms; Computational Biology; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Glioblastoma; Humans; Mitogen-Activated Protein Kinase Kinases; Mutation; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wnt Proteins

Colon cancer progression is characterized by activating mutations in Ras and by the emergence of the tumor-promoting effects of transforming growth factor-beta (TGF-beta) signaling. Ras-inducible rat intestinal epithelial cells (RIE:iRas) undergo a well-described epithelial to mesenchymal transition and invasive phenotype in response to H-RasV12 expression and TGF-beta treatment, modeling tumor progression. We characterized global gene expression profiles accompanying Ras-induced and TGF-beta-induced epithelial to mesenchymal transition in RIE:iRas cells by microarray analysis and found that the regulation of gene expression by the combined activation of Ras and TGF-beta signaling was associated with enrichment of a class of mRNAs containing 3' AU-rich element (ARE) motifs known to regulate mRNA stability. Regulation of ARE-containing mRNA transcripts was validated at the mRNA level, including genes important for tumor progression. Ras and TGF-beta synergistically increased the expression and mRNA stability of vascular endothelial growth factor (VEGF), a key regulator of tumor angiogenesis, in both RIE:iRas cells and an independent cell culture model (young adult mouse colonocyte). Expression profiling of human colorectal cancers (CRC) further revealed that many of these genes, including VEGF and PAI-1, were differentially expressed in stage IV human colon adenocarcinomas compared with adenomas. Furthermore, genes differentially expressed in CRC are also significantly enriched with ARE-containing transcripts. These studies show that oncogenic Ras and TGF-beta synergistically regulate genes containing AREs in cultured rodent intestinal epithelial cells and suggest that posttranscriptional regulation of gene expression is an important mechanism involved in cellular transformation and CRC tumor progression.

Topics: Animals; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclooxygenase 2; Disease Progression; Epithelium; Gene Expression Profiling; Gene Expression Regulation; Humans; Mesoderm; Mice; Oligonucleotide Array Sequence Analysis; Oncogene Protein p21(ras); Protein Binding; Rats; Regulatory Sequences, Ribonucleic Acid; Reproducibility of Results; RNA Stability; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-beta (TGF-beta) is a pluripotent cytokine that can have both tumor suppressing and tumor promoting effects on epithelial cells. It is unclear what determines when TGF-beta and its signaling pathway act predominantly as a tumor suppressor pathway or as a tumor-promoter pathway and whether TGF-beta can have both classes of effects concurrently on a cell. We investigated the effect of TGF-beta on anoikis in colorectal cancer cell lines sensitive to TGF-beta-mediated growth inhibition to determine if the context of the cells could be one of the factors that would affect whether TGF-beta exerts tumor suppressor or oncogene activity on colon cancer cells. We observed variable effects of TGF-beta on anoikis in these cell lines, even though they all are growth-inhibited by TGF-beta. Thus, we show that TGF-beta has variable effects on anoikis in colon cancer cell lines that likely reflects the effects of concurrent gene mutations in the cancer cells and the activation state of the signaling pathways controlled by these genes.

Topics: Anoikis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Culture Media, Serum-Free; Genes, Reporter; HCT116 Cells; Humans; Luciferases; Transforming Growth Factor beta

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

Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor-beta (TGF-beta) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-beta signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.

Topics: 3' Untranslated Regions; Adult; Aged; Aged, 80 and over; Alleles; Cell Line; Colorectal Neoplasms; Female; Gene Expression; Genetic Predisposition to Disease; Haplotypes; Heterozygote; Humans; Linkage Disequilibrium; Male; Middle Aged; Polymorphism, Single Nucleotide; Protein Serine-Threonine Kinases; Quantitative Trait, Heritable; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Risk Factors; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Blotting, Western; Cadherins; Cell Proliferation; Claudin-1; Claudin-4; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Membrane Proteins; Mice; Mice, Nude; Phosphorylation; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

The aim of this study is to identify gene expression signatures that accompany dedifferentiation at the cancer invasion front in colorectal cancer.. Two types of colorectal cancer were selected. Both types were well-differentiated adenocarcinomas at the superficial lesion. One type showed a dedifferentiated phenotype at the invasion front (type A, 13 samples); the other showed almost no dedifferentiated cancer cells at the invasion front (type B, 12 samples). Laser microdissection was combined with a cDNA microarray analysis to investigate the superficial lesions and the invasion front in colorectal cancers.. Eighty-three genes were differentially expressed between types A and B in the superficial lesions, and the samples of superficial lesions were divided correctly into two clusters by these genes. Interestingly, the samples of the invasion front were also divided into the two same clusters by these genes. The text mining method selected 10 genes involved in potential mechanisms causing dedifferentiation of cancer cells at the invasion front. The potential mechanisms include the networks of transforming growth factor-beta, Wnt, and Hedgehog signals. The expression levels of 10 genes were calculated by quantitative reverse transcription-PCR and 8 genes were confirmed to be significantly differentially expressed between two types (P < 0.05). The gene expression profiles of 8 genes divided 12 test cases into two clusters with one misclassification.. The molecular mechanisms constructed with 8 genes from three networks of transforming growth factor-beta, Wnt, and Hedgehog signals were found to correlate with dedifferentiation at the invasion front of colorectal cancer.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Cell Differentiation; Colorectal Neoplasms; Female; Gene Expression; Gene Expression Profiling; Hedgehog Proteins; Humans; Male; Microdissection; Middle Aged; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

The balance of activity between the endogenous enzyme inhibitors known as tissue inhibitors of metalloproteinases and their targets, the matrix metalloproteinases, in the extracellular matrix is thought to play an important role in tumour cell invasion. Supporting this notion, we have shown that colorectal cancer patients have increased plasma levels of the tissue inhibitor of metalloproteinases-1 (TIMP-1), and that high plasma TIMP-1 levels are associated with short colorectal cancer patient survival. However, although TIMP-1 has been extensively studied in cancer, very little is known about how it is regulated. To further elucidate potential mechanisms of regulation of this protein, we did a number of experiments to look at associations between the transcript profile of TIMP-1 with known matrix metalloproteinases (MMPs) as well as with expression profiles of other genes differentially regulated in human colorectal cancer (CRC) and the other TIMPs 2-4, which have also been associated with the progression of colorectal cancer. Genome-wide expression profiling of 172 CRC and normal mucosa samples was used to identify transcript changes for the genes under investigation. We found that TIMP-1 was up-regulated in CRC samples compared with normal tissue, while TIMP-2 was down-regulated. Eight MMPs were up-regulated in CRC compared with normal tissue. Correlating up-regulated genes with the TIMP-1 transcript, we identified 13 that were also up-regulated in cancerous tissue. Among these were genes associated with the synthesis of extracellullar matrix, genes involved in the TGF-beta signalling pathway, and genes that are likely transcribed by the tumour cells. These insights add to the complex picture emerging about the regulation of TIMPs in colorectal cancer.

Topics: Amino Acid Oxidoreductases; Cell Adhesion Molecules; Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibin-beta Subunits; Matrix Metalloproteinases; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Disruptions to the TGFbeta signaling pathway have been implicated in most human adenocarcinomas. In addition to its role in cancer cell migration and metastasis, TGFbeta has been implicated in tumor-mediated immunosuppression. Membrane-bound TGFbeta has previously been reported to be expressed on a subset of regulatory T cells and was shown to be critical to their immune suppressive function. In the present study, we document expression of a signaling competent, endogenously derived form of cell surface-bound TGFbeta on colorectal cancer cells. While antibodies against only the mature form of TGFbeta failed to label cells, surface-bound TGFbeta was clearly detected by antibodies specific for both the latent and mature forms of the cytokine. Confirming the notion that the surface TGFbeta was in latent form, brief acid pulsing of the cells increased the amount of detectable membrane-associated TGFbeta. In coculture assays, this cell-bound TGFbeta could be activated and utilized in a paracrine fashion both by other cancer cells and by CD8+ intraepithelial lymphocytes. This effect was abrogated by the use of a furin inhibitor which decreased the membranous expression of TGFbeta on the tumor cells. Signaling competent membrane-bound TGFbeta on cancer cells is thus likely to be a key player in regulating tumor cell interactions with each other as well as with other cells in their microenvironment.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immune Tolerance; Paracrine Communication; Transforming Growth Factor beta

Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFbeta signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases.. Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related to markers of Wnt (beta-catenin), Notch (HES1) and TGFbeta (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed.. When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear beta-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear beta-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear beta-catenin.. Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFbeta signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFbeta pathways is reduced in secondary colorectal tumors.

Topics: Adult; Aged; Brain Neoplasms; Cadherins; Colorectal Neoplasms; Female; Fibronectins; Humans; Liver Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Metastasis; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptors, Notch; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta; Vimentin; Wnt Proteins

Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor (TGF)-beta superfamily. Recent studies demonstrated that the expression patterns of BMPs are altered in several tumors. The purpose of the current study was to examine the expression of BMP7 in malignant and normal colorectal tissues, and to analyze whether BMP7 expression levels correlate with clinicopathological variables and prognosis in colorectal cancer.. Paired colorectal tissue samples from cancer and corresponding nonmalignant tissues were obtained from 65 patients who underwent surgical resection for colorectal cancer. The expression status of BMP7 mRNA was investigated by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and protein expression was analyzed by an immunohistochemical study.. Quantitative real-time RT-PCR showed that BMP7 mRNA expression in cancerous tissue was significantly higher than that in normal tissue (p < 0.0001). An immunohistochemical study revealed that BMP7 was predominantly expressed in cancer cells. Elevated BMP7 expression was significantly correlated with depth of tumor invasion, liver metastasis, liver recurrence, advanced Dukes' classification, and cancer-related death (p < 0.05, 0.001, 0.01, 0.05 and 0.01, respectively). Furthermore, patients with the highest levels of BMP7 expression showed the poorest prognosis (p < 0.01). A multivariate analysis showed that BMP7 expression status was an independent prognostic factor of overall survival (relative risk, 2.29; 95% confidence interval, 1.08-5.30; p < 0.05).. Expression of BMP7 in colorectal tumors correlates with parameters of pathological aggressiveness such as liver metastasis and poor prognosis. Thus, BMP7 could be a useful clinical marker for colorectal cancer patients.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Case-Control Studies; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Liver Neoplasms; Lymphatic Metastasis; Male; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Survival Rate; Transforming Growth Factor beta

The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC.. We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc.. BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss.. Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line, Tumor; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Male; Microsatellite Instability; Microsatellite Repeats; Middle Aged; Mutation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Smad4 Protein; Transforming Growth Factor beta

Chronic inflammation predisposes to cancer. Transforming growth factor (TGF)-beta, a multifunctional protein, suppresses the growth of normal colonic epithelial cells, whereas it stimulates the proliferation of cancer cells. Interleukin (IL)-10-deficient mice, which develop colitis and colorectal cancer, show an increased level of plasma TGF-beta. Although TGF-beta may be a key molecule in the development of colon cancer arising from chronic colitis in IL-10-deficient mice, the role of TGF-beta still remains unclear. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which converts the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). We studied C57BL/6-IL-10-deficient mice (n=18) at 4 to 32 weeks of age. We investigated histology, and pSmad2/3L, pSmad2/3C, and p53 by immunohistochemistry. pSmad3L staining was detected in the cancer cells in all 10 mice with colonic cancer and in the epithelial cells in 7 of 12 mice with colonic dysplasia, but not in the normal or colitic mice. pSmad3c was detected without any significant difference between stages. p53 was weakly stained in a few cancer cells in 5 out of 10 mice. Smad3L signaling plays an important role in the carcinogenesis of chronic colitis in IL-10-deficient mice.

Topics: Animals; Chronic Disease; Colitis; Colorectal Neoplasms; Immunoenzyme Techniques; Interleukin-10; Intestine, Large; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

To investigate genetic alterations involved in the TGF-beta signaling pathway in colorectal cancer, we assayed DNA synthesis rates after treating TGF-beta and checked for genetic alterations in TGF-betaRII, TGF-betaRI, Smad2, Smad3, and Smad4 in 12 colorectal cancer cell lines. Eleven lines, except SNU-61, show no significant change in DNA synthesis rate after TGF-beta treatment. In these 11 lines, several mutations were found in genes involved in the TGF-beta signaling pathway: (i) frameshift deletions in the poly(A)(10) tract of the TGF-betaRII gene in SNU-407, SNU-769A, SNU-769B, and SNU-1047 cell lines, (ii) a missense mutation of Smad2 (R321Q) in SNU-81, (iii) two missense mutations in TGF-betaRI (R487W in SNU-175 and A202V in SNU-1040), and (iv) a monoallelic loss at the Smad4 locus in three cell lines. Interestingly, a missense mutation (R373H) in Smad3 gene was found in SNU-769A. To our knowledge, this is the first report of Smad3 mutation in human malignancy. This mutation was found to result in the inhibition of translocation of Smad3 protein to the nucleus and a reduction in the activity of Smad3 during TGF-beta-induced transcriptional activation. These results indicate that the majority of cell lines, which are insensitive to TGF-beta, have alterations in genes involved in the TGF-beta signaling pathway in colorectal cancer cell lines.

Topics: Base Sequence; Cell Line, Tumor; Colorectal Neoplasms; DNA Primers; Frameshift Mutation; Humans; Mutagenesis, Site-Directed; Mutation, Missense; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Transcriptional Activation; Transforming Growth Factor beta

The prognostic importance of tumour infiltrating lymphocytes (TILs) in colorectal cancer (CRC) remains controversial. The present study was undertaken to evaluate the role of TILs as prognostic indicators and to investigate the role of transforming growth factor-beta (TGF-beta) in TIL infiltration. Immunohistochemical staining for components in the TGF-beta pathway was performed on a tissue microarray of 1420 unselected CRCs with complete clinico-pathological data. Statistical analyses were carried out on samples stratified by mismatch repair (MMR) proficiency status and TIL counts. TIL infiltration was found to correlate with multiple clinico-pathological features but was a prognostic marker only in MMR proficient CRCs. In all CRCs, findings indicative of insensitivity to TGF-beta and increased TGF-beta secretion were independent predictors of high TIL counts, suggesting that perturbations in the TGF-beta signalling pathway play an important role in the recruitment and retention of TILs within CRC epithelium.

Topics: Colorectal Neoplasms; DNA Mismatch Repair; Humans; Lymphocytes, Tumor-Infiltrating; Prognosis; Regression Analysis; ROC Curve; Survival Analysis; Transforming Growth Factor beta

We have recently reported that the expression of a tight junction protein, claudin-1, is increased during colon carcinogenesis and particularly metastatic colorectal cancer. Manipulation of claudin-1 levels in colon cancer cells showed a positive correlation between claudin-1 expression and tumor growth and metastasis. However, the mechanisms underlying the increased claudin-1 expression in colorectal cancer remains unknown. The tumor suppressor Smad4 is a central intracellular signal transduction component of the transforming growth factor-beta (TGF-beta) family of cytokines. Loss of Smad4 protein expression is correlated with poor prognosis and is frequently observed in invasive and metastatic colorectal carcinoma. In the present study, we report an inverse relationship between Smad4 and claudin-1 expression in human colorectal carcinoma tumor samples and in human colon cancer cell lines. We found that the expression of Smad4 in Smad4-deficient but claudin-1-positive SW480 or HT29 colon cancer cell lines down-regulates claudin-1 expression through transcriptional repression by modulating beta-catenin/T-cell factor/lymphocyte enhancer factor activity. Furthermore, this Smad4-dependent inhibition of claudin-1 expression is independent of TGF-beta signaling because Smad4 expression alone is insufficient to restore TGF-beta signaling in the SW480 cells, and the selective TGF-beta receptor kinase inhibitor LY364947 did not prevent the Smad4 suppression of claudin-1 protein expression in either SW480 or HT29 cells. Taken together, these findings suggest a novel mechanism underlying Smad4 tumor-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-beta-independent manner.

Topics: Animals; beta Catenin; Cell Line, Tumor; Claudin-1; Colorectal Neoplasms; HCT116 Cells; HT29 Cells; Humans; Membrane Proteins; Neoplasm Invasiveness; Rats; RNA, Messenger; Signal Transduction; Smad4 Protein; TCF Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Topics: Cell Line, Tumor; Colorectal Neoplasms; Cytoplasmic Dyneins; Dyneins; Humans; Mutation; Signal Transduction; Stomach Neoplasms; Transforming Growth Factor beta

An efficient cytolytic T cell function is essential for immune mediated rejection of colorectal cancer. However, the molecular mechanisms driving T cell mediated cancer rejection are still poorly understood. Here, we assessed the relevance of the T-box transcription factor eomesodermin in colorectal cancer. METHODS/ RESULTS: By analysing tissue probes from 88 different colorectal tumours, a significant (p<0.02) inverse correlation between eomesodermin expression in colorectal cancers and the presence of lymph node metastases could be shown, whereas no such correlation was noted for the master transcription factor of regulatory T cells, FoxP3 and CD8 alpha expression. To evaluate whether this effect might be due to effects of eomesodermin on tumour infiltrating CD8 T cells, we subsequently analysed the regulated expression and function of this transcription factor in human T cells. Whereas overexpression of this factor induced perforin but not granzyme expression, siRNA mediated suppression of eomesodermin expression led to significantly reduced IFN-gamma production, perforin levels and cytolytic activity of CD8 T cells. Furthermore, TGF-beta and IL4 could be identified as important inducer of eomesodermin expression.. These data define for the first time a regulatory role of eomesodermin for CD8 T cell activity in humans. Our findings are consistent with a model in which eomesodermin expression in tumour infiltrating T cells regulates cytolytic functions of CD8 T cells via perforin expression. These data provide novel insights into control mechanisms governing the functional activity of human CD8 T lymphocytes via T-box transcription factors in cancer.

Topics: CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-4; Lymphatic Metastasis; Male; Reverse Transcriptase Polymerase Chain Reaction; T-Box Domain Proteins; Transforming Growth Factor beta

The small GTPase Rac1 is involved in the regulation of critical cellular functions, such as transcription control, cell cycle, and organization of actin cytoskeleton. Rac1 signalling modulates cancer progression since its overexpression leads to an increased tumour growth of xenografts of human colorectal tumour cells, while a drastic reduction of Rac1 expression by siRNA interferes with cancer progression (Espina et al., unpublished results). We aimed to study the molecular basis for the specific contribution of Rac1 in the progression of colorectal cancer. Comparative microarray analysis of a human colorectal carcinoma cell line genetically engineered to display different levels of Rac1 identified novel target genes for this GTPase. These results suggest that Rac1 plays a critical role in signalling transduction pathways relevant to human colorectal tumour progression, such as activation of Wnt signalling, inhibition of TGF-beta signalling, and enhancement of metastasis-inducing genes.

Topics: Adaptor Proteins, Signal Transducing; beta Catenin; Calcium-Binding Proteins; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Colorectal Neoplasms; DNA-Binding Proteins; Galectin 1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Matrix Metalloproteinase 7; Membrane Proteins; Microfilament Proteins; Models, Biological; Oligonucleotide Array Sequence Analysis; rac1 GTP-Binding Protein; Repressor Proteins; Reproducibility of Results; RNA, Small Interfering; S100 Calcium Binding Protein A6; S100 Proteins; Signal Transduction; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Wnt Proteins

Disruptions to the TGFbeta signalling pathway have been implicated in most human adenocarcinomas. As cancers progress, many acquire resistance to the growth-suppressing properties of TGFbeta while retaining sensitivity to its tumour-promoting effects. Microsatellite unstable colorectal cancers (MSI-H CRCs) possess truncating mutations in the type II TGFbeta receptor (TGFbetaRII) gene that have been assumed to render these tumours insensitive to TGFbeta. However, numerous reports of TGFbetaRII bypass exist and this study was thus undertaken in order to clarify the true extent of TGFbeta sensitivity in MSI-H CRCs. Using stimulation with exogenous TGFbeta, we demonstrated that, while MSI-H CRCs are capable of binding soluble TGFbeta, two out of three cell lines examined remain refractory to its signalling effects. In contrast, use of a specific inhibitor of the type I TGFbeta receptor (TGFbetaRI) revealed that all remain sensitive to signalling by endogenously produced TGFbeta. Specifically, autocrine signalling via TGFbetaRI mediates constitutive activation of Smad2 as well as repression of Erk signalling. Real-time PCR confirmed that these effects are sufficient to affect the expression level of various TGFbeta-modulated genes. An invasion assay revealed that autocrine TGFbetaRI signalling also promotes the invasion capacity of MSI-H CRCs to an extent similar to that seen in their non-MSI-H counterparts. Independent TGFbetaRI signalling, however, has no effect on the rate of proliferation of MSI-H CRC cells. Together, these results demonstrate that MSI-H CRC cell lines are not completely refractory to TGFbeta, despite lacking functional TGFbetaRII. In addition to clarifying the true consequences of natural TGFbetaRII loss and the independent function of TGFbetaRI, our results highlight the selective nature of TGFbeta resistance developed by cancers.

Topics: Autocrine Communication; Benzamides; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Dioxoles; Drug Resistance, Neoplasm; Humans; Loss of Heterozygosity; Microsatellite Instability; Mutation; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Epidemiological evidence suggests that statins prevent colorectal cancer (CRC), but the biological mechanism remains obscure. Statins induce bone morphogenetic protein (BMP) expression in bone cells. We have previously shown that BMPs act as tumor suppressors in CRC. We hypothesized that the action of statins in CRC involves the induction of BMPs.. We investigated the effects of statins on CRC cell lines using immunoblotting, measurements of apoptosis and cell proliferation, and luciferase reporter assays. The effect of statins was confirmed in a xenograft mouse model.. CRC cell lines show widely differing sensitivities to statin treatment. Sensitive cell lines show induction of BMP2 protein levels and a BMP2 reporter construct, activation of the BMP pathway, and induction of the BMP target gene ID-2, whereas resistant cell lines do not. The addition of the specific inhibitor of BMPs, noggin, completely prevents lovastatin-induced apoptosis in sensitive cells. Sensitive cell lines express the central BMP pathway element SMAD4, whereas the resistant cell lines do not. Targeted knockout of SMAD4 leads to the loss of statin sensitivity and reconstitution with SMAD4, to the restoration of statin sensitivity. In a xenograft mouse model, tumors from sensitive and insensitive cell lines were treated with oral simvastatin. Significant inhibition of tumor growth using sensitive cells but increased tumor growth when using insensitive cells was observed.. Statins induce apoptosis in CRC cells through induction of BMP2. Statin therapy may only be effective in SMAD4-expressing CRCs and may have adverse effects in SMAD4-negative tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carrier Proteins; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Genes, Reporter; HCT116 Cells; HT29 Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Luciferases; Mice; Mice, Nude; Signal Transduction; Simvastatin; Smad4 Protein; Transfection; Transforming Growth Factor beta; Up-Regulation; Xenograft Model Antitumor Assays

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been implicated in metastasis of colorectal cancer (CRC). The present study aimed to clarify whether cancer-stromal interaction induces the production of VEGF.. Human colonic fibroblasts (CCD-18Co) and CRC (SW480, SW620) cells were analyzed in this study. The cell cycle of colonic fibroblasts during co-culture was analyzed by flow cytometry. VEGF and TGF-beta1 released into the conditioned media in co-culture models were measured. Northern blot with human specific VEGF probe was performed to identify the expression of VEGF in this model.. Co-culture of colonic fibroblasts with CRC cells increased the viability of fibroblasts during co-culture. Cell cycle analysis revealed that most of the fibroblasts co-cultured with CRC cells were arrested at G1 phase and few cells were in sub-G1 phase that indicates apoptosis. Although VEGF protein was detected in the culture media of all of the monocultures, co-cultivation of CRC with fibroblasts resulted in synergistic increase of VEGF production compared with monocultures. However TGF-beta1 protein was not detected in any conditioned medium. VEGF mRNA was detected in both CRC and fibroblasts. Under co-culture condition, an abundance of VEGF mRNA expression was noted in fibroblasts relative to CRC cells.. The present study suggests that CRC manipulates the host stroma to suppress apoptosis and up-regulate VEGF production.

Topics: Apoptosis; Blotting, Northern; Cell Cycle; Cell Line; Cell Line, Tumor; Coculture Techniques; Colon; Colorectal Neoplasms; Culture Media, Conditioned; Fibroblasts; Flow Cytometry; G1 Phase; Gene Expression; Humans; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

The development and progression of malignancies is a complex multistage process that involves the contribution of a number of genes giving growth advantage to cells when transformed. The role of transforming growth factor-beta (TGF-beta) in carcinogenesis is complex with tumor-suppressor or prooncogenic activities depending on the cell type and the stage of the disease. We have previously reported the identification of a novel WD-domain protein, STRAP, that associates with both TGF-beta receptors and that synergizes with the inhibitory Smad, Smad7, in the negative regulation of TGF-beta-induced transcription. Here, we show that STRAP is ubiquitously expressed and is localized in both cytoplasm and nucleus. STRAP is up-regulated in 60% colon and in 78% lung carcinomas. Stable expression of STRAP results in activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and in down-regulation of the cyclin-dependent kinase inhibitor p21(Cip1), which results in retinoblastoma protein hyperphosphorylation. In addition, we have observed that Smad2/3 phosphorylation, TGF-beta-mediated transcription, and growth inhibition are induced in STRAP-knockout mouse embryonic fibroblasts compared with wild-type cells. Ectopic expression of STRAP in A549 lung adenocarcinoma cell line inhibits TGF-beta-induced growth inhibition and enhances anchorage-independent growth of these cells. Moreover, overexpression of STRAP increases tumorigenicity in athymic nude mice. Knockdown of endogenous STRAP by small interfering RNA increases TGF-beta signaling, reduces ERK activity, increases p21(Cip1) expression, and decreases tumorigenicity. Taken together, these results suggest that up-regulation of STRAP in human cancers may provide growth advantage to tumor cells via TGF-beta-dependent and TGF-beta-independent mechanisms, thus demonstrating the oncogenic function of STRAP.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Transformation, Neoplastic; Chlorocebus aethiops; Colorectal Neoplasms; COS Cells; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mink; Mitogen-Activated Protein Kinase Kinases; Neoplasm Proteins; NIH 3T3 Cells; Oncogenes; Phosphorylation; Proteins; Retinoblastoma Protein; RNA-Binding Proteins; Transforming Growth Factor beta

To investigate the relationship between HIC-5 and colonrectal cancer.. Samples were obtained from 42 patients with colonrectal cancer, and the mRNA and protein expressions of HIC-5/ARA55 were determined by RT-PCR and Western-blot respectively. FCM was used to investigate the apoptosis of Lovo in the HIC-5/ARA55 transfection cells. The expression of apoptosis related proteins Bax and Bcl-2 was determined by Western-blot, and the MTT assay was used to test cell growth speed affected by HIC-5/ARA55.. The expression of HIC-5 significantly decreased on colonrectal cancer tissues as compared with adjacent normal tissues on mRNA level and protein level(P<0.05). The apoptosis rate increase significantly in the HIC-5/ARA55 transfected cells. HIC-5/ARA55 could restrain tumor cell grow speed.. HIC-5/ARA55 is the protein that can reduce tumor cell growth rate, and induce apoptosis in colonrectal cancer cells.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor beta

Human sporadic colorectal cancer is the result of a lengthy somatic evolutionary process facilitated by various forms of genomic instability. Such instability arises endogenously from mutations in genes whose role is to preserve genomic integrity, and exogenously from environmental agents that generate genomic damage. We have found that cigarette smoking shifts the genomic profiles and genomic instability patterns of colorectal carcinomas. The genomic profiles of 57 consecutive cancers were examined; 31 cases were current or former smokers and 26 were nonsmokers. Genome-wide allelotypes of 348 markers were examined, along with comparative genomic hybridization (CGH) on ordered BAC microarrays, microsatellite instability, and inter-(simple sequence repeat) polymerase chain reaction instability. Tumors from nonsmokers exhibited losses of heterozygosity, particularly on chromosomes 14 and 18, whereas tumors from smokers exhibited a more diffuse pattern of allelic losses. Tumors from smokers exhibited higher overall rates of loss of heterozygosity, but showed lower rates of background microsatellite instability (MSI-L). On BAC array CGH, higher levels of generalized amplifications and deletions were observed in tumors from smokers, differentially affecting male smokers. In the transforming growth factor-beta signaling pathway, MADH4 mutations were more common in tumors from smokers, whereas transforming growth factor-beta RII mutations were more common among nonsmokers.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Chromosomes, Artificial, Bacterial; Colorectal Neoplasms; DNA, Neoplasm; Female; Genome, Human; Genomic Instability; Humans; Male; Middle Aged; Mutation; Nicotiana; Sex Characteristics; Signal Transduction; Smoking; Transforming Growth Factor beta

Colorectal cancer, one of the most common human malignancies in the Western world, is often subdivided based on tumor location in either the distal or proximal colon. Several mouse models have been developed to study human colorectal cancer, but few display this clear distinction between the two colonic locations. By crossing Apc(Min/+) and Smad3 mutant mice, we showed that combined activation of the Wnt pathway and attenuation of the transforming growth factor-beta (TGF-beta) pathway causes high multiplicity and rapid onset of invasive tumorigenesis almost exclusively in the distal colon, closely mimicking the familial adenomatous polyposis (FAP) disease and consisting with distinct colorectal cancer etiologies based on tumor location. Transcriptional profiling revealed higher expression of several TGF-beta activators in the normal distal mucosa than in proximal mucosa, suggesting a stronger reliance on TGF-beta-mediated growth control in the distal than in the proximal colon. Apc(Min/+)Smad3(-/-) mice provide an alternative model to Apc(Min/+) mice to study FAP and distal sporadic colorectal cancer. This model will be useful in dissecting mechanistic and etiologic differences between proximal and distal colonic cancer, whereas the confinement of tumorigenesis to the distal colon offers unique advantages in monitoring tumor progression by in vivo imaging.

Topics: Adenomatous Polyposis Coli; Animals; Biopsy; Colonic Neoplasms; Colorectal Neoplasms; DNA; Gene Expression Profiling; Genes, APC; Humans; Intestinal Mucosa; Mice; Mice, Knockout; Mice, Transgenic; Neoplasm Invasiveness; Polymerase Chain Reaction; Rectal Neoplasms; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta

Colorectal cancers deficient in DNA mismatch repair (MMR) are often characterized by the presence of numerous intraepithelial lymphocytes (IELs). These CD8+ T cells selectively express CD103, which is upregulated locally by transforming growth factor (TGF)-beta, and adhere to E-cadherin expressed by mucosal epithelia. Many of these cancers also possess inactivating mutations in the type II TGF-beta receptor and are believed to be insensitive to TGF-beta. The present study aimed to explore whether such refractoriness to TGF-beta is an independently contributing factor to IEL retention.. A panel of colorectal cancers enriched for DNA MMR deficiency was examined by immunohistochemistry to explore the expression levels and localization of various components in the TGF-beta signalling pathway. Logistic regression was then carried out in order to identify predictors of elevated lymphocytic infiltration independent of DNA MMR status. Increases in Smad4 expression, tumour cell proliferation and TGF-beta secretion each emerged as independent predictors of marked lymphocyte infiltration.. These results strongly support the hypothesis that refractoriness to normal TGF-beta signalling in colorectal cancers plays a role in the retention of lymphocytes within tumour epithelium. Since IEL infiltration is an independent predictor of favourable prognosis, the TGF-beta pathway may represent an important therapeutic target.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Base Pair Mismatch; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; DNA Repair; Female; Humans; Immunohistochemistry; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Prognosis; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Human tumors constitutively release endosome-derived microvesicles, transporting a broad array of biologically active molecules with potential modulatory effects on different immune cells. Here, we report the first evidence that tumor-released microvesicles alter myeloid cell function by impairing monocyte differentiation into dendritic cells and promoting the generation of a myeloid immunosuppressive cell subset. CD14+ monocytes isolated from healthy donors and differentiated with interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in the presence of tumor-derived microvesicles turned into HLA-DR(-/low) cells, retaining CD14 expression and failing to up-regulate costimulatory molecules, such as CD80 and CD86. These phenotypic changes were paralleled by a significant release of different cytokines, including IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and a dose-dependent suppressive activity on activated T-cell-proliferation and cytolytic functions, which could be reversed by anti-TGF-beta-neutralizing antibodies. Microvesicles isolated from plasma of advanced melanoma patients, but not from healthy donors, mediated comparable effects on CD14+ monocytes, skewing their differentiation toward CD14+HLA-DR-/low cells with TGF-beta-mediated suppressive activity on T-cell-functions. Interestingly, a subset of TGF-beta-secreting CD14+HLA-DR- cells mediating suppressive activity on T lymphocytes was found to be significantly expanded in peripheral blood of melanoma patients compared with healthy donors. These data suggest the development in cancer patients of an immunosuppressive circuit by which tumors promote the generation of suppressive myeloid cells through the release of circulating microvesicles and without the need for cell-to-cell contact. Therapeutic interventions on the crucial steps of this pathway may contribute to restore tumor/immune system interactions favoring T-cell-mediated control of tumor growth in cancer patients.

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Colorectal Neoplasms; Dendritic Cells; Endosomes; HLA-DR Antigens; Humans; Leukocytes, Mononuclear; Lipopolysaccharide Receptors; Melanoma; Myeloid Cells; Secretory Vesicles; T-Lymphocytes; Transforming Growth Factor beta

Recent investigations support an important role for TGF-beta in the development of colorectal cancer. However, the molecular consequences of TGF-beta signaling in the colon remains incompletely understood. In a recent study in Immunity, we analyzed the role of TGF-beta in a murine model of colon cancer. Using transgenic mice overexpressing TGF-beta or a dominant negative TGF-beta receptor II under control of the CD2 minigene, we show that TGF-beta signaling in tumor infiltrating T lymphocytes regulates the growth of dysplastic colon epithelial cells, as determined by histology and a novel system for high resolution chromoendoscopy in vivo. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-dependent IL-6 trans-signaling prevented tumor progression in vivo. Similar to these observations in mice, here we show that human colon cancer tissue expressed only low amounts of membrane bound IL-6R. In contrast, expression and activity of the matrix metalloproteinase TACE were increased. In summary, our data provide novel insights into the role of TGF-beta signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on an inhibition of TGF-beta-dependent IL-6 trans-signaling.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Antineoplastic Agents; CD2 Antigens; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Interleukin-6; Mice; Mice, Transgenic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Interleukin-6; Receptors, Transforming Growth Factor beta; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes; Transforming Growth Factor beta

Conversion of normal epithelial cells to tumors is associated with a shift in transforming growth factor-beta (TGF-beta) function: reduction of tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal carcinogenesis remain to be elucidated. TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal adenocarcinomas. In particular, late-stage invasive and metastatic cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11 adenomas was intermediate between those in normal epithelial cells and adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive adenocarcinomas. In contrast, pSmad3C gradually decreased as the tumor stage progressed. Activated c-Jun NH(2)-terminal kinase in cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at codon 361 in one adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the type II TGF-beta receptor and Smad2 genes were observed in the tumors. In conclusion, pSmad3C, which favors tumor suppressor activity of TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal kinase tended to induce the phosphorylation of Smad2/3L in human colorectal adenoma-carcinoma sequence.

Topics: Adenocarcinoma; Binding Sites; Cell Transformation, Neoplastic; Colorectal Neoplasms; DNA-Binding Proteins; Humans; JNK Mitogen-Activated Protein Kinases; Neoplasm Staging; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

To evaluate the significance of transforming growth factor beta (TGF beta) expression, in correlation with histopathological parameters, at the front of invasion in T1 colorectal cancer (CRC) and presence of metastases.. TGF beta immunohistochemical expression was studied in 34 specimens of colorectal adenocarcinomas (pT1). A three-step avidin-biotinylated immuno-peroxidase (ABCu-NCL) staining technique was performed on 4-mum paraffin-embedded tissue sections with a monoclonal antibody to TGF beta (Novocastra, NCL-TGFB, clone TGFB 17, dilution 1:40).. Seventeen (50%) out of 34 lesions were positive for TGF beta expression. The TGF beta-positive rate in patients with vascular invasion was significantly higher than in those without vascular invasion (11/14 cases, P<0.01, P = 0.005). The TGF beta-positive rate was observed in 91.7% of patients with presence of tumor budding at the front of invasion (11/12 cases, P<0.01, P = 0.0003). A statistically significant correlation was found between the presence of lymph node metastases and positive expression of TGF beta (14/16 cases, P<0.01, P = 0.0001). We also observed that the TGF beta-positive rates in groups with distant and non-distant metastases were 92.8% and 20% respectively, and a significant correlation between TGF beta expression and distant metastasis was shown (P<0.01, P = 0.00003).. The evaluation of TGF beta expression of protein in association with histological parameters can be used as a parameter of the aggressiveness of pT1 CRC.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Humans; Immunohistochemistry; Lymphatic Metastasis; Neoplasm Invasiveness; Transforming Growth Factor beta

TGFBR1*6A is a common polymorphism of the type I transforming growth factor beta receptor (TGFBR1). Epidemiological studies suggest that TGFBR1*6A may act as a tumor susceptibility allele. How TGFBR1*6A contributes to cancer development is largely unknown.. To determine whether TGFBR1*6A is somatically acquired by primary tumors and metastases during cancer development and whether the 3-amino acid deletion that differentiates TGFBR1*6A from TGFBR1 is part of the mature receptor or part of the signal sequence and to investigate TGFBR1*6A signaling in cancer cells.. Tumor and germline tissues from 531 patients with a diagnosis of head and neck, colorectal, or breast cancer recruited from 3 centers in the United States and from 1 center in Spain from June 1, 1994, through June 30, 2004. In vitro translation assays, MCF-7 breast cancer cells stably transfected with TGFBR1*6A, TGFBR1, or the vector alone, DLD-1 colorectal cancer cells that endogenously carry TGFBR1*6A, and SW48 colorectal cancer cells that do not carry TGFBR1*6A.. TGFBR1*6A somatic acquisition in cancer. Determination of the amino terminus of the mature TGFBR1*6A and TGFBR1 receptors. Determination of TGF-beta-dependent cell proliferation.. TGFBR1*6A was somatically acquired in 13 of 44 (29.5%) colorectal cancer metastases, in 4 of 157 (2.5%) of colorectal tumors, in 4 of 226 (1.8%) head and neck primary tumors, and in none of the 104 patients with breast cancer. TGFBR1*6A somatic acquisition is not associated with loss of heterozygosity, microsatellite instability, or a mutator phenotype. The signal sequences of TGFBR1 and TGFBR1*6A are cleaved at the same site resulting in identical mature receptors. TGFBR1*6A may switch TGF-beta growth inhibitory signals into growth stimulatory signals in MCF-7 breast cancer cells and in DLD-1 colorectal cancer cells.. TGFBR1*6A is somatically acquired in 29.5% of liver metastases from colorectal cancer and may bestow cancer cells with a growth advantage in the presence of TGF-beta. The functional consequences of this conversion appear to be mediated by the TGFBR1*6A signal sequence rather than by the mature receptor. The results highlight a new facet of TGF-beta signaling in cancer and suggest that TGFBR1*6A may represent a potential therapeutic target in cancer.

Topics: Activin Receptors, Type I; Alleles; Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Genotype; Head and Neck Neoplasms; Humans; Neoplasm Metastasis; Phenotype; Polymorphism, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Sequence Deletion; Signal Transduction; Transforming Growth Factor beta

The Wnt signaling pathway is activated in most human colorectal tumors. Mutational inactivation in the tumor suppressor adenomatous polyposis coli (APC), as well as activation of beta-catenin, causes the accumulation of beta-catenin, which in turn associates with the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors and activates transcription of their target genes. Here we show that beta-catenin activates transcription of the BMP and activin membrane-bound inhibitor (BAMBI)/NMA gene. The expression level of BAMBI was found to be aberrantly elevated in most colorectal and hepatocellular carcinomas relative to the corresponding non-cancerous tissues. Expression of BAMBI in colorectal tumor cell lines was repressed by a dominant-negative mutant of TCF-4 or by an inhibitor of beta-catenin-TCF interaction, suggesting that beta-catenin is responsible for the aberrant expression of BAMBI in colorectal tumor cells. Furthermore, overexpression of BAMBI inhibited the response of tumor cells to transforming growth factor-beta signaling. These results suggest that beta-catenin interferes with transforming growth factor-beta-mediated growth arrest by inducing the expression of BAMBI, and this may contribute to colorectal and hepatocellular tumorigenesis.

Topics: Adenoviridae; Animals; beta Catenin; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Colorectal Neoplasms; COS Cells; Cytoskeletal Proteins; Dimerization; DNA Mutational Analysis; DNA-Binding Proteins; Genes, Reporter; Humans; Immunohistochemistry; Liver; Luciferases; Lymphoid Enhancer-Binding Factor 1; Membrane Proteins; Oligonucleotide Array Sequence Analysis; Plasmids; Promoter Regions, Genetic; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Smad4 is an essential signal transducer of the transforming growth factor beta (TGF-beta) signalling pathway and has been identified as a tumour suppressor, being mutated in approx. 50% of pancreatic cancers and approx. 15% of colorectal cancers. Two missense mutations in the C-terminal domain of Smad4, D351H (Asp351-->His) and D537Y (Asp537-->Tyr), have been described recently in the human colorectal cancer cell lines CACO-2 and SW948 respectively [Woodford-Richens, Rowan, Gorman, Halford, Bicknell, Wasan, Roylance, Bodmer and Tomlinson (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9719-9723]. Previous work in vitro suggested that only Asp-351 was required for interaction with Smad2 [Wu, Fairman, Penry and Shi (2001) J. Biol. Chem. 276, 20688-20694]. In the present study, we investigate the functional consequences of these point mutations in vivo. We demonstrate that neither of these colorectal cancer cells undergo growth arrest in response to TGF-beta, which can be explained, at least in part, by their inability to up-regulate cyclin-dependent kinase inhibitors p21 (CIP1 ) or p15 ( INK4b) after TGF-beta stimulation. Although the point-mutated Smad4s are expressed at normal levels in these colorectal cancer cells, they cannot interact with either TGF-beta-induced phosphorylated Smad2 or Smad3. As a result, these Smad4 mutants do not accumulate in the nucleus after TGF-beta stimulation, are not recruited to DNA by relevant Smad-binding transcription factors and cannot generate transcriptionally active DNA-bound complexes. Therefore both these colorectal tumour cells completely lack functional Smad4 activity owing to the missense mutations. Given the location of these mutations in the three-dimensional structure of the Smad4 C-terminal domain, the results also give us significant insights into Smad complex formation.

Topics: Active Transport, Cell Nucleus; ADP-Ribosylation Factor 1; Amino Acid Substitution; Cell Division; Cell Nucleus; Colorectal Neoplasms; Dimerization; DNA-Binding Proteins; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Macromolecular Substances; Models, Molecular; Mutation, Missense; Neoplasm Proteins; Nerve Growth Factors; Point Mutation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Signal Transduction; Smad Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Structure-Activity Relationship; Trans-Activators; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Xenopus Proteins

Breast cancer cells frequently metastasize to the skeleton, where they induce OCL formation and activity, resulting in extensive bone destruction. However, the mechanisms by which breast cancer cells mediate increased osteolysis remain unclear. To elucidate this point, we investigated how 3 human breast cancer cell lines, MDA-MB-231, MDA-MB-435 and MCF-7, induce OCL formation using a murine osteoblast-spleen cell coculture system and compared their effects with a human colorectal cancer cell line, HCT-15; a human lung cancer cell line, HT-1080; and a normal human breast cell line, HME. The breast cancer cell lines supported OCL formation only when osteoblasts were present in spleen cell cocultures, whilst the non-breast cancer cell lines and the normal breast cell line, HME, had no effect. Fractionation of BCCM by ultrafiltration established that osteoclastogenic activity was associated with factors having m.w. >3 kDa. Breast cancer cell lines produced primarily PTHrP, with lesser amounts of IL-6, IL-11 and TNF-alpha. The effect of BCCM on OCL formation in osteoblast-spleen cell cocultures was partially prevented by a neutralising antibody to human PTHrP and completely prevented by a neutralising antibody to either murine IL-11 or the murine IL-11 receptor; neutralising antibodies to human IL-6, IL-11 or TNF-alpha were without effect. BCCM or human PTHrP induced an increase in murine osteoblast IL-11 mRNA and protein production, effects that were prevented in the presence of a neutralising antibody to human PTHrP. The osteoclastogenic activity of IL-11 was mediated by enhancing osteoblast production of PGE(2) effects, which were abrogated by an inhibitor of cyclooxygenase. PGE(2) apparently enhanced OCL formation by downregulating GM-CSF production by spleen cells since recombinant murine GM-CSF inhibited OCL formation and a neutralising antibody to murine GM-CSF blocked these inhibitory effects. We conclude that breast cancer cells induce OCL formation by stimulating osteoblastic production of IL-11. The subsequent release of PGE(2) followed by inhibition of GM-CSF production by cells within the bone microenvironment plays an important part in mediating the effects of breast cancer cells on OCL formation and their resorptive activity.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Coculture Techniques; Colorectal Neoplasms; Dinoprostone; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-11; Interleukin-6; Lung Neoplasms; Mice; Osteoclasts; Parathyroid Hormone-Related Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional signalling molecule with a wide array of roles. Animal experiments suggest that TGFbeta1 plays a biphasic role in carcinogenesis by protecting against the early formation of benign epithelial growths, but promoting malignant transformation of those growths that do develop. A polymorphism in the signal peptide sequence of the TGFbeta1 gene (L10P) has been associated with increased levels of plasma TGFbeta1 in individuals with the P allele.. We investigated whether this polymorphism was associated with the risk of colorectal adenomatous or hyperplastic polyps in a case-control study of individuals from Minnesota. Risk of colorectal polyps was evaluated separately for individuals with adenomatous polyps (n = 513) and hyperplastic polyps (n = 191) relative to polyp-free controls (n = 606) using logistic regression analysis.. No overall association was seen between the L10P polymorphism and risk of colorectal adenomatous polyps. The age- and sex-adjusted odds ratios (OR) of developing colorectal hyperplastic polyps were 1.0 (95% CI: 0.7, 1.4) and 0.7 (95% CI: 0.4, 1.1) for individuals with the LP and PP genotypes, respectively, compared with individuals with the LL genotype. When stratified by smoking, evidence for a decreased risk of hyperplastic polyps associated with the P allele was seen only among ever smokers (P for trend = 0.05).. Whereas adenoma risk did not vary by TGFbeta1 L10P genotype, these results suggest that the L10P variant allele may have a protective role in the development of colorectal hyperplastic polyps, possibly consistent with its role as an inhibitor of epithelial growths.

Topics: Adenomatous Polyps; Adult; Age Distribution; Aged; Case-Control Studies; Colonic Polyps; Colorectal Neoplasms; Female; Genetic Predisposition to Disease; Genotype; Humans; Hyperplasia; Male; Middle Aged; Polymorphism, Genetic; Risk Factors; Smoking; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study investigates the expression of tumor growth factors TGFbeta1, TGFbeta2 and TGFbeta3 in tissue material from patients with colorectal carcinoma and evaluates their correlation with known prognostic markers and patient survival.. The study included 124 patients with colorectal carcinoma. According to the TNM classification of malignant tumors, 26 tumors were identified as being stage I, 30 stage II, 48 stage III, and 20 stage IV, whereas 106 tumors were low-grade and 18 high-grade malignancies. On paraffin sections, the streptavidin-biotin technique using antibodies against TGFbeta1, TGFbeta2 and TGFbeta3 was applied. Morphological and immunohistochemical results were correlated with clinicopathologic parameters.. TGFbeta1 protein was expressed in 88 out of 124 (71%) carcinomas, whereas TGFbeta2 and TGFbeta3 proteins were detected in all tumors examined. Normal colonic mucosal epithelial cells expressed TGFbeta2 (significantly less as compared to neoplastic cells; p < 0.01) and TGFbeta3 (p > 0.05 compared to neoplastic cells), but not TGFbeta1. Statistical analysis revealed a higher expression of TGFbeta1 in low-grade carcinomas (p = 0.009) and a higher presence of TGFbeta2 in advanced tumors (p = 0.008). TGFbeta1 expression was related with increased disease-free and overall survival (p < 0.05 each). The presence of TGFbeta2 was correlated with worse prognosis (p < 0.05). Cox analysis revealed that besides tumor grade and stage, TGFbeta1 expression constituted an independent prognostic factor.. This study shows that in adenocarcinomas of the colon, there is a differential expression of TGFbeta1, TGFbeta2 and TGF3. TGFbeta1 may be implicated in the pathogenesis of these tumors, since it is expressed only in neoplastic but not in normal cells. TGFbeta1 is related with an increased disease-free and overall survival and constitutes an independent prognostic factor. In advanced stages, TGFbeta2 seems to be involved in tumor progression and is related with worse prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Chemotherapy, Adjuvant; Colon; Colorectal Neoplasms; Combined Modality Therapy; Disease Progression; Disease-Free Survival; Female; Fluorouracil; Humans; Immunohistochemistry; Leucovorin; Male; Middle Aged; Neoplasm Staging; Postoperative Care; Prognosis; Proportional Hazards Models; Radiotherapy Dosage; Rectum; Survival Analysis; Time Factors; Transforming Growth Factor beta

NK cell function in cancer patients is severely impaired, but the mechanism underlying this impairment is not clearly understood. In this study we show evidence that TGF-beta1 secreted by tumors is responsible for the poor NK lytic activity via down-regulating an NK-activating receptor, NKG2D. The plasma level of TGF-beta1 in human lung cancer or colorectal cancer patients was elevated compared with that in normal volunteers, and this elevation was inversely correlated with surface expression of NKG2D on NK cells in these patients. Incubation of NK cells with plasma obtained from cancer patients specifically down-modulated surface NKG2D expression, whereas addition of neutralizing anti-TGF-beta1 mAbs completely restored surface NKG2D expression. Likewise, incubation of NK cells and lymphokine-activated killer cells with TGF-beta1 resulted in dramatic reduction of surface NKG2D expression associated with impaired NK cytotoxicity. Modulation of NKG2D by TGF-beta1 was specific, as expression of other NK receptors, CD94/NKG2A, CD44, CD16, 2B4, or CD56, was not affected by TGF-beta1. Impaired NK cytotoxicity by TGF-beta1 was not due to alteration of lytic moieties, such as perforin or Fas, or apoptotic pathway, but, rather, appeared to be due to lack of NKG2D expression. Taken together, our data suggest that impaired NK function in cancer patients can be attributed to down-modulation of activating receptors, such as NKG2D, via secretion of TGF-beta1.

Topics: Case-Control Studies; Cells, Cultured; Colorectal Neoplasms; Cytotoxicity, Immunologic; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Lung Neoplasms; NK Cell Lectin-Like Receptor Subfamily C; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, Natural Killer Cell; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF)-beta1 is associated with tumor progression and resistance to chemotherapy in established cancers, as well as host immune suppression. Here, we show that the serum glycoprotein alpha2-HS-glycoprotein (AHSG) blocks TGF-beta1 binding to cell surface receptors, suppresses TGF-beta signal transduction, and inhibits TGF-beta-induced epithelial-mesenchymal transition, suggesting that AHSG may play a role in tumor progression. In 66 consecutive sporadic human colorectal cancer specimens, we observed a 3-fold depletion of ASHG in tumor compared with normal tissue, whereas levels of other abundant plasma proteins, albumin and transferrin, were equivalent. Using the Multiple intestinal neoplasia/+ (Min/+) mouse model of intestinal tumorigenesis, we found twice as many intestinal polyps overall, twice as many large polyps (>3 mm diameter), and more progression to invasive adenocarcinoma in Min/+ Ahsg-/- mice than in littermates expressing Ahsg. Phosphorylated Smad2 was more abundant in the intestinal mucosa and tumors of Min/+ mice lacking Ahsg, demonstrating increased TGF-beta signaling in vivo. Furthermore, TGF-beta-mediated suppression of immune cell function was exaggerated in Ahsg-/- animals, as shown by inhibition of macrophage activation and reduction in 12-O-tetradecanoylphorbol 13-acetate-induced cutaneous inflammation. Reconstitution of Ahsg-/- mice with bovine Ahsg suppressed endogenous TGF-beta-dependent signaling to wild-type levels, suggesting that therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-beta.

Topics: Adult; Aged; Aged, 80 and over; alpha-2-HS-Glycoprotein; Animals; Binding, Competitive; Blood Proteins; Cattle; Colorectal Neoplasms; Female; Humans; Macrophage Activation; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

The aim of the study was to determine whether the expression of sulphotransferase enzymes could be affected by the presence of cytokines or peptide hormones. The effects of cytokines (TNF-alpha and TGF-beta) and insulin on sulphotransferase (SULT 1A1 and 1A3) activity were studied in a human neuronal cell line (SK-N-SH) and a human gastrointestinal tract cell line (HT-29). Cells were cultured with varying concentrations of TNF-alpha, TGF-beta or insulin for 24 h; the SULT 1A1 isoform in the 2 cell lines showed different optimal substrate concentrations. There were no direct effects of cytokines on enzyme activity. Culture with TNF-alpha increased activity of both SULT 1A1 and 1A3 in the HT-29 cells; TGF-beta also increased activities of both isoforms but to a lesser extent; insulin increased activity of SULT 1A1 only. The cytokines and insulin had relatively little effect on sulphotransferase activity in the neuronal cell line. These results suggest that, unlike neuronal cells, gastrointestinal cells may respond to physiological states by altering sulphotransferase activity. As certain substrates such as diet-derived heterocyclic amines are bioactivated by sulphation to produce carcinogenic metabolites this may be a factor in the increased incidence of colorectal cancer in patients with inflammatory bowel disease or diabetes.

Topics: Arylsulfotransferase; Cell Culture Techniques; Colon; Colorectal Neoplasms; Diabetes Mellitus; Diet; Humans; Hypoglycemic Agents; Inflammatory Bowel Diseases; Insulin; Neurons; Protein Isoforms; Sulfotransferases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Systemic and local immunological responses were studied in patients with or without preoperative administration of chemotherapeutic and/or immunotherapeutic drugs for colorectal cancer. The plasma TGFbeta and other cytokines such as IL-2, IL-4, IL-6, IL-10, IL-12, IFN-gamma in the supernatant fluid of culture of peripheral blood mononuclear cell (PBMC) and regional lymph node were measured by the ELISA method. A systemic response of cytokines was as follows: the production of plasma TGFbeta increased in many cases by chemotherapeutic drugs with a significant elevation of the mean production. Productions of IFN-gamma, IL-2, IL-12 in the supernatant fluid of culture of PBMC increased in many cases by immunotherapeutic drugs, and that of IL-4, IL-6 increased in many cases by chemotherapeutic drugs. A local response of cytokines was as follows: the production of IL-2 by immunotherapeutic drugs was greater than that without immunotherapeutic drugs whereas the production of IL-10 by immunotherapeutic drugs was smaller than that without immunotherapeutic drugs.

Topics: Adjuvants, Immunologic; Antineoplastic Combined Chemotherapy Protocols; Colorectal Neoplasms; Cytokines; Enzyme-Linked Immunosorbent Assay; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-2; Interleukin-4; Interleukin-6; Proteoglycans; Tegafur; Transforming Growth Factor beta; Uracil

Mutations of the Smad4 gene, a member of a group of TGF-beta signal transduction components, occur in several types of cancer suggesting that its inactivation significantly affects TGF-beta responsiveness in these tumors. To further investigate the role of Smad4 with respect to TGF-beta signaling and carcinogenesis, we re-expressed the Smad4 gene in the Smad4-deficient cancer cell line FaDu by microcell-mediated chromosome transfer (MMCT) and retroviral infection to closely approximate physiological protein levels. The Smad4-expressing FaDu clones were then evaluated for TGF-beta responsiveness to assess the role of Smad4 in TGF-beta-induced growth inhibition and target gene regulation. We found that the re-expression of the Smad4 gene by either method partially restored TGF-beta responsiveness in FaDu cells with respect to both growth inhibition and expression of p21WAF1/CIP1 and p15INK4B. However, only the microcell hybrids showed growth retardation in organotypic raft culture and an enhanced ability to upregulate fibronectin. In contrast, the re-expression of Smad4 by either method failed to suppress tumorigenicity. These results suggest that in addition to a homozygous deletion of Smad4, FaDu cells contain additional defects within the TGF-beta signaling pathway, thereby limiting the extent of TGF-beta responsiveness upon Smad4 re-expression and perhaps accounting for the inability to induce p15INK4B to a high level. They also demonstrate the advantages of providing a physiological extracellular environment, when assessing TGFbeta responsiveness.

Topics: Carcinogenicity Tests; Cell Cycle Proteins; Cell Division; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Fibronectins; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Hybrid Cells; Organ Culture Techniques; Phosphorylation; Retroviridae; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

Serrated adenomas, hyperplastic polyps, and admixed hyperplastic/adenomatous polyps form a distinct group of colorectal tumors, the molecular genetic basis of which is still poorly understood. We describe a novel mouse model for serrated adenomas and mixed polyposis, here referred to as Sad (serrated adenomas), caused by a spontaneously risen splice site mutation in the murine Smad4 gene. The Sad chromosomal region was identified by genetic linkage and loss of heterozygosity (LOH) analysis. Subsequently, several candidate genes were investigated by expression and mutation analysis. By use of genetic linkage and LOH analysis, we mapped the Sad candidate to mouse chromosome 18, 44-48 cM, syntenic to human chromosome band 18q21. Within this chromosomal interval, the Smad2, Smad4, and Smad7 genes were analyzed for the presence of a disease-causing mutation in affected animals. A single nucleotide (nt) deletion was identified in the intron 5/exon 6 splice acceptor site of the Smad4 gene. The single base deletion results in a frameshift and an early termination codon through activation of a cryptic splice site 4 nt downstream in exon 6. The resulting mRNA is unstable, and the Sad mutation is thus likely to represent a null allele. Identification of a Smad4 mutation in the Sad mouse model provides further support for the involvement of the Smad genes, and thus the TGFB pathway, in the serrated/hyperplastic route to colorectal cancer.

Topics: Adenomatous Polyps; Animals; Cell Line; Colonic Polyps; Colorectal Neoplasms; Disease Models, Animal; DNA-Binding Proteins; Female; Fetal Death; Gene Expression Profiling; Genes, Lethal; Homozygote; Hyperplasia; Loss of Heterozygosity; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; RNA Splice Sites; Sequence Deletion; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

The production of abundant connective tissue within malignant tumors, the so-called desmoplastic stromal reaction, is a hallmark of colorectal adenocarcinomas. This stroma is produced to a large extent by myofibroblasts and contains various amounts of collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. In this study we have established a monolayer coculture model between two different colorectal adenocarcinoma cell lines (HRT-18, and CX-2) and colonic fibroblasts (CCD-18) to investigate the mechanisms regulating (i) the production of extracellular matrix (ECM) components, (ii) the induction of myofibroblastic differentiation, and (iii) cellular proliferation. We found that TGFbeta1 and FGF-2 stimulated ECM synthesis of fibroblasts. Myofibroblastic differentiation was stimulated by TGFbeta1 but suppressed by FGF-2. There was a mutual stimulation of proliferation between fibroblasts and carcinoma cells. The analogies with ECM components expressed in cocultures and colorectal adenocarcinoma samples suggest that the coculture model used in this study is useful to study tumor cell-fibroblast interactions.

Topics: Adenocarcinoma; Cell Communication; Cell Culture Techniques; Cell Differentiation; Cell Division; Coculture Techniques; Colorectal Neoplasms; Connective Tissue; Extracellular Matrix Proteins; Fibroblast Growth Factor 2; Fibroblasts; Humans; Inflammation Mediators; Microscopy, Electron; Models, Biological; Stromal Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Topics: Activin Receptors, Type I; Adenoma; Adolescent; Adult; Aged; Bone Morphogenetic Protein Receptors, Type I; Colorectal Neoplasms; DNA Glycosylases; DNA Mutational Analysis; DNA-Binding Proteins; DNA, Neoplasm; Female; Germ-Line Mutation; Humans; Loss of Heterozygosity; Male; Middle Aged; N-Glycosyl Hydrolases; Neoplasms, Multiple Primary; Polymorphism, Single-Stranded Conformational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Growth Factor; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up-regulated by transforming growth factor beta (TGF-beta) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-beta, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression.

Topics: ADAM Proteins; ADAM12 Protein; Animals; Cell Division; Colorectal Neoplasms; COS Cells; Disintegrins; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Liver Cirrhosis; Liver Neoplasms; MAP Kinase Kinase Kinase 1; Matrix Metalloproteinase 2; Membrane Proteins; Metalloendopeptidases; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Transforming Growth Factor beta

Mechanism(s) for generation of the high tumor interstitial fluid pressure (TIFP) that is characteristic of carcinoma is not known. We investigated the role of hyaluronan, the major water-binding polysaccharide of the extracellular matrix, for the generation of a high TIFP. A human anaplastic thyroid carcinoma (KAT-4) xenografted to athymic mice and a syngeneic rat colon carcinoma (PROb) were used. Neither KAT-4 nor PROb cells produced hyaluronan (HA) in culture, however, both cell lines produced factors that stimulated HA-synthesis by cultured fibroblasts. Modulating hyaluronan levels by transfection of PROb carcinoma cells with hyaluronan synthase-2 revealed no correlation between hyaluronan content and TIFP. Furthermore, lowering of TIFP by treating KAT-4 tumors with a specific inhibitor of TGF-beta 1 and -beta 3 did not change the concentration of hyaluronan in the tumors. In summary, our results suggest that a modulation of hyaluronan content is not a major pathogenetic mechanism for the generation of the characteristically high TIFP in malignant carcinomas.

Topics: Animals; Carcinoma; Cell Line; Colorectal Neoplasms; Extracellular Space; Female; Fibroblasts; Humans; Hyaluronic Acid; Mice; Mice, Inbred C57BL; Mice, Nude; Pressure; Rats; Thyroid Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the tumor growth factor beta (TGF-beta) superfamily. Several observations suggest that it plays a role in colorectal carcinoma (CRC). In particular, MIC-1 is markedly up-regulated in colorectal cancers as well as in premalignant adenomas. This study examines the relationship of serum MIC-1 levels and genotypes to clinical and pathologic features of colonic neoplasia.. We confirmed the presence of MIC-1 in CRC tissue and the cell line CaCo-2. The normal range for serum MIC-1 levels was defined in 260 healthy blood donors, and the differences between normal subjects and 193 patients having adenomatous polyps or CRC were then determined. In a separate cohort of 224 patients, we evaluated the relationship of MIC-1 serum level and genotype to standard tumor parameters and outcome measures.. MIC-1 was expressed in CRC tissue and the cancer cell line CaCo-2. There was a progressive increase in serum MIC-1 levels between normal individuals [mean (M) = 495 pg/ml, SD = 210), those with adenomatous polyps (M = 681 pg/ml, SD = 410), and those with CRC (M = 783 pg/ml, SD = 491)]. Serum MIC-1 level was correlated with the extent of disease so that the levels were higher in patients with higher Tumor-Node-Metastasis stage. There were significant differences in time to relapse and overall survival between subjects with different MIC-1 levels and genotypes.. This study identifies a strong association between MIC-1 serum levels and neoplastic progression within the large bowel. We suggest that the measurement of serum MIC-1 levels and determination of MIC-1 genotype may have clinical use in the management of patients with CRC.

Topics: Adenoma; Adenomatous Polyps; Alleles; Carcinoma; Cell Line, Tumor; Cohort Studies; Colorectal Neoplasms; Cytokines; Disease-Free Survival; Female; Genotype; Growth Differentiation Factor 15; Humans; Immunohistochemistry; Logistic Models; Lymphocytes; Male; Neoplasm Metastasis; Prognosis; Time Factors; Transforming Growth Factor beta; Treatment Outcome; Up-Regulation

Cancer-related cytokines may interfere with the differentiation and migration of dendritic cells (DCs) and with the associated up-regulation of co-stimulatory molecules in vitro. We determined whether cytokines affected the distribution and activation of DCs in patients with colorectal cancer by measuring the levels of serum cytokines [transforming growth factor (TGF)-beta1 and vascular endothelial growth factor (VEGF)], DC numbers and phenotype from peripheral blood and mesenteric lymph nodes draining the cancer, and the infiltration of DCs into colorectal cancer. A significant increase in the serum level of TGF-beta1 correlated with a significant reduction in the level of circulating DCs in cancer patients that was associated with an increased infiltration of Langerhans cells into colorectal mucosa. The prevalence but not intensity of co-stimulatory molecule expression in circulating and mesenteric lymph node DCs was reduced in patients with colorectal cancer compared to patients with inflammatory bowel conditions. There was no correlation between co-stimulatory molecule expression and serum TGF-beta1. Thus the circulating DC depletion in colorectal cancer could be explained by a TGF-beta1-related DC redistribution from the circulation into the colorectal cancer and adjacent mucosa where DC levels were increased. There was an impairment of DC activation within colorectal cancer that was not related to serum level of cytokines.

Topics: Adult; Aged; Antigens, CD; Cell Movement; Colon; Colorectal Neoplasms; Dendritic Cells; Female; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Langerhans Cells; Leukocyte Count; Lymphatic Metastasis; Male; Mesentery; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

To investigate the role of TGFbeta1 in invasion and metastasis in colorectal cancer by analysing TGFbeta1 correlated with depth of tumor invasion, stage and metastasis.. Serum TGFbeta1 levels were determined in 50 patients with colorectal cancer and 30 healthy volunteers using a TGFbeta1 enzyme-linked immunosorbent assay. TGFbeta1 expression in primary and lymph node metastatic lesions were detected in 98 cases of colorectal cancer by immunohistochemical staining and in situ hybridization.. Serum levels of TGFbeta1 in patients with colorectal cancer(40+/-18 microg.L(-1)) were significantly higher than those in the healthy control group(19+/-8 microg.L(-1)), P<0.05. Elevated levels of serum TGFbeta1 were found in 60 % of patients with colorectal cancer when the mean +2 s was used as the upper limit of the normal range (35.1 microg.L(-1)). Increases in serum TGFbeta1 levels were significantly associated with Duke's stage (P<0.05), but there was no significant difference between Duke's stage B patients and Duke's stage C patients. In the cytoplasm of cancer cells, TGFbeta1 was immunostained in 37.8 % (37/98) of colorectal cancer, and this expression was confirmed by in situ hybridization. Among 35 cases of colorectal cancer with lymph node metastatic lesions, TGFbeta1 positive staining was found in 18 (51.4 %) cases of primary tumor, and 25 (71.4 %) cases with lymph node metastatic lesions, respectively. Of 17 cases w ith no staining in the primary lesion, 7 (41.2%) casesshowed TGFbeta1 staining in the metastatic lesion. Serum TGFbeta1 levels and TGFbeta1 expression in colorectal cancer tissues were correlated significantly with depth of tumor invasion, stage and metastasis. Patients in stage C-D,T3-T4 and with metastasis had significantly higher TGFbeta1 levels than patients in stage A-B,T1-T2 and without metastasis (P<0.05).. These results suggest that transforming growth factor-beta1 is closely related to the invasion and metastasis of colorectal cancer. It increased the invasive and metastatic potential of tumor by altering a tumor microenvironment. TGFbeta1 may be used as a possible biomarker.

Topics: Adult; Aged; Case-Control Studies; Colorectal Neoplasms; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cardiomyopathies; Carrier Proteins; Colorectal Neoplasms; Elastic Tissue; Extracellular Matrix; Gene Expression Regulation, Developmental; Gene Targeting; Humans; Introns; Latent TGF-beta Binding Proteins; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phenotype; Pulmonary Emphysema; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.

Topics: Animals; CD4 Antigens; Coculture Techniques; Colorectal Neoplasms; HLA-A1 Antigen; Humans; Lymphocyte Activation; Mice; Mice, Nude; Mice, SCID; Receptors, Interleukin-2; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Cells, Cultured

The gene for the transducer of transforming growth factor-beta/bone morphogenetic protein signalling SMAD4, a potential suppressor of colorectal carcinogenesis, is located at the chromosomal region 18q21. In order to evaluate the clinical relevance of SMAD4 deletion, gene copy alterations were determined by copy dosage using real-time quantitative PCR in 202 colorectal tumour biopsies from a previous randomised study of adjuvant chemotherapy. Patients with normal SMAD4 diploidy turned out to have a three-fold higher benefit of 5-fluorouracil-based adjuvant chemotherapy with a border line significance (overall survival: 3.23, P=0.056; disease-free survival: 2.89, P=0.045). These data are consistent with the previous observation that patients whose cancer had retention of the 18q21 region had a significantly higher benefit from 5-fluorouracil-based therapy. Moreover, these results may provide a refinement at the gene level of the clinical relevance of 18q21 deletion, thereby suggesting SMAD4 as a predictive marker in colorectal cancer. This data also indicate that integrity of this component of the transforming growth factor-beta/bone morphogenetic protein signalling pathway may be a critical factor for benefit of chemotherapy in patients with colorectal cancer.

Topics: Antimetabolites, Antineoplastic; Biomarkers; Chemotherapy, Adjuvant; Chromosome Deletion; Chromosomes, Human, Pair 18; Colorectal Neoplasms; DNA Primers; DNA-Binding Proteins; DNA, Neoplasm; Female; Fluorouracil; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Polymerase Chain Reaction; Predictive Value of Tests; Prognosis; Randomized Controlled Trials as Topic; Signal Transduction; Smad4 Protein; Survival Rate; Trans-Activators; Transforming Growth Factor beta

The expression of CD97, a member of the EGF-TM7 family with adhesive properties, is proportional to the aggressiveness and lymph node involvement in thyroid tumors. CD97 has never been systematically investigated in other tumors. First, we examined colorectal carcinoma cell lines (n = 18) for CD97 expression and regulation. All cell lines were CD97-positive. The level of CD97 in each line correlated with migration and invasion in vitro. This result was confirmed in CD97-inducible Tet-off HT1080 cells. Transforming growth factor-beta, which inhibits proliferation in transforming growth factor-beta-sensitive LS513 and LS1034 cells, down-regulated CD97 in these cell lines. Examining CD97 during sodium butyrate-induced cell differentiation of Caco-2 cells, we could demonstrate a CD97-decreasing effect. Second, we screened 81 colorectal adenocarcinomas by immunohistology for expression of CD97. Normal colorectal epithelium is CD97-negative. Seventy-five of 81 of the carcinomas expressed CD97. The strongest staining for CD97 occurred in scattered tumor cells at the invasion front compared to cells located within solid tumor formations of the same tumor. Carcinomas with more strongly CD97-stained scattered tumor cells showed a poorer clinical stage as well as increased lymph vessel invasion compared to cases with uniform CD97 staining. In summary, CD97 expression correlates with dedifferentiation, migration, and invasion in colorectal tumor cell lines. Moreover, more strongly CD97-stained tumor cells at the invasion front of colorectal carcinomas indicate the involvement of the molecule in tumor migration and invasion.

Topics: Androstadienes; Antigens, CD; Butyrates; Caco-2 Cells; Carcinoma; Cell Differentiation; Cell Movement; Colorectal Neoplasms; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Humans; Intestine, Large; Kinetics; Membrane Glycoproteins; Phosphoinositide-3 Kinase Inhibitors; Receptors, G-Protein-Coupled; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured; Wortmannin

Transforming growth factor beta1 (TGF-beta1) is thought to be involved in cancer growth and progression. TGF-beta1 changes to its active form after being secreted in its latent form. Our aim was to clarify the significance of plasma concentrations of active and total TGF-beta1 of patients with colorectal cancer. Plasma concentrations of active and total TGF-beta1 in 45 patients with colorectal cancer and 23 healthy volunteers were measured using ELISA and the activation rate (ratio of active to total TGF-beta1) was determined. Plasma concentrations of active TGF-beta1 (21.9 +/- 12.8 pg/ml) were significantly higher in patients with colorectal cancer than in healthy volunteers (9.9 +/- 5.9 pg/ml; p < 0.001, Welch's t-test). Concentration of total TGF-beta1 was also significantly higher for patients with colorectal cancer (18.0 +/- 13.0 ng/ml vs. 11.1 +/- 6.4 ng/ml; p < 0.01, Welch's t-test). However, there was no significant difference in the TGF-beta1 activation rate between the 2 groups. There was a correlation between Dukes' stage and plasma concentration of active or total TGF-beta1 (p < 0.01, Spearman's rank correlation test) and on day 7 the active TGF-beta1 levels for patients recovering from curative resection were similar to those of the control group of healthy volunteers. These results suggest that active TGF-beta1 might be used as a tumor marker for colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Inflammation; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Peptide Fragments; Prospective Studies; Protein Precursors; Transforming Growth Factor beta; Transforming Growth Factor beta1

To identify genes that mediate transforming growth factor-beta (TGF-beta) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-beta. Of the several genes identified in this screen, DEC1 was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the DEC1 locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF-beta in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-beta.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Colorectal Neoplasms; DNA-Binding Proteins; Gene Targeting; Homeodomain Proteins; Humans; Mice; Mice, Nude; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Repressor Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor TGF beta1 is involved in a variety of important cellular functions,including cell growth and differentiation, angiogenesis, immune function and extracellular matrix formation. However, the role of TGF beta(1) as an angiogenic factor in colorectal cancer is still unclear. We investigate the relationship between transforming growth factor beta(1) and angiogenesis by analyzing the expression of transforming growth factor TGF beta(1) in colorectal cancer, as well as its association with VEGF and MVD.. The expression of TGF beta(1),VEGF, as well as MVD were detected in 98 colorectal cancer by immunohistochemical staining. The relationship between the TGF beta(1) expression and VEGF expression,MVD was evaluated. To evaluate the effect of TGF beta(1) on the angiogenesis of colorectal cancers.. Among 98 cases of colorectal cancer,37 were positive for TGF beta(1) 37.8% 36 for VEGF 36.7% respectively. The microvessel counts ranged from 19 to 139.8, with a mean of 48.7(standard deviation,21.8). The expression of TGF beta(1) was correlated significantly with the depth of invasion, stage of disease, lymph node metastasis, VEGF expression and MVD. Patients in T3-T4, stage III-IV and with lymph node metastasis had much higher expression of TGF beta(1) than patients in T1-T2, stage I-II and without lymph node metastasis (P<0.05). The positive expression rate of VEGF(58.3%) in the TGF-beta(1) positive group is higher than that in the TGF-beta(1) negative group(41.7%, P<0.05). Also, the microvessel count (54+/-18) in TGF-beta(1) positive group is significantly higher than that in TGF-beta(1) negative group(46+/-15, P<0.05). The microvessel count in tumors with both TGF-beta(1) and VEGF positive were the highest (58+/-20 36-140, P<0.05). Whereas that in tumors with both TGF-beta(1) and VEGF negative were the lowest (38+/-16, 19-60, P<0.05).. TGF beta(1) might be associated with tumor progression by modulating the angiogenesis in colorectal cancer and TGF beta(1) may be used as a possible biomarker.

Topics: Adult; Aged; Colorectal Neoplasms; Endothelial Growth Factors; Female; Humans; Lymphokines; Male; Microcirculation; Middle Aged; Neovascularization, Pathologic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The antitumorigenic activity of nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase (COX) inhibitors, is well established, but responsible molecular mechanisms are not fully understood. NSAIDs stimulate apoptosis by COX dependent and independent mechanisms in colorectal cells in culture. Identification of genes regulated by COX inhibitors could lead to a better understanding of their proapoptotic and anti-neoplastic activities. Using subtractive hybridization, a cDNA which was designated as NSAID activated gene (NAG-1) was identified from NSAID-treated HCT-116, human colorectal cells. NAG-1 has an identical sequence with a novel member of the TGF-beta superfamily that has 5 different names. In the HCT-116 cells, NAG-1 expression is increased and apoptosis is induced by treatment with some NSAIDs in a concentration and time-dependent manner. NAG-1 transfected cells exhibited increased basal apoptosis, increased response to NSAIDs and reduced soft agar cloning efficiency. Furthermore, transplantable tumors derived from NAG-1 transfected HCT-116 cells showed reduced tumorigenicity in athymic nude mice compared with vector-transfected HCT-116 cells. The increased NAG-1 expression by NSAIDs provides a suitable explanation for COX-independent apoptotic effects of NSAIDs in cultured cells. These data demonstrate that NAG-1 is an antitumorigenic and proapoptotic protein, and its regulation by COX inhibitors may provide new clues for explaining their proapoptotic and antitumorigenic activities.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Cell Division; Colorectal Neoplasms; Cyclooxygenase Inhibitors; Cytokines; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 15; Humans; Male; Mice; Mice, Nude; Molecular Sequence Data; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Interleukin-10-deficient mice develop colitis and colorectal cancer similar to the inflammatory bowel disease associated cancer in humans. The aim of this study was to identify possible mutations of oncogenes and tumour suppressor genes involved in tumorigenesis in Interleukin-10 (IL-10)-deficient mice. Twenty colon carcinomas from IL-10-deficient mice were screened for mutations in the K-ras and p53 genes by 'cold' single-strand-conformation polymorphism. Immunohistochemical staining was performed to detect mutations in the proteins P53, APC and MSH2, and the transforming growth factor beta type II receptor. Microsatellite instability was analysed at eight chromosomal loci and plasma levels of transforming growth factor beta1 (TGF-beta1) were also measured. At 9 weeks, 14% of the animals developed colorectal cancer, and at 10-31 weeks the incidence of carcinoma was 65%. No mutations were detected in the analysed oncogene and tumour suppressor genes. Plasma TGF-beta1 levels in IL-10-deficient mice 10-31 weeks old were higher than in wild-type littermates e.g. 45.7 +/- 4.6 ng/ml versus 19.8 +/- 4.5 ng/ml (P<0.01). No alterations in K-ras, p53, APC: and Msh2 genes suggests that other genes are involved in the development of these tumours. Elevated TGF-beta1 plasma levels correspond to the high incidence of dysplasia and cancer. Normal expression of the TGF-beta II receptors hints at genetic alterations in other members of the TGF-beta receptor signal transduction pathway.

Topics: Adenomatous Polyposis Coli Protein; Animals; Carcinoma; Colonic Neoplasms; Colorectal Neoplasms; Cytoskeletal Proteins; DNA-Binding Proteins; Genes, p53; Genes, ras; Humans; Immunohistochemistry; Inflammatory Bowel Diseases; Interleukin-10; Intestine, Large; Intestine, Small; Mice; Mice, Inbred C57BL; Microsatellite Repeats; Mutation; MutS Homolog 2 Protein; Neoplasms; Polymorphism, Single-Stranded Conformational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Analysis, DNA; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

The transforming growth-beta receptor type II (TGF-beta RII) gene is one of the target genes of the DNA mismatch repair (MMR) defect. The human colorectal carcinoma cell line HCT116 has mutations in the hMLH1 gene and in the microsatellite region of the TGF-beta RII gene, both located on the short arm of chromosome 3. Introduction of the wild-type hMLH1 gene on transferred human chromosome 3 restores many characteristics of MMR-deficiency in HCT116. In this study, we determined whether transfer of chromosome 3 into HCT116 also complements the TGF-beta RII gene defect. We compared in vitro growth characteristics between HCT116 and HCT116 with a transferred chromosome 3 (HCT116 + ch3). The growth was suppressed in HCT116 + ch3 compared with parental HCT116. This suppression was abolished by frequent replacement with fresh medium, suggesting that the autocrine TGF-beta-TGF-beta RII system may be responsible for growth suppression. To explore this possibility, we determined several characteristics essential for the autocrine system. We found that HCT116 + ch3 expresses wild-type as well as mutated TGF-beta RII mRNA. In addition, phosphorylation of TGF-beta RI and growth inhibition were observed in HCT116 + ch3 but not in HCT116 by exposure to exogenous TGF-beta. The amount of TGF-beta1 in HCT116 + ch3 cultures was remarkably less than that in the HCT116, suggesting that TGF-beta produced by HCT116 + ch3 cells may be consumed by the cells. The conditioned medium from HCT116 cultures inhibits HCT116 + ch3 growth. This inhibition was neutralized by the anti-TGF-beta antibody. Taken together, these results strongly suggest that the TGF-beta RII gene defect in HCT116 is complemented by a wild-type gene on the transferred chromosome 3 and that HCT116 + ch3 gained the ability to respond to TGF-beta. Simultaneous complementation of defects of a responsible gene and a major target gene by the chromosome transfer is useful to prove the inactivated phenotypes acquired during colorectal tumorigenesis.

Topics: Activin Receptors, Type I; Antibodies; Autocrine Communication; Cell Division; Chromosomes, Human, Pair 3; Colorectal Neoplasms; Culture Media, Conditioned; Gene Expression; Gene Transfer Techniques; Humans; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Plasma transforming growth factor beta1 (TGF-beta1) has been reported to be correlated with the extent of disease in colorectal cancer, but it is not known whether measuring this cytokine can help predict liver metastasis after curative resection. We prospectively studied whether plasma TGF-beta1 levels could predict liver metastasis in 117 patients with colorectal cancer before and after curative resection. Blood samples were drawn before and 2 weeks after surgery to determine the cytokine levels. Abdominal ultrasonography or computed tomography was done every 3 months after surgery. The primary end point for follow-up was recurrence. Seventy-seven of 117 cases (66%) had preoperative levels of the cytokine higher than the borderline limit of 7.5 ng/ml. Postoperative levels were >7.5 ng/ml in 29 of 117 patients (25%). The median follow-up period was 42 months (range, 5--66 months), with follow-up of all 117 patients. No recurrence was observed in 13 patients with Dukes' stage A lesions. Liver metastasis occurred in 18 of 104 patients (17%) with Dukes' stage B or C disease. Fourteen of 18 patients (78%) who developed liver metastasis had shown a postoperative plasma TGF-beta1 level of >7.5 ng/ml. Cox proportional hazards regression analysis showed that the postoperative level was a significant predictive factor for liver metastasis (P < 0.001). A single point measurement of plasma TGF-beta1 levels at 2 weeks after curative resection seems to be able to predict liver metastasis in colorectal cancer. This finding suggests the value of a prospective trial of liver-targeted adjuvant therapy for patients with elevated postoperative plasma TGF-beta1 levels.

Topics: Aged; Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; Disease-Free Survival; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Postoperative Care; Preoperative Care; Prognosis; Recurrence; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF) beta1 is a growth factor with wide-ranging effects on proliferation, differentiation, immunosuppression, apoptosis and matrix remodelling. TGFbeta1 seems to have an antitumorigenic role in the gastrointestinal tract but may also be associated with the development of colorectal cancer. Initially, TGFbeta1 is produced in a latent (precursor) form in epithelial cells and then is activated by a not clearly understood multistep process. In this study, we analysed precursor TGFbeta1 protein expression (n=40) and TGFbeta1 gene expression (n=49) in human colorectal adenocarcinomas and 49 normal adjacent tissue. Out of these 49 normal tissues 40 were matched. Western blot analysis revealed that the precursor TGFbeta1 protein levels were generally lower in colorectal cancerous tissue compared to adjacent non-cancerous tissue (P<0.001). Furthermore, with real-time PCR our results cannot reflect a statistically significant difference in TGFbeta1 gene expression between the tumour tissue and normal tissue. These finds indicate that it is likely that there are mechanisms which control precursor TGFbeta1 protein expression by factor(s) at the level of pre-translation of the TGFbeta1 transcript and/or at the level of post-translation of the TGFbeta1 protein in the tumours. This process may be related to carcinogenesis and poses the question whether the suppression of the precursor TGFbeta1 is an early event, in vivo, in the human colorectal adenoma-carcinoma sequence.

Topics: Adenoma; Aged; Aged, 80 and over; Blotting, Western; Carcinoma; Colon; Colorectal Neoplasms; DNA, Complementary; Female; Humans; Immunohistochemistry; Male; Middle Aged; Polymerase Chain Reaction; Protein Biosynthesis; Protein Precursors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Deletions of chromosome band 18q21 appear with very high frequency in a variety of carcinomas, especially in colorectal cancer. Potent tumor suppressor genes located in this region encode transforming growth factor beta (TGF-beta) signal transducers SMAD2 and SMAD4, and inactivation of either one leads to impaired TGF-beta-mediated cell growth/apoptosis. Following the assignment of SMAD7 to 18q21, we first refined the SMAD7 gene position within this region by genetically mapping SMAD7 between SMAD2 and SMAD4. Further, to compare the respective frequencies of genetic alterations of these three SMAD genes in colorectal cancer, we undertook a large-scale evaluation of the copy status of each of these genes on DNA samples from colorectal tumor biopsy material. Among a subset of 233 DNA samples for which data were available for all four genes, SMAD4, SMAD2, and the nearby gene DCC showed high deletion rates (66%, 64%, and 59%, respectively), whereas SMAD7 was deleted in only 48% of the tumors. Unexpectedly, we found some gene duplications; SMAD7 appears to be more frequently amplified (10%) than the three other genes (4-7%). Compiled data for SMAD genes in each tumor show that the most common combination (26% of all the tumors) consists of the simultaneous deletions of SMAD2 and SMAD4 associated with normal diploidy or even duplication of SMAD7. Since SMAD7 normally counteracts SMAD2 and SMAD4 in TGF-beta signaling, we hypothesize that the tumor might not benefit from simultaneous SMAD7 inactivation, thereby exerting selective pressure to retain or even to duplicate the SMAD7 gene.

Topics: Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 18; Colorectal Neoplasms; DNA-Binding Proteins; Gene Dosage; Gene Order; Genes, Overlapping; Genes, Tumor Suppressor; Humans; Signal Transduction; Smad2 Protein; Smad4 Protein; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta

Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial-fibroblastoid conversion associated with loss of cell-cell adhesion receptors such as E-cadherin and beta-catenin. We report here that TGF-beta1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell-cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-beta1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-Ras(Val12) (RIE-iRas cells) were used in this study. Induced expression of activated Ha-Ras(Val12) caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of beta-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-beta1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of beta-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-beta receptor type II (TbetaRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-beta1, was strongly induced by TGF-beta1 despite the marked downregulation of TbetaRII. The induction of Ha-Ras(Val12) markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-beta1 treatment. Expression of a dominant-negative form of TbetaRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-beta1. Collectively, these results suggest that TGF-beta1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of beta-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-beta1 occurs via TbetaRII.

Topics: Adherens Junctions; beta Catenin; Cadherins; Carcinoma; Cell Line, Transformed; Colorectal Neoplasms; Cytoskeletal Proteins; Down-Regulation; Epithelial Cells; Intestinal Mucosa; Neoplasm Invasiveness; Oncogene Protein p21(ras); Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.

Topics: Adenocarcinoma; Blotting, Western; Cell Transformation, Neoplastic; Chromosome Deletion; Chromosomes, Human, Pair 18; Colorectal Neoplasms; DNA Mutational Analysis; DNA-Binding Proteins; DNA, Neoplasm; Gene Deletion; Gene Expression Profiling; Genes, DCC; Humans; Loss of Heterozygosity; Microsatellite Repeats; Mutation; Neoplasm Proteins; Ploidies; Polymorphism, Single-Stranded Conformational; Signal Transduction; Smad4 Protein; Time Factors; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

We tried to elucidate the effects of sulindac on human colorectal carcinoma.. Sulindac (300 mg/day) was administered for two weeks before operation to 33 patients with sporadic colorectal carcinoma (Sulindac Group). Resected specimens were used to detect apoptosis by terminal dUTP nick end labeling and transforming growth factor (TGF)-beta1 expression by immunohistochemistry. The results were compared with those from the historical Control Group. Twenty-nine available preoperative biopsies taken from carcinomas before sulindac prescription and 22 concurrent colorectal adenomas (9 and 13 in Sulindac and Control Groups, respectively) in the resected specimen were also examined regarding TGF-beta1 expression.. In the resected carcinomas and adenomas, more frequent apoptosis and higher TGF-beta1 scores were observed in the Sulindac Group than in the Control Group. Overexpression of TGF-beta1 and apoptosis occurred in the same region in adenomas but not in carcinomas. A positive correlation between TGF-beta1 scores and apoptotic frequency was found in adenomas (P = 0.01, rho = 0.91) but not in carcinomas (P = 0.89, rho = 0.03).. We conclude that sulindac induces apoptosis in human colorectal carcinomas as well as in adenomas. Also, one of the antineoplastic effects of sulindac might be mediated by upregulating TGF-beta1 expression, particularly in colorectal adenomas.

Topics: Adenoma; Aged; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma; Colorectal Neoplasms; Female; Humans; Male; Middle Aged; Preoperative Care; Sulindac; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Microsatellite instability (MSI) induces carcinoma through the alteration of target genes; TGF-beta RII, BAX, IGFIIR, hMSH3, and hMSH6. The grade of M

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Base Pair Mismatch; Base Sequence; bcl-2-Associated X Protein; Carcinoma; Colorectal Neoplasms; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; Female; Frameshift Mutation; Humans; Male; Microsatellite Repeats; Middle Aged; Multidrug Resistance-Associated Proteins; MutS Homolog 3 Protein; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 2; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Epidemiological, clinical, animal and laboratory studies have all provided evidence for the protective effects of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, against colorectal cancer. The main established target for NSAID action is cyclooxygenase (COX) and the inducible isoform, COX-2, is up-regulated in colorectal cancer. Rat intestinal epithelial cells transfected with a COX-2 expression vector have previously been found to be resistant to butyrate-induced apoptosis. Butyrate, a by-product of dietary fibre fermentation, is known to induce differentiation and apoptosis in colorectal tumour cells in vitro. In recent years there has been considerable interest in the possible role of dietary fibre/resistant starch in the prevention of colorectal cancer. In this study we investigated whether inhibition of COX-2 with a highly selective COX-2 inhibitor (NS-398) would sensitize human colorectal carcinoma cells to the growth inhibitory effect of butyrate. HT29 and S/KS colorectal carcinoma cell lines were treated for 72 h with 2 mM butyrate and/or 10 microM NS-398. Addition of 10 microM NS-398 alone (to inhibit COX-2 activity) did not result in detectable growth inhibition in either of the cell lines. NS-398 enhanced sensitivity to the growth inhibitory effect of butyrate in HT29 cells expressing COX-2 protein. In contrast, NS-398 did not sensitize S/KS cells lacking detectable COX-2 protein and function (as determined by prostaglandin E(2) production) to the growth inhibitory effect of butyrate. In addition, we report that butyrate treatment of carcinoma (HT29) and adenoma (PC/AA/C1) cells leads to up-regulation of COX-2 protein. Thus NS-398 only appears to sensitize human colorectal carcinoma cells expressing COX-2 protein to the growth inhibitory effect of butyrate. As COX-2 is up-regulated in colorectal carcinogenesis, this could have important implications for the selective inhibition of cells expressing COX-2 protein over those lacking COX-2 protein expression and for dietary modification to be considered alongside NSAIDs in the prevention, and possibly treatment, of colorectal cancer.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Butyrates; Colorectal Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Drug Synergism; Humans; Isoenzymes; Membrane Proteins; Nitrobenzenes; Prostaglandin-Endoperoxide Synthases; Rats; Sulfonamides; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The cell biology of intravascular tumor cells is clinically important but the many important variables of this environment have proved difficult to model. We studied the effects of repetitive mechanical deformation, a phenomenon affecting all intravascular cells, on human colon cancer cell line HCT 116 in vitro. Cell proliferation, assessed by [3H]-thymidine incorporation and cell count, increased by about 30% at two days in cells subjected to deformation at 30 cycles/min as compared to controls; levels of the nuclear proliferation antigen detected by monoclonal antibody MIB-1 were also increased. Deformation increased transforming growth factor beta1 (TGF-beta1) and plasminogen activator inhibitor-1 gene expression sevenfold at two days, but mannose-6-phosphate did not affect cell proliferation, indicating that endogenous TGF-beta is not involved in the proliferative response. HCT 116 cells lack TGF-beta type II receptors, but stable transfection of TGF-beta type II receptor cDNA did not alter the cellular response to mechanical deformation, as assessed by cell proliferation, morphology, or gene expression. Mechanical deformation affects several important aspects of HCT 116 cell biology, suggesting that the intravascular environment may regulate tumor cell biology in general. Endogenous TGF-beta and TGF-beta receptor-mediated signaling are not responsible for the deformation-induced proliferative response in HCT 116.

Topics: Cell Division; Colorectal Neoplasms; Humans; Immunohistochemistry; Plasminogen Activator Inhibitor 1; RNA, Messenger; Stress, Mechanical; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured

Butyrate, a short-chain fatty acid, modulates proliferation and differentiation of normal and neoplastic colonocytes. We examined the expression of 5-lipoxygenase (5-LO) and its metabolites in human colorectal carcinoma (Caco-2) cells, exposed to differentiation-inducing doses of butyrate. Treatment with butyrate significantly increased 5-lipoxygenase mRNA and protein in comparison to nontreated cells. Cyclooxygenases (COX)-1 and COX-2 mRNA were not significantly influenced by the treatment. However, 5-LO activity, low in nontreated cells, increased only minimally after butyrate, and its metabolic product (5-HETE) was detectable neither in control nor in treated cells. In contrast, 15-HETE (a product of 15-LO, which is also upregulated by butyrate) rose significantly. We conclude that, although being overexpressed by butyrate on mRNA and protein level, 5-LO remains inactive in differentiating Caco-2 cells. This is likely to be due either to some associated actions of butyrate, or to 5-LO-inhibition by 15-HETE, concomitantly induced by butyrate treatment.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Butyric Acid; Caco-2 Cells; Carrier Proteins; Cell Differentiation; Cell Division; Colorectal Neoplasms; Cyclooxygenase Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Membrane Proteins; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Up-Regulation

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

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.

Topics: Apoptosis; Bioreactors; Carcinoembryonic Antigen; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Size; Colorectal Neoplasms; Culture Media; ErbB Receptors; Humans; Rotation; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Weightlessness

The type II transforming growth factor (TGF)-beta receptor gene (TGFBR2) is often mutated in nucleotide repeat sequences in colorectal cancers that are replication error positive (RER+). These mutations are thought to be selected for escape from growth inhibition by TGF-beta rather than representing bystander events because of an increased mutation rate. We investigated the role of TGFBR2 mutations in 12 colorectal cancer cell lines. Six of these were RER+, and these were shown to have homozygous TGFBR2 mutations. All cell lines then were tested for changes in proliferation in response to TGF-beta stimulation. Despite homozygous mutation of the type II TGF-beta receptor, two RER+ cell lines, Lovo and SW48, showed statistically significant growth inhibition when stimulated by TGF-beta1 in serum-free conditions. This shows that the type II TGF-beta receptor can be bypassed in certain cases to maintain growth inhibition. We next investigated whether there was any alternative mode through which TGFBR2 mutation may give a selective advantage, such as a change in adhesion molecule expression. All cell lines were stimulated with TGF-beta1 and adhesion molecules detected by ELISA. No consistent changes were identified between the RER+ and the RER- cell lines, although changes in E-cadherin, beta-catenin, and gamma-catenin were identified in individual cell lines. We conclude that (i) type II TGF-beta receptor activity can be bypassed and thus TGFBR2 mutations in RER+ cancers may, at least sometimes, be just "bystander" events and (ii) TGF-beta can affect adhesion molecule expression so that TGFBR2 mutation may give rise to a selective advantage through an effect other escape from growth inhibition.

Topics: Cell Adhesion; Cell Adhesion Molecules; Cell Division; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Mutation; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Disease Progression; Humans; Prognosis; Transforming Growth Factor beta

Escape from transforming growth factor-beta (TGF-beta)-induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common-mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts.

Topics: Amino Acid Sequence; Carcinoembryonic Antigen; Carcinoma; Colorectal Neoplasms; Disease Progression; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, APC; Genes, DCC; Humans; In Situ Nick-End Labeling; Molecular Sequence Data; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Smad1 Protein; Smad2 Protein; Smad3 Protein; Smad5 Protein; Smad6 Protein; Smad7 Protein; Smad8 Protein; Trans-Activators; Transforming Growth Factor beta

Many colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-beta (TGF-beta). TGF-beta also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-beta and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-beta1, type I and type II receptor mRNA and immunohistochemical staining of TGF-beta1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-beta receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-beta receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-beta1 mRNA and the protein were detected in all colorectal cancers. TGF-beta receptor mRNAs and TGF-beta1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-beta stimulation. The escape of human colon cancer from TGF-beta-mediated growth inhibition by down-regulation of TGF-beta receptors as well as the effects of TGF-beta on stroma formation and angiogenesis indicate a possible role for TGF-beta in the progression of colon cancer in an intact host.

Topics: Adenoma; Adult; Aged; Blotting, Northern; Colorectal Neoplasms; Down-Regulation; Female; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Carcinoma cells frequently invade the surrounding tissue as coherent clusters or nests of cells. We have called this type of movement "cohort migration." We have previously presented an in vitro two-dimensional cohort migration model, in which highly metastatic variant L-10 cells of human rectal adenocarcinoma cell line RCM-1 moved as coherent cell sheets when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor/scatter factor (HGF/SF). Pericellular deposition of EDA-containing fibronectin (EDA+FN) was essential for TPA-induced cohort migration. In this study, we investigated how colon-derived fibroblasts could affect the induction of cohort migration of colorectal carcinoma cells by HGF/SF, since carcinoma cell-fibroblast interactions frequently regulate biological events during cancer cell invasion. Fibroblasts co-cultured with L-10 carcinoma cells stimulated HGF/SF-induced cohort migration of L-10 cells up to 2 to 3-fold. Conditioned medium (CM) from fibroblasts that were cultured alone was not effective but CM from fibroblasts cocultured with carcinoma cells enhanced HGF/SF-induced cohort migration, and this effect in CM was found to be mediated by TGF-beta1 upregulated in co-cultured conditions. Among the motogenic growth factors examined, only TGF-beta1 synergistically stimulated HGF/SF-induced L-10 cell cohort migration, although TGF-beta1 alone did not induce cohort migration. TGF-beta1 also exhibited synergistic effect in several other human colorectal carcinoma cell lines. The synergistic stimulation of L-10 cell cohort migration by HGF/SF and TGF-beta1 was associated with increased production of motility-enhancing EDA+FN by L-10 cells, and blocking FN with a specific antibody effectively inhibited the synergistic effect.

Topics: Adenocarcinoma; Cell Movement; Colorectal Neoplasms; Drug Synergism; Fibroblasts; Fibronectins; Hepatocyte Growth Factor; Humans; Neoplasm Invasiveness; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Topics: Colorectal Neoplasms; Humans; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Smad4 (DPC4) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) beta and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-beta, as well as from the TGF-beta family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-beta unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis.

Topics: Activins; Cell Division; Colorectal Neoplasms; DNA Primers; DNA-Binding Proteins; Forkhead Transcription Factors; Gene Deletion; Genes, Tumor Suppressor; Humans; Inhibins; Nerve Growth Factors; Polymerase Chain Reaction; Recombination, Genetic; Restriction Mapping; Signal Transduction; Smad Proteins; Smad4 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Xenopus Proteins

Familial juvenile polyposis is an autosomal dominant disease characterized by a predisposition to hamartomatous polyps and gastrointestinal cancer. Here it is shown that a subset of juvenile polyposis families carry germ line mutations in the gene SMAD4 (also known as DPC4), located on chromosome 18q21.1, that encodes a critical cytoplasmic mediator in the transforming growth factor-beta signaling pathway. The mutant SMAD4 proteins are predicted to be truncated at the carboxyl-terminus and lack sequences required for normal function. These results confirm an important role for SMAD4 in the development of gastrointestinal tumors.

Topics: Cell Membrane; Cell Nucleus; Chromosome Mapping; Chromosomes, Human, Pair 18; Colorectal Neoplasms; DNA-Binding Proteins; Female; Frameshift Mutation; Gastrointestinal Neoplasms; Genes, DCC; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Germ-Line Mutation; Hamartoma Syndrome, Multiple; Humans; Intestinal Polyps; Male; Pedigree; Polymerase Chain Reaction; Sequence Deletion; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Microsatellite instability occurs in 15% of colorectal carcinomas and may be due to replication errors (RER). The pattern of instability--'severe' vs 'mild'--and the tumorigenic pathway, as reflected by the involvement of functionally important genes, may vary according to the underlying gene(s). We defined 'mild' RER as mono- or tetranucleotide repeat instability in the absence of widespread instability at dinucleotide repeats and studied 15 colorectal tumors with this phenotype for mutations in the DNA mismatch repair genes MSH2, MLH1, MSH3, and MSH6. No mutations were found, suggesting that these genes were not implicated. We then compared colorectal cancers with 'mild' RER (n = 15), and those with 'severe' RER without (n = 11) or with (n = 22) detectable mutations in MSH2 or MLH1 to assess the involvement of mononucleotide repeats contained in the coding regions of MSH3, MSH6, BAX, and TGFbeta RII. The combined mutation rates of the above mentioned loci varied significantly between the three groups of tumors, being 0%, 25% and 52%, respectively. Furthermore, the individual genes showed specific patterns of involvement; for example, among tumors with 'severe' RER, TGFbeta RII displayed uniformly high mutation rates while MSH3, MSH6, and BAX were more frequently altered in tumors that also showed MSH2 or MLH1 mutations. Our findings suggest that different subcategories exist among unstable tumors, defined by the RER pattern on the one hand and tumorigenic pathway on the other, and structural changes of MSH2 and MLH1 are likely to explain only a proportion of these cases.

Topics: Adaptor Proteins, Signal Transducing; bcl-2-Associated X Protein; Carrier Proteins; Colorectal Neoplasms; DNA Repair; DNA Replication; DNA-Binding Proteins; DNA, Neoplasm; Humans; Microsatellite Repeats; Multidrug Resistance-Associated Proteins; Mutagenesis; MutL Protein Homolog 1; MutS Homolog 2 Protein; MutS Homolog 3 Protein; Neoplasm Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta

Bcl-2 expression is confined to the base of the colonic crypt, whereas transforming growth factor beta (TGFbeta) is expressed in the upper crypt, as are the apoptotic death promoters, Bak and Bax. In colonic adenoma cells, TGFbeta induces a growth arrest. In some adenoma cell lines, this is accompanied by apoptosis and in others it is not. In this study, we used two human colonic adenoma cell lines: RG/C2, in which TGFbeta induces a G1 arrest without apoptosis, and BH/C1, in which TGFbeta induces both a G1 arrest and apoptosis. TGFbeta does not induce apoptosis in RG/C2 cells even if hydrocortisone and insulin are removed from the culture medium. In BH/C1 cells, TGFbeta induces apoptosis in the presence of insulin and hydrocortisone. Apoptosis induced by TGFbeta is preceded by a reduction in p26-Bcl-2 protein levels. There was no change in the levels of the p30 phosphorylated form of Bcl-2 or in levels of the proapoptotic proteins Bax or Bak. RG/C2 cells did not show decreased Bcl-2 levels in response to TGFbeta-induced growth inhibition. Therefore, TGFbeta regulates Bcl-2 expression in colonic adenoma cells which undergo apoptosis in response to TGFbeta, but not in those which are growth inhibited, but resistant to TGFbeta-induced apoptosis. TGFbeta may play an important role in the colonic epithelium, not only in the inhibition of cell proliferation, but also in the regulation of apoptosis.

Topics: Adenoma; Apoptosis; Colorectal Neoplasms; Humans; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Tumor Cells, Cultured

A subset of hereditary and sporadic colorectal carcinomas is defined by microsatellite instability (MSI), but the spectra of gene mutations have not been characterized extensively. Thirty-nine hereditary nonpolyposis colorectal cancer syndrome carcinomas (HNPCCa) and 57 sporadic right-sided colonic carcinomas (SRSCCa) were evaluated. Of HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa (P < 0.000001), but instability tended to be more widespread in SRSCCa (P = 0.08). Absence of nuclear hMSH2 mismatch repair gene product by immunohistochemistry was associated with germline hMSH2 mutation (P = 0.0007). The prevalence of K-ras proto-oncogene mutations was similar in HNPCCa and SRSCCa (30% (11/37) and 30% (16/54)), but no HNPCCa from patients with germline hMSH2 mutation had codon 13 mutation (P = 0.02), and two other HNPCCa had multiple K-ras mutations attributable to subclones. 18q allelic deletion and p53 gene product overexpression were inversely related to MSI (P = 0.0004 and P = 0.0001, respectively). Frameshift mutation of the transforming growth factor beta type II receptor gene was frequent in all MSI-positive cancers (85%, 46/54), but mutation of the E2F-4 transcription factor gene was more common in HNPCCa of patients with germline hMSH2 mutation than in those with germline bMLH1 mutation (100% (8/8) versus 40% (2/5), P = 0.04), and mutation of the Bax proapoptotic gene was more frequent in HNPCCa than in MSI-positive SRSCCa (55% (17/31) versus 13% (2/15), P = 0.01). The most common combination of mutations occurred in only 23% (8/35) of evaluable MSI-positive cancers. Our findings suggest that the accumulation of specific genetic alterations in MSI-positive colorectal cancers is markedly heterogeneous, because the occurrence of some mutations (eg, ras, E2F-4, and Bax genes), but not others (eg, transforming growth factor beta type II receptor gene), depends on the underlying basis of the mismatch repair deficiency. This genetic heterogeneity may contribute to the heterogeneous clinical and pathological features of MSI-positive cancers.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma, Mucinous; Adult; Aged; Aged, 80 and over; bcl-2-Associated X Protein; Carrier Proteins; Cell Cycle Proteins; Chromosomes, Human, Pair 18; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA Mutational Analysis; DNA-Binding Proteins; DNA, Neoplasm; E2F Transcription Factors; E2F4 Transcription Factor; Female; Genes, APC; Humans; Male; Microsatellite Repeats; Middle Aged; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nuclear Proteins; Point Mutation; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The insulin-like growth factor II receptor (IGFIIR) gene has been identified as a coding region target of microsatellite instability in human gastrointestinal (GI) tumors. IGFIIR normally has two growth-suppressive functions: it binds and stimulates the plasmin-mediated cleavage and activation of the latent transforming growth factor-beta1 (LTGF-beta1) complex, and it mediates the internalization and degradation of IGFII ligand, a mitogen. We used an immunohistochemical approach to determine whether IGFIIR mutation affected expression of these proteins in GI tumors. Four highly specific antibodies were used: LC(1-30), which recognizes the active form of TGF-beta1; anti-LTGF-beta1, which detects the LTGF-beta1 precursor protein; anti-IGFIIR; and anti-IGFII ligand. Twenty GI tumors either with (6 of 20) or without (14 of 20) known IGFIIR mutation were examined, along with matching normal tissues. Results were statistically significant in the following categories: (a) decreased active TGF-beta1 protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues; (b) increased LTGF-beta1 protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues; and (c) increased IGFII ligand protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues. These data suggest that in genetically unstable GI tumors, mutation of a microsatellite within the coding region of IGFIIR functionally inactivates this gene, causing both diminished growth suppression (via decreased activation of TGF-beta1) and augmented growth stimulation (via decreased degradation of the IGFII ligand).

Topics: Colorectal Neoplasms; Humans; Immunohistochemistry; Insulin-Like Growth Factor II; Membrane Glycoproteins; Microsatellite Repeats; Mutation; Receptor, IGF Type 2; Receptors, Fibroblast Growth Factor; Stomach Neoplasms; Transforming Growth Factor beta

Activation of the receptor tyrosine kinase c-kit by the kit-ligand, also known as stem cell factor (SCF), is essential to melanocyte and germ cell development and during the early stages of hematopoiesis. Deregulated expression of c-kit has been reported in malignancies affecting these lineages, i.e., myeloid leukemias, melanomas, and germ cell tumors. In addition, c-kit and SCF are coexpressed in some breast and colorectal cancer (CRC) cells, raising the question of whether c-kit serves an autocrine role in normal or malignant epithelial tissues. In this study, we demonstrate that human colorectal carcinomas, but not normal colorectal mucosa cells, coexpress SCF and c-kit in situ. Expression of c-kit was also observed in mucosa adjacent to colorectal tumor tissue. Consistent with a growth-regulatory role of SCF in CRC cells, exogenous SCF stimulated anchorage-dependent and anchorage-independent growth in four out of five CRC cell lines. Exogenous transforming growth factor (TGF)-beta 1 added at nanomolar concentrations to HT-29 CRC cells, which express the type I, II, and III TGF-beta receptors, downregulated c-kit expression to background levels and inhibited c-kit-dependent proliferation. Similarly, TGF-beta 1 inhibited SCF-dependent proliferation of three first-passage CRC cell lines. In summary, expression of the potential autocrine SCF/ c-kit axis is a tumor-associated phenomenon in colorectal cancer that can be suppressed by TGF-beta 1 in TGF-beta-responsive CRC cells.

Topics: Adult; Aged; Carcinoma; Cell Adhesion; Cell Division; Colorectal Neoplasms; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Male; Middle Aged; Proto-Oncogene Proteins c-kit; RNA, Messenger; RNA, Neoplasm; Stem Cell Factor; Transforming Growth Factor beta

Tumor samples from five patients with metastatic colorectal cancer who demonstrated tumor regressions in clinical trials of interleukin (IL)-1 beta, IL-2, and adoptive cellular therapy were analyzed for oncogene and cytokine mRNA expression. Tumors from eight nonresponding patients were also studied. Mutations of the ras protooncogene and overexpression of c-myc protooncogene were observed in both responding and nonresponding tumors. In contrast, none of the responding tumors expressed transforming growth factor (TGF)-beta 1 mRNA, whereas nonresponding tumors did. The expression of IL-1, IL-6, IL-8, IL-10, tumor necrosis factor-alpha, granulocyte macrophage-colony-stimulating factor, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, macrophage chemotactic protein, and RANTES was variable between responding and nonresponding patients. Although we cannot conclude that a pattern of oncogene and/or cytokine mRNA expression specifically characterizes sensitive colorectal cancers, these analyses-the assessment of TGF-beta 1 mRNA in particular-merit further evaluation as biomarkers prognostic of immunotherapy response.

Topics: Colorectal Neoplasms; Genes, myc; Genes, ras; Humans; Immunotherapy, Adoptive; Interleukin-1; Interleukin-2; Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Messenger; Transforming Growth Factor beta

Recent animal studies demonstrate that prothymosin alpha 1 (ProT alpha) enhances the antitumor response by stimulation of mononuclear phagocyte functions. The present study was aimed at characterizing the in vitro effects by ProT alpha on blood monocytes from human colon cancer patients. Purified peripheral blood monocytes were studied in terms of tumor cytostatic ability and cytokine production after incubation with ProT alpha or interferon (rIFN-gamma) and transforming growth factor-beta (TGF beta), used as reference substances. SW620 colon carcinoma cells were used as tumor target cells in growth inhibition experiments. The level of baseline growth inhibitory activity of unstimulated patient's monocytes was significantly lower than that of normal monocytes. The defective antitumor activity of patient monocytes was associated with a higher production of the inhibitory monokines prostaglandin E2 (PGE2) and TGF beta. The stimulation of monocytes by ProT alpha and/or rIFN-gamma elevated the average antitumor activity in all donor groups. The ProT alpha-induced increase was associated with a significantly higher monocytic secretion of IL-1 beta and TNF-alpha. Moreover, the concentrations of TGF beta and PGE2 in the culture supernatants decreased significantly, when patient's monocytes were treated with ProT alpha and/or rIFN-gamma. Additionally, ProT alpha enhanced the diminished antitumor activity of TGF beta-treated normal monocytes. These results suggest that ProT alpha selectively regulates distinct functions of blood monocytes, the effect of this cytokine varying with the parameter and donor population examined. These data provide a rational and biological endpoint for further studies with ProT alpha as an activator of mononuclear function in colon cancer.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoembryonic Antigen; Colorectal Neoplasms; Cytokines; Dinoprostone; Humans; In Vitro Techniques; Interleukin-1; Middle Aged; Monocytes; Protein Precursors; Thymosin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Contribution of transforming growth factor beta 1 (TGF-beta 1) to tumor progression has been suggested. However, little is known about the role of TGF-beta 1 in colorectal cancer. Plasma TGF-beta 1 levels and its expression were analyzed in patients with colorectal cancer.. Plasma TGF-beta 1 levels were measured in 22 patients with colorectal cancer using a TGF-beta 1 enzyme-linked immunosorbent assay. Expression of TGF-beta 1 messenger RNA and immunohistochemical distribution of the protein in colorectal cancer tissues were examined.. Plasma TGF-beta 1 levels in patients with colorectal cancer (14.8 +/- 8.4 ng/mL) were significantly higher than in normal controls (1.9 +/- 1.4; n = 22) (P < 0.001). After curative surgical resection, plasma TGF-beta 1 levels decreased in examined patients from 11.9 +/- 6.7 to 3.8 +/- 1.2 ng/mL (P < 0.01). TGF-beta 1 messenger RNA was about 2 1/2 times more abundant in colorectal cancer tissues than in control (P < 0.01). TGF-beta 1 was detected in the cytoplasm of colorectal cancer cells immunohistochemically. Both TGF-beta 1 messenger RNA expression in colorectal adenocarcinoma tissues and its plasma levels were associated with tumor stage of Dukes' classification (P < 0.05).. These results suggest that plasma TGF-beta 1 levels may reflect overexpression of the gene in colon cancer tissues and are associated with disease progression.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Blotting, Northern; Colorectal Neoplasms; Cytoplasm; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; RNA, Messenger; Transforming Growth Factor beta

We have compared the effect of interleukin-4 and transforming growth factor beta 1 on proliferation and gene expression in two colorectal carcinoma cell lines, LS513 and LS1034. Transforming growth factor beta 1 was a potent inhibitor for both cells lines and virtually abolished de novo DNA synthesis. Interleukin-4 inhibited thymidine incorporation up to 60 and 45%, respectively. While both cytokines exerted a comparable cyto-inhibitory activity they displayed differential effects on proto-oncogene expression. Transforming growth factor beta 1 markedly down-regulated c-myc in LS1034 but not in LS513 cells. In contrast, expression of c-fos was induced by interleukin-4 in LS513 but not in LS1034 cells. Interestingly, in agreement with their cyto-inhibitory activity both cytokines suppressed the expression of insulin-like growth factor II in LS1034, which is an autocrine growth factor for these cells.

Topics: Cell Division; Colorectal Neoplasms; Gene Expression; Genes, fos; Genes, myc; Humans; Insulin-Like Growth Factor II; Interleukin-4; Proto-Oncogene Mas; Proto-Oncogenes; Transforming Growth Factor beta; Tumor Cells, Cultured

Resistance to the growth inhibitory effects of TGF-beta is common in human cancers. However, the mechanism(s) by which tumour cells become resistant to TGF-beta are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-beta family members. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-beta-like ligands.

Topics: Amino Acid Sequence; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Chromosome Mapping; Chromosomes, Human, Pair 18; Colorectal Neoplasms; DNA Mutational Analysis; DNA-Binding Proteins; Genes, Tumor Suppressor; Humans; Molecular Sequence Data; Repressor Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Smad1 Protein; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

The MAD-related (MADR) family of proteins are essential components in the signaling pathways of serine/threonine kinase receptors for the transforming growth factor beta (TGFbeta) superfamily. We demonstrate that MADR2 is specifically regulated by TGFbeta and not bone morphogenetic proteins. The gene for MADR2 was found to reside on chromosome 18q21, near DPC4, another MADR protein implicated in pancreatic cancer. Mutational analysis of MADR2 in sporadic tumors identified four missense mutations in colorectal carcinomas, two of which display a loss of heterozygosity. Biochemical and functional analysis of three of these demonstrates that the mutations are inactivating. These findings suggest that MADR2 is a tumor suppressor and that mutations acquired in colorectal carcinomas may function to disrupt TGFbeta signaling.

Topics: Amino Acid Sequence; Animals; Base Sequence; Chromosomes, Human, Pair 18; Cloning, Molecular; Colorectal Neoplasms; DNA-Binding Proteins; DNA, Complementary; Genes, Tumor Suppressor; Humans; Molecular Sequence Data; Oligonucleotide Probes; Phosphoproteins; Phosphorylation; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Smad2 Protein; Structure-Activity Relationship; Trans-Activators; Transforming Growth Factor beta; Xenopus laevis; Xenopus Proteins

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine and is thought to be involved in colorectal tumorigenesis as a regulator of cell growth and differentiation. This role is mainly supported by in vitro studies while its role in vivo remains unclear. The aim of the present study was to investigate whether the TGF-beta 1 precursor (beta 1-LAP) and the latent TGF-beta 1 binding protein (LTBP) are expressed in colorectal adenomas, the presumed precursors of most of colorectal adenocarcinomas. TGF-beta 1 precursor and LTBP were examined in 35 adenomas and 10 normal colonic mucosa specimens by immunohistochemistry, using specific polyclonal antibodies. In normal colonic mucosa, beta 1-LAP was moderately expressed in epithelial crypt cells and in the stromal cells in the lamina propria. In adenomas, beta 1-LAP was localized in epithelial cells with an heterogeneous pattern and was also present in stromal cells around the adenomatous glands. LTBP was not detected in epithelial cells but was observed in stromal cells and in the extracellular matrix (ECM). beta 1-LAP expression in epithelial cells did not correlate with the grade of dysplasia, while LTBP localized in stromal cells and ECM appeared to be closely associated with areas of higher grade of dysplasia. This study is the first demonstration of both beta 1-LAP and LTBP in colorectal adenomas with different dysplasia grades. Our results suggest that TGF-beta 1 might be involved in the mechanisms controlling in vivo colorectal tumorigenesis and support a role for the stromal-associated TGF-beta 1.

Topics: Adenomatous Polyps; Carrier Proteins; Colorectal Neoplasms; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Peptide Fragments; Protein Precursors; Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Members of the transforming growth factor beta (TGF-beta family, in particular TGF-beta 1, are some of the most potent inhibitory growth factors in a variety of cell types. Resistance to TGF-beta 1-induced growth inhibition is frequently observed in colorectal carcinomas and is associated with tumour progression. Perturbations of TGF-beta 1 expression and function, therefore, may contribute to the loss of some constraints on tumour cell growth. In this study we have examined the expression of TGF-beta 1 and its precursor latency-associated peptide (LAP)-TGF-beta in human colorectal tumours using immunohistochemical techniques. In 86% of the tumours the LAP-TGF-beta complex was present in both the stromal and epithelial cells, whereas the mature TGF-beta 1 peptide was expressed in the glandular epithelium of 58.3% of these tumours. Intense staining for TGF-beta 1 was positively associated with advanced Dukes' stage. Furthermore, there was a significant correlation between the presence of TGF-beta 1 in the tumours and a shorter post-operative survival. This was most significant in a subgroup of patients who had received only a palliative operation. These results suggest that TGF-beta 1 expression may be useful as an independent prognostic indicator for a subgroup of patients who have a particularly poor prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Chi-Square Distribution; Colorectal Neoplasms; Disease Progression; Female; Humans; Immunohistochemistry; Male; Middle Aged; Peptide Fragments; Prognosis; Protein Precursors; Proteins; Survival Rate; Transforming Growth Factor beta; Transforming Growth Factor beta1

Unlike normal intestinal cells, colorectal-carcinoma cell lines are usually not responsive to transforming growth factor beta1. The cyclin-dependent kinase inhibitor p21 that is induced by X irradiation in cells expressing normal p53 can also be induced by TGF-beta1 by a p53-independent pathway. We have investigated possible interactions between ionizing radiation and TGF-beta1, using a panel of 8 human colorectal-cancer cell lines varying in p53 status and sensitivity to the cyto-inhibitory effect of TGF-beta1. Heterogeneity in the radiosensitivity of these cell lines was observed, with SF2 (surviving fraction after irradiation with 2 Gy) ranging from 0.19 to 0.82. Radioresistance (high SF2 values) was in general associated with abnormal expression of p53. An effect of TGF-beta1 treatment on radiosensitivity was observed with one cell line only (LS513), and manifested by enhancement of the cytotoxic effect of radiation. In an experiment with fractionated irradiation during continuous exposure to TGF-beta1, there was no change in the intrinsic radiosensitivity of LS513 cells, though irradiated cells treated with TGF-beta1 were more sensitive to the first radiation dose. Irradiated LS513 colorectal-cancer and Mv-1-Lu epithelial cells were significantly more sensitive to TGF-beta1 than were unirradiated controls, whereas no change was observed in the TGF-beta1 sensitivity of irradiated LS1034 cells. Radio-induced modulation of TGF-beta1 sensitivity was transitory and declined before the decline to baseline level of p21 mRNA expression. On the basis of these results, we postulate that radiation-induced sensitization to TGF-beta1 occurs in TGF-beta1-sensitive cells expressing wild-type p53.

Topics: Cell Survival; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Epithelium; Gene Expression; Genes, p53; Humans; Kinetics; Radiation-Sensitizing Agents; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Colorectal Neoplasms; DNA-Binding Proteins; Drosophila Proteins; Humans; Insect Proteins; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Gastric cancers, sporadic colorectal cancers, and ulcerative colitis (UC)-associated colorectal carcinomas and dysplasias manifest microsatellite instability (MI); however, esophageal carcinomas rarely exhibit MI. Recently, a transforming growth factor-beta 1 type II receptor (TGF-beta 1RII) mutation in a coding microsatellite was described in primary colorectal carcinomas demonstrating MI. No previous studies of TGF-beta 1RII have addressed mechanisms of inactivation other than MI in human tumors; furthermore, MI-negative tumors have not been examined for TGF-beta 1RII mutation. We evaluated 138 primary human neoplasms for mutation in the poly-A microsatellite tract of TGF-beta 1RII. Additionally, a group of esophageal tumors was evaluated for the expression of TGF-beta 1RII messenger RNA (mRNA).. First, we determined whether MI was present at other chromosomal loci in these lesions. The poly-deoxyadenine (poly-A) microsatellite tract within the TGF-beta 1RII coding region was then PCR-amplified. In a group of MI-negative esophageal tumors, RT-PCR was performed to determine the expression of TGF-beta 1RII mRNA.. Among 17 MI+ UC specimens, 3 (18%) demonstrated TGF-beta 1RII poly-A tract mutation (2 cancers and 1 dysplasia), while 2 (4%) of 44 MI-negative UC specimens (1 dysplasia and 1 tumor), and 13 (81%) of 16 MI+ sporadic colorectal cancers, contained TGF-beta 1RII poly-A mutation. No gastric or esophageal tumors contained TGF-beta 1RII mutation. Among 21 MI-negative esophageal carcinomas. 6 cases (28.5%) had TGF-beta 1RII transcripts that were low or undetectable by RT-PCR.. Mutation within the poly-A microsatellite tract of TGF-beta 1RII occurs early in a subset of UC-neoplasms and commonly in sporadic colorectal cancers, but may be rare in MI+ gastric tumors. Diminished expression of TGF-beta 1RII mRNA in esophageal tumors suggests that mechanisms of inactivation in this gene other than MI play a role in esophageal carcinogenesis.

Topics: Colitis, Ulcerative; Colorectal Neoplasms; Esophageal Neoplasms; Humans; Microsatellite Repeats; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Stomach Neoplasms; Transforming Growth Factor beta

We macroscopically classified 25 gastric and 23 colorectal advanced cancers into "contracted" and "uncontracted" types, and found immunohistochemically that integrin subunit alpha 3 was more frequently expressed in the extracellular matrix (ECM) in the former than in the latter (75%:9/12 vs. 38%: 5/13 in gastric and 86%:6/7 vs. 25%:4/16 in colorectal cancers, respectively). Integrin subunit alpha 3 was also expressed more frequently in cancers producing transforming growth factor-beta (TGF-beta), which is related to ECM deposition, integrin expression and cell mobility, than in those which did not produce TGF-beta (67%:10/15 vs. 40%:4/10 in gastric and 57%:4/7 vs. 38%:6/16 in colorectal cancers, respectively). In addition, integrin subunit alpha 3 was not expressed in 2 benign gastric ulcers combined with gastric cancer elsewhere in the stomach. On the other hand, a retrospective analysis of 107 cases of rectal cancer which recurred after a curative operation revealed that local recurrence was more frequent in "contracted" than "uncontracted" types (44%:11/25 vs. 26%:21/82). These results may suggest that the abundant interstitial fibrosis which leads to remarkable gastric or colorectal wall contraction is a result of the interaction between cancer cells and ECM, along with the expression of integrin and/or the production of TGF-beta. This fibrosis may also be closely related to the mode of gastric and colorectal cancer invasion.

Topics: Antigens, CD; Cell Adhesion Molecules; Colorectal Neoplasms; Female; Humans; Immunoenzyme Techniques; Integrin alpha3; Integrins; Male; Recurrence; Retrospective Studies; Stomach Neoplasms; Transforming Growth Factor beta

To determine whether a single mutational event in one p53 gene is sufficient to confer a significant growth advantage on a colonic epithelial cell, the 143(Ala) p53 mutation was previously expressed in the human colonic adenoma derived cell line AA/C1 (which is wild type for p53) and shown to have no effect on it's in vitro or in vivo growth characteristics. In this investigation, by expressing the 175(His), 248(Trp) or 273(His) mutations in the same AA/C1 cell line, we have shown that this failure to affect the growth of the cells was not mutant specific. We have also demonstrated, using induction of MDM2 protein and the ability of the cells to undergo a p53 dependent G1 arrest, that the 143(Ala), 175(His) or 248(Trp) transfected cells retain functional endogenous wild type p53 activity, and suggest that these p53 mutations would not have a fully dominant negative mode of action in vivo. In contrast, one of the two AA/C1 cell lines transfected with the 273(His) mutation did fail to cell cycle arrest after gamma irradiation, indicating that this mutation can act as a dominant negative. However even loss of wild type p53 function in this cell line was insufficient to directly effect the growth rate of the AA/C1 cells, suggesting that acquisition of the 273(His) mutation may contribute to malignant progression through genomic instability (by inhibiting the G1 arrest) and that other mutations are required before outgrowth of the cell population containing the p53 mutation.

Topics: Adenoma; Base Sequence; Cell Division; Colorectal Neoplasms; DNA Primers; G1 Phase; Gamma Rays; Genes, Dominant; Genes, p53; Humans; Molecular Sequence Data; Mutation; Neoplasm Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation. On the basis of the crystal structure of TGF-beta 2, we have designed and synthesized two mutant TGF-beta s, TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73). Although both of these molecules inhibited the growth of Mv1Lu mink lung epithelial cells and LS1034 colorectal cancer cells, which are affected equally by TGF-beta 1 and TGF-beta 2, TGF-beta 1 (delta 69-73) was much less potent than TGF-beta 1 or TGF-beta 1 (71 Trp) at inhibiting the growth of LS513 colorectal cancer cells which are growth-inhibited by TGF-beta 1 but not TGF-beta 2. Both TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73) increased levels of mRNAs for fibronectin and plasminogen activator inhibitor with Mv1Lu cells, whereas only TGF-beta 1 (71 Trp) and not TGF-beta 1 (delta 69-73) up-regulated the mRNA level of carcinoembryonic antigen in LS513 cells. The expression level of carcinoembryonic antigen mRNA in LS1034 cells was not altered by either wild-type or mutant TGF-beta s. Receptor labeling experiments demonstrated that TGF-beta 1 (71 Trp) bound with high affinity to the cell-surface receptors of Mv1Lu, LS1034, and LS513 cells while TGF-beta 1 (delta 69-73) bound effectively to the receptors of Mv1Lu and LS1034 cells but much less to the receptors on LS513 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Binding Sites; Blotting, Northern; Carcinoembryonic Antigen; Cell Division; Cell Line; CHO Cells; Colorectal Neoplasms; Cricetinae; Cross-Linking Reagents; Crystallography, X-Ray; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Feasibility Studies; Gene Expression Regulation, Neoplastic; Humans; Mink; Molecular Sequence Data; Mutation; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Umbilical Veins; Up-Regulation

Many tumors are surrounded by a highly fibrous stroma composed of fibroblasts and extracellular matrix. This desmoplastic response has been suggested to both inhibit and favor tumor progression. The present study deals with the effects of tumor cells on the fibroblastic reactions they cause and relates this to progression or regression of tumors. Two rat colon carcinoma cell lines, one which develops progressive tumors when injected s.c. in syngeneic animals (PROb cell line) and the other which develops regressive tumors in similar conditions (REGb cell line), were compared by the fibroblastic reaction which they cause. Comparative histological analysis of progressive and regressive tumors developed by the two cell lines showed a significant but opposite response of fibroblastic compartment. The progressive tumor nodules were observed to grow within a loose tissue, whereas the regressive tumor cells were surrounded by a fibrous capsule. Immunohistological labelings revealed the presence of alpha-smooth muscle actin-positive myofibroblasts during the tumor expansion, while these specific cells disappeared during the tumor regression. Immunostainings of transforming growth factor beta 1 showed an increasing staining of the progressive tumor cells during tumor development but a slight expression by tumor cells and stroma during the tumor regression. This growth factor was demonstrated to facilitate initial steps of the tumor progression by addition of active transforming growth factor beta 1 at the time of s.c. injection of PROb cells in syngeneic rat models. In vitro experimental analysis with the use of neutralizing antibody showed that active transforming growth factor beta produced by the progressive cells inhibited fibroblast proliferation and facilitated their differentiation into myofibroblasts. Since the number of myofibroblasts increased with time in progressive tumors, their presence may constitute a potential growth advantage for tumor growth. In contrast, our results indicated involvement of platelet-derived growth factor-like protein(s) in fibroblast proliferation under the control of regressive cells and the presence of an important sheath of alpha-smooth muscle actin-negative fibroblasts in regressive tumors may support a role for this growth factor in vivo. Thus, the ability of tumor cells to produce or induce the production of transforming growth factor beta or platelet-derived growth factor may give rise to a specific fibroblast reaction, w

Topics: Actins; Animals; Cell Differentiation; Colorectal Neoplasms; Culture Media, Conditioned; Desmin; Fibroblasts; Male; Muscle, Smooth; Platelet-Derived Growth Factor; Rats; Transforming Growth Factor beta; Vimentin

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.

Topics: Alleles; Base Sequence; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 5; Colorectal Neoplasms; DNA, Neoplasm; Genes, Tumor Suppressor; Humans; Kinetics; Molecular Sequence Data; Mutagenicity Tests; Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We analysed the effect of interleukin-1 (IL-1), IL-6 and transforming growth factor beta 1 (TGF beta 1) on the growth of a panel of eight colorectal carcinoma cell lines. IL-1 stimulated growth of two lines (LS411N and LS1034) up to 20-fold and IL-6 enhanced proliferation of LS1034 more than 5-fold. Both cytokines also augmented colony-formation of LS1034 in methylcellulose. Under both growth conditions IL-1 was the most potent stimulator. However, the addition of IL-6 to IL-1 synergistically enhanced proliferation of LS1034 in monolayer culture and additively augmented the number of colonies formed in methylcellulose. Furthermore, TGF beta 1 strongly reduced the growth rate of LS1034. Low amounts of TGF beta 1 markedly inhibited the response of LS1034 to IL-1 and totally abrogated proliferation induced by IL-6. We conclude that different cytokines can provide distinct signals for the regulation of growth of colorectal carcinoma cells.

Topics: Cell Division; Colorectal Neoplasms; Drug Synergism; Humans; Interleukin-1; Interleukin-2; Mitosis; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

We have established 3 new human colorectal cancer cell lines (LS411N, LS513, and LS1034) from clinical biopsy samples. These lines are tumorigenic and grow s.c. as adenocarcinomas in nude mouse xenografts. Specific marker chromosomes are observed in each line. Carcinoembryonic antigen is expressed at the surface of all 3 lines, but with marked quantitative differences. Indeed, less than 10% of the cells from the HT-29 line used as a reference express carcinoembryonic antigen while more than 90% of the LS1034 cells do so. LS513 and LS1034 consistently express HLA class I antigens and intercellular adhesion molecule 1 which are not detected at the surface of the LS411N cells. No expression of HLA class II antigens DR, DQ, and DP has been measured on any of the lines. All three lines grow well in 5% fetal calf serum medium without addition of exogenous growth factors. The LS1034 line has been adapted to growth in serum-free conditions and exhibits increased clonogenicity when cells are seeded in serum-free methylcellulose medium, as compared with medium containing 5% fetal calf serum. The LS513 and LS1034 lines have proved to be of particular interest since they respond to the growth-inhibitory action of TGF-beta 1 and TGF-beta 2 in both liquid and semisolid medium. Both factors were, at pM concentrations, equipotent inhibitors of LS1034 cell proliferation. In contrast, higher concentrations of TGF-beta 1 are inhibitory for proliferation of LS513 cells, whereas TGF-beta 2 has no effect on the growth of these cells in liquid assay. On this basis, using appropriate anti-TGF-beta 1 and anti-TGF-beta 1 IgY, we developed a bioassay for TGF-beta 1 and TGF-beta 2. Two of the three lines have indeed been shown to produce latent-TGF-beta 1 activity.

Topics: Adult; Antigens, Surface; Colorectal Neoplasms; Humans; Karyotyping; Male; Middle Aged; Neoplasm Transplantation; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

The growth of three non-tumorigenic human colonic adenoma cell lines, designated AA/C1, RG/C2 and RR/C1, was inhibited by low concentrations of transforming growth factor beta (TGF-beta) (0.05-0.5 ng ml-1). However, the growth of five human colon cancer cell lines under identical conditions was resistant to high concentrations of TGF-beta (2-10 ng ml-1). This is the first report of well-characterized premalignant human colonic cells showing sensitivity to TGF-beta. The TGF-beta-sensitive adenoma cell line AA/C1 was derived from a relatively large adenoma with a K-ras gene mutation and represents a relatively late-stage adenoma, indicating that loss of response to TGF-beta occurs at a relatively late stage in colorectal carcinogenesis and that the presence of a ras gene mutation does not necessarily confer resistance to TGF-beta. Of further interest, the RG/CZ cell line has a p53 mutation showing that p53 mutations do not necessarily lead to TGF-B insensitivity. Furthermore, in this paper we show that the conversion of the AA/C1 adenoma cell line to a tumorigenic phenotype [Williams et al., (1990) Cancer Res., 50, 4724] is accompanied by a reduced response to the growth-inhibitory effects of TGF-beta up to 10 ng ml-1. Reduced responsiveness to the inhibitory effects of TGF-beta may be an important event in the loss of growth control in colorectal carcinogenesis.

Topics: Adenoma; Carcinoma; Cell Line, Transformed; Cell Transformation, Neoplastic; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Resistance; Genes, ras; Humans; Mutation; Phenotype; Precancerous Conditions; Transforming Growth Factor beta; Tumor Cells, Cultured