epidermal-growth-factor and Neoplasm-Metastasis

Intravasation is a key step in cancer metastasis during which tumor cells penetrate the vessel wall and enter circulation, thereby becoming circulating tumor cells and potential metastatic seeds. Understanding the molecular mechanisms of intravasation is critically important for the development of therapeutic strategies to prevent metastasis. In this article, we review current data on the mechanisms of cancer cell intravasation into the blood and lymphatic vessels. The entry of mature thymocytes into the circulation and of dendritic cells into the regional lymph nodes is considered as example of intravasation under physiologically normal conditions. Intravasation in a pathophysiological state is illustrated by the reverse transendothelial migration of leukocytes into the bloodstream from the sites of inflammation mediated by the sphingosine 1-phosphate interaction with its receptors. Intravasation involves both invasion-dependent and independent mechanisms. In particular, mesenchymal and amoeboid cell invasion, as well as neoangiogenesis and vascular remodeling, are discussed to play a significant role in the entry of tumor cells to the circulation. Special attention is given to the contribution of macrophages to the intravasation via the CSF1/EGF (colony stimulating factor 1/epidermal growth factor) paracrine signaling pathway and the TMEM (tumor microenvironment of metastasis)-mediated mechanisms. Other mechanisms including intravasation of tumor cell clusters surrounded by the vessel wall elements, cooperative intravasation (entry of non-invasive tumor cells to the circulation following invasive tumor cells), and intravasation associated with the vasculogenic mimicry (formation of vascular channels by tumor cells) are also discussed. Novel intravasation-specific mechanisms that have not yet been described in the literature are suggested. The importance of targeted therapeutic strategies to prevent cancer intravasation is emphasized.

Topics: Capillary Permeability; Epidermal Growth Factor; Humans; Macrophage Colony-Stimulating Factor; Macrophages; Neoplasm Invasiveness; Neoplasm Metastasis; Paracrine Communication; Transendothelial and Transepithelial Migration; Tumor Microenvironment; Vascular Remodeling

Within tumor microenvironment, a lot of growth factors such as hepatocyte growth factor and epidermal growth factor may induce similar signal cascade downstream of receptor tyrosine kinase (RTK) and trigger tumor metastasis synergistically. In the past decades, the intimate relationship of RTK-mediated receptor endocytosis with signal transduction was well established. In general, most RTK undergoes clathrin-dependent endocytosis and/or clathrin-independent endocytosis. The internalized receptors may sustain the signaling within early endosome, recycling to plasma membrane for subsequent ligand engagement or sorting to late endosomes/lysosome for receptor degradation. Moreover, receptor endocytosis influences signal transduction in a temporal and spatial manner for periodical and polarized cellular processes such as cell migration. The endosomal signalings triggered by various metastatic factors are quite similar in some critical points, which are essential for triggering cell migration and tumor progression. There are common regulators for receptor endocytosis including dynamin, Rab4, Rab5, Rab11 and Cbl. Moreover, many critical regulators within the RTK signal pathway such as Grb2, p38, PKC and Src were also modulators of endocytosis. In the future, these may constitute a new category of targets for prevention of tumor metastasis.

Topics: Disease Progression; Endocytosis; Endosomes; Epidermal Growth Factor; Fibroblast Growth Factors; Hepatocyte Growth Factor; Humans; Neoplasm Metastasis; Neoplasms; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta

Despite significant improvements in diagnosis, surgical techniques, and advancements in general patient care, the majority of deaths from cancer are caused by the metastases. There is an urgent need for an improved understanding of the cellular and molecular factors that promote cancer metastasis. The process of cancer metastasis depends on multiple interactions between cancer cells and host cells. Studies investigating the TGF α-EGFR signaling pathways that promote the growth and spread of cancer cells. Moreover, the signaling activates not only tumor cells, but also tumor-associated endothelial cells. TGF α-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGF α-positive cancers. In this review, we describe the recent advances in our understanding of the molecular basis of cancer metastasis.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Metastasis; Neoplasms; Signal Transduction; Tumor Microenvironment

The KRAS oncogene is mutated in approximately 35%-45% of colorectal cancers, and KRAS mutational status testing has been highlighted in recent years. The most frequent mutations in this gene, point substitutions in codons 12 and 13, were validated as negative predictors of response to anti-epidermal growth factor receptor antibodies. Therefore, determining the KRAS mutational status of tumor samples has become an essential tool for managing patients with colorectal cancers. Currently, a variety of detection methods have been established to analyze the mutation status in the key regions of the KRAS gene; however, several challenges remain related to standardized and uniform testing, including the selection of tumor samples, tumor sample processing and optimal testing methods. Moreover, new testing strategies, in combination with the mutation analysis of BRAF, PIK3CA and loss of PTEN proposed by many researchers and pathologists, should be promoted. In addition, we recommend that microsatellite instability, a prognostic factor, be added to the abovementioned concomitant analysis. This review provides an overview of KRAS biology and the recent advances in KRAS mutation testing. This review also addresses other aspects of status testing for determining the appropriate treatment and offers insight into the potential drawbacks of mutational testing.

Topics: Alleles; Biomarkers; Biomarkers, Tumor; Colorectal Neoplasms; DNA Mutational Analysis; Early Detection of Cancer; Epidermal Growth Factor; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Microsatellite Repeats; Mutation; Neoplasm Metastasis; Predictive Value of Tests; Prognosis; Signal Transduction

Metastatic, rather than primary tumours are responsible for ninety percent cancer deaths. Despite significant advances in the understanding of molecular and cellular mechanisms in tumour metastases, there are limitations in preventive treatment of metastatic tumours. Much evidence arising from laboratory and clinical studies suggests that growth factors and their receptors are implicated in cancer metastases development. We review the origin and production of growth factors and their receptors in all stages of cancer metastases including epithelial-mesenchymal transition, cancer cell invasion and migration, survival within the circulation, seeding at distant organs and metastatic tumour angiogenesis. The functions of growth factors and their receptors are also discussed. This review presents the efforts made in understanding this challenge to aid in the development of new treatment strategies for cancer metastases.

Topics: Angiopoietins; Animals; Apoptosis; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Glucose-6-Phosphate Isomerase; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Interleukin-8; Multienzyme Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Seeding; Neoplasms; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptor, IGF Type 1; Receptors, Growth Factor; Ribonuclease, Pancreatic; Smad Proteins; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The most ominous stage of cancer progression is metastasis, or the dissemination of carcinoma cells from the primary site into distant organs. Metastases are often resistant to current extirpative therapies and even the newest biological agents cure only a small subset of patients. Therefore a greater understanding of tumor biology that integrates properties intrinsic to carcinomas with tissue environmental modulators of behavior is needed. In no aspect of tumor progression is this more evident than the acquisition of cell motility that is critical for both escape from the primary tumor and colonization. In this overview, we discuss how this behavior is modified by carcinoma cell phenotypic plasticity that is evidenced by reversible switching between epithelial and mesenchymal phenotypes. The presence or absence of intercellular adhesions mediate these switches and dictate the receptivity towards signals from the extracellular milieu. These signals, which include soluble growth factors, cytokines, and extracellular matrix embedded with matrikines and matricryptines will be discussed in depth. Finally, we will describe a new mode of discerning the balance between epithelioid and mesenchymal movement.

Topics: Cadherins; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Cytokines; Desmosomes; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Gap Junctions; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Integrins; Neoplasm Metastasis; Neoplasms; Phenotype; Signal Transduction; Tight Junctions; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Ovarian cancer is a highly metastatic disease and the leading cause of death from gynecologic malignancy. Hence, and understanding of the molecular changes associated with ovarian cancer metastasis could lead to the identification of targets for novel therapeutic interventions. The conversion of an epithelial cell to a mesenchymal cell plays a key role both in the embryonic development and cancer invasion and metastasis. Cells undergoing epithelial-mesenchymal transition (EMT) lose their epithelial morphology, reorganize their cytoskeleton and acquire a motile phenotype through the up- and down-regulation of several molecules including tight and adherent junctions proteins and mesenchymal markers. EMT is believed to be governed by signals from the neoplastic microenvironment including a variety of cytokines and growth factors. In ovarian cancer EMT is induced by transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and endothelin-1 (ET-1). Alterations in these cellular pathways candidate them as useful target for ovarian cancer treatment.

Topics: Bone Morphogenetic Protein 4; Cadherins; Cell Differentiation; Endothelin-1; Epidermal Growth Factor; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Mesoderm; Neoplasm Metastasis; Ovarian Neoplasms; Transforming Growth Factor beta

Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.

Topics: Animals; Breast Neoplasms; Cell Dedifferentiation; Cell Differentiation; Cell Transdifferentiation; Embryonic Development; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Ligands; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Mesenchymal Stem Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Receptor Protein-Tyrosine Kinases; Signal Transduction

Topics: Animals; Antibodies, Monoclonal; Biomarkers, Tumor; Breast Neoplasms; Drug Delivery Systems; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; GPI-Linked Proteins; Humans; Immunotherapy; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins

Topics: Carcinoma; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-1; Humans; Ligands; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; Prognosis

Cancer-related mortality is caused in a large part by the metastasis of primary tumor. Each cancer has a particular way of spreading cancerous cells. Recently, genetic and pharmacological analysis identified the set of genes, such as epidermal growth factor receptor ligand epiregulin (EREG), cyclooxygenase-2 (COX2) and matrix metalloproteinases 1 and 2 (MMP-1 and MMP-2) that have been found to be associated with metastasis of breast cancer to lung. Inhibition of EGFR and COX2 could minimize lung metastasis of breast cancer in a clinical setting. In this review, we summarized the current knowledge on EREG, COX2, MMP-1 and MMP-2 in tumor development and metastasis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Disease Progression; Epidermal Growth Factor; Epiregulin; Gene Expression Regulation, Neoplastic; Humans; Lung; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Models, Biological; Models, Genetic; Neoplasm Invasiveness; Neoplasm Metastasis

Patients with metastatic pancreatic cancer have poor prognosis and short survival due to lack of effective therapy and aggressiveness of the disease. Pancreatic cancer has widespread chromosomal instability, including a high rate of translocations and deletions. Upregulated EGF signaling and mutation of K-RAS are found in most pancreatic cancers. Therefore, inhibitors that target EGF receptor, K-RAS, RAF, MEK, mTOR, VEGF and PDGF, for example, have been evaluated in patients with pancreatic cancer. Although significant activities of these inhibitors have not been observed in the majority of pancreatic cancer patients, an enormous amount of experience and knowledge has been obtained from recent clinical trials. With a better inhibitor or combination of inhibitors, and improvement in the selection of patients for available inhibitors, better therapy for pancreatic cancer is on the horizon.

Topics: Epidermal Growth Factor; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms; Signal Transduction

Advances in the medical treatment of colorectal cancer patients have resulted in considerable improvements through the introduction of new cytotoxic drugs. The significant progress in molecular and tumour biology has produced a great number of targeted, tumour-specific, monoclonal antibodies that are now in various stages of clinical development. Two of these antibodies, cetuximab (Erbitux) und bevacizumab (Avastin), directed against the epidermal growth factor receptor (EGFR) and the vascular epithelial growth factor (VEGF), respectively, have recently been approved for use in metastatic colorectal cancer. The combination of well-known and newly developed cytotoxic agents with monoclonal antibodies makes the medical treatment of colorectal cancer patients considerably more complex, but also provides additional therapeutic strategies for patients in advanced stages of disease.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Bevacizumab; Camptothecin; Cetuximab; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Colorectal Neoplasms; Drug Therapy, Combination; Epidermal Growth Factor; ErbB Receptors; Fluorouracil; Humans; Irinotecan; Leucovorin; Neoplasm Metastasis; Panitumumab; Phthalazines; Pyridines; Randomized Controlled Trials as Topic; Vascular Endothelial Growth Factor A; Vitamin B Complex

In this review, we describe the critical functions assumed by the interplay of epidermal growth factor, hedgehog, Wnt/beta-catenin, tumor growth factor-beta and integrin signaling cascades in tumorigenic and migrating cancer progenitor cells and activated stromal cells during carcinogenesis. These growth factors provide an important role for the sustained growth and survival of tumorigenic cancer progenitor cells and their progeny by up-regulating numerous mitotic and antiapoptotic signaling cascades. Furthermore, these potent morphogens may cooperate for inducing the molecular events associated with the epithelial-mesenchymal program in cancer cells including the alterations in epithelial cell shape and motility through the dissociation of intercellular adherens junctions. Of therapeutic interest, new strategies for the development of more effective clinical treatments against the locally aggressive and invasive cancers based on the molecular targeting of deregulated signaling elements in tumorigenic and migrating cancer cells and their local microenvironment are also described.

Topics: Antineoplastic Combined Chemotherapy Protocols; beta Catenin; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cytokines; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Growth Substances; Hedgehog Proteins; Humans; Integrins; Mesoderm; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Signal Transduction

Recent evidence indicates that metastatic capacity is an inherent feature of breast tumours and not a rare, late acquired event. This has led to new models of metastasis. The interpretation of expression-profiling data in the context of these new models has identified the cofilin pathway as a major determinant of metastasis. Recent studies indicate that the overall activity of the cofilin pathway, and not that of any single gene within the pathway, determines the invasive and metastatic phenotype of tumour cells. These results predict that inhibitors directed at the output of the cofilin pathway will have therapeutic benefit in combating metastasis.

Topics: Actin Depolymerizing Factors; Animals; Breast Neoplasms; Cell Line, Tumor; Drosophila melanogaster; Epidermal Growth Factor; Female; Humans; Models, Biological; Models, Molecular; Morphogenesis; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction

Metastasis development requires the migratory activity of tumor cells. It is therefore important to understand the molecular mechanisms of this migration in order to prevent metastasis development, which is the pernicious step in most solid tumor diseases. A lot of methods have been invented to investigate tumor cell migration, but not all are equally suited and no method alone is able to deliver a complete picture of tumor cell migration. We herein suggest a combination of three-dimensional in vitro and in vivo methods for the investigation of tumor cell migration and summarize the knowledge, which has been reached so far.

Topics: Animals; Cell Adhesion; Cell Movement; Epidermal Growth Factor; Humans; In Vitro Techniques; Luciferases; Mice; Mice, Inbred BALB C; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Rats; Signal Transduction

Although cancer of the prostate (CaP) is the most commonly occurring cancer in males, there are major limitations in its diagnosis and long-term cure. Consequently, understanding the molecular mechanisms involved in the progression of CaP is of particular importance for production of pharmacological and biological agents to manage the disease. The development of the normal prostate is regulated by stromal-epithelial interactions via endocrine and paracrine factors, such as androgens and growth factors, which act as precise homeostatic regulators of cellular proliferation. Importantly, after a period of hormonal therapy, CaP shifts from an androgen-dependent to an androgen-independent state with a concomitant switch from paracrine to autocrine growth factor stimulation and subsequent upregulation of growth factor expression. Thus, growth factors and their receptors have a pivotal role in CaP. This is emphasized by current evidence obtained from clinical specimens as well as several in vitro and in vivo models strongly suggesting that epidermal growth factor and the neurotrophins (nerve growth factor, brain derived neurotrophin factor, neurotrophin-3 and neurotrophin-4/5) together with their tyrosine kinase receptors could play a very significant role in CaP progression.

Topics: Androgens; Brain-Derived Neurotrophic Factor; Cell Division; Epidermal Growth Factor; Epithelial Cells; Humans; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Nerve Growth Factors; Prostatic Neoplasms; Stromal Cells

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

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

Recent advances in the treatment of colorectal cancer have lead to significant gains in response rates and survival. The combination of newer agents such as irinotecan and oxaliplatin with 5-fluorouracil/leucovorin using various dosing schedules in the metastatic setting has resulted in a steady improvement in the outcome of patients with colorectal cancer. Experimental therapies such as epidermal growth factor receptor inhibitors, vascular endothelial growth factor inhibitors and cyclooxygenase-2 inhibitors, have shown promise in early clinical trials and have acceptable toxicity profiles. Efforts towards improving risk-stratification of stage II colorectal cancer patients and optimising therapy in patients with advanced disease, have focused on molecular and genetic markers. It is hoped that the addition of new therapies to existing drug combinations, as well as further advances in the understanding of colorectal cancer biology, will lead to further improvement in survival and quality of life for patients.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Cancer Vaccines; Clinical Trials as Topic; Colorectal Neoplasms; Cyclooxygenase 2; Epidermal Growth Factor; Humans; Isoenzymes; Membrane Proteins; Neoplasm Metastasis; Prostaglandin-Endoperoxide Synthases; ras Proteins

Even when diagnosed at the earliest stages, non-small-cell lung cancer (NSCLC) has often begun to metastasize, leading to frequent systemic relapses and a poor prognosis. There is an urgent need for effective adjuvant systemic therapy in conjunction with surgery to reverse or control further growth of micrometastases in early-stage NSCLC. Several approaches have been investigated in the search for such a therapy, including various chemotherapies, applied preoperatively or postoperatively, chemopreventive agents, and molecular-targeted agents. This article presents an overview of clinical trials for these adjunctive therapies, including completed trials and trials whose results are anticipated. Although standard postoperative chemotherapy has been found to offer little benefit, likely because of the acquisition of resistance in advanced tumors, some clinical trials with neoadjuvant or alternative chemotherapies have produced encouraging results. Targeted agents such as the epidermal growth factor receptor/tyrosine kinase inhibitor gefitinib have shown early promise for effective disease control. A combination of these new approaches and standard therapy for NSCLC may improve long-term survival in patients with lung cancer

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Chemoprevention; Chemotherapy, Adjuvant; Drug Resistance; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Neoadjuvant Therapy; Neoplasm Metastasis; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Radiotherapy, Adjuvant; Selenium; Survival Analysis

The processes of cellular proliferation and progressive acquisition of a specialized phenotype show a remarkable degree of coordination that involves both intracellular programming and intercellular communication. One of the major incentives for studying factors that regulate the processes of cellular proliferation and differentiation is the recognition of their potential contribution to tumorigenesis. In normal cells, stimulatory and inhibitory events are believed to be under the control of growth factors and growth inhibitory factors, which are known to be protooncogene products. Growth regulatory mechanisms usually involve the binding of a growth factor to a specific receptor on the cell surface, which then through an intracellular biochemical cascade leads to cell division. The cell regulation pathways initiated by growth factors may be subverted at several distinct levels in cancer cells. Studies of oncogenes have shown that they may function as abnormal growth factors or abnormal receptors, induce expression of potential signal regulators or encode proteins which modulate gene transcription. The purpose of the present paper is to examine the role of growth factors, growth factor receptors and intracellular proteins involved in signal transduction (with particular regard to the epidermal growth factor receptor system) in the control of normal growth and differentiation, and their contribution to transformation and tumorigenesis. We also review the classical theories of neoplasia and various other models. Chemical carcinogenesis and Vogelstein-Lane model are presented.

Topics: Amino Acid Sequence; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Genes, Tumor Suppressor; Growth; Growth Substances; Humans; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Oncogenes; Phenotype; Receptors, Growth Factor; Signal Transduction; Transcription, Genetic; Transforming Growth Factor alpha

The plateau in survival rates from head and neck cancer as well as the increasing incidence of disease among various populations demands the need for new perspectives in head and neck oncology. In pursuit of that goal, investigators have been developing improved biologic markers for metastatic risk of head and neck cancer. Such markers can be placed into categorical groupings, of which markers for cellular differentiation may be the most relevant. Among the growth factors relevant to head and neck cancer, epidermal growth factor and its receptor have received the most attention. Those tumors with unregulated growth factor control tend towards a more dedifferentiated state. Additionally, the degree of cellular differentiation and resulting risk of metastases may be predetermined in an individual through constitutively expressed susceptibility genes. Polymorphisms of the L-myc oncogene identified within peripheral blood lymphocytes may represent such a marker. Certain polymorphisms of this gene will identify individuals likely to express dedifferentiated head and neck cancer. Finally, the expression of cell-surface differentiation antigens may govern the capacity of cell-mediated host immune systems to control metastatic growth.

Topics: Antigens, Differentiation; Biomarkers, Tumor; Cell Differentiation; Epidermal Growth Factor; ErbB Receptors; Genes, myc; Head and Neck Neoplasms; Histocompatibility Antigens Class I; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Polymorphism, Genetic; Prognosis

The incidence of breast cancer is 25-30% of all malignant female tumors and represents the highest rate of mortality. To define those breast cancer patients at higher risk several prognostic factors are routinally evaluated in the primary tumor. Among them, the presence and degree of tumor involvement in axillary lymph node is one of the most powerful prognostic indicator. However, around 30% of node negative patients (good prognosis group) have recurrences and die within the next 10 years from diagnosis. Therefore, there is a need for markers to better discriminate biologic differences in the primary tumors. The techniques of molecular biology are shedding new insight into the subcellular pathology of malignancy. The crucial events of carcinogenesis, tumor progression, and metastatic spread are coming into focus at a molecular level. In some tumors, these advances in the laboratory are beginning to have applications at the bedside. Recently, abnormalities in the copy number and expression of several genes have been correlated with prognosis of individual patients with selected types of cancer. Molecular biology laboratories can provide useful predictive information that can be used to influence decision on the selection of treatment and to estimate a better risk stratification. Among these new markers are: oncongene amplification and/or over-expression, growth factors, cellular proliferation rate and ploidy, estrogens induced proteins, expression of metastasis related molecules, drug resistance associated proteins, etc. In this article we attempt to present a brief evaluation of a new technology that promises to add greater precision in evaluating molecular tumor markers and we mention some of its clinical applications.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Humans; Molecular Probe Techniques; Neoplasm Metastasis; Oncogenes; Prognosis; Receptors, Cell Surface

Recently, expectations have been raised that molecular biological studies of human tumours may be of value in helping to predict future clinical behaviour, in terms of therapeutic response and long-term survival. The epidermal growth factor receptor (EGFr) is a cell surface receptor for EGF and transforming growth factor-alpha which is overexpressed by a number of human tumours. This article principally reviews previous investigations of the role of the epidermal growth factor receptor in bladder cancer and examines methods of detection, the correlation between EGFr status and known prognostic indicators and the value of assessing EGFr status in predicting clinical outcome in patients with bladder cancer. Recent studies of the c-erbB-2 proto-oncogene in bladder cancer and of cell cycling using Ki-67 are included.

Topics: Biomarkers, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Prognosis; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

The process of metastasis is not random. Rather, the outcome depends on the interaction of unique metastic cells with different organ microenvironments. Although the ability of some tumor cells to reach and proliferate in the parenchyma of some organs is responsible for the development of site-specific metastasis, the molecular basis of organ-specific metastasis is unclear. This article will address the molecular mechanisms involved in the interaction between favored tumor cells and a compatible organ environment in terms of growth factors and those receptors participating in paracrine and autocrine signal transduction pathways.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factors; Growth Substances; Humans; Mice; Neoplasm Metastasis; Platelet-Derived Growth Factor; Receptor, Insulin; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; Receptors, Somatomedin; Signal Transduction; Somatomedins; Transforming Growth Factor beta

Topics: Animals; Cell Adhesion; Cell Division; Cell Movement; Endothelium, Vascular; Epidermal Growth Factor; Extracellular Matrix; Humans; Insulin-Like Growth Factor I; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Cells, Circulating; Transferrin

Chemotherapeutic assays, using nitrosoureas, performed on tumor bearing rats have shown a regression of local tumor, accompanied with an amplification of pulmonary metastases, demonstrating that the treatment of metastasis differs from the treatment of a local tumor. Cells organizing a tumor are heterogeneous for their drug resistance, and for a series of properties including their ability to form metastasis. Metastatic cells have to leave the tumoral tissue, to traverse biological barriers, to resist to immune system, to implant and growth in the target tissue. An experimental model has been used to characterize metastatic cells. Metastatic potential has been defined as the ability to invade lungs. Highly metastatic cloned cell lines, such as subline 6, were strongly stimulated to proliferate by EGF, expressed fibronectin, actively degraded the extracellular matrix, rapidly attached to endothelial vascular cells, and resisted to natural killer lymphocytes. Inversely, weakly metastatic lines, such as subline 8, were preferentially stimulated by FGF and EDGF, poorly expressed fibronectin, did not degrade extracellular matrix, slowly attached to vascular cells, and were killed by NK lymphocytes. Studies on a large series of clones showed a diversity between them, and that no one property was determinant. Modulation of these characters by growth factors, hormones and immune state of the host is discussed, and leads to conclude that the expression of metastatic potential of a tumor depends on genetically defined characters and also on influences excerted by the host.

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Cytotoxicity, Immunologic; Epidermal Growth Factor; Extracellular Matrix; Fibroblast Growth Factors; Fibronectins; Growth Substances; Killer Cells, Natural; Laminin; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Staging; Rats; Rats, Inbred Strains; Rhabdomyosarcoma

It is now widely accepted that therapeutic antibodies targeting epidermal growth factor receptor (EGFR) can have efficacy in KRAS wild-type advanced colorectal cancer (CRC) patients. What remains to be ascertained is whether a subgroup of KRAS-mutated CRC patients might not also derive benefit from EGFR inhibitors. Metalloproteinase inhibitor 1 (TIMP-1) is a pleiotropic factor predictive of survival outcome of CRC patients. Levels of TIMP-1 were measured in pre-treatment plasma samples (n = 426) of metastatic CRC patients randomized to Nordic FLOX (5-fluorouracil and oxaliplatin) +/- cetuximab (NORDIC VII study). Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 protein levels, KRAS status and treatment with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (> 3rd quartile) showing a significantly longer overall survival if treated with cetuximab (HR, 0.48; 95% CI, 0.25 to 0.93). To gain mechanistic insights into this association we analyzed a set of five different CRC cell lines. We show here that EGFR signaling induces TIMP-1 expression in CRC cells, and that TIMP-1 promotes a more aggressive behavior, specifically in KRAS mutated cells. The two sets of data, clinical and in vitro, are complementary and support each other, lending strength to our contention that TIMP- 1 plasma levels can identify a subset of patients with KRAS-mutated metastatic CRC that will have benefit from EGFR-inhibition therapy.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cetuximab; Colorectal Neoplasms; Epidermal Growth Factor; Female; Fluorouracil; Humans; Male; Mutation; Neoplasm Metastasis; Organoplatinum Compounds; Oxaliplatin; Phenotype; Proto-Oncogene Proteins p21(ras); Signal Transduction; Survival Analysis; Tissue Inhibitor of Metalloproteinase-1

The antiepidermal growth factor receptor (EGFR) antibody cetuximab shows activity in multiple epithelial tumor types; however, responses are seen in only a subset of patients. This study was conducted to identify markers that are associated with disease control in patients treated with cetuximab.. One hundred ten patients with metastatic colorectal cancer were enrolled onto a cetuximab monotherapy trial. Transcriptional profiling was conducted on RNA from mandatory pretreatment metastatic biopsies to identify genes whose expression correlates with best clinical responses. EGFR and K-ras mutation analyses and EGFR gene copy number analyses were performed on DNA from pretreatment biopsies.. Gene expression profiles showed that patients with tumors that express high levels of the EGFR ligands epiregulin and amphiregulin are more likely to have disease control with cetuximab (EREG, P = .000015; AREG, P = .000025). Additionally, patients whose tumors do not have K-ras mutations have a significantly higher disease control rate than patients with K-ras mutations (P = .0003). Furthermore, patients with tumors that have high expression of EREG or AREG also have significantly longer progression-free survival (PFS) than patients with low expression (EREG: P = .0002, hazard ratio [HR] = 0.47, and median PFS, 103.5 v 57 days, respectively; AREG: P < .0001, HR = 0.44, and median PFS, 115.5 v 57 days, respectively).. Patients with tumors that have high gene expression levels of epiregulin and amphiregulin and patients with wild-type K-ras are more likely to have disease control on cetuximab treatment. The identified markers could be developed further to select patients for cetuximab therapy.

Topics: Adult; Aged; Aged, 80 and over; Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biomarkers, Tumor; Cetuximab; Colorectal Neoplasms; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Profiling; Genes, ras; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Mutation; Neoplasm Metastasis; Predictive Value of Tests; Proportional Hazards Models; Survival Rate; Treatment Outcome

Epidermal growth factor receptor (EGFR) appears to play an important role in the pathogenesis of colorectal cancer. We have performed a Phase I/II study of the EGFR tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients in which serial biopsies were taken pre- and posttreatment to assess biological activity.. Paired biopsies were obtained from colorectal cancer patients before and after treatment. Proliferation and apoptosis were assessed using Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated nick end labeling assays, respectively. Immunohistochemistry for EGFR, activated EGFR, phosphorylated Akt, phosphorylated ERK, p27(Kip1), and beta-catenin was also performed.. Posttreatment samples showed a statistically significant reduction in the cancer cell proliferation index (mean proliferation index pretreatment 31%; posttreatment 21%; P = 0.047). The mean cancer cell apoptosis index also increased from 6 to 12% in posttreatment samples, although this difference did not achieve statistical significance. All pretreatment samples showed strong staining for EGFR. Loss of immunohistochemical staining for activated EGFR, phosphorylated Akt, and phosphorylated ERK in cancer cells was observed in some patients after treatment. p27(Kip1) was absent in the cancer cells of most pretreatment biopsies; two patients showed a marked increase in staining for nuclear p27(Kip1) after treatment with ZD1839. These two patients also showed large increases in apoptotic index.. ZD1839 inhibits EGFR signaling and proliferation in the cancer cells of patients with metastatic colorectal cancer. ZD1839 may also induce cancer cell apoptosis in a subset of colorectal cancer patients via up-regulation of p27(Kip1).

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Cell Cycle Proteins; Cell Division; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Cytoskeletal Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Gefitinib; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Ki-67 Antigen; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; Signal Transduction; Time Factors; Trans-Activators; Tumor Suppressor Proteins

Even when diagnosed at the earliest stages, non-small-cell lung cancer (NSCLC) has often begun to metastasize, leading to frequent systemic relapses and a poor prognosis. There is an urgent need for effective adjuvant systemic therapy in conjunction with surgery to reverse or control further growth of micrometastases in early-stage NSCLC. Several approaches have been investigated in the search for such a therapy, including various chemotherapies, applied preoperatively or postoperatively, chemopreventive agents, and molecular-targeted agents. This article presents an overview of clinical trials for these adjunctive therapies, including completed trials and trials whose results are anticipated. Although standard postoperative chemotherapy has been found to offer little benefit, likely because of the acquisition of resistance in advanced tumors, some clinical trials with neoadjuvant or alternative chemotherapies have produced encouraging results. Targeted agents such as the epidermal growth factor receptor/tyrosine kinase inhibitor gefitinib have shown early promise for effective disease control. A combination of these new approaches and standard therapy for NSCLC may improve long-term survival in patients with lung cancer

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Chemoprevention; Chemotherapy, Adjuvant; Drug Resistance; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Neoadjuvant Therapy; Neoplasm Metastasis; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Radiotherapy, Adjuvant; Selenium; Survival Analysis

The incidence of colorectal cancer (CRC) has increased significantly in the past decade. Early diagnosis and new therapeutics are still urgently needed for CRC in clinical practice. Human α-defensin 6 (HD6) plays a defense role against microbes in the gastrointestinal tract. However, the role and mechanism of HD6 in CRC is still unresolved. Specimens from CRC patients with higher HD6 showed better outcomes. Overexpressed HD6 in CRC cells caused a reduction of cell proliferative, migratory, and invasive ability

Topics: alpha-Defensins; Animals; Biomarkers, Tumor; Cell Cycle Checkpoints; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression; Humans; Kaplan-Meier Estimate; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Plasminogen Activator Inhibitor 1; S Phase; Tumor Cells, Cultured

The effect of targeted therapy for metastatic hepatocellular carcinoma (HCC) is still unsatisfactory. Exploring the underlying mechanism of HCC metastasis is favorable to provide new therapeutic strategies. T-box (TBX) transcription factor family genes, which are crucial regulators in embryo and organ development, are vital for regulating tumor initiation, growth and metastasis. Here we explored the role of TBX19 in HCC metastasis, which is one of the most upregulated TBX family genes in human HCC tissues. TBX19 expression was markedly upregulated in HCC tissues and elevated TBX19 expression predicted poor prognosis. Overexpression of TBX19 enhanced HCC metastasis through upregulating epidermal growth factor receptor (EGFR) and Rac family small GTPase 1 (RAC1) expression. Downregulation of EGFR and RAC1 inhibited TBX19-mediated HCC metastasis, while upregulation of EGFR and RAC1 restored inhibition of HCC metastasis mediated by TBX19 knockdown. Furthermore, epidermal growth factor (EGF)/EGFR signaling upregulated TBX19 expression via the extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-kB axis. Besides, the combined application of EGFR inhibitor Erlotinib and RAC1 inhibitor NSC23766 markedly inhibited TBX19-mediated HCC metastasis. In HCC cohorts, TBX19 expression was positively associated with EGFR and RAC1 expression. Patients with positive coexpression of TBX19/EGFR or TBX19/RAC1 displayed the poorest prognosis. In conclusion, EGF/EGFR signaling upregulated TBX19 expression via ERK/NF-kB pathway and TBX19 fostered HCC metastasis by enhancing EGFR and RAC1 expression, which formed an EGF-TBX19-EGFR positive feedback loop. Targeting this signaling pathway may offer a potential therapeutic strategy to efficiently restrain TBX19-mediated HCC metastasis.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Liver Neoplasms; Neoplasm Metastasis; rac1 GTP-Binding Protein; T-Box Domain Proteins; Transcription Factors

Colorectal cancer (CRC) is an extremely malignant tumor with a high mortality rate. Little is known about the mechanism by which forkhead Box q1 (FOXQ1) causes CRC invasion and metastasis through the epidermal growth factor receptor (EGFR) pathway.. To illuminate the mechanism by which FOXQ1 promotes the invasion and metastasis of CRC by activating the heparin binding epidermal growth factor (HB-EGF)/EGFR pathway.. We investigated the differential expression and prognosis of FOXQ1 and HB-EGF in CRC using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of FOXQ1 and HB-EGF in cell lines and tissues, and we constructed a stable low-expressing FOXQ1 cell line and verified it with the above method. The expression changes of membrane-bound HB-EGF (proHB-EGF) and soluble HB-EGF (sHB-EGF) in the low-expressing FOXQ1 cell line were detected by flow cytometry and ELISA. Western blotting was used to detect changes in the expression levels of HB-EGF and EGFR pathway-related downstream genes when exogenous recombinant human HB-EGF was added to FOXQ1 knockdown cells. Proliferation experiments, transwell migration experiments, and scratch experiments were carried out to determine the mechanism by which FOXQ1 activates the EGFR signaling pathway through HB-EGF, and then to evaluate the clinical relevance of FOXQ1 and HB-EGF.. GEPIA showed that the expression of FOXQ1 in CRC tissues was relatively high and was related to a lower overall survival rate. PCR array results showed that FOXQ1 is related to the HB-EGF and EGFR pathways. Knockdown of FOXQ1 suppressed the expression of HB-EGF, and led to a decrease in EGFR and its downstream genes

Topics: Cell Line, Tumor; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Forkhead Transcription Factors; Heparin-binding EGF-like Growth Factor; Humans; Neoplasm Invasiveness; Neoplasm Metastasis

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Neoplasms; Zebrafish

During pregnancy, trophoblast cell invasion needs to be finely controlled. Aberrant trophoblast cell invasion is associated with placental diseases. Epidermal growth factor (EGF) and its receptor, EGFR, are expressed in trophoblast cells. Although the pro-invasive effect of EGF on trophoblast cells has been reported, the underlying mechanism remains largely unknown.. In the present study, we conducted an RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to EGF and identified KISS1 as a target gene of EGF. The human KISS1 gene encodes kisspeptin, also known as metastin, which can suppress tumor metastasis. Our results showed that EGF treatment downregulated KISS1 expression and secretion by activating the EGFR-mediated PI3K/AKT signaling pathway. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) was downregulated by EGF and that was required for the EGF-suppressed KISS1 expression. Functionally, transwell invasion assays demonstrated that EGF stimulated human trophoblast cell invasion by downregulating KISS1 expression. Preeclampsia (PE) is a placental disease characterized by insufficient trophoblast cell invasion. Our clinical results revealed that serum levels of EGF were downregulated while serum and placental levels of KISS1 were upregulated in PE patients.. This study demonstrates that downregulation of EGF can lead to poor trophoblast cell invasion by increasing KISS1 expression which subsequently contributes to the pathogenesis of PE. Video Abstract.

Topics: Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Inhibitor of Differentiation Proteins; Kisspeptins; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Placenta; Placenta Diseases; Pre-Eclampsia; Pregnancy; Proto-Oncogene Proteins c-akt; Signal Transduction; Trophoblasts

Chondroitin sulfate (CS) chains containing GlcUAβ1-3GalNAc(4S,6S) (E unit) have been shown to be involved in various physiological and pathological processes. However, commercial E unit-rich CS (CS-E) is difficult to produce on a large scale due to expensive and limited squid cartilage resources. In this study, a novel CS-E (CS-nE) was isolated from the cheap and abundant cartilage of the giant squid Dosidicus gigas. The CS-nE has a surprisingly large molecular mass of 696 kDa and a relatively high E unit proportion (44.5 %). It can interact with various growth factors, including HGF, bFGF, pleiotrophin, and HB-EGF, with high affinity, and exhibits dose-dependent anti-metastatic activity. Furthermore, the E unit-rich decasaccharide selectively prepared from CS-nE has been shown to be the minimal functional domain with the strongest antitumor metastatic activity. Taken together, CS-nE will be a very promising candidate for the development of CS-E-based pharmaceutical products.

Topics: Animals; Antineoplastic Agents; Carrier Proteins; Cartilage; Cell Line, Tumor; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Cytokines; Decapodiformes; Disaccharides; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Heparin-binding EGF-like Growth Factor; Hepatocyte Growth Factor; Mice; Neoplasm Metastasis

Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer mainly owing to its proclivity to early metastasis and the lack of effective targeted therapeutic drugs. Hence, understanding the molecular mechanisms underlying early invasion and metastasis by PDAC is imperative for improving patient outcomes. The present study identified that upregulation of TSPAN8 expression in PDAC facilitates metastasis in vivo and in vitro. We found SOX9 as a key transcriptional regulator of TSPAN8 expression in response to EGF stimulation. SOX9 modulation was sufficient to positively regulate endogenous expression of TSPAN8, with concomitant in vitro phenotypic changes such as loss of cell-matrix adherence and increased invasion. Moreover, increased SOX9 and TSPAN8 levels were shown to correlate in human pancreatic cancer specimens and downregulated in vitro by EGFR tyrosine kinase inhibitors. High expression of SOX9 and TSPAN8 has been associated with tumor stage, poor prognosis and poor patient survival in PDAC. In conclusion, this study highlights the importance of the EGF-SOX9-TSPAN8 signaling cascade in the control of PDAC invasion and implies that TSPAN8 may be a promising novel therapeutic target for the treatment of PDAC.

Topics: Biomarkers, Tumor; Cell Movement; Disease Progression; Disease Susceptibility; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; Promoter Regions, Genetic; Protein Binding; SOX9 Transcription Factor; Tetraspanins

Nasopharyngeal carcinoma (NPC) is a unique head and neck malignancy with highly metastatic cell-biological characteristics, for which latent EBV-infection is responsible. Our earlier studies showed that EGF-stimulated Ca

Topics: Animals; Calcium Signaling; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Epstein-Barr Virus Infections; Female; Humans; Mice; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; Stromal Interaction Molecule 1; Up-Regulation; Zebrafish

Stemness and epithelial-mesenchymal transition (EMT) are two fundamental characteristics of metastasis that are controlled by diverse regulatory factors, including transcription factors. Compared with other subtypes of breast cancer, basal-type or triple-negative breast cancer (TNBC) has high frequencies of tumor relapse. However, the role of alpha-globin transcription factor CP2 (TFCP2) has not been reported as an oncogenic driver in those breast cancers. Here, we show that TFCP2 is a potent factor essential for EMT, stemness, and metastasis in breast cancer. TFCP2 directly bound promoters of EGF and TGFα to regulate their expression and stimulate autocrine signaling via EGFR. These findings indicate that TFCP2 is a new antimetastatic target and reveal a novel regulatory mechanism in which a positive feedback loop comprising EGF/TGFα and AKT can control malignant breast cancer progression. SIGNIFICANCE: TFCP2 is a new antimetastatic target that controls TNBC progression via a positive feedback loop between EGF/TGFα and the AKT signaling axis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Progression; DNA-Binding Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Feedback, Physiological; Female; Gene Knockdown Techniques; Heterografts; Humans; MCF-7 Cells; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Transcription Factors; Transforming Growth Factor alpha; Up-Regulation

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models.

Topics: Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Humans; Lung Neoplasms; Neoplasm Metastasis; Oncogene Proteins; Phosphorylation; Prognosis; Promoter Regions, Genetic; Protein Stability; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Transcription, Genetic

Interactions between cells and their environment influence key physiologic processes such as their propensity to migrate. However, directed migration controlled by extrinsically applied electrical signals is poorly understood. Using a novel microfluidic platform, we found that metastatic breast cancer cells sense and respond to the net direction of weak (∼100 µV cm

Topics: Actins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Down-Regulation; Electromagnetic Fields; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

The molecular mechanisms driving metastatic progression in triple-negative breast cancer (TNBC) patients are poorly understood. In this study, we demonstrate that epidermal growth factor-like 9 (EGFL9) is significantly upregulated in basal-like breast cancer cells and associated with metastatic progression in breast tumor samples. Functionally, EGFL9 is both necessary and sufficient to enhance cancer cell migration and invasion, as well as distant metastasis. Mechanistically, we demonstrate that EGFL9 binds cMET, activating cMET-mediated downstream signaling. EGFL9 and cMET co-localize at both the cell membrane and within the mitochondria. We further identify an interaction between EGFL9 and the cytochrome c oxidase (COX) assembly factor COA3. Consequently, EGFL9 regulates COX activity and modulates cell metabolism, promoting a Warburg-like metabolic phenotype. Finally, we show that combined pharmacological inhibition of cMET and glycolysis reverses EGFL9-driven stemness. Our results identify EGFL9 as a therapeutic target for combating metastatic progression in TNBC.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Electron Transport Complex IV; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glucose; Glycolysis; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondrial Proteins; Neoplasm Metastasis; Proto-Oncogene Proteins c-met; Signal Transduction; Triple Negative Breast Neoplasms

Tumor microenvironments expose cancer cells to heterogeneous, dynamic environments by shifting availability of nutrients, growth factors, and metabolites. Cells integrate various inputs to generate cellular memory that determines trajectories of subsequent phenotypes. Here we report that short-term exposure of triple-negative breast cancer cells to growth factors or targeted inhibitors regulates subsequent tumor initiation. Using breast cancer cells with different driver mutations, we conditioned cells lines with various stimuli for 4 hours before implanting these cells as tumor xenografts and quantifying tumor progression by means of bioluminescence imaging. In the orthotopic model, conditioning a low number of cancer cells with fetal bovine serum led to enhancement of tumor-initiating potential, tumor volume, and liver metastases. Epidermal growth factor and the mTORC1 inhibitor ridaforolimus produced similar but relatively reduced effects on tumorigenic potential. These data show that a short-term stimulus increases tumorigenic phenotypes based on cellular memory. Conditioning regimens failed to alter proliferation or adhesion of cancer cells in vitro or kinase signaling through Akt and ERK measured by multiphoton microscopy in vivo, suggesting that other mechanisms enhanced tumorigenesis. Given the dynamic nature of the tumor environment and time-varying concentrations of small-molecule drugs, this work highlights how variable conditions in tumor environments shape tumor formation, metastasis, and response to therapy.

Topics: Animals; Carcinogenesis; Cell Adhesion; Cell Count; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Disease Progression; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Luminescent Measurements; Mechanistic Target of Rapamycin Complex 1; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Serum Albumin, Bovine; Sirolimus; Triple Negative Breast Neoplasms; Tumor Microenvironment

The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Lysine; Mice, Inbred BALB C; Mice, Nude; Mutation; Neoplasm Metastasis; Neoplastic Stem Cells; Phenotype; Phosphoproteins; Proteasome Endopeptidase Complex; Protein Binding; Protein Interaction Domains and Motifs; Proteolysis; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sumoylation; Thiolester Hydrolases; Time Factors; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; YAP-Signaling Proteins

The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study, we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs), that occurs between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long-distance tumor cell streaming

Topics: Animals; Biological Transport; Breast Neoplasms; Cell Communication; Cell Line, Tumor; Cell Movement; Embryo, Nonmammalian; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Macrophages; Mammary Neoplasms, Animal; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Primary Cell Culture; Rats; RAW 264.7 Cells; Signal Transduction; Tumor Necrosis Factors; Zebrafish

The triple-negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA-ZEB2-AS1 was dramatically up-regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA-ZEB2-AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA-ZEB2-AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple-negative breast cancer. It is mainly endo-nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF-induced F-actin polymerization in MDA231 cells can be suppressed by reducing lncRNA-ZEB2-AS1 expression. The migration and invasion of triple-negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA-ZEB2-AS1 is an important factor affecting the development of triple-negative breast cancer and thus a potential oncogene target.

Topics: Actins; Animals; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycogen Synthase Kinase 3 beta; Humans; Mice, SCID; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Polymerization; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Survival Analysis; Triple Negative Breast Neoplasms; Up-Regulation; Zinc Finger E-box Binding Homeobox 2

FBXW2 inhibits proliferation of lung cancer cells by targeting SKP2 for degradation. Whether and how FBXW2 regulates tumor invasion and metastasis is previously unknown. Here, we report that FBXW2 is an E3 ligase for β-catenin. FBXW2 binds to β-catenin upon EGF-AKT1-mediated phosphorylation on Ser

Topics: A549 Cells; beta Catenin; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; F-Box Proteins; Gene Knockdown Techniques; HEK293 Cells; HeLa Cells; Humans; In Vitro Techniques; Lung Neoplasms; Lymphatic Metastasis; Matrix Metalloproteinases; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Survival Rate; Ubiquitination

Patients with advanced hepatocellular carcinoma (HCC) will almost always develop acquired tolerance after sorafenib therapy, and the molecular mechanism of sorafenib tolerance remains poorly characterized. Here, using our established sorafenib-resistant HCC cell and xenograft models, we identified a novel gene, KIAA1199, which was markedly elevated among the differentially expressed genes involved in sorafenib tolerance. Moreover, elevated expression of KIAA1199 was positively correlated with a high risk of recurrence and metastasis and advanced TNM stage in HCC patients. Functionally, loss- and gain-of-function studies showed that KIAA1199 promoted the migration, invasion, and metastasis of sorafenib-resistant HCC cells. Mechanistically, KIAA1199 is required for EGF-induced epithelial-mesenchymal transition (EMT) in sorafenib-resistant HCC cells by aiding in EGFR phosphorylation. In summary, our data uncover KIAA1199 as a novel sorafenib-tolerant promoting gene that plays an indispensable role in maintaining sorafenib-resistant HCC cell metastasis.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Hep G2 Cells; Heterografts; Humans; Hyaluronoglucosaminidase; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Phosphorylation; Sorafenib

Reseptor tyrosine kinases (cMET and EGFR) are important in lung cancer targeted therapy. We believe if we can use them as markers for clinicians to help decide the diagnosis of lung cancer. This parameter will be important in serum samples of patients with lung cancer diagnosis and treatment. The aim of this study is aimed to evaluate the clinical utility of serum protein and circulating mRNA of cMET and HGF in lung cancer patients. We also analyzed the correlation of mRNA expression with clinicopathologic parameters.. We performed enzyme-linked immunosorbent assay (ELISA) to measure and compare serum protein and circulating mRNA of cMET and HGF levels in peripheral blood from 60 lung cancer patients and 40 healthy control group.. We found that both protein and gene expression levels of serum c-MET, HGF and EGFR were significantly higher in patients with lung cancer than control group. There was no association between HGF, cMET, EGF, EGFR (both protein and gene) expression levels with age, gender, smoking habit, COPD, pathological types or tumor size, stage, metastatic-non metastatic adenocarcinoma-squamous carcinoma, SCLC-NSCLC. As a result of ROC analysis, serum cMET (AUC: 0.892) and HGF protein (AUC: 0.784) were diagnosed in lung cancer patients (Fig. 1). The AUC values of serum EGF and EGFR proteins were calculated to be 0.631 and 0.692, respectively.. To our knowledge this is the first study comparing the levels of protein and mRNA in the serum material of HGF, c-MET, EGF and EGFR parameters in lung cancer patients' blood samples. Further prospective studies with more participants for better understanding of mechanism and effect for HGF and c-MET inhibitors in lung cancer will help us to identify of these biomarkers role for guiding us to sellect individualized itargeted therapies.

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Circulating Tumor DNA; Epidermal Growth Factor; ErbB Receptors; Female; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Precision Medicine; Proto-Oncogene Proteins c-met; ROC Curve

BACKGROUND The mammalian cyclic guanosine monophosphate (cGMP)-dependent protein kinases type II (PKG II) plays critical physiological or pathological functions in different tissues. However, the biological effects of PKG II are dependent on cGMP. Published data indicated that L-arginine (L-Arg) promoted NO production, NO can activate soluble guanylate cyclase (sGC), and catalyzes guanosine triphosphate (GTP) into cGMP, which suggested L-Arg could activate PKG II. Therefore, the present work was performed to address: (i) whether L-Arg could be a potential alternative in PKG II activation, and (ii) whether L-Arg also contributes to PKG II against cancer. MATERIAL AND METHODS Nude BALB/c mice were inoculated with human MCF-7, HepG2, and SW480 cell lines via subcutaneous (s.c.) injecting. After 7 days of inoculation, Ad-PKG II was injected into the cancer tissues every 4 days, and the next day 10 μmol/mouse L-Arg was administered. Western blotting and immunohistochemistry were used to assess protein expression. RESULTS Our results demonstrated that L-Arg significantly activated PKG II and effectively ameliorated xenograft tumor development through inhibiting cancer growth, angiogenesis, and metastasis, which was partially dependent on blocking of epidermal growth factor receptor (EGFR) activity, as well as downstream signaling pathways such as Erk1/2. CONCLUSIONS Our results provide an exciting new insight: L-Arg is a potential alternative to PKG II activation.

Topics: Animals; Arginine; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

BACKGROUND Epithelial-mesenchymal transition (EMT) is responsible for metastasis of cancers, and NF-κB can promote tumor progression. Ezrin is an important molecule participating in EMT. However, whether Ezrin mediates NF-κB in EGF-induced osteosarcoma is unknown. MATERIAL AND METHODS Ezrin phosphorylation, NF-κB activation, and EGF-induced EMT were studied in MG63 and U20S cells with NF-κB inhibition, silencing, or over-expressing Ezrin. Cell morphology, proliferation, migration, and motility were analyzed. An osteosarcoma model was established in mice by injecting MG63 and U20S and reducing Ezrin. RESULTS With EGF induction in vitro, Ezrin Tyr353 and Thr567 were phosphorylated, and EMT, proliferation, migration, and motility of osteosarcoma cells were promoted. Silencing Ezrin suppressed and over-expressing Ezrin promoted the nuclear translocation of p65 and phosphorylated IκBα (p-IκBα) in EGF-induced osteosarcoma cells. NF-κB inhibitor blocked EGF-induced EMT in both cell types, as well as reserving cell morphology and suppressing proliferation, migration, and motility. In vivo, reducing Ezrin significantly suppressed metastasis of osteosarcoma xenografts, increased liver and lung weights, and activated NF-κB, which were both induced by EGF. CONCLUSIONS Ezrin/NF-κB regulated EGF-induced EMT, as well as progression and metastasis of osteosarcoma in vivo and in vitro. Ezrin/NF-κB may be a new therapeutic target to prevent osteosarcoma from deterioration.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Disease Models, Animal; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Heterografts; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; NF-kappa B; NF-KappaB Inhibitor alpha; Osteosarcoma; Phosphorylation; Signal Transduction

The loss of miR-200 family, through DNA methylation, results in cancer cells undergoing an epithelial to mesenchymal transition (EMT), and metastasis. In this study, we established that the transcriptional repressor Kaiso directly binds methylated regions of the miR-200 family, and this is reversed with 5-aza treatment. sh-Kaiso PC-3 cells display increased miR-200-a/b/c, miR-141, and miR-429 expression, with miR-200c demonstrating the most significant increase. Interestingly, overexpression of EGFR or treatment with EGF decreases miR-200c expression and this is reversed after treatment with EGFR specific kinase inhibitor PD153035. However, EGF did not have a significant effect on miR-200c in sh-Kaiso DU-145 or PC-3 cell lines, suggesting Kaiso silences miR-200c through the activation of EGFR signaling. Overexpression of Kaiso in LNCaP cells results in decreased expression of miR-200-a/b/c, miR-141, and miR-429, along with increased expression of ZEB1, p-EGFR and total EGFR levels. Overexpression of miR200c in PC-3 cells results in decreased expression of EGFR, ZEB1, ERK1/2 and Kaiso. Additionally, sh-Kaiso PC-3 demonstrates reduced in vivo tumor formation and metastasis. Thus, our data suggests that EGFR signaling regulates the silencing of miR-200 family through Kaiso binding to methylated regions in the promoter.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Male; Mice; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Neoplasm Transplantation; Prostatic Neoplasms; Quinazolines; Transcription Factors

Chemotactic cell migration is a central mechanism during cancer cell invasion and hence metastasis. In order to mimic in vivo conditions, we used a three-dimensional hydrogel matrix made of collagen I and a stable gradient-generating chemotaxis assay system, which is commercially available (μ-Slide Chemotaxis) to characterize epidermal growth factor (EGF)-induced chemotaxis of the human breast cancer cell line MDA-MB-231. Surprisingly, chemotactic effects of EGF on MDA-MB-231 cells could neither be observed in the standard growth medium DMEM/F-12 supplemented with 10% serum nor in starvation medium. In contrast, after adapting the cells to the serum-free growth medium UltraCULTURETM, significant chemotactic effects could be measured with high sensitivity. The extremely time-stable linear gradients, generated in the chemotaxis chamber, led to consistent directional migration of MDA-MB-231 cells. Dose-response experiments showed increased directional and kinetic response of MDA-MB-231 cells towards stable gradients of EGF. While EGF-guided directional migration (chemotaxis) was highly concentration-dependent with the highest response at 1.5 nM/mm EGF, we found that the chemokinetic effect induced by EGF was concentration-independent. Both, blocking the ligand-binding domain of the EGF receptor by an antibody (monoclonal anti-EGFR antibody 225) and inhibition of its kinase domain by a small molecule inhibitor (AG1478) led to a reduction in EGF-induced directed migration. The high sensitivity of the assay even allowed us to observe synergistic effects in EGF-receptor inhibition using a combination of low doses of both inhibitor types. Those results validate the fact that EGF is a potent guidance cue for MDA-MB-231 cell migration and help to understand the mechanism behind chemotaxis-driven cancer metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Chemotaxis; Collagen; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrogels; Neoplasm Metastasis; Peptide Fragments; Tissue Scaffolds

The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer.. Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed.. Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data.. These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; Proteomics; Signal Transduction; Triple Negative Breast Neoplasms

Lysyl oxidase (LOX) remodels the tumour microenvironment by cross-linking the extracellular matrix. LOX overexpression is associated with poor cancer outcomes. Here, we find that LOX regulates the epidermal growth factor receptor (EGFR) to drive tumour progression. We show that LOX regulates EGFR by suppressing TGFβ1 signalling through the secreted protease HTRA1. This increases the expression of Matrilin2 (MATN2), an EGF-like domain-containing protein that traps EGFR at the cell surface to facilitate its activation by EGF. We describe a pharmacological inhibitor of LOX, CCT365623, which disrupts EGFR cell surface retention and delays the growth of primary and metastatic tumour cells in vivo. Thus, we show that LOX regulates EGFR cell surface retention to drive tumour progression, and we validate the therapeutic potential of inhibiting this pathway with the small molecule inhibitor CCT365623.

Topics: Aminopropionitrile; Animals; Biosensing Techniques; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Disease Progression; Dogs; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; High-Temperature Requirement A Serine Peptidase 1; Humans; Matrilin Proteins; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms; Protein-Lysine 6-Oxidase; Rats; Signal Transduction; Transforming Growth Factor beta1

Cultured cancer cells serve as important models for preclinical testing of anti-cancer compounds. However, the optimal conditions for retaining original tumor features during in vitro culturing of cancer cells have not been investigated in detail. Here we show that serum-free conditions are critical for maintaining an immature phenotype of neuroblastoma cells isolated from orthotopic patient-derived xenografts (PDXs). PDX cells could be grown either as spheres or adherent on laminin in serum-free conditions with retained patient-specific genomic aberrations as well as tumorigenic and metastatic capabilities. However, addition of serum led to morphological changes, neuronal differentiation and reduced cell proliferation. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were central for PDX cell proliferation and MYCN expression, and also hindered the serum-induced differentiation. Although serum induced a robust expression of neurotrophin receptors, stimulation with their cognate ligands did not induce further sympathetic differentiation, which likely reflects a block in PDX cell differentiation capacity coupled to their tumor genotype. Finally, PDX cells cultured as spheres or adherent on laminin responded similarly to various cytotoxic drugs, suggesting that both conditions are suitable in vitro screening models for neuroblastoma-targeting compounds.

Topics: Animals; Biomarkers, Tumor; Biopsy; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Culture Media, Conditioned; Disease Models, Animal; Epidermal Growth Factor; Fibroblast Growth Factor 2; Heterografts; Humans; Immunohistochemistry; Mice; N-Myc Proto-Oncogene Protein; Neoplasm Metastasis; Neural Stem Cells; Neuroblastoma

EGFR signaling is implicated in NF-κB activation. However, the concrete mechanisms by which the core transducer of NF-κB signaling pathway, RelA/p65 is regulated under EGFR activation remains to be further clarified. Here, we show that EGF stimulation induces PKCε-dependent phosphorylation of migration and invasion inhibitory protein (MIIP) at Ser303; this phosphorylation promotes the interaction between MIIP and RelA in the nucleus, by which MIIP prevents histone deacetylase 6 (HDAC6)-mediated RelA deacetylation, and thus enhances transcriptional activity of RelA and facilitates tumor metastasis. Meanwhile PP1, which functions as a phosphatase, is found to mediate MIIP-S303 dephosphorylation and its expression level inversely correlates with metastatic capability of tumor cells. Moreover, clinical analyses indicate the level of MIIP-S303 phosphorylation correlates with colorectal cancer (CRC) metastasis and prognosis. These findings uncover an unidentified mechanism underlying the precise regulation of NF-κB by EGF, and highlight the critical role of nuclear MIIP in tumor metastasis.In colorectal cancer, EGFR signalling is implicated in metastasis. Here, the authors unravel a mechanism through which EGF stimulation induces MIIP phosphorylation, leading to MIIP interacting with RelA-this prevents RelA deactylation and enhances transcriptional activity, facilitating metastasis.

Topics: Acetylation; Animals; Caco-2 Cells; Carrier Proteins; Cell Line, Tumor; Colorectal Neoplasms; Epidermal Growth Factor; Female; HCT116 Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Phosphorylation; Protein Binding; Protein Kinase C-epsilon; RNA Interference; Serine; Transcription Factor RelA; Transplantation, Heterologous

Epidermal growth factor (EGF) is important for cancer cell proliferation, angiogenesis and metastasis in many types of cancer. However, the mechanisms involved in EGF-induced head and neck squamous cell carcinoma (HNSCC) metastasis remain largely unknown. In this study, we reveal that angiopoietin-like 4 (ANGPTL4) plays an important role in the regulation of EGF-induced cancer metastasis. We showed that EGF-induced ANGPTL4 expression promoted anoikis resistance and cancer cell migration and invasion in HNSCC. In addition, depletion of ANGPTL4 inhibited EGF-induced cancer cell invasion. Autocrine production of EGF-induced ANGPTL4 regulated the expression of matrix metalloproteinases (MMPs). The induction of MMP-1 gene expression by ANGPTL4-activated integrin β1 signalling occurred through the AP-1 binding site in the MMP-1 gene promoter. Furthermore, down-regulation of MMP-1 impeded EGF- and recombinant ANGPTL4-enhanced HNSCC cell migration and invasion. Depletion of ANGPTL4 significantly blocked EGF-primed extravasation and metastatic seeding of tumour cells and MMP-1 expression in lungs. However, no effect of ANGPTL4 on tumour growth was observed. These results suggest that EGF-induced expression and autocrine production of ANGPTL4 enhances HNSCC metastasis via the up-regulation of MMP-1 expression. Inhibition of ANGPTL4 expression may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Anoikis; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Genes, jun; Head and Neck Neoplasms; Humans; Integrin beta1; Matrix Metalloproteinase 1; Neoplasm Metastasis; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Epidermal Growth Factor; Female; Humans; Lysophospholipids; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; RNA, Small Interfering; Signal Transduction; Sphingosine; Triple Negative Breast Neoplasms

Solid lipid-polymer hybrid nanocarrier (LPN) was previously reported to achieve high siRNA transfection efficiency and induce sustained RNAi-based chemosensitizing effect at cellular level. In this study, our objectives were to evaluate the in vivo biodistribution of LPNs in a prostate cancer model and determine the factors that potentially affect tumor penetration by LPNs. The LPN formulation with the highest transfection efficiency (64%) and stability was selected for the study. Mice bearing tumors of PC-3Mcells were treated with LPNs labeled with IR780 or AF647-siRNA. Near infrared imaging showed that LPNs achieved favorable in vivo biodistribution with high tumor/low organ ratios. LPN accumulation was also observed in liver metastatic tissue. Result of extravasation study confirmed that encapsulated siRNA molecules were able to escape into the tumor tissue at the extravascular area. When LPN levels in large (volume>750mm

Topics: Animals; Cell Line, Tumor; Docetaxel; Epidermal Growth Factor; Genetic Therapy; Humans; Lipids; Liver Neoplasms; Male; Mice; Nanomedicine; Nanoparticles; Neoplasm Metastasis; Neoplasm Transplantation; Polyethyleneimine; Prostatic Neoplasms; Random Allocation; RNA Interference; RNA, Small Interfering; Taxoids; Transfection

Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Base Sequence; Cell Differentiation; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation; Humans; Molecular Sequence Data; Neoplasm Metastasis; Phosphorylation; Prognosis; Proto-Oncogene Proteins; Signal Transduction; Stomach Neoplasms; Wnt Proteins; Wnt-5a Protein

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

Topics: Animals; C-Reactive Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Heterografts; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, SCID; Neoplasm Metastasis; NF-kappa B; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Serum Amyloid P-Component; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transcription Factor AP-1; Transfection

Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, has been reported to exhibit a wide range of physiological effects, including the potential effect against cancer. However, the anti-prostate cancer (PCa) efficacy of Lycorine remains unrevealed. In this context, we figured out Lycorine's anti-proliferative and anti-migratory properties for PCa treatment. Lycorine inhibited proliferation of various PCa cell lines, induced cell apoptosis and cell death. Here we showed that Lycorine decreased proliferation, migration, invasion, survival and EMT of prostate cancer cell lines. Subcutaneous and orthotopic xenotransplantations by ectopic implantation of the human hormone-refractory PC-3M-luc cells were used to confirm in vivo anticancer effects of Lycorine. Lycorine inhibited both growth and metastasis in multiple organs (liver, lung, kidney, spleen and bone) in vivo and improved mice survival. Lycorine prevented EGF-induced JAK/STAT signaling. Importantly, anti-cancer effects of Lycorine were dependent on STAT expression. We suggest that Lycorine is a potential therapeutic in prostate cancer.

Topics: Amaryllidaceae Alkaloids; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Phenanthridines; Prostatic Neoplasms; Signal Transduction; STAT3 Transcription Factor; Transplantation, Heterologous

The normal process of epithelial mesenchymal transition (EMT) is subverted by carcinoma cells to facilitate metastatic spread. Cancer cells rarely undergo a full conversion to the mesenchymal phenotype, and instead adopt positions along the epithelial-mesenchymal axis, a propensity we refer to as epithelial mesenchymal plasticity (EMP). EMP is associated with increased risk of metastasis in breast cancer and consequent poor prognosis. Drivers towards the mesenchymal state in malignant cells include growth factor stimulation or exposure to hypoxic conditions.. We have examined EMP in two cell line models of breast cancer: the PMC42 system (PMC42-ET and PMC42-LA sublines) and MDA-MB-468 cells. Transition to a mesenchymal phenotype was induced across all three cell lines using epidermal growth factor (EGF) stimulation, and in MDA-MB-468 cells by hypoxia. We used RNA sequencing to identify gene expression changes that occur as cells transition to a more-mesenchymal phenotype, and identified the cell signalling pathways regulated across these experimental systems. We then used inhibitors to modulate signalling through these pathways, verifying the conclusions of our transcriptomic analysis.. We found that EGF and hypoxia both drive MDA-MB-468 cells to phenotypically similar mesenchymal states. Comparing the transcriptional response to EGF and hypoxia, we have identified differences in the cellular signalling pathways that mediate, and are influenced by, EMT. Significant differences were observed for a number of important cellular signalling components previously implicated in EMT, such as HBEGF and VEGFA. We have shown that EGF- and hypoxia-induced transitions respond differently to treatment with chemical inhibitors (presented individually and in combinations) in these breast cancer cells. Unexpectedly, MDA-MB-468 cells grown under hypoxic growth conditions became even more mesenchymal following exposure to certain kinase inhibitors that prevent growth-factor induced EMT, including the mTOR inhibitor everolimus and the AKT1/2/3 inhibitor AZD5363.. While resulting in a common phenotype, EGF and hypoxia induced subtly different signalling systems in breast cancer cells. Our findings have important implications for the use of kinase inhibitor-based therapeutic interventions in breast cancers, where these heterogeneous signalling landscapes will influence the therapeutic response.

Topics: Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Everolimus; Female; Humans; Immunosuppressive Agents; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In the extracellular space, the gonadotropin-releasing hormone (GnRH) is metabolized by the zinc metalloendopeptidase EC3.4.24.15 (EP24.15) to form the pentapeptide, GnRH-(1-5). GnRH-(1-5) diverges in function and mechanism of action from GnRH in the brain and periphery. GnRH-(1-5) acts on the orphan G protein-coupled receptor 101 (GPR101) to sequentially stimulate epidermal growth factor (EGF) release, phosphorylate the EGF receptor (EGFR), and facilitate cellular migration. These GnRH-(1-5) actions are dependent on matrix metallopeptidase (MMP) activity. Here, we demonstrated that these GnRH-(1-5) effects are dependent on increased MMP-9 enzymatic activity in the Ishikawa and ECC-1 cell lines. Furthermore, the effects of GnRH-(1-5) mediated by GPR101 and the subsequent increase in MMP-9 enzymatic activity lead to an increase in cellular invasion. These results suggest that GnRH-(1-5) and GPR101 regulation of MMP-9 may have physiological relevance in the metastatic potential of endometrial cancer cells.

Topics: Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gonadotropin-Releasing Hormone; Humans; Matrix Metalloproteinase 9; Neoplasm Metastasis; Peptide Fragments; Phosphorylation; Receptors, G-Protein-Coupled; Signal Transduction

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.

Topics: Actins; Breast Neoplasms; Cell Adhesion; Cell Movement; Cytoskeletal Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Microfilament Proteins; Neoplasm Metastasis; Phosphorylation; Protein Isoforms; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Receptor Protein-Tyrosine Kinases; Signal Transduction

Sur8 (also known as Shoc2) is a Ras-Raf scaffold protein that modulates signaling through extracellular signal-regulated kinase (ERK) pathway. Although Sur8 has been shown to be a scaffold protein of the Ras-ERK pathway, its interaction with other signaling pathways and its involvement in tumor malignancy has not been reported. We identified that Sur8 interacts with the p110α subunit of phosphatidylinositol 3-kinase (PI3K), as well as with Ras and Raf, and these interactions are increased in an epidermal growth factor (EGF)- and oncogenic Ras-dependent manner. Sur8 regulates cell migration and invasion via activation of Rac and matrix metalloproteinases (MMPs). Interestingly, using inhibitors of MEK and PI3K we found Sur8 mediates these cellular behaviors predominantly through PI3K pathway. We further found that human metastatic melanoma tissues had higher Sur8 content followed by activations of Akt, ERK, and Rac. Lentivirus-mediated Sur8-knockdown attenuated metastatic potential of highly invasive B16-F10 melanoma cells indicating the role of Sur8 in melanoma metastasis. This is the first report to identify the role of scaffold protein Sur8 in regulating cell motility, invasion, and metastasis through activation of both ERK and PI3K pathways.

Topics: Animals; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma, Experimental; Mice; Neoplasm Metastasis; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; ras Proteins; Signal Transduction

Epithelial-to-mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors. However, the molecular mechanisms underlying this transition are poorly understood. In this study, we demonstrate that interferon regulatory factor 4 binding protein (IBP) regulates EMT and the motility of breast cancer cells through Rac1, RhoA and Cdc42 signaling pathways. We found that increased expression of IBP was associated with the progression of breast cancer and that IBP protein levels were significantly elevated in matched distant metastases. High IBP levels also predict shorter overall survival of breast cancer patients. Furthermore, the forced expression of IBP decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing IBP in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. IBP silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2). Moreover, we identified a role for IBP in endogenous EMT induced by epidermal growth factor (EGF) and deletion of IBP attenuated EGF receptor (EGFR) signaling in breast cancer cells. Furthermore, IBP regulates the migration, invasion and matrix metalloprotease production in breast cancer cells as well as actin cytoskeleton rearrangement and the activation of GTP-Rac1, GTP-RhoA and GTP-Cdc42. Taken together, our findings demonstrate an oncogenic property for IBP in promoting the metastatic potential of breast cancer cells.

Topics: Breast Neoplasms; Cadherins; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Homeodomain Proteins; Humans; Interferon Regulatory Factors; Neoplasm Invasiveness; Neoplasm Metastasis; rac1 GTP-Binding Protein; Repressor Proteins; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

Allyl isothiocyanate (AITC) has been found to present sources from consumed cruciferous vegetables. AITC is known to possess pharmacological and anticancer activities. The present study was designed to test the hypothesis that AITC suppressed the invasion and migration of epidermal growth factor (EGF)-stimulated HT29 cells and to elucidate the mechanisms for the antimetastatic abilities in vitro. The invasion and migration of EGF-stimulated HT29 cells were determined individually by Transwell cell invasion and wound-healing assays. Our results showed that AITC effectively inhibited both the invasive and migratory ability of HT29 cells. Furthermore, AITC downregulated the protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and mitogen-activated protein kinases (MAPKs) (p-JNK, p-ERK and p-p38) by western blot analysis in HT29 cells following EGF induction. Thus, the metastatic responses in AITC-treated HT29 cells after EGF stimulation were mediated by the MMP-2/-9 and MAPK signaling pathways. We also used gene expression microarrays to investigate the gene levels related to cell growth, G-protein coupled receptor, angiogenesis, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, transcription and translation, EGFR and PKB/mTOR signals. In summary, it is possible that AITC suppresses the invasion and migration of EGF-induced HT29 cells, resulting from MMP-2/-9 and MAPKs. Hence, AITC may be beneficial in the treatment of human colorectal adenocarcinoma in the future.

Topics: Adenocarcinoma; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Down-Regulation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Food Preservatives; Humans; Isothiocyanates; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Wound Healing

Epithelial-mesenchymal transition (EMT) is a crucial programme in cancer metastasis. Epidermal growth factor (EGF) is a key inducer of EMT, and Ezrin has an important role in this process. However, how Ezrin is activated and whether it mediates EGF-induced EMT in tongue squamous cell carcinomas (TSCCs) through activating NF-κB remains obscure.. We used two TSCC cell lines as a cell model to study invasion and EMT in vitro, and used nude mice xenografts model to evaluate metastasis of TSCC cells. Finally, we evaluated the level of pEzrin Tyr353, nuclear p65 and EMT markers in TSCC clinical samples.. Ezrin Tyr353 was phosphorylated through Akt (but not ERK1/2, ROCK1) pathway, and lead to the activation of NF-κB in EGF-treated TSCC cells. Akt and NF-κB inhibitors blocked EGF-induced EMT, and suppressed invasion and migration of TSCC cells. In vivo, silencing Ezrin significantly suppressed EGF-enhanced metastasis of TSCC xenografts. Finally, high levels of expression of pEzrin Tyr353, nuclear p65, vimentin and low level of expression of E-cadherin were correlated with cancer metastasis and poor patient prognosis.. Our data suggest that Akt/Ezrin Tyr353/NF-κB pathway regulates EGF-induced EMT and metastasis inTSCC, and Ezrin may serve as a therapeutic target to reverse EMT in tongue cancers and prevent TSCC progression.

Topics: Animals; Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cytoskeletal Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Metastasis; NF-kappa B; Proto-Oncogene Proteins c-akt; Signal Transduction; Tongue Neoplasms; Xenograft Model Antitumor Assays

TrkB mediates the effects of brain-derived neurotrophic factor (BDNF) in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR) signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC). TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.

Topics: Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Polarity; Epidermal Growth Factor; Humans; Lung Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; Protein-Tyrosine Kinases; Receptor, trkB; Signal Transduction

Approximately 70% of prostate cancer patients will develop bone metastasis in axial and other regions of the skeleton. Epidermal growth factor (EGF) generated from bone tissue contributes to prostate cancer metastasis. In a previous study, penta-O-galloyl-β-D-glucose (PGG) suppressed androgen-independent prostate cancer bone metastasis by transcriptionally repressing EGF-induced MMP-9 expression. This study utilized proteomics to analyze the effects of PGG in EGF-induced prostate cancer bone metastasis. This study showed that PGG suppressed EGF-induced eIF3i expression in PC-3 cells. By transfection of eIF3i shRNA, it was observed that reduced eIF3i expression suppressed the invasion of PC-3 cells in vitro. PGG reduced EGF-induced eIF3i expression through inhibition of the PI3K/AKT/mTOR pathway. Therefore, PGG may be able to be used as a potential new therapeutic drug for prostate cancer bone metastasis.

Topics: Bone Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Eukaryotic Initiation Factor-3; Gene Expression; Humans; Hydrolyzable Tannins; Male; Neoplasm Metastasis; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; TOR Serine-Threonine Kinases; Transfection

Inhalation of chemotherapeutic drugs directly into the lungs augments the drug exposure to lung cancers. The inhalation of free drugs however results in over exposure and causes severe adverse effect to normal cells. In the present study, epidermal growth factor (EGF)-modified gelatin nanoparticles (EGNP) was developed to administer doxorubicin (DOX) to lung cancers.. The EGNP released DOX in a sustained manner and effectively internalized in EGFR overexpressing A549 and H226 lung cancer cells via a receptor-mediated endocytosis. In vitro cytotoxicity assay showed that EGNP effectively inhibited the growth of A549 and H226 cells in a dose-dependent manner. In vivo biocompatibility study showed that both GNP and EGNP did not activate the inflammatory response and had a low propensity to cause immune response. Additionally, EGNP maintained a high therapeutic concentration in lungs throughout up to 24 h comparing to that of free drug and GNP, implying the effect of ligand-targeted tumor delivery. Mice treated with EGNP remarkably suppressed the tumor growth (~90% tumor inhibition) with 100% mice survival rate. Furthermore, inhalation of EGNP resulted in elevated levels of cleaved caspase-3 (apoptotic marker), while MMP-9 level significantly reduced comparing to that of control group.. Overall, results suggest that EGF surface-modified nanocarriers could be delivered to lungs via inhalation and controlled delivery of drugs in the lungs will greatly improve the therapeutic options in lung cancer therapy. This ligand-targeted nanoparticulate system could be promising for the lung cancer treatment.

Topics: Animals; Antineoplastic Agents; Caspase 3; Cell Line, Tumor; Doxorubicin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Neoplasm Metastasis; Xenograft Model Antitumor Assays

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-β (IFNβ) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNβ induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.

Topics: Adenocarcinoma; Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Histones; Humans; Interferon-beta; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Promyelocytic Leukemia Protein; RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factors; Transcriptional Activation; Tumor Suppressor Proteins; Up-Regulation

MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

Topics: Animals; Breast Neoplasms; Cell Growth Processes; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Mice, Inbred BALB C; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Protein Tyrosine Phosphatases, Non-Receptor; Receptor-Like Protein Tyrosine Phosphatases, Class 2; Signal Transduction; Transgenes

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Migration Assays; Cell Movement; Disease Progression; Epidermal Growth Factor; Female; Humans; Lysophospholipids; Neoplasm Metastasis; Phenotype; Tumor Stem Cell Assay

The causes for malignant progression of disseminated tumors and the reasons recurrence rates differ in women with different breast cancer subtypes are unknown. Here, we report novel mechanisms of tumor plasticity that are mandated by microenvironmental factors and show that recurrence rates are not strictly due to cell-intrinsic properties. Specifically, outgrowth of the same population of incipient tumors is accelerated in mice with triple-negative breast cancer (TNBC) relative to those with luminal breast cancer. Systemic signals provided by overt TNBCs cause the formation of a tumor-supportive microenvironment enriched for EGF and insulin-like growth factor-I (IGF-I) at distant indolent tumor sites. Bioavailability of EGF and IGF-I enhances the expression of transcription factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Combinatorial therapy with EGF receptor and IGF-I receptor inhibitors prevents malignant progression. These results suggest that plasticity and recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for patients with TNBC.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Transplantation; Receptor, IGF Type 1; Stromal Cells; Transcription Factors; Triple Negative Breast Neoplasms; Tumor Microenvironment

MET amplification as a mechanism of acquired resistance to EGF receptor (EGFR)-targeted therapies in non-small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by coexpression of ERBB3. EGFR-MET cross-talk was not direct, but occurred by a combined regulation of MET levels and intermediary signaling through mitogen-activated protein kinases (MAPK). In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAPK reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Finally, EGFR-MET signaling was enhanced in a highly metastatic EGFR-mutant cell subpopulation, compared with the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR-MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression.

Topics: Animals; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasm Metastasis; Oncogene Proteins v-erbB; Phosphorylation; Proto-Oncogene Proteins c-met; Receptor, ErbB-3

Epidermal growth factor (EGF)-induced EGFR tyrosine kinase receptor activation in cancer cell survival responses has become a strategic molecular-targeting clinical therapeutic intent, but the failures of these targeted approaches in the clinical setting demand alternate strategies. Here, we uncover a novel neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with GPCR neuromedin B, which is essential for EGF-induced receptor activation and cellular signaling. Neu1 and MMP-9 form a complex with EGFR on the cell surface. Tamiflu (oseltamivir phosphate), anti-Neu1 antibodies, broad range MMP inhibitor galardin (GM6001), neuromedin B GPCR specific antagonist BIM-23127, the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 and MMP-9 specific inhibitor dose-dependently inhibited Neu1 activity associated with EGF stimulated 3T3-hEGFR cells. Tamiflu, anti-Neu1 antibodies and MMP9i attenuated EGFR phosphorylation associated with EGF-stimulated cells. Preclinical data provide the proof-of-evidence for a therapeutic targeting of Neu1 with Tamiflu in impeding human pancreatic cancer growth and metastatic spread in heterotopic xenografts of eGFP-MiaPaCa-2 tumors growing in RAGxCγ double mutant mice. Tamiflu-treated cohort exhibited a reduction of phosphorylation of EGFR-Tyr1173, Stat1-Tyr701, Akt-Thr308, PDGFRα-Tyr754 and NFκBp65-Ser311 but an increase in phospho-Smad2-Ser465/467 and -VEGFR2-Tyr1175 in the tumor lysates from the xenografts of human eGFP-MiaPaCa-2 tumor-bearing mice. The findings identify a novel promising alternate therapeutic treatment of human pancreatic cancer.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Matrix Metalloproteinase 9; Mice; Neoplasm Metastasis; Neuraminidase; NIH 3T3 Cells; Oseltamivir; Pancreas; Pancreatic Neoplasms; Signal Transduction

ADF/cofilin proteins are key modulators of actin dynamics in metastasis and invasion of cancer cells. Here we focused on the roles of ADF and cofilin-1 individually in the development of polarized migration of rat mammary adenocarcinoma (MTLn3) cells, which express nearly equal amounts of each protein. Small interference RNA (siRNA) technology was used to knockdown (KD) the expression of ADF and cofilin-1 independently.. Either ADF KD or cofilin KD caused cell elongation, a reduction in cell area, a decreased ability to form invadopodia, and a decreased percentage of polarized cells after 180 s of epidermal growth factor stimulation. Moreover, ADF KD or cofilin KD increased the rate of cell migration and the time of lamellipodia protrusion but through different mechanisms: lamellipodia protrude more frequently in ADF KD cells and are more persistent in cofilin KD cells. ADF KD cells showed a significant increase in F-actin aggregates, whereas cofilin KD cells showed a significant increase in prominent F-actin bundles and increased cell adhesion. Focal adhesion area and cell adhesion in cofilin KD cells were returned to control levels by expressing exogenous cofilin but not ADF. Return to control rates of cell migration in ADF KD cells was achieved by expression of exogenous ADF but not cofilin, whereas in cofilin KD cells, expression of cofilin efficiently rescued control migration rates.. Although ADF and cofilin have many redundant functions, each of these isoforms has functional differences that affect F-actin structures, cell adhesion and lamellipodial dynamics, all of which are important determinants of cell migration.

Topics: Actin Cytoskeleton; Actins; Adenocarcinoma; Animals; Cell Adhesion; Cell Movement; Cofilin 1; Destrin; Epidermal Growth Factor; Female; Focal Adhesions; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Animal; Neoplasm Metastasis; Rats; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

Breast cancer progression is strongly linked to inflammatory processes, aggravating disease course. The impacts of the inflammatory cytokine TNF α on breast malignancy are not fully substantiated, and they may be affected by cooperativity between TNF α and other protumoral mediators. Here, we show that together with representatives of other important arms of the tumor microenvironment, estrogen (hormonal) and EGF (growth-supporting), TNF α potently induced metastasis-related properties and functions in luminal breast tumor cells, representing the most common type of breast cancer. Jointly, TNFα + Estrogen + EGF had a stronger effect on breast cancer cells than each element alone, leading to the following: (1) extensive cell spreading and formation of FAK/paxillin-enriched cellular protrusions; (2) elevated proportion of tumor cells coexpressing high levels of CD44 and β 1 and VLA6; (3) EMT and cell migration; (4) resistance to chemotherapy; (5) release of protumoral factors (CXCL8, CCL2, MMPs). Importantly, the tumor cells used in this study are known to be nonmetastatic under all conditions; nevertheless, they have acquired high metastasizing abilities in vivo in mice, following a brief stimulation by TNFα + Estrogen + EGF. These dramatic findings indicate that TNF α can turn into a strong prometastatic factor, suggesting a paradigm shift in which clinically approved inhibitors of TNFα would be applied in breast cancer therapy.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Survival; Disease Progression; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Estrogens; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Integrin beta1; MCF-7 Cells; Mice; Mice, Nude; Microscopy, Confocal; Neoplasm Metastasis; Neoplasm Transplantation; Spheroids, Cellular; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Metastatic breast adenocarcinomas display activation signatures for signaling pathways that trigger cell motility and tissue invasion. Here, we report that the adaptor protein transducer of Cdc42-dependent actin assembly-1 (Toca-1) is expressed in highly invasive breast cancers and regulates their metastatic phenotypes. We show that Toca-1 localizes to the filamentous actin-rich core of invadopodial protrusions actively degrading the extracellular matrix (ECM). Toca-1 colocalizes with Cortactin, and we show that this interaction is mediated by the SH3 domain of Toca-1. Stable knockdown (KD) of Toca-1 expression in MDA-MB-231 cells led to a significant defect in epidermal growth factor (EGF)-induced cell migration and invasion. Toca-1 KD cells also showed significant defects in EGF- and Src-induced ECM digestion and formation of invadopodial membrane protrusions. To test the role of Toca-1 in metastasis, we achieved stable Toca-1 KD in both human and rat metastatic breast adenocarcinoma cell lines. Orthotopic tumor xenografting of control and Toca-1 KD cells in natural-killer /B-/T-cell-deficient mice revealed a significant defect in spontaneous lung metastases with Toca-1 silencing in vivo. In contrast, no defects in primary tumor growth or lung seeding following tail vein injection of Toca-1 KD cells was observed, suggesting that Toca-1 functions at an early step in the dissemination of metastatic breast tumor cells. Taken together, our results identify Toca-1 as a proinvasive protein in breast adenocarcinoma and a potential therapeutic target to limit tumor metastasis.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Cortactin; Epidermal Growth Factor; Extracellular Matrix; Female; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Rats

Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.

Topics: Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Male; Neoplasm Metastasis; Neoplasm Proteins; Prostatic Neoplasms; Proteolysis; Signal Transduction; Transfection

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Topics: Animals; Carcinoma, Squamous Cell; Caveolin 1; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Enzyme Activation; Epidermal Growth Factor; Gene Knockdown Techniques; Humans; Keratin-18; Keratinocytes; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Serum; Skin Neoplasms; Transcription Factor AP-1

Since epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and in maintaining cancer stem cell properties, EMT is emerging as a therapeutic target for inhibiting the metastatic progression of cancer cells. 2'-Hydroxycinnamaldehyde (HCA) and its derivative, 2'-benzoyloxycinnamaldehyde, have recently been suggested as promising therapeutic candidates for cancer treatment. The purpose of this study is to investigate the anti-metastatic effect of HCA on breast cancer and the molecular mechanisms by which HCA regulates the transcriptional program during EMT. HCA induces epithelial reversion at nanomolar concentrations by suppressing Snail via the nuclear translocalization of GSK-3β, which results in the transcriptional upregulation of E-cadherin. HCA also activates the transcription factor KLF17, which suppresses Id-1, indicating that HCA inhibits EMT by multiple transcriptional programs. Further, HCA treatment significantly inhibits lung metastasis in a mouse orthotopic breast cancer model. This study demonstrates the anti-metastatic effect of the non-toxic natural compound HCA through attenuation of EMT in a breast cancer model.

Topics: Acrolein; Animals; Antineoplastic Agents; Benzoates; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Survival; Cinnamates; Disease Models, Animal; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Protein 1; MCF-7 Cells; Mice; Neoplasm Metastasis; Snail Family Transcription Factors; Transcription Factors; Transcriptional Activation; Wnt Signaling Pathway

Perioperative blood transfusion in pancreatic cancer patients is linked to decreased survival; however, a causal mechanism has not been determined. Previously we have shown that the plasma fraction of stored packed red blood cells (pRBCs) promotes pancreas cancer progression and associated morbidity. We hypothesize these untoward effects will be mitigated by use of a hemoglobin-based oxygen carrier (HBOC).. Cytokines and growth factors were measured in the plasma fraction from stored pRBCs and in an HBOC via cytokine array followed by formal enzyme-linked immunosorbent assay (ELISA). In an immunocompetent murine model, pancreas cancer progression was determined in vivo by bioluminescence, tumor weight, and number of metastases.. Elevated levels of epidermal growth factor (EGF), platelet-derived growth factor BB (PDGF-BB), and regulated upon activation, normal T cell expressed and secreted (RANTES) were present in the plasma fraction of stored pRBCs, but were not found in the HBOC. Intravenous delivery of plasma fraction to mice with pancreatic cancer resulted in increased bioluminescence activity compared with mice that received HBOC. Metastatic events and pancreatic primary tumor weights were significantly higher in animals receiving plasma fraction from stored pRBCs compared with animals receiving HBOC.. Intravenous receipt of the acellular plasma fraction of stored pRBCs promotes pancreatic cancer progression in an immunocompetent mouse model. These untoward events are mitigated by use of an HBOC.

Topics: Analysis of Variance; Animals; Becaplermin; Blood Substitutes; Chemokine CCL5; Cytokines; Disease Progression; Epidermal Growth Factor; Erythrocyte Transfusion; Hemoglobins; Humans; Mice; Neoplasm Metastasis; Pancreatic Neoplasms; Plasma; Protein Array Analysis; Proto-Oncogene Proteins c-sis

Piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) is a polyphenol detected in grapes, red wine and Rheum undulatum; it has also been demonstrated to exert anticarcinogenic effects. In this study, in order to determine whether piceatannol inhibits the lung metastasis of prostate cancer cells, MAT-Ly-Lu (MLL) rat prostate cancer cells expressing luciferase were injected into the tail veins of male nude mice. The oral administration of piceatannol (20 mg/kg) significantly inhibited the accumulation of MLL cells in the lungs of these mice. In the cell culture studies, piceatannol was demonstrated to inhibit the basal and epidermal growth factor (EGF)-induced migration and invasion of DU145 cells, in addition to the migration of MLL, PC3 and TRAMP-C2 prostate cancer cells. In DU145 cells, piceatannol attenuated the secretion and messenger RNA levels of matrix metalloproteinase-9, urokinase-type plasminogen activator (uPA) and vascular endothelial growth factor (VEGF). Piceatannol increased the protein levels of tissue inhibitor of metalloproteinase-2 in a concentration-dependent fashion. Additionally, piceatannol inhibited the phosphorylation of signal transducer and activator of transcription (STAT) 3. Furthermore, piceatannol effected reductions in both basal and EGF-induced interleukin (IL)-6 secretion. An IL-6 neutralizing antibody inhibited EGF-induced STAT3 phosphorylation and EGF-stimulated migration of DU145 cells. Interleukin-6 treatment was also shown to enhance the secretion of uPA and VEGF, STAT3 phosphorylation and the migration of DU145 cells; these increases were suppressed by piceatannol. These results demonstrate that the inhibition of IL-6/STAT3 signaling may constitute a mechanism by which piceatannol regulates the expression of proteins involved in regulating the migration and invasion of DU145 cells.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation; Humans; Interleukin-6; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Phosphorylation; Prostatic Neoplasms; Signal Transduction; STAT3 Transcription Factor; Stilbenes; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A

We have recently shown that the adaptor protein p140Cap regulates tumor properties in terms of cell motility and growth. Here, by using the highly metastatic rat adenocarcinoma cell line MTLn3-epidermal growth factor receptor (EGFR), we assess the role of p140Cap in metastasis formation. Orthotopic transplantation of MTLn3-EGFR cells over-expressing p140Cap in Rag2(-/-)γ(c)(-/-) mice resulted in normal primary tumor growth compared with the controls. Strikingly, p140Cap over-expression causes an 80% inhibition in the number of lung metastases. p140Cap over-expressing cells display a 50% reduction in directional cell migration, an increased number and size of focal adhesions, and a strong impairment in the ability to invade in a 3D matrix. p140Cap over-expression affects EGFR signaling and tyrosine phosphorylation of cortactin in response to EGF stimulation. Intriguingly, p140Cap associates with cortactin via interaction with its second proline-rich domain to the cortactin SH3 domain. The phosphomimetic cortactin tyrosine 421 mutant rescues migration and invasive properties in p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility.

Topics: Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Cortactin; Epidermal Growth Factor; ErbB Receptors; Green Fluorescent Proteins; HEK293 Cells; Humans; Immunohistochemistry; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Microscopy, Fluorescence, Multiphoton; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Protein Binding; Rats; RNA Interference; Transplantation, Heterologous

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Carcinoma; Colorectal Neoplasms; Epidermal Growth Factor; Gastroenterology; Humans; Medical Oncology; Molecular Targeted Therapy; Neoplasm Metastasis

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show dramatic antitumor activity in a subset of patients with non-small cell lung cancer who have an active mutation in the epidermal growth factor receptor (EGFR) gene. On the other hand, some lung cancer patients with wild type EGFR also respond to EGFR-TKIs, suggesting that EGFR-TKIs have an effect on host cells as well as tumor cells. However, the effect of EGFR-TKIs on host microenvironments is largely unknown. A multiple organ metastasis model was previously established in natural killer cell-depleted severe combined immunodeficient mice using human lung cancer cells. This model was used to investigate the therapeutic efficacy of erlotinib, an EGFR-TKI, on multiple organ metastases induced by human small cell lung cancer cells (SBC-5 cells) that did not express EGFR. Although erlotinib did not have any effect on the proliferation of SBC-5 cells in vitro, it significantly suppressed bone and lung metastases in vivo, but not liver metastases. An immunohistochemical analysis revealed that, erlotinib significantly suppressed the number of osteoclasts in bone metastases, whereas no difference was seen in microvessel density. Moreover, erlotinib inhibited EGF-induced receptor activator of nuclear factor kappa-B expression in an osteoblastic cell line (MC3T3-E1 cells). These results strongly suggested that erlotinib prevented bone metastases by affecting host microenvironments irrespective of its direct effect on tumor cells.

Topics: Animals; Bone Neoplasms; Carcinoma, Small Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Male; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Osteoblasts; Osteoclasts; Protein Kinase Inhibitors; Quinazolines; RANK Ligand

The presence of lymph node metastases is one of the most important prognostic indicators in head and neck squamous cell carcinomas (HNSCCs). An alteration of the E-cadherin-catenins complex and EGFR is essential for the invasiveness of cancer cells. Caveolin-1, the major structural protein of the caveolae, represents a scaffolding molecule for several signaling proteins including EGFR. Although caveolin-1 has been shown to play a role in inducing the invasive phenotype of cancer cells, its role appears to be cell-type specific and for some tumors it has not been defined yet. In this study we used 57 HNSCC specimens to investigate whether the abnormal expression of caveolin-1 was associated with the derangement of the E-cadherin-catenins complex and with the overexpression of ErbB receptors. We demonstrate that in HNSCCs caveolin-1 overexpression is associated with the simultaneous abnormal expression of at least one member of the E-cadherin/α-β catenins complex and multiple ErbB receptors as well as with lymph node metastases. We also demonstrate that chronic stimulation of a human hypopharyngeal carcinoma cell line (FaDu) with EGF induced the internalization of β-catenin and caveolin-1 and their co-localization with EGFR. Moreover, EGF treatment induced an increased physical interaction between EGFR/β-catenin/caveolin-1 and between E-cadherin/β-catenin/caveolin-1. These molecular events were associated with an increased directional motility of FaDu cells in vitro. These findings may provide new insight into the biology of HNSCC progression and help to identify subgroups of primary HNSCCs with a more aggressive behavior.

Topics: alpha Catenin; beta Catenin; Cadherins; Carcinoma, Squamous Cell; Caveolin 1; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Hypopharyngeal Neoplasms; Lymph Nodes; Neoplasm Invasiveness; Neoplasm Metastasis; Receptor, ErbB-2

Metastasis is the major cause of morbidity and mortality from breast cancer. Cell motility and chemotaxis play important roles in the metastatic cascade of cancer cells. Protein kinase C ζ (PKCζ) mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the current study, we investigated the inhibitory effect of a compound called J-4 targeting PKCζ. J-4 was tested with inhibitory concentration (IC(50)) of 10 µmol/l using a Z'-LYTE™ Kinase Assay-Ser/Thr 7 Peptide Kit. Our results show that J-4 inhibited spontaneous migration and epidermal growth factor (EGF)-induced chemotaxis of human breast cancer cell MDA-MB-231. Through an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the drug designated as J-4 had no obvious cytotoxicity in vitro. Meanwhile, in the presence of J-4, the cells showed defects in EGF-induced actin polymerization and adhesion. Furthermore, J-4 dampened EGF-induced phosphorylation and recycling of cofilin. Taken together, our data demonstrate that J-4 is a new and typical inhibitor that blocks the PKCζ pathway. Moreover, a better understanding of the mechanism of action of J-4 may provide a novel medical therapeutic strategy for cancer treatment that would block metastasis, thereby reducing the proliferation and dissemination of cancer cells and increasing patient survival.

Topics: Actins; Amidines; Antineoplastic Agents; Benzene Derivatives; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Migration Inhibition; Cell Movement; Cell Proliferation; Chemotaxis; Epidermal Growth Factor; Humans; Neoplasm Metastasis; Protein Kinase C

Receptor tyrosine kinases and integrins play an essential role in tumor cell invasion and metastasis. We previously showed that EGF and other growth factors induce human carcinoma cell invasion and metastasis mediated by integrin αvβ5 that is prevented by Src blockade. MUC1, a transmembrane glycoprotein, is expressed in most epithelial tumors as a heterodimer consisting of an extracellular and a transmembrane subunit. The MUC1 cytoplasmic domain of the transmembrane subunit (MUC1.CD) translocates to the nucleus where it promotes the transcription of a metastatic gene signature associated with epithelial to mesenchymal transition. Here, we demonstrate a requirement for MUC1 in carcinoma cell metastasis dependent on EGFR and Src without affecting primary tumor growth. EGF stimulates Src-dependent MUC1 cleavage and nuclear localization leading to the expression of genes linked to metastasis. Moreover, expression of MUC1.CD results in its nuclear localization and is sufficient for transcription of the metastatic gene signature and tumor cell metastasis. These results demonstrate that EGFR and Src activity contribute to carcinoma cell invasion and metastasis mediated by integrin αvβ5 in part by promoting proteolytic cleavage of MUC1 and highlight the ability of MUC1.CD to promote metastasis in a context-dependent manner. Our findings may have implications for the use and future design of targeted therapies in cancers known to express EGFR, Src, or MUC1.

Topics: Animals; Carcinoma; Cell Line, Tumor; Chick Embryo; CSK Tyrosine-Protein Kinase; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Mucin-1; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Receptors, Vitronectin; Signal Transduction; src-Family Kinases

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.

Topics: Analysis of Variance; Blotting, Western; Cell Line, Tumor; Cell Movement; Cobra Cardiotoxin Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Neoplasm Metastasis; Phosphorylation; Quinazolines; Signal Transduction; Tyrphostins

Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined.. We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration.. Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Analysis of Variance; Butadienes; Carcinoma, Hepatocellular; Cell Movement; Epidermal Growth Factor; Guanine Nucleotide Exchange Factors; Hep G2 Cells; Humans; Immunoblotting; In Vitro Techniques; Microscopy, Fluorescence; Mutation, Missense; Neoplasm Metastasis; Nitriles; rac1 GTP-Binding Protein; RNA, Small Interfering; Signal Transduction

In this study, we investigated the role of the kallikrein-kinin-system in head and neck squamous cell carcinoma (HNSCC) and its implication on tumour survival, invasion, migration and response to radiotherapy.. The expression of BKB2R was studied in a series of 180 tumour samples to determine the functional significance of BKB2R in HNSCC. Additionally, four different HNSCC cell lines were treated with an irradiation dose of 8Gy following bradykinin receptor stimulation or blockage. Tumour cell survival was tested using a colony formation assay. The invasive potential of tumour cells was assessed using Matrigel invasion chambers, the cells' ability to migrate was determined with a wound-healing assay. To examine the biochemical activation of BKB2R, the epidermal growth factor receptor (EGFR) and its downstream pathways, western blot analyses were conducted.. Immunohistochemistry revealed an over-expression of BKB2R in HNSCC tumour cells in comparison to normal peritumoural tissue. Blocking the BKB2R at irradiated tumour cells led to a reduced response to radiotherapy of tumour cells and led to an activation of the EGFR and its downstream pathways, known mediators of tumour cell survival, migration and invasion. Bradykinin stimulation also resulted in a better tumour cell survival, but these effects were achieved via an EGFR-independent signalling.. Our results demonstrate that the kallikrein-kinin-system is involved in survival, invasion and migration of HNSCC cells.

Topics: Adult; Aged; Blotting, Western; Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Line, Tumor; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Kallikrein-Kinin System; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction

Versican is detected in the interstitial tissues at the invasive margins of breast carcinoma, is predictive of relapse, and negatively impacts overall survival rates. The versican G3 domain is important in breast cancer cell growth, migration and bone metastasis. However, mechanistic studies evaluating versican G3 enhanced breast cancer bone metastasis are limited.. A versican G3 construct was exogenously expressed in the 66c14 and the MC3T3-E1 cell line. Cells were observed through light microscopy and viability analyzed by Coulter Counter or determined with colorimetric proliferation assays. The Annexin V-FITC apoptosis detection kit was used to detect apoptotic activity. Modified Chemotactic Boyden chamber migration invasion assays were applied to observe tumor migration and invasion to bone stromal cells and MC3T3-E1 cells. Alkaline phosphatase (ALP) staining and ALP ELISA assays were performed to observe ALP activity in MC3T3-E1 cells.. In the four mouse breast cancer cell lines 67NR, 66c14, 4T07, and 4T1, 4T1 cells expressed higher levels of versican, and showed higher migration and invasion ability to MC3T3-E1 cells and primary bone stromal cells. 4T1 conditioned medium (CM) inhibited MC3T3-E1 cell growth, and even lead to apoptosis. Only 4T1 CM prevented MC3T3-E1 cell differentiation, noted by inhibition of alkaline phosphatase (ALP) activity. We exogenously expressed a versican G3 construct in a cell line that expresses low versican levels (66c14), and observed that the G3-expressing 66c14 cells showed enhanced cell migration and invasion to bone stromal and MC3T3-E1 cells. This observation was prevented by selective EGFR inhibitor AG1478, selective MEK inhibitor PD 98059, and selective AKT inhibitor Triciribine, but not by selective JNK inhibitor SP 600125. Versican G3 enhanced breast cancer cell invasion to bone stromal cells or osteoblast cells appears to occur through enhancing EGFR/ERK or AKT signaling. G3 expressing MC3T3-E1 cells showed inhibited cell growth and cell differentiation when cultured with TGF-β1 (1 ng/ml), and expressed enhanced cell apoptosis when cultured with TNF-α (2 ng/ml). Enhanced EGFR/JNK signaling appears to be responsible for G3 enhanced osteoblast apoptosis and inhibited osteoblast differentiation. Whereas repressed expression of GSK-3β (S9P) contributes to G3 inhibited osteoblast growth. Versican G3 functionality was dependent on its EGF-like motifs. Without the structure of EGF-like repeats, the G3 domain would not confer enhancement of tumor cell migration and invasion to bone with concordant inhibition of osteoblast differentiation and promotion of osteoblast apoptosis.. Versican enhances breast cancer bone metastasis not only through enhancing tumor cell mobility, invasion, and survival in bone tissues, but also by inhibiting pre-osteoblast cell growth, differentiation, which supply favorable microenvironments for tumor metastasis.

Topics: Amino Acid Motifs; Animals; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Culture Media, Conditioned; Epidermal Growth Factor; Female; Gene Expression; Humans; Mice; Models, Biological; Neoplasm Metastasis; Osteoblasts; Protein Interaction Domains and Motifs; Versicans

ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70-100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6β4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6β4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6β4 and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Humans; Integrin alpha6beta4; Lapatinib; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasm Metastasis; Nitriles; Phosphorylation; Quinazolines; Receptor, ErbB-2; Tetraspanin 24; Transendothelial and Transepithelial Migration

The transmembrane mucin MUC1 is overexpressed in most ductal carcinomas, and its overexpression is frequently associated with metastatic progression. MUC1 can drive tumor initiation and progression via interactions with many oncogenic partners, including β-catenin, the EGF receptor (EGFR) and Src. The decoy peptide protein transduction domain MUC1 inhibitory peptide (PMIP) has been shown to inhibit the tumor promoting activities of MUC1 in breast and lung cancer, including cell growth and invasion, and its usage suppresses metastatic progression in mouse models of breast cancer. To further characterize the reduced metastasis observed upon PMIP treatment, we conducted motility assays and observed that PMIP inhibits cell motility of breast cancer cells. To determine the mechanism by which PMIP inhibits motility, we evaluated changes in global gene transcription upon PMIP treatment, and identified a number of genes with altered expression in response to PMIP. Among these genes is the metastatic mediator, c-Met, a transmembrane tyrosine kinase that can promote cell scattering, migration, and invasion. To further investigate the role of c-Met in MUC1-dependent metastatic events, we evaluated the effects of MUC1 expression and EGFR activation on breast cancer cell scattering, branching, and migration. We found that MUC1 strongly promoted all of these events and this effect was further amplified by EGF treatment. Importantly, the effect of MUC1 and EGF on these phenotypes was dependent upon c-Met activity. Overall, these results indicate that PMIP can block the expression of a key metastatic mediator, further advancing its potential use as a clinical therapeutic.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease Progression; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mucin-1; Neoplasm Metastasis; Peptides; Proto-Oncogene Proteins c-met; Transcription, Genetic

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid Precursor Protein Secretases; beta-Cyclodextrins; Cell Line, Tumor; Cell Movement; Cholesterol; Epidermal Growth Factor; Glioma; Humans; Hyaluronan Receptors; Hyaluronic Acid; Membrane Microdomains; Membrane Proteins; Neoplasm Metastasis; Neoplasm Proteins

The cause of most cancer deaths is incurable dissemination of cancer cells into vital organs. Current systemic therapies for disseminated cancers provide limited efficacy and are often accompanied by toxic side effects. We have recently shown that local application of epidermal growth factor receptor (EGFR)-targeted polyinosine-cytosine (polyIC) eradicates preestablished EGFR-overexpressing tumors. Here we show for the first time the high efficiency of systemic application of polyIC/melittin-polyethyleneimine-polyethyleneglycol-EGF (polyIC/MPPE) in combination with human immune cells.. Cancer-targeted activation of immune cells was examined in vitro and in vivo following transfection with polyIC/MPPE. The therapeutic efficiency of the strategy was then examined on disseminated EGFR-overexpressing tumors grown in severe combined immunodeficient (SCID) mice.. Intravenous delivery of polyIC/MPPE followed by intraperitoneal injection of peripheral blood mononuclear cells induced the complete cure of SCID mice with preestablished disseminated EGFR-overexpressing tumors, with no adverse toxic effects. The immune cells and the cytokines they produce are localized to the tumor site of the treated animal and contribute decisively to the demise of the tumor cells. The immune system homes to the tumors, due to the chemokines produced by the internalized polyIC.. The EGFR-homing vector loaded with polyIC can be used to treat and possibly cure patients with disseminated EGFR-overexpressing tumors. The possibility of adopting this strategy to treat other tumors that express a protein capable of ligand induced internalization is discussed.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Melitten; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasms, Experimental; Poly I-C; Polyethylene Glycols; Polyethyleneimine; RNA, Double-Stranded; Xenograft Model Antitumor Assays

MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.

Topics: Animals; Butadienes; Carcinoma, Pancreatic Ductal; Cell Cycle; Cell Growth Processes; Epidermal Growth Factor; Humans; Intermediate Filament Proteins; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Mucin-1; Neoplasm Metastasis; Nerve Tissue Proteins; Nestin; Nitriles; Pancreatic Neoplasms; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Tubulin

Expression of ID1 (inhibitor of differentiation) has been correlated with the progression of a variety of cancers, but little information is available on its role in non-small cell lung cancer (NSCLC). Here we show that ID1 is induced by nicotinic acetylcholine receptor (nAChR) and epidermal growth factor receptor (EGFR) signaling in a panel of NSCLC cell lines and primary cells from the lung. ID1 induction was Src dependent and mediated through the α7 subunit of nAChR; transfection of K-Ras or EGFR to primary cells induced ID1. ID1 depletion prevented nicotine- and EGF-induced proliferation, migration, and invasion of NSCLC cells and angiogenic tubule formation of human microvascular endothelial cells from lungs (HMEC-Ls). ID1 could induce the expression of mesenchymal markers such as vimentin and fibronectin by downregulating ZBP-89, a zinc finger repressor protein. ID1 levels were elevated in tumors from mice that were exposed to nicotine. Further, human lung tissue microarrays (TMAs) showed elevated levels of ID1 in NSCLC samples, with maximal levels in metastatic lung cancers. Quantitative reverse transcription-PCR (RT-PCR) performed on patient lung tumors showed that ID1 levels were elevated in advanced stages of NSCLC and correlated with elevated expression of vimentin and fibronectin, irrespective of smoking history.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Progression; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Protein 1; Lung; Lung Neoplasms; Neoplasm Metastasis; Neovascularization, Pathologic; Nicotine; Nicotinic Agonists; Protein Subunits; Receptors, Nicotinic; RNA, Small Interfering; Signal Transduction; src-Family Kinases; Transcription Factors; Vimentin

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.

Topics: Bacterial Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Migration Assays; Cell Movement; Collagen; Drug Combinations; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Gene Knockdown Techniques; Genes, Neoplasm; Humans; Laminin; MAP Kinase Signaling System; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proteoglycans; RNA Interference; Streptolysins

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Estrogens; Female; Fibronectins; Humans; Lymphatic Metastasis; Matrix Metalloproteinases; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Neoplasm Metastasis

Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

Topics: Animals; Calcium; Calcium Channels; Cell Cycle; Cell Movement; Cell Proliferation; Disease Models, Animal; Epidermal Growth Factor; Female; Focal Adhesions; Humans; Membrane Proteins; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; ORAI1 Protein; Signal Transduction; Stromal Interaction Molecule 1; Treatment Outcome; Uterine Cervical Neoplasms

ARF6 GTPase is an important regulator of membrane trafficking and actin-based cytoskeleton dynamics active at the leading edge of migrating cells. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Our recent proteomic analyses of ARF6 effectors have identified a novel ARF6 GTPase-activating protein, ACAP4, essential for EGF-induced cell migration. However, molecular mechanisms underlying ACAP4-mediated cell migration have remained elusive. Here, we show that ACAP4 regulates integrin β1 dynamics during EGF-stimulated cell migration by interaction with Grb2. Our biochemical study shows that EGF stimulation induces phosphorylation of tyrosine 733, which enables ACAP4 to bind Grb2. This interaction of ACAP4 with Grb2 regulates integrin β1 recycling to the plasma membrane. Importantly, knockdown of ACAP4 by siRNA or overexpression of ACAP4 decreased recycling of integrin β1 to the plasma membrane and reduced integrin-mediated cell migration. Taken together, these results suggest a novel function for ACAP4 in the regulation of cell migration through controlling integrin β1 dynamics.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Animals; Cell Membrane; Cell Movement; Chlorocebus aethiops; COS Cells; Cytoskeletal Proteins; Cytoskeleton; Epidermal Growth Factor; GRB2 Adaptor Protein; GTPase-Activating Proteins; HeLa Cells; Humans; Integrin beta1; Integrins; Neoplasm Metastasis; Phosphorylation; Tyrosine

Teratocarcinoma-derived growth factor 1 (TDGF1) is a member of the epidermal growth factor-cripto FRL1 cryptic protein family and is involved in the activation of several different signaling pathways during embryonic development and cellular transformation. Previous reports show that TDGF1 regulates the activation of several signaling pathways and controls cellular transformation in embryonic status, whereas its significance in colorectal cancer (CRC) is not yet fully understood. The present study comprised 55 patients who underwent surgery for CRC, as well as two cell lines derived from human CRC. The correlation of gene expression with clinical parameters in patients was assessed. The biological significance of TDGF1 expression was evaluated by knockdown experiments in the cell lines. Seventeen of 55 (30.9%) cases exhibited a higher TDGF1 expression in cancerous regions than in marginal non-cancerous regions. Patients with high TDGF1 expression were statistically susceptible to a recurrence of the disease, and showed poorer disease-free survival than those with low expression. The assessment of TDGF1 knock-down in the 2 cell lines demonstrated that the siRNA inhibition resulted in a statistically significant reduction in cell growth and invasion. In conclusion, the present data strongly suggest the usefulness of TDGF1 as a predictive marker for metachronous metastasis in CRC patients.

Topics: Aged; Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; Disease-Free Survival; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; RNA, Messenger; Signal Transduction

Identification of molecular mechanisms responsible for brain metastatic breast cancer (BMBC) is imperative to develop novel therapies. However, current understanding of the molecular circuitry that governs BMBC dissemination remains fragmentary. Heparanase (HPSE) is the only functional mammalian endoglycosidase whose activity correlates with cancer metastasis, angiogenesis, and the reduced postoperative survival of cancer patients, making it an active target for anticancer therapeutics. We hypothesized that human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) activation promotes HPSE function in human BMBC. To address this, we examined HPSE content, activity, and intracellular trafficking in a HER2/EGFR-expressing BMBC model system and show that HPSE is present, functional, and correlates with HER2 status. Further, we showed that EGF induced nucleolar translocation of HPSE in these cells in a dose- and time-dependent manner upon activation of HER2/EGFR. Knockdowns of HER2/EGFR by small interference RNA abolished EGF-induced HPSE nucleolar translocalization. It was also noted that nucleolar HPSE modulates DNA topoisomerase I (Topo I), an enzyme that is highly present in nucleoli, essential for DNA replication and transcription in a variety of tumors, and inhibited by heparan sulfate. Evidence is provided that HPSE can regulate Topo I activity, which subsequently affects BMBC cell proliferation. Finally, we showed that the nucleolar presence of HPSE with Topo I colocalization is detected only in HER2-overexpressing BMBC patient specimens. Altogether, these findings support the notion that HPSE is a critical downstream target of HER2 mechanisms driving BMBC and is potentially relevant for BMBC therapeutic interventions.

Topics: Active Transport, Cell Nucleus; Animals; Brain Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Nucleolus; DNA Topoisomerases, Type I; Down-Regulation; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Glucuronidase; Humans; Mice; Mice, Nude; Models, Biological; Neoplasm Metastasis; Protein Transport; Receptor, ErbB-2; RNA Interference; Signal Transduction

The diagnosis of multiple primary lung cancer is sometimes difficult when multiple lung tumors with the same histologic type are identified. We now present a case of synchronous double primary lung adenocarcinomas (one in the right upper lobe and another in the right middle lobe) diagnosed based on mutational analysis of the epidermal growth factor receptor (EGFR) gene, although clinico-pathological findings suggested the diagnosis of intrapulmonary metastasis. After complete resection, pathological sections revealed the similar pathological features of two adenocarcinomas and unexpected subcarinal nodal metastasis. As the L858R mutation within exon 21 of the EGFR gene was identified in the middle-lobe tumor and the subcarinal node but not in the upper-lobe tumor, we diagnosed as double primary cancers. Local mediastinal recurrence after operation has been well-controlled with administration of gefitinib, a EGFR-tyrosine kinase inhibitor, and mutational analysis of the EGFR gene provided important information not only in the diagnosis of double primary cancers but also in decision-making of selection of chemotherapeutic agent.

Topics: Adenocarcinoma; Aged; Diagnosis, Differential; DNA Mutational Analysis; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Multiple Primary; Pneumonectomy; Quinazolines; Radiography, Thoracic

Deciphering the molecular basis of esophageal cancer metastasis requires adequate experimental models. Epithelial-mesenchymal transition (EMT) is the hallmark of tumor metastasis. As a promoter of the malignant progression of esophageal cancer, epidermal growth factor (EGF) has been shown to induce EMT in several cell lines. In this study we examined the effects of EGF on esophageal carcinoma EC109 cells. We found that EGF at high concentration induced the cells to undergo morphological change, exhibit higher invasive and metastatic potential, as well as change in the expression of lineage markers. This EMT model might facilitate mechanistic studies of esophageal cancer metastasis.

Topics: Cell Dedifferentiation; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Esophageal Neoplasms; Gelatin; Humans; Keratins; Mesoderm; Neoplasm Invasiveness; Neoplasm Metastasis; Recombinant Proteins; Vimentin; Wound Healing

Ewing's sarcoma family of tumours (ESFT) is a malignant small round-cell tumour of the bone and soft tissues. It is characterised by a strong tendency to invade and form metastases. The microenvironment of the bone marrow is a large repository for many growth factors, including the basic fibroblast growth factor (bFGF). However, the role of bFGF in the invasive and metastatic phenotype of ESFT has not been investigated.. The motility and invasion of ESFT cells were assessed by a wound-healing assay, chemotaxis assay, and invasion assay. The expression and activation of FGF receptors (FGFRs) in ESFT cell lines and clinical samples were detected by RT-PCR, western blotting, and immunohistochemistry. The morphology of ESFT cells was investigated by phase-contrast microscopy and fluorescence staining for actin. Activation of Rac1 was analysed by a pull-down assay.. bFGF strongly induced the motility and invasion of ESFT cells. Furthermore, FGFR1 was found to be expressed and activated in clinical samples of ESFT. Basic FGF-induced cell motility was mediated through the FGFR1-phosphatidylinositol 3-kinase (PI3K)-Rac1 pathway. Conditioned medium from bone marrow stromal cells induced the motility of ESFT cells by activating bFGF/FGFR1 signalling.. The bFGF-FGFR1-PI3K-Rac1 pathway in the bone microenvironment may have a significant role in the invasion and metastasis of ESFT.

Topics: Bone Marrow; Cell Line, Tumor; Cell Movement; Cytoskeleton; Enzyme Activation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; rac1 GTP-Binding Protein; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 3; Recombinant Proteins; Sarcoma, Ewing; Wound Healing

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis.

Topics: Animals; Binding Sites; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 2; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Serine; Signal Transduction; Transfection; Versicans

The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development.. To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice.. Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching.. Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.

Topics: Animals; Antigens, Polyomavirus Transforming; Cytoskeletal Proteins; Disease Progression; Epidermal Growth Factor; Female; Gene Expression; Lung Neoplasms; Macrophages; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, SCID; Mice, Transgenic; Microfilament Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; Polymerase Chain Reaction; Tumor Microenvironment

Isoliquiritigenin (ISL, 4,2',4'-trihydroxychalcone), which is found in licorice, shallot and bean sprouts, is a potent antioxidant with anti-inflammatory and anti-carcinogenic effects. The purpose of this study was to investigate the effects of ISL treatment on the migration, invasion and adhesion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0-20 micromol/L ISL with or without 10 microg/L epidermal growth factor (EGF). ISL inhibited basal and EGF-induced cell migration, invasion and adhesion dose dependently. ISL decreased EGF-induced secretion of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF), but increased TIMP-2 secretion in a concentration-dependent manner. In addition, ISL decreased the protein levels of integrin-alpha2, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), and mRNA levels of uPA, MMP-9, VEGF, ICAM and integrin-alpha2. Furthermore, basal and EGF-induced activator protein (AP)-1 binding activity and phosphorylation of Jun N-terminal kinase (JNK), c-Jun and Akt were decreased after ISL treatment. However, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase was not altered. The JNK inhibitor SP600125 inhibited basal and EGF-induced secretion of uPA, VEGF, MMP-9 and TIMP-1, as well as AP-1 DNA binding activity and cell migration. These results provide evidence for the role of ISL as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of prostate cancer cells. The inhibition of JNK/AP-1 signaling may be one of the mechanisms by which ISL inhibits cancer cell invasion and migration.

Topics: Androgens; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Movement; Chalcones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Humans; JNK Mitogen-Activated Protein Kinases; Male; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Tissue Inhibitor of Metalloproteinase-2; Transcription Factor AP-1; Vascular Endothelial Growth Factors

By using proteomic technology, the authors previously observed the substantial down-regulation of mammary serine protease inhibitor (maspin) in esophageal squamous cell carcinoma and metastases. In the current study, they examined the effects of maspin re-expression in a maspin-null esophageal cancer cell line EC109 and also investigated the underlying mechanism.. A cell line with stable maspin expression was established. An epithelial growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) model was used to mimic some aspects of the metastatic process in vitro. The effects of maspin reintroduction on EGF-induced EMT and cell growth characteristics were evaluated. Comparative proteomic analysis of transfected cells versus parental cells was then performed to explore the potential mechanism.. The introduction of maspin into EC109 cells was able to inhibit EGF-induced EMT and altered cell growth characteristics, including the serum dependence, proliferative response to EGF stimulation, and colony formation ability in soft agar, indicating a conversion from a malignant phenotype to a benign phenotype. Proteomic analysis revealed a significant down-regulation of a group of glycolytic enzymes in maspin-transfected cells. In addition, maspin-transfected cells expressed much lower levels of hypoxia-inducible factor 1alpha than parental cells or empty vector transfected cells.. Maspin exhibited a metastasis-suppressive effect, which may be a consequence of the reversal of the malignant phenotype of EC109 cells. The switch of cellular metabolic phenotype to low glycolysis by the gain of maspin function may play a key role in the process. This finding provides additional evidence of the tumor metastasis-suppressive activity of maspin and may indicate a new direction for future studies of the mechanism of maspin.

Topics: Cell Differentiation; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Esophageal Neoplasms; Humans; Neoplasm Metastasis; Serine Proteinase Inhibitors; Serpins; Transfection

ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

Topics: Amino Acid Motifs; Animals; Chlorocebus aethiops; Clathrin; Clathrin-Coated Vesicles; COS Cells; Cysteine Proteinase Inhibitors; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Leupeptins; Nedd4 Ubiquitin Protein Ligases; Neoplasm Metastasis; Neoplasms; Oxidoreductases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-cbl; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination; WW Domain-Containing Oxidoreductase

The effect of anti-epidermal growth factor receptor (EGFR) antibodies (mAb) in metastatic colorectal cancer seems limited to KRAS wild-type (wt) tumours, but still a major fraction of KRASwt patients are nonresponders and supplementary selection criteria are needed. We investigated methodological aspects of KRAS testing and the predictive and prognostic value of KRAS status combined with three EGFR-related gene polymorphisms [single-nucleotide polymorphisms (SNPs)] in patients treated with cetuximab and irinotecan.. The study included 71 patients referred to third-line cetuximab-irinotecan. Blood samples were analysed for SNPs. KRAS analysis was carried out by sequencing analysis and quantitative PCR (DxS kit) in primary tumour and distant metastases.. There was a clear correlation between KRAS status in primary tumours and metastasis. The DxS kit presented the highest sensitivity. Response was confined to KRASwt patients (40% response rate versus 0%, P < 0.1(-3)), which translated into a significant difference in PFS. The EGF61A>G polymorphism showed relation to clinical outcome. A combined biomarker analysis showed a 19% progression rate in KRASwt-EGF61 homozygote patients and 60% in the EGF61A/G patients (P = 0.006) and a significant increase in overall survival (17.1 versus 5.9 months, log-rank, P = 0.002).. The combined biomarker analysis maybe an attractive approach to selection of patients for third-line treatment including anti-EGFR mAbs.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Camptothecin; Cetuximab; Colorectal Neoplasms; Disease-Free Survival; DNA Mutational Analysis; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Irinotecan; Kaplan-Meier Estimate; Male; Middle Aged; Mutation; Neoplasm Metastasis; Patient Selection; Polymorphism, Single Nucleotide; Proportional Hazards Models; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Retrospective Studies; Risk Assessment; Time Factors; Treatment Outcome

Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.

Topics: Animals; Carcinoma; Cell Movement; Chick Embryo; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Gene Knockdown Techniques; Humans; Inverted Repeat Sequences; Lung; Lung Neoplasms; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Polymerase Chain Reaction; Receptor Cross-Talk; Receptors, Vitronectin; RNA, Neoplasm; Tumor Cells, Cultured

Lung cancer has become increasingly common in women, and gender differences in the physiology and pathogenesis of the disease have suggested a role for estrogens. In the lung recent data have shown local production of estrogens from androgens via the action of aromatase enzyme and higher levels of estrogen in tumor tissue as compared with surrounding normal lung tissue. High levels of aromatase expression are also maintained in metastases as compared with primary tumors. Consistent with these findings, clinical studies suggest that aromatase expression may be a useful predictive biomarker for prognosis in the management of non-small cell lung cancer (NSCLC), the most common form of lung malignancy. Low levels of aromatase associate with a higher probability of long-term survival in older women with early stage NSCLC. Treatment of lung NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. Further, lung cancer progression is also governed by complex interactions between estrogen and growth factor signaling pathways to stimulate the growth of NSCLC as well as tumor-associated angiogenesis. We find that combination therapy with the multitargeted growth factor receptor inhibitor vandetanib and the estrogen receptor antagonist fulvestrant inhibit tumor growth more effectively than either treatment administered alone. Thus, incorporation of antiestrogen treatment strategies in standard antitumor therapies for NSCLC may contribute to improved patient outcome, an approach that deserves to be tested in clinical trials.

Topics: Animals; Antineoplastic Agents; Aromatase; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Line, Tumor; Disease Progression; Epidermal Growth Factor; Estrogen Receptor Modulators; Estrogens; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Signal Transduction; Transplantation, Heterologous

Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non-small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif -190/-171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Cetuximab; Chick Embryo; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; MAP Kinase Signaling System; Neoplasm Metastasis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Receptors, Urokinase Plasminogen Activator

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Epidermal Growth Factor; Female; Focal Adhesions; Humans; Integrin-Binding Sialoprotein; Male; Neoplasm Metastasis; Prostatic Neoplasms; Sialoglycoproteins; Signal Transduction; Transforming Growth Factor beta1

The epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas and promotes cellular responses that contribute to ovarian cancer pathobiology. In addition to modulation of mitogenic and motogenic behavior, emerging data identify EGFR activation as a novel mechanism for rapid modification of the cell surface proteome. The transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a major contributor to pericelluar proteolysis in the ovarian carcinoma microenvironment and is subjected to extensive posttranslational regulation. In the present study, the contribution of EGFR activation to control of MT1-MMP cell surface dynamics was investigated. Unstimulated ovarian cancer cells display caveolar colocalization of EGFR and MT1-MMP, whereas EGFR activation prompts internalization via distinct endocytic pathways. EGF treatment results in phosphorylation of the MT1-MMP cytoplasmic tail, and cells expressing a tyrosine mutated form of MT1-MMP (MT1-MMP-Y(573)F) exhibit defective MT1-MMP internalization. As a result of sustained cell surface MT1-MMP activity, a phenotypic epithelial-mesenchymal transition is observed, characterized by enhanced migration and collagen invasion, whereas growth within three-dimensional collagen gels is inhibited. These data support an EGFR-dependent mechanism for regulation of the transition between invasive and expansive growth of ovarian carcinoma cells via modulation of MT1-MMP cell surface dynamics.

Topics: Cell Line, Tumor; Cell Proliferation; Collagen; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Humans; Matrix Metalloproteinase 14; Microscopy, Fluorescence; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Protein Transport; Tissue Scaffolds

High expression of 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been detected in various invasive cancers. In the current study, we investigated its role in cancer cell migration and experimental metastasis. Down-regulation of PDK1 expression by small interference RNA markedly inhibited spontaneous migration and epidermal growth factor (EGF)-induced chemotaxis of human breast cancer cells. The defects were rescued by expressing wild-type PDK1. PDK1-depleted cells showed impaired EGF-induced actin polymerization and adhesion, probably due to a decrease in phosphorylation of LIM kinase/cofilin and integrin beta1. Confocal microscopy revealed that EGF induced cotranslocation of PDK1 with Akt and protein kinase Czeta (PKCzeta), regulators of LIM kinase, and integrin beta1. Furthermore, PDK1 depletion dampened EGF-induced phosphorylation and translocation of Akt and PKCzeta, suggesting that Akt and PKCzeta functioned downstream of PDK1 in the chemotactic signaling pathway. In severe combined immunodeficiency mice, PDK1-depleted human breast cancer cells formed more slowly growing tumors and were defective in extravasation to mouse lungs after i.v. injection. Our results indicate that PDK1 plays an important role in regulating the malignant behavior of breast cancer cells, including their motility, through activation of Akt and PKCzeta. Thus, PDK1, which increases its expression in cancer cells, can be used as a target for the development of novel therapies.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Actins; Animals; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chemotaxis; Down-Regulation; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Mice, SCID; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Kinase C-delta; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14(+)/KDR(+) angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14(+)/KDR(+) cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14(+)/KDR(+) cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14(+)/KDR(+) cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14(+)/KDR(+) population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.

Topics: Adenocarcinoma; Adult; Aged; Antigens, CD; Cell Differentiation; Cell Division; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Female; Flow Cytometry; Humans; Hydrocortisone; Lipopolysaccharide Receptors; Mesoderm; Middle Aged; Monocytes; Neoplasm Metastasis; Neovascularization, Pathologic; Ovarian Diseases; Ovarian Neoplasms

Besides current clinicopathologic staging system extensively used in clinic, more information of molecular staging is need for more accurate staging of colorectal cancer (CRC). This study was to evaluate the prognostic value of metastasis-related tumor markers in CRC.. The expression of CD44v6, matrix matalloproteinase-2 (MMP-2), cyclooxygenase-2 (COX-2), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR) and vascular epidermal growth factor (VEGF) in a tissue microarray containing 95 specimens of CRC were detected by immunohistochemistry (IHC). The correlations of these tumor markers to the prognosis of CRC patients were analyzed.. In patients with Dukes' A/B disease, the 5-year recurrence rates were significantly higher in CD44v6-, EGF-and EGFR-positive groups than in negative groups (30.9% vs. 8.3%,P=0.045; 38.1% vs. 8.8%, P=0.022; 27.5% vs. 11.8%, P=0.047, respectively). In patients with Dukes' C disease, the 5-year recurrence rates were significantly higher in MMP-2-, COX-2-and VEGF-positive group than in negative groups (73.3% vs. 37.5%, P=0.045; 69.2% vs. 25.0%, P=0.017; 62.5% vs. 25.0%, P=0.03, respectively). In patients with Dukes' A/B disease, there were a significantly higher 5-year recurrence rate and a lower 5-year survival rate in those with more than three positive markers than in those with 1-3 positive markers (P=0.019, P=0.03). However, there was no significant difference in patients with Dukes' C disease in such condition.. Over-expression of CD44v6, EGF and EGFR are related to poor prognosis of Dukes' A/B CRC, while over-expression of MMP-2, COX-2 and VEGF are related to poor prognosis of Dukes' C CRC. For patients with Dukes' A/B CRC, the more positive markers, the higher 5-year recurrence rate and the poorer 5-year survival.

Topics: Adult; Aged; Biomarkers, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Epidermal Growth Factor; ErbB Receptors; Female; Follow-Up Studies; Humans; Hyaluronan Receptors; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Rectal Neoplasms; Survival Rate; Vascular Endothelial Growth Factor A; Young Adult

To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE(38)KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo.. A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE(38)). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231.. EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by approximately 90% with no apparent loss in in vitro or in vivo activity.. Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials.

Topics: Animals; Breast Neoplasms; Carcinoma; Drug Resistance, Neoplasm; Epidermal Growth Factor; Female; Humans; Immunotoxins; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis, Site-Directed; Neoplasm Metastasis; Receptors, Interleukin-4; Recombinant Fusion Proteins; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor beta1 (TGFbeta1) and transcription factors including Snail and Slug. We utilized the ARCaP(E)/ARCaP(M) prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaP(E) cells, the highly tumorigenic mesenchymal ARCaP(M) and ARCaP(M1) variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaP(M) and ARCaP(M1) expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-kappaB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFbeta1 treatment and Snail overexpression induced EMT in ARCaP(E) and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Topics: Animals; Biomarkers, Tumor; Bone Remodeling; Cadherins; Carcinoma; Cell Dedifferentiation; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelial Cells; Humans; Male; Mesoderm; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Osteoclasts; Prostatic Neoplasms; RANK Ligand; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta1; Vimentin

Abnormalities in the expression and signaling pathways downstream of epidermal growth factor receptor (EGFR) contribute to progression, invasion, and maintenance of the malignant phenotype in human cancers. Accordingly, biological agents, such as the EGFR-blocking antibody IMC-C225 have promising anticancer potential and are currently in various stages of clinical development. Because use of IMC-C225 is limited, at present, only for treatment of cancer with high EGFR expression, the goal of the present study was to determine the effect of IMC-C225 on the invasiveness of breast cancer cells with high and low levels of EGFR expression.. The effect of IMC-C225 on invasion was studied using breast cancer cell lines with high and low levels of EGFR expression.. The addition of EGF led to progressive stress fiber dissolution. In contrast, cells treated with IMC-C225 showed reduced invasiveness and increased stress-fiber formation. Interestingly, IMC-C225 pretreatment was accompanied by EGFR phosphorylation, as detected using an anti-phosphorylated tyrosine antibody (PY99), which correlated with phosphorylation of Vav2 guanine nucleotide exchange factor and activation of RhoA GTPase irrespective of EGFR level, and Vav2 interacted with EGFR only in IMC-C225-treated cells. The underlying mechanism involved an enhanced interaction between beta1 integrins and EGFR upon IMC-C225 treatment.. Here, we defined a new mechanism for IMC-C225 that cross-links integrins with EGFR, leading to activation of RhoA and inhibition of breast cancer cell invasion irrespective of the level of EGFR in the cells, thus providing a rationale for using IMC-C225 in the metastatic setting independent of the levels of EGFR.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cetuximab; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-vav; rho GTP-Binding Proteins; rhoA GTP-Binding Protein

The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena(INV). Here we show that Mena and Mena(INV) promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena(INV) potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena(INV) is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena(INV) could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels.

Topics: Alternative Splicing; Animals; Carcinoma; Cell Movement; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Macrophages; Mice; Microfilament Proteins; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Protein Isoforms

Epithelial-to-mesenchymal transition (EMT) is a fundamental biological process whereby epithelial cells lose their polarity and undergo a transition to a mesenchymal phenotype. When cancer cells invade adjacent tissues, they use a mechanism akin to EMT, and understanding the molecular mechanisms that drive this transition will facilitate studies into new targets for prevention of metastasis. Extracellular stimuli, such as growth factors, and their cytosolic effectors cooperate to promote EMT. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules, including collagen, can initiate signals that promote EMT. Here, we present data showing that collagen I induces EMT in non-small cell lung cancer cell lines, which is prevented by blocking transforming growth factor (TGF)-beta3 signaling. In addition, we show that collagen I-induced EMT is prevented by inhibitors of phosphoinositide 3-kinase and extracellular signal-related kinase signaling, which promotes transcription of TGF-beta3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces EMT in lung cancer cells by activating autocrine TGF-beta3 signaling. Epidermal growth factor also seems to initiate EMT via a TGF-dependent mechanism.

Topics: Animals; Autocrine Communication; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Polarity; Collagen Type I; Epidermal Growth Factor; Epithelial Cells; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Neoplasm Metastasis; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta3

CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been reported to mediate the metastasis of many solid tumors including ovarian, breast, lung and prostate. The over-expression of the epidermal growth factor receptor (EGFR) is associated with the majority of ovarian cancer and has been implicated in the process of malignant transformation by promoting cell proliferation, survival, and motility. In this research, the result first showed that epidermis growth factor (EGF) enhanced the expression of CXCR4 and the migration of ovarian cancer cells, moreover, both stromal cell derived factor-1alpha (SDF-1alpha) and EGF-induced high matrix metallopeptidase 9 (MMP9) expressions. Molecular analysis indicated that augmented CXCR4 and MMP9 expression was regulated by phosphatidylinositol-3-kinase(PI3K)/Akt signal transduction pathway. These results suggested a possible important "cross-talk" between CXCR4 and EGFR intracellular pathways that might link signals of tumor deteriorated and provided a plausible explanation for the poor overall survival rate of patients whose co-expression of CXCR4 and EGFR was detected in their tissue sections. It enlightened that, compared to the respective inhibition of the EGFR or CXCR4 signaling, the simultaneous inhibition of them might be a more useful therapeutic strategy of cancer.

Topics: Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 9; Neoplasm Metastasis; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, CXCR4; Signal Transduction

Chemotaxis plays an important role in metastasis of cancer cells. In the current study, we investigated the role of PTEN, a tumor suppressor, in chemotaxis of human breast cancer cells. Over-expression of PTEN inhibited EGF-induced chemotaxis, probably due to an overall reduction of PIP(3) levels. Disruption of PTEN by siRNA caused a marked decrease in chemokinesis, cell adhesion, and membrane spreading, resulting in a severe defect in chemotaxis. In PTEN disrupted cells, PDK1, AKT, and PKCzeta exhibited elevated basal activities, which prevented EGF-induced further activation of these molecules. In the absence of EGF, active PDK1 was detected on multiple directions of the plasma membranes of PTEN disrupted cells, which competed against EGF-induced gradient sensing. To confirm the biological relevance of in vitro studies, both PTEN disrupted cells and its parental human breast cancer cells were injected into tail veins of SCID mice. Mice injected with PTEN disrupted cancer cells showed a marked decrease in lung metastasis. Taken together, our data show that PTEN plays a non-redundant role in EGF-induced chemotaxis of human breast cancer cells, and an optimal level of PTEN is required in these responses.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Membrane; Chemotaxis; Epidermal Growth Factor; Gene Expression; Humans; Lung Neoplasms; Mice; Mice, SCID; Neoplasm Metastasis; PTEN Phosphohydrolase; RNA, Small Interfering; Signal Transduction

We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.

Topics: Actin Depolymerizing Factors; Actins; Animals; Cell Line, Tumor; Cell Movement; Cell Size; Chemotaxis; Epidermal Growth Factor; Female; Mammary Neoplasms, Experimental; Microscopy, Video; Models, Biological; Neoplasm Metastasis; RNA, Small Interfering; Time Factors; Transfection

Although a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.. Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion). Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied.. 1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

Topics: Cell Line, Tumor; Cell Movement; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Microscopy, Confocal; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; RNA, Messenger; Sodium Channels; Tetrodotoxin

Capillaries expressing the laminin alpha2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin alpha2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin alpha2 chain rather than through the laminin beta1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the alpha6beta1 integrin, which is one of the laminin alpha2 chain receptors. EGF up-regulated alpha6beta1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-alpha6 integrin chain monoclonal antibody. These data indicate that laminin alpha2 chain and alpha6beta1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin alpha2 chain-positive vessels may favor metastasis of EGF-dependent tumors.

Topics: Antibodies, Monoclonal; Capillaries; Carcinoid Tumor; Carcinoma, Neuroendocrine; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Gene Expression; Humans; Laminin; Lung Neoplasms; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Up-Regulation

Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine-threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119-1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868-36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247-7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349-359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Chemotaxis; Cofilin 1; Epidermal Growth Factor; Female; Green Fluorescent Proteins; Lim Kinases; Lung Neoplasms; Mammary Neoplasms, Experimental; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Protein Kinases; Rats; Rats, Inbred F344; RNA, Small Interfering; Survival Analysis; Transfection

A cell responds to a chemotactic signal by activating actin polymerization and forming a protrusion oriented towards the source. Recent work shows that the activity of cofilin, a protein that creates new barbed ends for actin filament elongation, amplifies and specifies the direction of the response in carcinoma cells.

Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Actins; Animals; Breast Neoplasms; Chemotactic Factors; Chemotaxis; Epidermal Growth Factor; Lim Kinases; Neoplasm Metastasis; Protein Kinases; Type C Phospholipases

Non-small cell lung cancer (NSCLC) expresses a particularly aggressive metastatic phenotype, and patients with this disease have a poor prognosis. CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung, breast, kidney, and prostate. In addition, overexpression of the epidermal growth factor receptor (EGFR) is associated with the majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility. Here we show for the first time that activation of the EGFR by EGF increases CXCR4 expression and the migratory capacity of NSCLC cells. Furthermore, many solid tumors are associated with low oxygen tension, and when NSCLC cells were cultured with EGF under hypoxic conditions, CXCR4 expression was dramatically enhanced. A molecular analysis of these events indicated that augmented CXCR4 expression was regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signal transduction pathway, activation of hypoxia inducible factor (HIF) 1alpha, and ultimately HIF-1-dependent transcription of the CXCR4 gene. Thus, a combination of low oxygen tension and overexpression of EGFR within the primary tumor of NSCLC may provide the microenvironmental signals necessary to upregulate CXCR4 expression and promote metastasis.

Topics: Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cell Survival; Chemokine CXCL12; Chemokines, CXC; Chemotaxis; Dose-Response Relationship, Drug; Epidermal Growth Factor; Flow Cytometry; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Neoplasm Metastasis; Oxygen; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, CXCR4; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Suppressor Proteins; Up-Regulation

We have reported that the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib ('Iressa', ZD1839), suppressed intrahepatic metastasis of hepatocellular carcinoma CBO140C12 cells. In this study, we focused on the tumour necrosis factor-alpha (TNF-alpha) signalling pathways. Real-time reverse transcription-polymerase chain reaction showed that TNF-alpha mRNA was expressed in large quantities in the implanted tumour. Gefitinib inhibited EGF- but not hepatocyte growth factor (HGF)-induced activation of mitogen-activated protein kinase (MAPK) cascades, suggesting selectivity of the inhibitor. However, gefitinib inhibited the TNF-alpha-induced activation of MAPKs and Akt. In addition, TNF-alpha-induced metastatic properties including adhesion to fibronectin, mRNA expression of integrin alpha v, production of matrix metalloproteinase-9 and invasion were inhibited by gefitinib without affecting cell proliferation. Furthermore, the TNF-alpha-induced responses except for NF-kappaB activation were blocked by metalloprotease inhibitors, suggesting that gefitinib inhibited the transactivation of EGFR induced by TNF-alpha. These results suggest that the TNF-alpha signalling pathway is a possible target of gefitinib in suppressing the intrahepatic metastasis of hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Epidermal Growth Factor; Female; Gefitinib; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Protein Kinase Inhibitors; Quinazolines; Tumor Necrosis Factor-alpha

Urokinase-type plasminogen activator receptor (uPAR) and Epidermal Growth Factor Receptor (EGFR) are ubiquitous receptors involved in the control of a variety of cellular processes frequently found altered in cancer cells. The EGFR has been recently described to play a transduction role of uPAR stimuli, mediating uPA-induced proliferation in highly malignant cells that overexpress uPAR. We compared the uPA production, the presence of uPAR, AR, EGFR and Her2 with the chemotaxis and the Matrigel invasion in ten human PCa cell lines and observed that: (1) the levels of Her2, but not of EGFR, as well as the uPA secretion, cell motility and Matrigel invasion were statistically higher in AR negative than in AR positive PCa cells; (2) the uPA secretion and uPA Rexpression were positively related to Matrigel invasion; (3) the EGF was able to stimulate chemotaxis and Matrigel invasion in a dose-dependent manner; (4) the EGF-induced cell migration was statistically higher inAR negative than in AR positive cells with a similar increase with respect to basal value (about 2.6 fold); (5) the Matrigel invasion was statistically higher in AR negative than in AR positive PCa cells also if the increment of Matrigel invasion after EGF treatment was statistically higher in AR positive respect to AR negative cells; (6) the EGF induced uPA secretion and its membrane uptake through the increment of uPAR; and (7) these effects were blocked by EGFR/Her2 tyrosine kinase inhibitors with IC(50) lower than those needed to inhibit cell proliferation and required PI3K/Akt, MAPK and PI-PLC activities as verified by inhibition experiments. These enzymatic activities were regulated in different manners in PTEN positive and negative cells. In fact, the inhibition of PI3K blocked the EGF-induced invasiveness in PTEN positive cells but not in PTEN negative cells, in which PI3K activity was not influenced by EGFR/Her2 activation, whereas the inhibition of MAPK was able to block the invasive phenomena in both cell types. Taken together, our data suggest that the blockade of EGFR could attenuate the invasive potential of PCa cells. In addition, considering that the EGFR expression is related to higher Gleason grade of PCa and that EGFR levels are increased after anti androgenic therapy, this therapeutic approach could slow down the metastasis formation which represents the most dramatic event of PCa progression.

Topics: Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Inhibitory Concentration 50; Laminin; Male; MAP Kinase Signaling System; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Monoester Hydrolases; Phosphorylation; Prostatic Neoplasms; Proteoglycans; PTEN Phosphohydrolase; Quinazolines; Receptor, ErbB-2; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Proteins; Urokinase-Type Plasminogen Activator

Cell lines pairs were established from a primary squamous carcinoma of tongue and a lymph node metastasis and their biological behavior characterized. HN12 cells, derived from metastatic SCC, formed tumors upon subcutaneous transplantation to athymic mice, whereas HN4, derived from a primary lesion in the same individual, were non-tumorigenic in this assay. EGF stimulated proliferation of HN4 cells; in comparison, not only were metastatic HN12 cells refractory to the stimulatory effects of this growth factor but showed inhibition at higher growth factor concentrations. However, in contrast to the effects on proliferation, EGF (10 ng/ml) readily induced HN12 cells to invade in Boyden chamber assays whereas HN4 were non-invasive under these conditions. The invasive properties of HN12 cells were apparently independent of MMP-2 activity, as levels of active MMP-2 were higher in the non-invasive cells. However, EGF stimulated MMP-9 activity in invasive cells. Additionally, HN12 cells expressed constitutively high levels of active MMP-7 and MMP-3/10. The pharmacological agents LY294002, PD098059, SP600125, or SB202190 inhibited invasion of HN12 cells, suggesting requirement for phosphoinositide 3-OH kinase- and mitogen activated protein kinase-dependent pathways in the process. The data indicate that distinct biochemical differences distinguish metastatic squamous carcinoma cells from those derived from corresponding primary tumors, resulting in their contrasting biological properties.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Lymphatic Metastasis; Mice; Mice, SCID; Neoplasm Invasiveness; Neoplasm Metastasis; Tongue Neoplasms

Endocrine therapy is the most important treatment option for women with hormone-receptor-positive breast cancer. The potential mechanisms for endocrine resistance involve estrogen receptor (ER)-coregulatory proteins and crosstalk between ER and other growth factor signaling networks. However, the factors and pathways responsible for endocrine resistance are still poorly identified.. Using immunohistochemical techniques, we focused on the expression and phosphorylation of hormone receptors themselves and examined the phosphorylation of ER-alpha Ser118 and ER-alpha Ser167 and the expression of ER-alpha, ER-beta1, ER-betacx/beta2, progesterone receptor (PR), PRA, and PRB in the primary breast carcinomas of 75 patients with metastatic breast cancer who received first-line treatment with endocrine therapy after relapse.. Phosphorylation of ER-alpha Ser118, but not Ser167, was positively associated with overexpression of HER2, and HER2-positive tumors showed resistance to endocrine therapy. The present study has shown for the first time that phosphorylation of ER-alpha Ser167, but not Ser118, and expression of PRA and PRB, as well as ER-alpha and PR in primary breast tumors are predictive of response to endocrine therapy, whereas expression of ER-beta1 and ER-betacx/beta2 did not affect response to the therapy. In addition, patients with either high phosphorylation of ER-alpha Ser167, or high expression of ER-alpha, PR, PRA, or PRB had a significantly longer survival after relapse.. These data suggest that phosphorylation of ER-alpha Ser167 is helpful in selecting patients who may benefit from endocrine therapy and is a prognostic marker in metastatic breast cancer.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chlorocebus aethiops; COS Cells; Epidermal Growth Factor; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Phosphorylation; Phosphoserine; Prognosis; Receptor, ErbB-2; Receptors, Progesterone; Recombinant Proteins; Recurrence; Survival Analysis; Transfection

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related beta-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not beta-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.

Topics: Adenoviridae; Adherens Junctions; Animals; beta Catenin; Blotting, Western; Cell Adhesion; Cells, Cultured; Desmosomes; Detergents; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; gamma Catenin; Green Fluorescent Proteins; Immunoprecipitation; Keratinocytes; Mice; Mice, Knockout; Microscopy, Fluorescence; Neoplasm Metastasis; Octoxynol; Phosphorylation; Protein Structure, Tertiary; Signal Transduction; Time Factors; Tyrosine; Wound Healing

Cytokines and growth factors in malignant ascites are thought to modulate a variety of cellular activities of cancer cells and normal host cells. The motility of cancer cells is an especially important activity for invasion and metastasis. Here, we examined the components in ascites, which are responsible for cell motility, from patients and cancer cell-injected mice. Ascites remarkably stimulated the migration of pancreatic cancer cells. This response was inhibited or abolished by pertussis toxin, monoglyceride lipase, an enzyme hydrolyzing lysophosphatidic acid (LPA), and Ki16425 and VPC12249, antagonists for LPA receptors (LPA1 and LPA3), but not by an LPA3-selective antagonist. These agents also inhibited the response to LPA but not to the epidermal growth factor. In malignant ascites, LPA is present at a high level, which can explain the migration activity, and the fractionation study of ascites by lipid extraction and subsequent thin-layer chromatography indicated LPA as an active component. A significant level of LPA1 receptor mRNA is expressed in pancreatic cancer cells with high migration activity to ascites but not in cells with low migration activity. Small interfering RNA against LPA1 receptors specifically inhibited the receptor mRNA expression and abolished the migration response to ascites. These results suggest that LPA is a critical component of ascites for the motility of pancreatic cancer cells and LPA1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells.

Topics: Adult; Animals; Ascites; Blotting, Northern; Cell Adhesion; Cell Division; Cell Line, Tumor; Cell Movement; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Isoxazoles; Lipids; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Middle Aged; Monoacylglycerol Lipases; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Pancreatic Neoplasms; Pertussis Toxin; Propionates; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection

The MUC1 tumor antigen is overexpressed on most breast tumors and metastases. It interacts with signaling proteins such as the ErbB kinases and beta-catenin, and is involved in mammary gland oncogenesis and tumor progression. Herein, we report a novel interaction between MUC1 and adenomatous polyposis coli (APC), a tumor suppressor involved in downregulating beta-catenin signaling. Initially identified in colorectal cancer, APC is also downregulated in breast tumors and presumably involved in mammary carcinogenesis. MUC1 and APC co-immunoprecipitate from the ZR-75-1 human breast carcinoma cell line and co-localize in mouse mammary glands and tumors. These studies also indicate that the association of MUC1 and APC may be increased by epidermal growth factor stimulation. Intriguingly, the co-immunoprecipitation of MUC1 and APC increases in human breast tumors and metastases as compared to adjacent normal tissues, indicating that this association may play a role in the formation and progression of breast tumors.

Topics: Adenomatous Polyposis Coli Protein; Animals; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Female; Humans; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Mucin-1; Neoplasm Metastasis; Precipitin Tests

Although the effects of progesterone on cell cycle progression are well known, its role in spreading and adhesion of breast cancer cells has not attracted much attention until recently. Indeed, by controlling cell adhesion proteins, progesterone may play a direct role in breast cancer invasion and metastasis. Progesterone has also been shown to modulate epidermal growth factor (EGF) effects in neoplasia, although EGF effects on progesterone pathways and targets are less well understood. In the present study we identify an effect of EGF on a progesterone target, namely desmoplakin.. Initially flow cytometry was used to establish the growing conditions and demonstrate that the T47D breast cancer cell line was responding to progesterone and EGF in a classical manner. Differential display RT-PCR was employed to identify differentially expressed genes affected by progesterone and EGF. Western and Northern blotting were used to verify interactions between EGF and progesterone in three breast cancer cell lines: T47D, MCF-7, and ZR-75.. We found the cell adhesion protein desmoplakin to be upregulated by progesterone - a process that was suppressed by EGF. This appears to be a general but not universal effect in breast cancer cell lines.. Our findings suggest that progesterone and EGF may play opposing roles in metastasis. They also suggest that desmoplakin may be a useful biomarker for mechanistic studies designed to analyze the crosstalk between EGF and progesterone dependent events. Our work may help to bridge the fields of metastasis and differentiation, and the mechanisms of steroid action.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Size; Cytoskeletal Proteins; Desmoplakins; Epidermal Growth Factor; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Progesterone; Promegestone; S Phase

The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Actins; Animals; Antibodies; Carcinoma; Cell Line, Tumor; Chemotaxis; Enzyme Inhibitors; Epidermal Growth Factor; Microfilament Proteins; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Pseudopodia; Rats; RNA Interference; Type C Phospholipases

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Chemotaxis; Collagen; Diffusion Chambers, Culture; Dose-Response Relationship, Drug; Drug Combinations; Epidermal Growth Factor; Extracellular Matrix; Female; Humans; Laminin; Neoplasm Metastasis; Nonlinear Dynamics; Proteoglycans; Vitronectin

LIM kinase (LIMK) plays a critical role in stimulus-induced remodeling of the actin cytoskeleton by linking signals from the Rho family GTPases to changes in cofilin activity. Recent studies have shown an important role for LIMK1 signaling in tumor cell invasion through regulating actin dynamics. In this study, we investigate the role of LIMK1 in intracellular vesicle trafficking of lysosomes/endosomes. We analyzed by confocal immunofluorescence microscopy the cellular distribution of lysosomal proteins and the endocytosis of an endocytic tracer, epidermal growth factor (EGF), in LIMK1-transfected cells. We found in these cells an abnormal dispersed translocation of lysosomes stained for LIMPII and cathepsin D throughout the cytoplasm. The small punctate structures that stained for these lysosomal proteins were redistributed to the periphery of the cell. Computational 3D-image analysis of confocal immunofluorescence micrographs further demonstrated that these vesicles did not colocalize with the transferrin receptor, an early endosomal marker. Furthermore, LIMPII-positive lysosomes did not colocalize with early endosomes labeled with endocytosed Texas red-transferrin. These results indicate that there is no mixing between dispersed lysosomes and early endosomes in the LIMK1-transfected cells. Moreover, LIMK1 overexpression resulted in a marked retardation in the receptor-mediated internalization of Texas red-labeled EGF in comparison with mock-transfected cells. At 30 min after internalization, most of the Texas red-EGF staining overlapped with LIMPII-positive late endosomes/lysosomes in mock-transfected cells, whereas in LIMK1 transfectants only a small fraction of internalized EGF colocalized with LIMPII-positive structures in the perinuclear region. Taken together, the findings presented in this paper suggest that LIMK1 has a role in regulating vesicle trafficking of lysosomes and endosomes in invasive tumor cells.

Topics: Actins; Biological Transport, Active; Breast Neoplasms; Cathepsin D; Cell Line, Tumor; Cytoskeleton; Endocytosis; Endosomes; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Lim Kinases; Lysosomal Membrane Proteins; Lysosomes; Membrane Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Neoplasm Metastasis; Protein Kinases; Proteins; Receptors, Scavenger; Sialoglycoproteins; Signal Transduction

Malignant tumors are characterized by excessive growth, immortalization, and metastatic spread, whereas benign tumors do not express gene products that mediate invasion. The molecular basis for this difference is incompletely understood. We have screened signal transduction molecules associated with the epidermal growth factor (EGF) receptor and have identified constitutive phosphorylation, indicative of activation, of Akt kinase in MT2994 breast cancer cells. In contrast, cells of the benign breast epithelial cell lines Comma-D and FSK-7 are immortalized through pathways that are independent of the EGF-phosphatidylinositol 3-kinase-Akt kinase cascade, but this is not associated with invasiveness. Transfection of constitutively active Akt kinase causes accelerated cell division and osteopontin expression. Conversely, dominant-negative Akt kinase slows cell cycle progression and suppresses osteopontin expression. The manipulation of osteopontin expression in this setting by transfection of the gene or its antisense does not affect the growth rate of the cells but alters cell motility and anchorage independence. Therefore, Akt kinase activates two distinct genetic programs: the program of growth and survival, which is independent of osteopontin expression, and the program of invasiveness and anchorage independence, which is mediated by osteopontin. These studies define Akt kinase as a molecular bridge between cell cycle progression and dissemination.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line, Transformed; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Osteopontin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sialoglycoproteins; Signal Transduction

Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy. Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases. Thus, novel effective therapies are urgently needed to improve the outcome of the patients. Overexpression of the epidermal growth factor receptor (EGFR) and its ligands has been implicated in the oncogenesis of pancreatic carcinoma and associated with an unfavorable prognosis. Consequently, the EGFR represents a specific target antigen suitable for immunotherapy. We generated a recombinant immunotoxin by fusing the anti-EGFR single chain fragment 425(scFv) to a truncated mutant of Pseudomonas Exotoxin A (ETA'). Using the expression vector pBM1.1, functional 425(scFv)-ETA' was periplasmically expressed under osmotic stress conditions in the presence of compatible solutes. The 72 kDa His10-tagged fusion protein was purified by a combination of metal-ion affinity and molecular size chromatography. Binding activity and specificity of the immunotoxin to the EGFR-positive pancreatic carcinoma cell line L3.6pl was confirmed by flow cytometry and ELISA. Finally, 425(scFv)-ETA' showed significant toxicity toward this cell line reaching 50% inhibition of cell proliferation at a concentration (IC50) of 7.5 ng/ml. This is the first report documenting the specific cytotoxicity of a recombinant immunotoxin towards metastatic pancreatic carcinoma cells, suggesting that EGFR-specific antibody toxins may become valuable therapeutic reagents for the treatment of pancreatic carcinoma.

Topics: Carcinoma; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Escherichia coli; Flow Cytometry; Humans; Immunoglobulin Variable Region; Immunotherapy; Immunotoxins; Inhibitory Concentration 50; Ligands; Models, Genetic; Mutation; Neoplasm Metastasis; Pancreatic Neoplasms; Plasmids; Prognosis; Recombinant Fusion Proteins; Recombinant Proteins; Single-Chain Antibodies

EGF receptor (EGFR) overexpression correlates with metastasis in a variety of carcinomas, but the underlying mechanisms are poorly understood. We demonstrated that EGF disrupted cell-cell adhesion and caused epithelial-to-mesenchymal transition (EMT) in human tumor cells overexpressing EGFR, and also induced caveolin-dependent endocytosis of E-cadherin, a cell-cell adhesion protein. Chronic EGF treatment resulted in transcriptional downregulation of caveolin-1 and induction of the transcriptional repressor Snail, correlating with downregulation of E-cadherin expression. Caveolin-1 downregulation enhanced beta-catenin-TCF/LEF-1 transcriptional activity in a GSK-3beta-independent manner. Antisense RNA-mediated reduction of caveolin-1 expression in EGFR-overexpressing tumor cells recapitulated these EGF-induced effects and enhanced invasion into collagen gels. We propose that EGF-induced negative regulation of caveolin-1 plays a central role in the complex cellular changes leading to metastasis.

Topics: beta Catenin; Cadherins; Caveolae; Caveolin 1; Caveolins; Cell Adhesion; Cell Movement; Cell Nucleus; Cytoskeletal Proteins; DNA-Binding Proteins; Down-Regulation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Intercellular Junctions; Neoplasm Metastasis; RNA, Antisense; Signal Transduction; Snail Family Transcription Factors; Trans-Activators; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Current antibody-based immunotherapeutic approaches under evaluation for breast carcinoma are limited in target scope. For example, administration of the human epidermal growth factor receptor (EGFR) antibody, alone or in combination with a chemotherapeutic drug, is thought to primarily inhibit tumor cell proliferation. The aim of this study was to assess the effects of a combined blockade designed to inhibit tumor growth by inhibition of proliferation rate and the proinflammatory effects of interleukin (IL) 8.. A human breast carcinoma cell line that produces high levels of IL-8 was injected s.c. into severe combined immunodeficient mice. IL-8 has been reported to augment the progression of some human tumors; thus, we used a human IL-8 antibody, ABXIL8, in combination with anti-EGFR, ABXEGFR, to inhibit the metastasis of MDA231 tumors.. Whereas anti-IL-8 alone had no appreciable antimetastatic effect, the combination of ABXIL8 significantly enhanced the antitumor effects of ABXEGFR, resulting in greater survival of SCID tumor-bearing mice. This effect on survival was correlated with decreased metastatic spread and decreased tumor size in mice receiving both antibodies. Intriguingly, in vitro studies indicate that this antibody combination markedly inhibited matrix metalloproteinase activity associated with MDA-231 cells to a greater degree than either antibody alone.. Combined administration of these two human antibodies using growth factor blockade in conjunction with chemokine blockade may thus provide a more effective approach for treatment of metastatic human breast carcinoma.

Topics: Animals; Antibodies; Antineoplastic Agents; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Gelatinases; Humans; Immunoblotting; Interleukin-8; Matrix Metalloproteinase 9; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Precipitin Tests; Time Factors; Tumor Cells, Cultured

Tumor metastasis represents a complex multistep process that requires migration, invasion, and angiogenesis. In this study, we examined the impact of molecular blockade of the epidermal growth factor receptor on the invasive and metastatic capacity of human squamous cell carcinoma (SCC) of the head and neck using in vitro and in vivo model systems. Treatment with the anti-epidermal growth factor receptor antibody C225 attenuated the migration of SCC-1 tumor cells through a chemotaxis chamber in a dose-dependent manner. Incubation of SCC cells with 10-100 nM C225 for 4 h resulted in 40-60% inhibition of cell migration. Furthermore, in the presence of C225, the capacity of SCC-1 to invade across a layer of extracellular matrix (Matrigel) was significantly inhibited. Using an in vivo orthotopic floor-of-mouth xenograft model, locoregional tumor invasion of SCC-1 into muscle, vessel, bone, and perineural tissues was inhibited in C225-treated mice. This inhibition was additionally characterized by down-regulation in the expression of matrix metalloproteinase-9. These data suggest that inhibition of metastatic potential by C225 may be mediated via decreased migration and invasion of SCC cells. Regarding angiogenesis in vitro, we first studied human umbilical vascular endothelial cells, which established a capillary-like network structure (tube formation) in the presence of reconstituted Matrigel. Treatment with C225 reduced cell-to-cell interaction of human umbilical vascular endothelial cells, resulting in disruption of tube formation. The effect of C225 was additionally examined using an in vivo tumor xenograft neovascularization model of angiogenesis. Systemic treatment with C225 not only reduced tumor growth and the number of blood capillaries but also hindered the growth of established vessels toward the tumor. Taken together, these results provide evidence that C225 can suppress tumor-induced neovascularization and metastasis in SCC of the head and neck.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Cell Movement; Cells, Cultured; Cetuximab; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Drug Combinations; Endothelium, Vascular; Epidermal Growth Factor; Extracellular Matrix; Fibronectins; Head and Neck Neoplasms; Humans; Immunohistochemistry; Laminin; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Proteoglycans; Time Factors; Tumor Cells, Cultured

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor (EGF) receptor in many cancers is associated with progression of superficial to invasive forms of the disease and is sometimes found in tumors that also have activating Ras mutations, suggesting that both events contribute to tumor invasion. Here we show that EGF stimulates motility in human tumor cell lines, which harbor activating Ha-RasV12 via a novel signal transduction pathway mediated by the small GTP-binding proteins RalA and RhoA but independent of Rac1 and Cdc42. On EGF stimulation, RalA localizes to the cell membrane. In addition, activation of RalA and expression of Rho were increased by EGF stimulation in both the nonmetastatic and metastatic variants of the same cell line. However, elevated levels of constitutively activated RalA were only found in the metastatic variant. This is the first demonstration of an essential role for Ral in EGF-mediated cell motility and its potential contribution to tumor metastasis in human cancer.

Topics: Animals; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Neoplasm Metastasis; Quinazolines; ral GTP-Binding Proteins; Tumor Cells, Cultured; Tyrphostins; Urinary Bladder Neoplasms

In human androgen-independent prostate cancer (PCa), epidermal growth factor receptor (EGFR) regulates angiogenesis, tumor growth, and progression. In this study, we evaluated whether the blockade of EGFR by the anti-EGFR antibody ImClone C225 (IMC-C225) inhibited tumor growth and metastasis by inhibiting angiogenesis, and whether paclitaxel enhanced the results of therapy in androgen-independent PCa. PC-3M-LN4 PCa cells were implanted orthotopically in athymic nude mice and treated with i.p. IMC-C225 (1 mg twice a week) and/or paclitaxel (200 microg once a week). In vitro treatment of PC-3M-LN4 with IMC-C225 inhibited EGFR autophosphorylation without any significant antiproliferative effect. In contrast, in vivo therapy with IMC-C225 alone (P < 0.05) or in combination with paclitaxel (P < 0.005) significantly inhibited PCa growth and metastasis. Serum levels of interleukin (IL) 8 were lower after therapy, and IL-8 mRNA expression was down-regulated within the tumors after therapy. The down-regulation of IL-8 correlated with reduced microvessel density. IMC-C225 reduced tumor cell proliferation, enhanced p27(kip1) expression, and induced tumor and endothelial cell apoptosis. These studies indicate that IMC-C225 has significant antitumor effect in this murine model, mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. The simultaneous administration of paclitaxel enhanced this effect.

Topics: Androgens; Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Apoptosis; Blood Vessels; Cell Cycle Proteins; Cell Division; Cetuximab; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Interleukin-8; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Paclitaxel; Phosphorylation; Prostatic Neoplasms; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins; Tyrosine; Up-Regulation; Xenograft Model Antitumor Assays

Progression to an invasive, metastatic tumour requires the coordinated expression and function of a number of gene products, as well as their regulation in the context of invasion. The transcription factor AP-1 regulates expression of many of those genes necessary for implementation of the invasion programme. Two such gene products, CD44 and ezrin, are both upregulated in fibroblasts transformed by v-fos and are commonly implicated in cell motility and invasion. Here we report that CD44 and ezrin colocalise to membrane ruffles and microvilli of A431 cells after treatment with EGF. However, A431 cells expressing dominant-negative c-Jun (TAM67), and which as a consequence fail to invade in response to EGF, also fail to correctly localise CD44 and ezrin. CD44 and ezrin are both substrates for Protein Kinase C, and we show that their EGF-dependent colocalisation requires Protein Kinase C activity. Associated with TAM67 expression and disrupted CD44 and ezrin colocalisation is the increased expression and activation of the novel PKC theta isoform. Expression of PKC theta in A431 cells results in the inhibition of cell motility and disrupted localisation of CD44 and ezrin. We propose that AP-1 regulates the integrity of Protein Kinase C signalling and identifies PKC theta as a potential suppressor of the invasion programme.

Topics: Animals; Cell Compartmentation; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeletal Proteins; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Fluorescent Antibody Technique; Humans; Hyaluronan Receptors; Isoenzymes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phenotype; Phorbol Esters; Phosphoproteins; Protein Isoforms; Protein Kinase C; Protein Kinase C-theta; Protein Transport; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Up-Regulation

Signals from the epidermal growth factor (EGF) receptor and integrin-dependent adhesion to laminin contribute to the progression and metastasis of colonic tumors. However, little is know about the mechanisms by which these signals cooperate. Recently, we have reported that the colon cancer cell line LIM1215 secretes and adhere to autocrine laminin-10 via multiple integrin receptors and that EGF stimulates spreading of these cells on the same substrate. In this report, we investigate the effect of EGF and laminin-10 on colon cancer cell migration in vitro. EGF stimulates migration of LIM1215 cells in a wound healing assay. The response to EGF is inhibited by anti-EGF receptor antibody 528, the EGF receptor kinase inhibitor AG-1478, or the MAP kinase kinase inhibitor PD98059 but not the PI3-K inhibitor wortmannin. Using Transwell migration chambers, we demonstrate that laminin-10 but not collagen-I, collagen-IV, or a commercial preparation of human placental laminin is a potent motility factor for LIM1215 cells. The migration response to laminin-10 is increased upon stimulation of the cells with EGF and correlates with the up-regulation of alpha(6)beta(4) integrin expression as measured by analysis of Triton X-100-soluble cellular extracts. The results from integrin inhibition experiments indicate that basal migration on laminin-10 is mediated by alpha(3)beta(1) but not alpha(2)beta(1) nor alpha(6)beta(4) integrins. Alpha(3) blocking antibodies also inhibited EGF-stimulated chemokinetic migration of LIM1215 cells on laminin-10. However, in contrast to unstimulated cells, alpha(6) or beta(4) integrin-blocking antibodies inhibited the migration of EGF-stimulated cells by up to 50%. Taken together, these results support the cooperative role of EGF receptor and laminin-10 on colon cancer cell motility and suggest a critical role for both the alpha(3)beta(1) and the alpha(6)beta(4) integrins in this process.

Topics: Actins; Antigens, Surface; Biological Assay; Carcinoma; Cell Movement; Colonic Neoplasms; Cytoskeleton; Epidermal Growth Factor; Humans; Integrin alpha3beta1; Integrin alpha6beta4; Integrins; Laminin; Neoplasm Metastasis; Time Factors; Tumor Cells, Cultured

Cripto-1 is an EGF-CFC protein that performs an important role during early vertebrate development and is overexpressed in several types of human cancer. In the present study mouse EpH4, NMuMG, and TAC-2 mammary epithelial cells that are negative for endogenous cripto-1 expression were transfected with the murine cripto-1 cDNA. Cripto-1-transfected cell lines exhibited functional and physiological differences from the original cell lines including enhanced anchorage-independent growth in soft agar (EpH4 cells), growth in serum-free medium, increased proliferation, and formation of branching, duct-like structures when grown in a three-dimensional collagen type I matrix. Furthermore, cripto-1-expressing cell lines showed elevated migration in vitro in Boyden chamber and wound-healing assays. These results indicate that cripto-1 can function through an autocrine pathway that enables mammary epithelial cells to undergo an epithelial to mesenchymal transition.

Topics: Animals; Biological Assay; Breast; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Cell Movement; Cell Size; Cells, Cultured; Culture Media, Serum-Free; Epidermal Growth Factor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; RNA, Messenger; Transgenes

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.

Topics: 3T3 Cells; Alu Elements; Animals; Base Sequence; Cell Adhesion; Cell Movement; Cytoskeletal Proteins; DNA Primers; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Paxillin; Phenotype; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Retinoblastoma-Like Protein p130; Tumor Cells, Cultured

The expressions of Lewis (Le) antigens, alpha-1,3/1,4 fucosyltransferases (alpha-1,3/1,4 FuTs), and metastatic potential after the treatment of 2 differentiation inducers, all- trans retinoic acid (ATRA), 8-bromo-cyclic 3',5'adenosine monophosphate (8-Br-cAMP); and 2 proliferation inducers, epidermal growth factor (EGF) and phobol-12-myristate-13-acetate (PMA), on 7721 human hepatocarcinoma cell line were studied. Cell adhesion to human umbilical vein endothelial cells (HUVEC), cell migration through transwell and invasion through matrigel were selected as the indexes of metastatic potential-related phenotypes. Using fluorescence-labelled antibodies and flow-cytometric analysis, it was found that 7721 cells mainly expressed sialyl Lewis X (SLe(x)) and a less amount of sialyl dimeric Lewis X (SDLe(x)) antigens on the cell surface. Their expressions were down-regulated by ATRA, and up-regulated by EGF. SLe(x)antigen was also decreased and increased by the treatment of 8-Br-cAMP and PMA respectively. With Northern blot to detect the mRNAs of alpha-1,3/1,4 FuTs, the main enzymatic basis for the change in SLe(x)expression was found to be the alteration of the expression of alpha-1,3 FuT-VII. It was evidenced by the observations that alpha-1,3 FuT-VII was the main alpha-1,3/1,4 FuT in 7721 cells, while alpha-1,3/1,4 FuT-III and alpha-1,3 FuT-VI were expressed rather low. The changes in the expressions of SLe(x)antigen and alpha-1,3 FuT-VII resulted in the altered cell adhesion to tumour necrosis factor-alpha stimulated HUVEC, since only the monoclonal antibody of the SLe(x), but not other monoclonal antibodies blocked the adhesion of 7721 cells to HUVEC. The migration and invasion of 7721 cells were also reduced by the treatment of ATRA or 8-Br-cAMP, and elevated by EGF or PMA. The above findings indicate that the metastatic potential of 7721 cells is suppressed by differentiation-inducers and promoted by proliferation-inducers.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Division; Cell Movement; Epidermal Growth Factor; Fucosyltransferases; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Liver Neoplasms; Neoplasm Metastasis; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.

Topics: Animals; Carcinoma, Squamous Cell; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Substances; I-kappa B Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-1; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Transforming Growth Factor beta

Ezrin is a member of the ezrin, radixin, and moesin family. These proteins are membrane-actin cross-linking proteins. Furthermore, ezrin is an important signal transduction protein that undergoes phosphorylation and translocation on stimulation by growth factors. Ezrin phosphorylation and translocation are thought to be correlated with cell motility, invasion, and carcinoma metastasis. Recently, the authors reported that an ezrin antisense phosphorothionate could significantly inhibit endometrial carcinoma cells' penetration in the Matrigel membrane cancer invasion assay. In the current study, the authors measured ezrin content in clinical ovarian epithelial carcinoma (OVCA) specimens and cell lines and investigated whether interleukin (IL)-1alpha and epidermal growth factor (EGF) induce an invasive phenotype in OVCA cells via ezrin phosphorylation and translocation.. Twenty-five normal ovary, 25 primary OVCA, 21 metastatic OVCA tissue (7 in omentum, 16 in ascites), and 3 OVCA cell lines were collected for Western blot detection of ezrin content. The OVCA cell line SKOV3 was treated with IL-1alpha or EGF. Indirect immunofluorescence staining followed by confocal laser scanning and double-staining electron microscopic immunohistochemistry were used to investigate changes in the intracellular distribution of ezrin and cell morphology after IL-1alpha or EGF treatment. The content of ezrin was measured by Western blotting and analyzed by the National Institutes of Health Image computer program. Immunoprecipitation and Western blot techniques were used for ezrin phosphorylation studies. Genistein was used to block tyrosine phosphorylation.. (1) Ezrin was overexpressed in OVCA, with the highest values in metastases. (2) Interleukin-1alpha and EGF significantly increased OVCA tyrosine phosphorylation, ezrin translocation, and cell growth. (3) These effects were abolished by treatment with the tyrosine kinase inhibitor, genistein. (4) Treatment with IL-1alpha or EGF induced an invasive phenotype, i.e., membrane ruffling, and process formation.. High expression and activation of ezrin appear to be related to OVCA metastatic behavior. Interleukin-1alpha and EGF may regulate OVCA invasive behavior by activating ezrin tyrosine phosphorylation, translocation, and cancer cell proliferation. The authors' results may partially explain why OVCA patients with positive macrophage colony stimulating factor (a chemoattractant of IL-1alpha secreting monocytes) or EGF receptors (c-erb B-2) have a poor prognosis.

Topics: Blotting, Western; Carcinoma; Cell Division; Cytoskeletal Proteins; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-1; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Phenotype; Phosphoproteins; Phosphorylation; Translocation, Genetic; Tumor Cells, Cultured; Tyrosine

The effects of all-trans retinoic acid (ATRA) and epidermal growth factor (EGF) on the expression of nm23-H1, a metastasis suppressor gene, were studied in a human 7721 hepatocarcinoma cell line. It was discovered that the expression of nm23-H1 mRNA was up-regulated by ATRA. This was compatible with the observation that the metastasis-associated phenotypes, such as chemotaxic cell migration and invasion, were both reduced in the ATRA-treated and nm23-H1-cDNA-transferred 7721 cells. However, ability of cells to adhere to fibronectin and laminin was not altered identically in the ATRA-treated and nm23-H1-cDNA-transfected 7721 cells. In contrast, the expression of nm23-H1 mRNA in 7721 cells was down-regulated both by the treatment with EGF and by the transfection of c-erbB-2/neu cDNA, which codes a protein homologous to the EGF receptor. EGF is a compound with biological effects opposite to those of ATRA, and c-erbB-2/neu is known to be a metastasis-promoting gene. These results reveal that the metastasis-preventing effect of ATRA may partly result from the up-regulation of nm23-H1, and the metastasis-promoting effects of EGF and c-erbB-2/neu were probably mediated in part by the down-regulation of nm23-H1.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Curative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF121 and VEGF165. Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bg(c)) IL-2 receptor. Cytokine-induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum.

Topics: Biomarkers, Tumor; Carrier Proteins; Epidermal Growth Factor; Epithelium; Fibroblast Growth Factors; Gastrointestinal Neoplasms; Gene Expression Regulation, Neoplastic; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Neoplasm Metastasis; Peritoneum; RNA, Messenger

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Division; Cetuximab; Deoxycytidine; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Gemcitabine; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-8; Lymphokines; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Pancreas; Pancreatic Neoplasms; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Proliferating Cell Nuclear Antigen; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This study evaluates the expression of cripto (CR-1) protein in matched sets of non-neoplastic cervical epithelium, primary cervical carcinoma and metastatic tumours in the lymph nodes to investigate its role in uterine cervical cancer development and progression. Ninety-four primary cervical carcinomas in an early clinical stage and having the same surgical treatment modality were analysed. Immunoreactivity in the primary tumour was compared with that of non-neoplastic cervical epithelium and metastatic lymph nodes. The conventional clinicopathological prognostic variables for cervical carcinomas such as grade, tumour size, depth of invasion, parametrial and endometrial extension, lymphovascular space involvement and lymph node metastasis status were also compared with CR-1 expression of the primary tumour. Strong CR-1 immunopositivity was significantly correlated with tumour size and lymphovascular space involvement (P < 0.05). Furthermore, a significant relationship was found between CR-1 immunoreactivity and endometrial extension as well as parametrial involvement (P < 0.05). Interestingly, the CR-1 expression level was increased in metastatic lymph nodes compared with their primary tumours. These results suggest that CR-1 may contribute to disease progression in cervical carcinomas.

Topics: Epidermal Growth Factor; Female; Follow-Up Studies; GPI-Linked Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Membrane Glycoproteins; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Analysis; Uterine Cervical Neoplasms

In this study, we report that needles containing chemoattractants can be used to collect the subpopulation of motile and chemotactic tumor cells from a primary tumor in a live rat as a pure population suitable for further analysis. The most efficient cell collection requires the presence of chemotactic cytokines, such as epidermal growth factor and serum components, and occurs with 15-fold higher efficiency in metastatic tumors compared with nonmetastatic tumors. Although tumor cells of the nonmetastatic tumors show a motility response to serum, they were not collected with high efficiency into needles in vivo in response to serum, indicating that additional factors besides motility are required to explain differences in cell collection efficiencies between metastatic and nonmetastatic tumors. The results reported here indicate that needles filled with growth factors and matrigel, when inserted into the primary tumor, can faithfully mimic the environment that supports invasion and intravasation in vivo. Furthermore, the results indicate that the same cell behaviors that contribute to chemotaxis in vitro also contribute to invasion in vivo.

Topics: Animals; Cell Movement; Cell Separation; Chemotactic Factors; Chemotaxis; Collagen; Drug Combinations; Epidermal Growth Factor; Female; Laminin; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Neoplasm Transplantation; Proteoglycans; Rats; Rats, Inbred F344

We have identified a novel 3845 bp cDNA differentially expressed in a human melanoma metastasis model. Northern blot analysis showed expression in the poorly and intermediately metastasizing cell lines and a marked downregulation in the highly metastatic cell lines. Using RT-PCR expression was also seen in several other tumor cell lines and normal cell types of human origin. cDNA sequence analysis revealed an ORF of 687 amino acids containing seven putative transmembrane domains C-terminally and a long N-terminus. The gene was mapped to 16q13. Highest homology was observed with members of the EGF-TM7 subfamily of the secretin/calcitonin receptor family. We propose the delineation of a subfamily of TM7 proteins, LN-TM7, containing seven transmembrane proteins with a long N-terminal extracellular part.

Topics: Amino Acid Sequence; Base Sequence; Chromosome Mapping; Cloning, Molecular; DNA, Complementary; Epidermal Growth Factor; Gene Expression; Humans; Melanoma; Molecular Sequence Data; Neoplasm Metastasis; RNA, Messenger; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

Urokinase (u-PA) and the u-PA receptor (u-PAR) influence tumor invasion and metastasis.. The purpose of this study is to determine whether u-PAR display in 4 human bladder cancer cell lines (RT4, 253J, EJ and T24) can be modulated with substances including phorbol 12-myristate 13-acetate, interferon gamma, epidermal growth factor, and transforming growth factor beta and to correlate changes with u-PAR expression with the ability of these cells to invade artificial basement membrane (Matrigel).. u-PAR display was determined using flow cytometry and immunohistochemical staining with an anti-u-PAR monoclonal antibody (Mab3936). Matrigel invasion chamber assays were used to assess the invasive capacity of the cell lines.. The T24, EJ and 253J cells expressed the u-PAR while the RT4 cells did not. The EJ cells (expressing the highest u-PA antigen levels and the u-PAR) invaded Matrigel. The T24 cells, which expressed the u-PAR but do not produce u-PA, invaded Matrigel only when pretreated with high molecular weight urokinase (HMW u-PA). HMW u-PA pretreatment of EJ and 253J cells also enhanced their invasive potential. Blocking u-PA/u-PAR attachment with an anti-u-PAR monoclonal antibody (Mab3936) inhibited invasion. Modulation of u-PAR expression on cell lines displaying the u-PAR directly affected in vitro invasiveness of the cell lines. The RT4 cells which lack the u-PAR were not invasive under conditions tested. Thus, bladder cancer cell lines require both u-PA and the u-PAR to invade Matrigel and modulation of u-PAR display directly affects their in vitro invasive capacity.. This study shows that bladder tumor cells produce u-PA and express the u-PAR and require both for in vitro invasion to occur. When bladder cancer cells express the u-PAR, invasiveness can be enhanced by exogenous u-PA and inhibited by anti-u-PAR antibodies. Modulation of u-PAR expression on the cell surface of bladder cancer cells can also affect their ability to invade Matrigel.. Histologically similar bladder tumors show differences in their propensity to invade locally or metastasize. Intrinsic differences in the tumor cells such as the production of u-PA antigen and u-PA receptor on the cell surface and extrinsic differences in the tumor cell environment such as substances influencing u-PAR display or antibodies blocking the u-PAR may affect the biological potential of human bladder cancers and offer one explanation for the aggressive or indolent tumor behavior observed in individual patients.

Topics: Basement Membrane; Collagen; Drug Combinations; Epidermal Growth Factor; Humans; Interferon-gamma; Laminin; Neoplasm Invasiveness; Neoplasm Metastasis; Proteoglycans; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urokinase-Type Plasminogen Activator

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.

Topics: Antibodies; Antineoplastic Agents; Benzamidines; Bombesin; Cell Division; Cell Membrane; Collagen; Collagenases; Culture Media, Conditioned; Drug Combinations; Enzyme Activation; Enzyme Induction; Epidermal Growth Factor; Extracellular Matrix; Fibrinolysin; Gastrin-Releasing Peptide; Humans; Laminin; Male; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Peptide Hydrolases; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; Proteoglycans; Serine Proteinase Inhibitors; Tissue Inhibitor of Metalloproteinase-1; Urokinase-Type Plasminogen Activator

We report here the first direct observation of chemotaxis to EGF by rat mammary carcinoma cells. When exposed to a gradient of EGF diffusing from a micropipette, MTLn3 cells displayed typical ameboid chemotaxis, extending a lamellipod-like protrusion and moving toward the pipette. Using a homogeneous upshift in EGF to model stimulated lamellipod extension (J. E. Segall et al., 1996, Clin. Exp. Metastasis 14, 61-72), we analyzed the relationship between adhesion and chemoattractant-stimulated protrusion. Exposure to EGF led to a rapid remodeling of the adhesive contacts on adherent cells, in synchrony with extension of a flat lamellipod over the substratum. EGF-stimulated lamellipods still extended in the presence of adhesion-blocking peptides or over nonadhesive surfaces. They were, however, slightly shorter and retracted rapidly under those conditions. The major protrusive structure observed on well-spread, adherent cells, after EGF stimulation was a flat broad lamellipod, whether or not in contact with the substratum, while cells in suspension showed transient protrusive activity over the entire cell surface. We conclude that the initial adhesive status of the cell conditions the shape of the outcoming protrusion. Altogether our results suggest that, although adhesive contacts are not necessary for lamellipod extension, they play a role in stabilizing the protrusion as well as in the control of its final shape and amplitude.

Topics: Animals; Carcinoma; Cell Adhesion; Cell Size; Chemotactic Factors; Chemotaxis; Epidermal Growth Factor; Mammary Neoplasms, Animal; Neoplasm Metastasis; Rats; Tumor Cells, Cultured

A number of molecules involved in the process of invasion and metastasis of cancer cells have been demonstrated as a biological prognostic parameter. In esophageal cancer, overexpression of the oncogenes (c-erbB, int-2/hst-1/cyclin D1, MDM2), altered expression of suppressor genes (p 16, DCC), and abnormal expression of adhesion molecules (E-cadherin, alpha-catenin) has been reported as markers of high malignant potential. Proliferation markers (Ki-67, AgNORs, PCNA) and angiogenetic factors (intratumoral microvessel density, VEGF) are also related to the prognosis of the patients with various cancers including esophageal cancer. Prognostic significance of p53 is still controversial. In addition to the clinicopathological parameters, combination of these biological markers would be important to predict the clinical outcome of the cases and to establish an individualized strategy of the treatment of each case according to the biological behavior of the cancer cells.

Topics: Cell Division; Cyclin D1; Endothelial Growth Factors; Epidermal Growth Factor; Esophageal Neoplasms; Genes, Tumor Suppressor; Humans; Lymphatic Metastasis; Lymphokines; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Proliferating Cell Nuclear Antigen; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To understand the role of NM23/NDPK in the transmembrane signal transduction proccess in tumor metastasis.. The NDPK activity, IP3 contents, the invasive potentials of the cells of mouse forestomach carcinoma(MFC) substrains with different metastatic potentials and the effect of LN and EGF were observed.. Both the NDPK activity and the contents of IP3 of the cells of the subclone with lower metastatic potential was higher than that with higher metastatic potential. Also, the cells with lower metastatic potential had lower invasive potential and higher responsibility to the challenge of EGF compared with that higher metastatic potential. However, no different effect of LN on the cells of those subclones was found.. NDPK might regulate the metastasis of FMC cells by impacting the G protein efficacy with the selection of exotic signal ligands.

Topics: Animals; Epidermal Growth Factor; Inositol 1,4,5-Trisphosphate; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Nucleoside-Diphosphate Kinase; Signal Transduction; Stomach Neoplasms; Tumor Cells, Cultured

The ability of TGF-beta 1 (transforming growth factor-beta 1) to suppress growth factor induced proliferation of many cell types in vitro is well documented; however, TGF-beta 1 increases within a similar time frame as the hepatocyte mitogens HGF (hepatocyte growth factor), EGF (epidermal growth factor), and TGF-alpha (transforming growth factor-alpha) prior to hepatocyte proliferation during liver regeneration. This has raised the issue that TGF-beta 1 may have effects on hepatocytes additional to mito-inhibition and that these effects may be relevant to the regenerative process. To this end, we examined the effect of TGF-beta 1 on both the mitogenesis and the motility of growth factor stimulated primary rat hepatocytes and the hepatoblastoma cell line HepG2 in vitro. TGF-beta 1 significantly enhanced the chemotactic motility of EGF or TGF-alpha, and not HGF, stimulated hepatocytes on a collagen I substratum. TGF-beta 1 was not chemotactic when added alone and decreased the DNA synthesis of all hepatocyte cultures to near control levels. HepG2 cells were chemotactic toward HGF, EGF, and TGF-beta 1 alone and displayed an additive chemotactic response when TGF-beta 1 was added to either HGF or EGF. Additionally, HepG2 cells were refractory to the growth stimulatory effects of HGF or EGF and the growth inhibitory effects of TGF-beta 1. Hepatocytes plated onto other collagen-containing substrates (collagen IV, Matrigel, or ECL, an entactin-collagen IV-laminin matrix), but not on fibronectin or laminin alone, also displayed enhanced EGF stimulated motility by TGF-beta 1. The data indicate that an additional, novel role for TGF-beta 1 during liver tissue remodeling following PHx may include the synergistic enhancement EGF stimulated hepatocyte motility responses, and this enhancement is observed only on collagen-containing extracellular matrices.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Movement; Collagen; DNA; Drug Synergism; Epidermal Growth Factor; Extracellular Matrix; Hepatocyte Growth Factor; Liver Regeneration; Neoplasm Metastasis; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), amphiregulin (AR) and heregulin (HRG-beta 1). These polypeptides regulate proliferation in breast cancer cells through interaction with membrane receptors. It has been previously shown that high EGF receptor number correlates with aggressive behavior and increased metastasis in human breast cancer. In the present study, we investigated the association between EGF and EGF-like ligand-induced DNA synthesis and secretion of MMP-9 and MMP-2 in metastatic SKBR-3 and non-metastatic MCF-7 breast cancer cells. Exposure of SKBR-3 cells to EGF or AR induces expression of MMP-9 but has no effect on MMP-2 secretion. In contrast to EGF and AR, HRG had no effect on gelatinase induction. None of the EGF polypeptides had any effect on gelatinase induction in MCF-7 non-metastatic breast cancer cells. While a relatively specific inhibitor of EGF receptor tyrosine kinase, PD 153035, inhibited EGF-, AR- and HRG-induced cell proliferation, it had no effect on MMP-9 induced by EGF and AR. Experimental evidence suggests that signaling mechanisms for cell proliferation and MMP-9 induction are mediated by different pathways down-stream of EGF receptor autophosphorylation or that low levels of EGF-induced signal that escape inhibition are sufficient to induce MMP-9 but unable to support cell proliferation. In addition, our results suggest that EGF and AR may modulate invasion of metastatic breast cancer cells by increasing the expression of MMPs.

Topics: Amphiregulin; Antineoplastic Agents; Breast Neoplasms; Cell Division; Collagenases; EGF Family of Proteins; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gelatinases; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Metastasis; Quinazolines; Tumor Cells, Cultured; Up-Regulation

We measure EGF in saliva and plasma from 52 patients with active breast cancer, 22 breast cancer patients in follow-up (non-active) and 33 healthy women. EGF concentrations in saliva were significantly higher in patients with active and non-active breast cancer than healthy women, whereas the opposite results were found in plasma. The highest values of EGF in saliva were found in the local recurrence subgroup.

Topics: Adult; Aged; Breast Neoplasms; Epidermal Growth Factor; Female; Humans; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Saliva

Cancer cell adhesion to the subendothelial extracellular matrix (ECM) is an important step in metastasis formation. The effect of epidermal growth factor (EGF) and eicosapentaenoic acid (EPA) on adhesion of the highly metastatic MDA-MB-231 human breast cancer cell line to ECM components was examined in the present study. MDA-MB-231 cells exhibited a dose-dependent decrease in adhesion to Matrigel when treated with EGF. EGF and EPA, alone or in combination, decreased adhesion to Matrigel, fibronectin and type IV collagen. These results suggest that decreased adhesion to ECM substrates by combined EPA and EGF treatment may be the result of a common post-receptor signaling pathway.

Topics: Breast Neoplasms; Cell Adhesion; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Eicosapentaenoic Acid; Epidermal Growth Factor; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibronectins; Humans; Laminin; Neoplasm Metastasis; Proteoglycans; Tumor Cells, Cultured

The interplay between cyclic AMP (cAMP)-dependent protein kinase A (PKA)- and p21ras-mediated signaling pathways is expected to determine further loss, maintenance, or modulation of differentiation and proliferation of a particular cell. Therefore, the relationship and nature of the cross-talk between these two major signaling systems are of utmost importance to the understanding of these processes in both normal and neoplastic cells. In view of their paramount physiological importance, one would expect the existence of a well-controlled bidirectional interaction between these pathways, which would be more appropriate and in agreement with basic principles of cellular homeostasis. However, based on the discovery that activated PKA may inhibit ras-mediated translocation of c-Raf-1 to the plasma membrane, it is generally accepted that the cross-talk between cAMP/PKA and p21ras-mediated signal transduction pathways is unilateral, i.e., that the activation of PKA regulates growth factor receptor protein tyrosine kinase-mediated signaling. To challenge the validity of a unilateral approach, we decided to test the possible existence of cross-talk of a bidirectional nature between the aforementioned signaling pathways at different stages of malignant differentiation. For that purpose, we investigated the nature of the cross-talk existing between a known receptor protein tyrosine kinase-epidermal growth factor receptor (EGFR) and PKA in highly metastatic and nonmetastatic cloned variants of a murine fibrosarcoma (T-10). Our study revealed the existence of principal differences in PKA activity between metastatic and nonmetastatic cloned fibrosarcoma variants that may be due to the differential expression and membrane translocation of the p21(Ki-ras) small mass G-protein. Most importantly, our experiments have demonstrated the existence of a novel character of interactions between EGFR and PKA, because the ligation of the EGFR by epidermal growth factor in the metastatic variant induced a high activity of PKA. These findings are of prime importance, because they reveal the existence of a new relationship between two major signal transduction pathways in mammalian cells, i.e., the existence of a bilateral interaction between the ras- and cAMP/PKA-mediated signal transduction pathways. Furthermore, the fact that two tumor cell variants originating in the same tumor and differing in their metastatic capacity differ as well in the nature of the cross-talk between major

Topics: 3T3 Cells; Animals; Benzylidene Compounds; Bucladesine; Clone Cells; Culture Media, Serum-Free; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factors; Fibrosarcoma; Genetic Variation; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasm Metastasis; Nitriles; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured; Tyrphostins

A model has been established using primary human cell culture to study the cell biology of breast cancer metastasis to bone marrow. Mammary epithelia were obtained in single cell suspension from tumour (macroscopically involved), benign (macroscopically uninvolved) and normal (reduction mammoplasty) breast tissue as well as from locally involved lymph nodes. Stromal layers were generated from long-term cultures of human bone marrow or from mammary fibroblasts derived from normal or malignant tissue. The interaction between epithelia and stroma has been studied in terms of adhesion of the epithelia to the stroma and their subsequent growth in co-culture. Our results show that when assayed up to 9 hr after plating, epithelial cells from malignant tissue (14 primary tumours and 9 metastases in lymph nodes) displayed a significant preference for adhesion to bone marrow stroma compared with mammary fibroblasts. In contrast, epithelial cells from 4 normal and 2 of 4 benign samples showed no significant preferential adherence. Subsequent co-culture of mammary epithelia with each of the 3 stromal layers revealed that under serum-free, in vitro conditions, bone marrow stromal layers did not provide an advantageous environment for colony growth, in contrast to their ability to provide a preferential substratum for adhesion.

Topics: Animals; Bone Marrow Cells; Breast; Breast Neoplasms; Cell Adhesion; Cell Communication; Cell Division; Cells, Cultured; Coculture Techniques; Epidermal Growth Factor; Epithelial Cells; Female; Fibroblasts; Humans; Immunohistochemistry; Lymph Nodes; Models, Biological; Neoplasm Metastasis; Stromal Cells; Tumor Cells, Cultured

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.

Topics: Colonic Neoplasms; Colonic Polyps; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, src; Humans; Neoplasm Metastasis; Neoplasm Proteins; Phenotype; Phosphorylation; Protein Kinases; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Tumor Cells, Cultured

To examine the IP3 amounts, invasive potentials, and response to LN and EGF of the substrains with high or low metastatic ability from two kinds of carcinoma cell lines (MFC and CNZ-2Z).. Radio-ligand binding assay and matrigel invasive assay.. The low metastatic substrains from the two lines had higher amounts of IP3 and stronger response to EGF than their responsive high ones, but high metastatic substrains from CNZ-2Z had stronger response to LN than the low ones while MFC substrains had not significant difference in response to LN.. There is internal difference in signal transduction between the high and low metastatic cancer cells, and it is significant to study the difference in detail.

Topics: Animals; Epidermal Growth Factor; Humans; Inositol 1,4,5-Trisphosphate; Laminin; Mice; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Signal Transduction; Stomach Neoplasms; Tumor Cells, Cultured

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 microgram/ml anti-hEGF monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

Topics: Alkaloids; Antibodies, Monoclonal; Carbazoles; Carcinogens; Carcinoma; Cell Movement; Dimethyl Sulfoxide; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Indole Alkaloids; Male; Neoplasm Metastasis; Phosphorylation; Prostatic Neoplasms; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of 16 metastatic sites of human prostatic carcinoma. Exogenous ET-1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like growth factor I, insulin-like growth factor II, platelet-derived growth factor, basic fibroblast growth factor, and epidermal growth factor in serum-free conditions in vitro. The ETA-selective receptor antagonist A-127722 inhibits ET-1-stimulated growth, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer growth. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced.

Topics: Apoptosis; Atrasentan; Base Sequence; Cell Division; Cell Line; Culture Media, Serum-Free; DNA Primers; DNA, Neoplasm; Endothelin Receptor Antagonists; Endothelins; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Growth Substances; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Male; Mitotic Index; Molecular Sequence Data; Neoplasm Metastasis; Platelet-Derived Growth Factor; Polymerase Chain Reaction; Prostate; Prostatectomy; Prostatic Hyperplasia; Prostatic Neoplasms; Pyrrolidines; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Tumor Cells, Cultured

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

Topics: Bone Marrow; Brain Neoplasms; Carcinoma; Cell Division; Culture Media; Dihydrotestosterone; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Topics: Cell Adhesion; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Integrins; Neoplasm Metastasis

Metastasis is a multistep process that involves alterations in a tumour cell's invasion, motility and adhesive capabilities. This study examined the effect of EGF on the in vitro invasion, motility and adhesion of the primary renal adenocarcinoma cell line, A704. Stimulation of the tumour cells by EGF (40 ng/ml) for a period of 24 h increased the in vitro invasion (P = 0.040) and motility (P = 0.039). Cell adhesion was examined on fibronectin, laminin, collagen IV and a 1:1:1 mix of the three extracellular matrix components. After EGF (40 ng/ml) stimulation, adhesion was significantly decreased on fibronectin (P = 0.022) and collagen type IV (P = 0.026), but increased on the 1:1:1 mix of extracellular matrix components (P = 0.022). The 92 kDa matrix metalloproteinase (MMP-9) present in the cell-conditioned medium was also increased after a 24 h stimulation with EGF (40 ng/ml) when measured. Hence, EGF can modulate the in vitro invasion, motility, adhesiveness and matrix metalloproteinase production in the A704 cell line, and subsequently may have a role in the metastatic potential of some renal carcinomas.

Topics: Adenocarcinoma; Cell Adhesion; Cell Movement; Collagenases; Epidermal Growth Factor; Humans; Kidney Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Tumor Cells, Cultured

Five autonomous sublines, T4-O1320, T4-O1320CY, T4-O1165, T4-O1145 and T4-O196, were established from the transplantable hormone-dependent mouse mammary tumor, TPDMT-4, by passaging under different conditions. These autonomous tumors were characterized by rapid growth in DDD virgin mice and the parental TPDMT-4 by no growth in these mice. Thus, 10(5) T4-O1320, 2 x 10(4) T4-O1320CY, 2 x 10(3) T4-O1165, 2 x 10(3) T4-O1145 and 10(3) T4-O196 cells were co-injected with 5 x 10(5) TPDMT-4 cells into virgin mice to determine whether or not hormone-dependent tumor cells influence the growth of autonomous tumor cells. TPDMT-4 cells retarded the growth of T4-O1320 and T4-O1320CY tumors but accelerated that of T4-O1165, T4-O1145 and T4-O196. Irradiated TPDMT-4 cells stimulated the growth of all the sublines except T4-O1320. In 3-dimensional collagen-gel culture, T4-O1320 and T4-O1320CY cells formed branched or stellate structures similar to normal mammary glands, as did TPDMT-4, but T4-O1165, T4-O1145 and T4-O196 cells grew as rounded masses with knobs and showed a completely different morphology. T4-O1165, T4-O1145 and T4-O196 cells, but not the others, had lung-colonizing ability. Chromosomal aberration was found in T4-O1320CY and T4-O196 but not in the others. Thus, the susceptibility of autonomous subline tumor cells to growth-inhibitory regulation from the parental hormone-dependent tumor cells correlated well with growth morphology within collagen gels and metastatic ability, but not with chromosomal aberration. The results suggest that metastatically-competent tumor-cell variants, once they appear, may have a growth advantage in hormone-dependent mammary tumors.

Topics: Animals; Cell Aggregation; Cell Communication; Cell Division; Chromosome Aberrations; Collagen; Epidermal Growth Factor; Female; In Vitro Techniques; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Tumor Cells, Cultured

Increased expression of EGF receptor (EGFR) in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive causative link, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of functional receptors. A full length cDNA of the human EGFR (HER) was introduced by infection with a retroviral vector into MTC cells. Expression of HER was stable and receptors were functional with respect to surface expression, ligand binding and EGF-stimulated phosphorylation. Independent clones of the transfectants were isolated and characterized. Ligand stimulation of MTC HER cells and derived clones led to enhanced adhesion of cells to extracellular matrix proteins. Implantation of cells intravenously into female nu/nu mice revealed ligand-dependent enhancement of lung colonizing potential of EGFR-expressing cells.

Topics: Adenocarcinoma; Animals; Cell Adhesion; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix; Female; Ligands; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Rats; Transfection

The metastatic variants of human lung adenocarcinoma cell line DMS4C were established by selection in vivo. Lung, brain, spleen and liver metastatic tumors derived from intracarotid inoculation of athymic BALB/c mice were collected, and their corresponding variant cell lines established. The epidermal growth factor (EGF) receptor expression of the parental cell line as well as the metastatic variant cell lines were investigated. 125I-labeled EGF binding assays showed that there were two types of EGF receptors in both parental and metastatic variants. Compared to DMS4C, the EGF binding capacities were found to be down-regulated by 70, 79, 85 and 89% for lung, spleen, liver and brain variant, respectively. The dissociation constants of spleen, liver and brain EGF receptors were distinct from that of the parental cell line. The EGF receptor autophosphorylation activity of lung variant was shown to be down-regulated as shown by immune complex kinase assay that corresponded to EGF receptor numbers whereas kinase activities of the liver, spleen and brain variants EGF receptors were completely abolished. However, the 170 kilodalton EGF receptor was shown to be unaltered during metastasis. The results indicated that, during metastasis progression, the proliferation of adenocarcinoma cells may have adopted a different growth regulation that is independent of EGF receptor kinase-modulated autocrine pathway. The result also implies that other oncogene may emerge as the major growth regulator for distant metastasis of adenocarcinoma cancer cells. This work provides a model for understanding tumor metastasis progression of human lung cancer.

Topics: Adenocarcinoma; Animals; Brain; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Liver; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Spleen; Tumor Cells, Cultured

The aggressiveness of follicular thyroid cancer (FTC) varies widely, and metastasis is the primary cause of death. Uncontrolled proliferation of cancer cells may be associated with loss of growth factor control. We investigated the effects of stimulating (epidermal growth factor [EGF]; thyreotropin [TSH] in low concentrations) and inhibiting growth factors (transforming growth factor beta 1 [TGF beta 1]; TSH in high concentrations) on invasion and growth of FTC cell lines from the thyroid tumor (FTC133) and from the lymph node (FTC236) and lung (FTC238) metastases of the same patient. Invasion-penetration through an 8 microns pore membrane, covered by Matrigel (basement membrane)-and growth were measured using the MTT-method. EGF (10 ng/ml) and TSH in low concentrations (1 mU/ml) stimulated invasion and growth of all FTC cell lines, but the amplitude of stimulation differed significantly. The parental cell line FTC133 was considerably more responsive to growth factor stimulation than the metastatic clones. Invasion of FTC133 was enhanced by 42% (EGF; p < 0.02) and 21% (TSH; p < 0.01), invasion of FTC236 by 8% (EGF; p < 0.02) and 8% (TSH; p < 0.01), and invasion of FTC238 by 9% (EGF; p < 0.02) and 8% (TSH; p < 0.01). Conversely, invasion and growth of FTC133 were significantly more inhibited by TGF beta 1 (10 ng/ml) and supraphysiologic concentrations of TSH (100 mU/ml) than the cell lines from the lymph node and lung metastases. At day 7, invasion of FTC133 was inhibited by 32% (TGF beta 1; p < 0.02) and 21% (TSH; p < 0.01), invasion of FTC236 by 18% (TGF beta 1; p < 0.02) and 11% (TSH; p < 0.01), and invasion of FTC238 by 16% (TGF beta 1; p < 0.02) and 12% (TSH; p < 0.01). Moreover, we analyzed growth factor independence in minimally supplemented or unsupplemented medium. Growth, but no invasion was evident when cells were cultured completely unsupplemented over 7 days. These results suggest that metastatic FTCs may have developed by escaping from the normal control of TSH and other growth factors.

Topics: Adenocarcinoma, Follicular; Cell Division; Culture Media; Epidermal Growth Factor; Growth Substances; Humans; In Vitro Techniques; Neoplasm Invasiveness; Neoplasm Metastasis; Thyroid Neoplasms; Thyrotropin; Transforming Growth Factor beta; Tumor Cells, Cultured

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Topics: Adenocarcinoma; Animals; Aorta; Cattle; Cell Division; Collagenases; Culture Media, Conditioned; Endothelium, Vascular; Epidermal Growth Factor; Glycoproteins; Humans; Matrix Metalloproteinase Inhibitors; Neoplasm Metastasis; Salivary Gland Neoplasms; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured

A long-term continuous exposure to epidermal growth factor (EGF) enhanced the tumorigenicity of spontaneously transformed cells arising in a clonal population of normal cultured rat liver epithelial cells propagated in a selective growth condition. Lengthy EGF exposure also induced the expression of several phenotypes that differed from the phenotypes of rat liver epithelial cells transformed spontaneously in the absence of EGF. Epidermal growth factor treatment caused consistently an enhancement of the constitutive mRNA expression of transforming growth factor-alpha (TGF-alpha), but not of the EGF receptor and transforming growth factor-beta. The overexpression of TGF-alpha persisted in cell lines derived from tumors formed by the EGF-treated transformed cells. These tumors also exhibited high metastatic incidence and ductal cell differentiation. In contrast, untreated spontaneously transformed cells formed non-metastatic tumors with hepatocellular differentiation. These results suggest that long-term, continuous exposure to EGF/TGF-alpha may modulate the phenotypic expressions of neoplastic transformation.

Topics: Animals; Blotting, Northern; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Epithelium; ErbB Receptors; Gene Expression Regulation, Neoplastic; Liver; Liver Neoplasms; Neoplasm Metastasis; Phenotype; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Rats; Rats, Inbred F344; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

Insulin-like growth factor I (IGF-I) stimulates the proliferation of highly metastatic NL-17 cells to a greater extent than poorly metastatic NL-44 cells, both of which are derived from mouse colon carcinoma 26. The NL-17 cells have been compared with NL-44 cells for the signal transduction pathway of IGF-I. IGF-I receptors of both cell types were identified by affinity labeling, and there was no significant difference between the two cell types in the amount or the autophosphorylation activity of the IGF-I receptors. However, when IGF-I-dependent tyrosine phosphorylation of cellular components was examined, remarkable tyrosine phosphorylation of proteins with molecular weights of 150,000 (pp150) and 160,000 (pp160) was found in NL-17 cells. In contrast, this phosphorylation stayed at significantly lower levels in NL-44 cells than in NL-17 cells. The phosphorylation of pp150 and pp160 was induced within 10 s after the addition of IGF-I and reached its maximal level by 30 s. After the removal of IGF-I, the phosphorylation of pp150 and pp160 was reduced to the basal level within 30 min. This phosphorylation was not induced by platelet-derived or epidermal growth factor. The pp150 and pp160 were not absorbed by wheat germ agglutinin-agarose. They were found in the soluble fraction of cytoplasm but not in the membrane or the cytoskeleton. The pp150 and pp160 might be endogenous substrates of IGF-I receptor kinase. These results suggest that tyrosine phosphorylation of pp150 and pp160 mediates the higher proliferative response of NL-17 cells to IGF-I.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; Insulin; Insulin-Like Growth Factor I; Kinetics; Mice; Molecular Weight; Neoplasm Metastasis; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Somatomedin; Sepharose; Tyrosine

Thirty-two surgical specimens and three cell lines of human gastric cancers were used for subcutaneous transplantation into nude mice, resulting in the establishment of eight (25%) xenografts from the surgical specimens and two (67%) from the cell lines. The localization of epidermal growth factor (EGF) in the surgical specimens and cell lines of the gastric cancers and their xenografts in nude mice was then investigated immunohistochemically. Epidermal growth factor was stained in the cytoplasm of the cancer cells, being detected in 16 (50%) of the 32 surgical specimens and in all of the cell lines. Seven (44%) of the sixteen EGF-positive surgical specimens and one (6%) of the 16 EGF-negative ones were tumorigenic in nude mice. All of the xenografts in nude mice were positive for EGF. The tumorigenicity of human gastric cancer xenografts in nude mice may, therefore, be correlated with the presence of EGF in cancer cells.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Animals; Carcinoma, Renal Cell; Epidermal Growth Factor; Female; Humans; Kidney Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Neoplasm Transplantation; Staining and Labeling; Stomach Neoplasms; Tumor Cells, Cultured; Wilms Tumor

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth of paired murine melanoma cell clones that differ with respect to their experimental metastatic potential. Neither poorly (clone 16) nor highly (clone M2) metastatic cells were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium in the absence of serum. However, both clones were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium containing 10% calf serum. Colony formation in the presence of 10% calf serum was enhanced in a dose-dependent manner by TGF-beta 1 (half-maximal dose, 0.1 ng/ml) and was 5- to 10-fold greater than colony formation in the presence of 10% calf serum alone. Under anchorage-dependent (monolayer) conditions, neither clone grew in the absence of serum or in medium containing less than 1% calf serum. The monolayer growth of poorly metastatic cells (clone 16) was enhanced in a dose-dependent manner by TGF-beta 1 in medium supplemented with calf serum. Growth was 3.5-fold and 2.3-fold greater than untreated controls after 5 days in submitogenic (0.5%) and mitogenic (10%) concentrations of calf serum, respectively. In contrast, TGF-beta 1 had no effect on the monolayer growth of highly metastatic cells (clone M2) either in submitogenic (0.5%) or mitogenic (10%) concentrations of serum. TGF-beta 1 did not directly stimulate DNA synthesis by either poorly or highly metastatic cells when measured 24 h after TGF-beta 1 treatment. The ability of TGF-beta 1 to stimulate the anchorage-independent growth of metastatic melanoma cells suggests that this potent growth factor may play a role in the growth of these cells in vivo. In addition, the differential sensitivity of poorly and highly metastatic cells to TGF-beta 1 may be relevant to their metastatic potential in vivo. While the mechanism(s) by which TGF-beta 1 stimulates the growth of these cells remains unknown, these differentially metastatic clones of the K-1735 murine melanoma should provide a useful model in which to study the effects of transforming growth factor beta on the metastatic phenotype.

Topics: Animals; Cell Division; Culture Media; DNA, Neoplasm; Epidermal Growth Factor; Melanoma; Mice; Neoplasm Metastasis; Transforming Growth Factors; Tumor Cells, Cultured

Pancreatic carcinoma is usually a fatal disease with most patients dying of metastases. We have developed several pancreatic carcinoma cell lines that have varying metastatic abilities in a splenic injection/liver metastasis model. Epidermal growth factor (EGF) is a mitogen to most cell types with increased levels of EGF receptor. The parent cell line (COLO-357) of the pancreatic carcinoma cell lines used in this study has been shown to have a high number of EGF receptors per cell. We studied the relationship between the mitogenic responsiveness to EGF and the metastatic rate of each of the cell lines. Three of the six cell lines were significantly stimulated by EGF as determined by an increase in cell number over the course of 4 days, and three of the cell lines were not. There was no correlation between metastatic rate and EGF responsiveness. Further work will be needed to determine if there is any relationship between growth factors and their receptors and tumor metastasis with these pancreatic cancer cell lines.

Topics: Animals; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Pancreatic Neoplasms; Tumor Cells, Cultured

Epidermal growth factor receptor (EGFr) and cytosolic (cER) and nuclear (nER) estradiol receptors were quantified in 220 primary breast cancers. The EGFr was significantly more frequent (X2 = 5.9; P less than 0.025) and its concentration was significantly higher (P less than 0.001) among ER- tumors than in ER+ tumors. There was a significantly greater proportion (X2 = 6.4; P less than 0.05) of node involvement in EGFr+/ER+ tumors than in EFGr/ER+. Increases in the proportion of EGFr+ in ER- tumors are parallel to Scarff-Bloom scores (X2 = 6.1; P less than 0.05) and there is a significant trend towards increased EGFr concentrations with histologic dedifferentiation. In ER+ tumors the median concentrations of EGFr in the different age groups show linear correlation and follow a parallel profile with the medians of nER. These findings support the hypothesis that considers EGFr as a bad prognosis factor and suggest that EGFr expression and concentration in ER+ tumors might be considered an estrogenic action mediated through the binding of ER to their nuclear acceptors.

Topics: Age Factors; Breast Neoplasms; Cell Nucleus; Cytosol; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Metastasis; Prognosis; Receptors, Estradiol

A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.

Topics: Amino Acids; Animals; Antigens, Neoplasm; Carcinoma; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Female; Insulin; Integrin alpha6beta4; Integrins; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Phosphorylation

The effect of epidermal growth factor (EGF) on the in vitro growth of human malignant tumors was compared in serum-supplemented (n = 54) and serum-free (N = 41) media at clonal density to determine the true EGF dependency of tumors. In the complete absence of serum at a 1,000 cells/cm2 seeding inoculation (approximately 100-200 adherent cells), EGF increased growth by greater than 50% in 27 of 41 specimens (66%), and growth increased by 100% or more in 18 of these EGF-sensitive tumors. In 12 serum-free cultures (29%), in vitro growth failed to occur without EGF. With 10% serum supplementation and a lower cell density (250 cells/cm2), EGF increased growth by greater than 50% in 34 of 54 specimens (63%), of which 25 had more than a 100% increase. The maximum growth induced by EGF in serum was usually seen in those tumors already capable of moderate in vitro growth. No difference in response to EGF was detected between specimens from primary tumors (n = 24) and those from metastases (n = 30). Under the stringent culture conditions of complete absence of serum and with tumors seeded at a low cell number, EGF stimulated most primary or metastatic human tumors to establish and sustain short-term in vitro growth successfully.

Topics: Blood; Epidermal Growth Factor; Humans; Neoplasm Metastasis; Tumor Cells, Cultured

The clinical implications of the epidermal growth factor (EGF) and its receptor (EGF-R) were studied in 52 squamous cell carcinomas of the uterine cervix. In comparison to 40 biopsies of the normal cervix EGF-R capacity was significantly increased in the carcinomas, while the affinity was unchanged. The amount of EGF-like substances extracted from the tumors was increased in patients with lymph node metastases, in whom 5-year survival is reduced. Irrespective of tumor stage patients with a very high level of EGF-R (greater than 100 fmole/mg protein) were more likely to have recurrences later or to die from disease: recurrence or death occurred in 5 of 7 patients with high capacity and in 2 of 45 patients with low capacity. Our data suggest that the level of EGF-R is indicative of the biological aggressiveness of cervical carcinomas.

Topics: Adult; Carcinoma, Squamous Cell; Cervix Uteri; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lymphatic Metastasis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Uterine Cervical Neoplasms

Using a rat rhabdomyosarcoma 9-4/0, we investigated the role of epidermal growth factor (EGF) in tumor dissemination. In vitro, we detected high-affinity EGF receptors on tumor cells and stimulation of their proliferation by EGF. When injected iv, EGF-pretreated cells demonstrated an increased capacity to form lung colonies and to invade lymphatic tissue. In vivo, EGF treatment led to increased metastatic spread of subcutaneous tumors. When primary tumors were ablated, and the treatment was given from the time of graft until ablation (seeding step), no effect on metastatic spread was noticed. When treatment was given from the time of ablation until death (growth step), EGF increased the number of lung metastases and of invaded lymph node sites.

Topics: Animals; Cell Division; Epidermal Growth Factor; Female; Lung Neoplasms; Neoplasm Metastasis; Rats; Rhabdomyosarcoma

The fate of exogenous glycosaminoglycans in cultures of strongly (RMS 0) and weakly (RMS 8) metastatic rat rhabdomyosarcoma cells was studied. The time course and concentration dependence of binding and internalization of the radiolabeled sulfated glycosaminoglycans were determined. Weakly metastatic cells took up heparin, heparan and dermatan sulfates into their pericellular compartment at a higher rate than the strongly metastatic RMS 0 cells. The RMS 8 cells exhibited about two times more binding sites for these iduronic acid containing glycosaminoglycans, and internalized higher amounts of them than the RMS 0 cells. The uptake of the chondroitin sulfate into the peri- and intracellular compartments of both cell types was about 5-15% of that of the other glycosaminoglycans studied. The specificity of displacement of the pericellular heparin and dermatan sulfate by the unlabeled glycosaminoglycans indicates the involvement of specific structural features of the polysaccharide chains in the interactions of glycosaminoglycans with the surface of rhabdomyosarcoma cells, beside ionic forces due to the polyanionic character of the glycosaminoglycans. Heparin and heparan sulfate degradation products, mainly large oligosaccharides, were recovered from the surface of RMS 0 cells but were absent on the surface of the RMS 8 cells. About 30% of the internalized heparin and heparan sulfate was present in the partially degraded form in both cell types. Oligosaccharides derived from glycosaminoglycans were not released into the medium. The decrease in the amount of iduronic acid containing glycosaminoglycans internalized by the highly invasive cells seems to be correlated with an increased cell-associated degradation and with an apparent loss of glycosaminoglycan binding sites on the cell surface.

Topics: Animals; Binding Sites; Cell Line; Cell Membrane; Endocytosis; Epidermal Growth Factor; Glycosaminoglycans; Kinetics; Molecular Weight; Neoplasm Metastasis; Rats; Rhabdomyosarcoma; Temperature; Tumor Cells, Cultured

Abnormally high expression of epidermal growth factor receptors (EGF-receptors) may contribute to the unregulated growth of some tumors. We here report the EGF-receptor numbers and the effects of epidermal growth factor (EGF) on two human cell lines. The glioblastoma cell line T-MG1 had 135,000 EGF-receptors per cell, was slightly growth stimulated by EGF and showed no obvious change in morphology after exposure to EGF. The carcinoma cell line T-CAR1, derived from a brain metastasis of a carcinoma of the adrenal cortex, had approximately 7 million EGF-receptors per cell. EGF had a significant antiproliferative effect on these cells and caused rounding and detachment of cells in adherent cultures. The cell lines may become useful in future studies concerning the role of the EGF-receptors in malignant growth.

Topics: Brain Neoplasms; Carcinoma; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Neoplasm Metastasis; Time Factors; Tumor Cells, Cultured

Epidermal growth factor receptors (EGFr) have been measured on primary human bladder tumor membranes by 125I-EGF ligand binding. High affinity receptors were detected on both superficial (Kd 0.2-1.45 nM; mean, 0.86 nM; median, 0.88 nM) and invasive tumors (Kd 0.19-2.38 nM; mean, 0.9 nM, median, 0.79 nM). There was one class of binding sites and EGFr concentration was quantified by competitive binding and Scatchard analysis. The EGFr was further characterized and shown to be cleaved at the major autophosphorylation site by a calcium-activated mechanism. Thus the EGFr from primary bladder tumors exhibits similar biochemical characteristics to those in established cell lines. Tumors classified as invasive on the basis of muscle invasion had higher EGFr levels [EGF binding, 99 +/- 252 (SD) fmol/mg protein; median, 21; n = 24] than superficial tumors (12 +/- 12 fmol/mg protein; median, 11; n = 23) or normal bladder mucosa (9 +/- 12 fmol/mg protein; median, 6; n = 6) (P = 0.05). When the two largest subgroups of superficial and invasive tumors were compared (15 pTa, 16 T3), the invasive tumors had significantly higher EGFr levels (P less than 0.05). EGFr may therefore be involved in mechanisms of tumor progression. EGFr may be a target for selective therapy with EGF-linked drugs in a subset of invasive bladder cancers.

Topics: Aged; Biomarkers, Tumor; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Male; Molecular Weight; Neoplasm Invasiveness; Neoplasm Metastasis; Urinary Bladder Neoplasms

Cell lines with varying tumorigenic and metastatic potentials have been obtained by transformation of 10T 1/2 fibroblasts using radiation or transfection with T-24 H-ras. We have observed an inverse relationship between metastatic potential and dependence on serum for growth. The effects of basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta 1) on these lines were then examined to determine if the changes in the serum dependence of metastatic cells may be due to altered responsiveness to specific growth factors (GFs). Cells were grown in monolayer culture and DNA synthesis was measured by [CH3-3H]thymidine incorporation experiments. Both metastatic and nonmetastatic cells were shown to be equivalent in their diminished responsiveness to basic fibroblast growth factor, platelet-derived growth factor, and epidermal growth factor as compared to their nontransformed, parental 10T 1/2 cells. However, a unique response of metastatic cells to TGF-beta 1 was identified. While TGF-beta 1 inhibited DNA synthesis in 10T 1/2 cells and a nonmetastatic tumor, cells with intermediate to high metastatic ability were stimulated up to 5.8-fold by TGF-beta 1. Interestingly, epidermal growth factor abrogated the TGF-beta 1 inhibition of the parental 10T 1/2 cells, but had no effect on the TGF-beta 1 response of any metastatic line. Therefore, metastatic but not nonmetastatic cells, demonstrated a dramatically altered sensitivity to TGF-beta 1, a response which may be important in determining metastatic potential.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; DNA Replication; Epidermal Growth Factor; Fibroblast Growth Factors; Fibroblasts; Fibrosarcoma; Genes, ras; Growth Substances; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Platelet-Derived Growth Factor; Transforming Growth Factors

Two hundred and twenty one gastric carcinomas were immunohistochemically stained for h-EGF and we examined the correlation between h-EGF immunoreactivities and histologic findings. Regarding macroscopic and histologic types, incidence of h-EGF immunoreactivities in infiltrating type and in poorly differentiated type was significantly higher than those in localized type and in differentiated type, respectively. In addition, h-EGF producing carcinomas showed high positive rate in prognostic serosal involvement and scirrhous type in stroma. Prognosis in patients with h-EGF producing tumors was poorer than that in those with h-EGF non-producing tumors, especially in stages II and III. These results suggest that h-EGF immunoreactivities serves as a biological marker of high malignancy.

Topics: Adenocarcinoma; Biomarkers, Tumor; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Stomach Neoplasms

The v-fps oncoprotein was expressed in a pre-neoplastic, growth factor-dependent Chinese hamster lung fibroblast line (CCL39) to study its effect on growth controls and on the induction of malignancy. Two transfectants were characterized which expressed low (39FPS-8) or high (51FPS-6) levels of P130gag-f ps protein-tyrosine kinase activity. 39FPS-8 cells still arrested in quiescence when deprived of growth factors, but developed an increased sensitivity to the mitogenic actions of epidermal growth factor (20-fold) and alpha-thrombin (50-fold), although not to insulin. In contrast, 51FPS-6 cells completely escaped growth controls, divided in serum-free medium, and were insensitive to further growth factor stimulation. Both transfectants produced rapidly growing tumors in nude mice that formed pulmonary metastases from a subcutaneous site, unlike the parental cells which are non-metastatic. 51FPS-6 cells were comparatively more efficient than 39FPS-8 cells in colonizing the lungs after intravenous inoculation. The v-fps tyrosine kinase therefore induces a partial to complete relaxation of growth factor-mediated controls on the CCL39 cell cycle, with the extent of factor independence reflecting the amount of P130gag-f ps synthesized. This reduction in growth factor requirements correlates with the capacity of v-fps to confer the attributes of metastatic tumors upon preneoplastic CCL39 fibroblasts. We speculate that increased sensitivity to growth factor stimulation represents a common mechanism by which tumor cells acquire metastatic properties.

Topics: Animals; Cell Division; Cell Line; Cricetinae; Epidermal Growth Factor; Growth Substances; Insulin; Interphase; Lung; Lung Neoplasms; Neoplasm Metastasis; Oncogene Proteins, Viral; Precancerous Conditions; Protein-Tyrosine Kinases; Thrombin

A radioimmunoassay for transforming growth factor alpha (TGF-alpha) using synthetic rat sarcoma transforming growth factor and its rabbit polyclonal antibody has been developed. Using radioimmunoassays, the urinary TGF-alpha and epidermal growth factor (EGF) concentrations in 31 patients with hepatocellular carcinoma (HCC), 15 probable HCC, four metastatic liver cancer, and 33 age, sex-matched healthy controls were determined. For the first time, we have shown that the average TGF-alpha concentration for HCC patients was 21.5 +/- 20.3 micrograms per g creatinine, significantly higher than that of healthy subjects, 4.9 +/- 2.8 micrograms per g creatinine (P less than 0.001). There was no statistical difference in the level of EGF between HCC patients and controls (40.9 +/- 29.3 versus 46.2 +/- 16.6 micrograms per g creatinine; P greater than 0.05). The ratio of EGF/TGF-alpha between HCC patients (3.37 +/- 4.42) and controls (15.5 +/- 13.0) was significantly different (P less than 0.001). Among patients, 65% (20 of 31) of HCC cases and 87% (13 of 15) of probable HCC cases showed a marked elevation of TGF-alpha levels. We found only 16% (five of 31) of HCC cases with increased EGF level. EGF excretion was inversely age related. Serum total protein concentration and alkaline phosphatase activity were positively correlated to EGF concentration (r = 0.522, P less than 0.01 and rt = 0.393, P less than 0.05, respectively). There was no correlation between biochemical functions of liver and TGF-alpha concentration in HCC patients. Our results also suggested that TGF-alpha may be a useful complementary tumor marker for management of patients with clinical manifestation of HCC who have low alpha-fetoprotein levels.

Topics: Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Creatinine; Epidermal Growth Factor; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Peptides; Reference Values; Transforming Growth Factors

Topics: Animals; Antibodies, Monoclonal; Epidermal Growth Factor; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Nerve Growth Factors; Submandibular Gland

The hormone-responsive mammary tumor 13762NF of the F344 rat was cultured in a collagen gel matrix with the use of a serum-free medium supplemented with hormones and epidermal growth factor (EGF). Hydrocortisone (F) had the greatest effect on cell growth. EGF had no growth-promoting activity when used alone, but it had a significant effect when used with F. There was also a population of cells responsive to progesterone (P) and prolactin (PRL). P synergized with EGF as well as with PRL to promote growth. 17 beta-Estradiol alone or in combination with other hormones had no growth-promoting activity. Receptor levels in the tumor were high for glucocorticoids, intermediate for P and EGF, and low for estrogens. Metastasis in the lung and lymph node showed the same basic hormonal responses as the parent tumor. Cultured cells produced tumors with the same histopathology as the parent tumor when transplanted back into female rats; they had the same receptor levels and when placed back in culture showed a growth response to the same set of hormones. The tumor cells formed colonies that were spherical, which was different from the branching structures formed by normal mammary cells in collagen gel.

Topics: Animals; Basement Membrane; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Gels; Gonadal Steroid Hormones; Hormones; Hydrocortisone; Mammary Neoplasms, Experimental; Microscopy, Electron; Neoplasm Metastasis; Neoplasm Transplantation; Prolactin; Rats; Receptors, Cell Surface; Receptors, Estradiol; Receptors, Glucocorticoid; Receptors, Progesterone

c-Ha-ras oncogene product in human gastric carcinomas was examined by Western blotting and immunohistochemistry using anti-Ha-ras p21 antibody. In Western blotting, high levels of c-Ha-ras p21s were found in gastric carcinomas. Immunohistochemically, c-Ha-ras p21 was detected in 3 (11.1%) of 27 early carcinomas and in 63 (43.8%) of 144 advanced carcinomas. In advanced carcinomas, c-Ha-ras p21-immunoreactivity was correlated with the depth of tumor invasion and was stronger in metastatic tumors than in primary tumors. Patients with c-Ha-ras p21-positive carcinomas had a significantly worse prognosis than those with p21-negative carcinomas.

Topics: Amino Acid Sequence; Carcinoma; Epidermal Growth Factor; Histocytochemistry; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Oncogenes; Prognosis; Proto-Oncogene Proteins; Stomach Neoplasms

Epidermal growth factor (EGF) receptor is expressed selectively by human melanoma cells which show the presence of an extra copy of chromosome 7. None of the cells of benign pigmented lesions (nevi) or radial growth phase (nonmetastatic) primary melanoma expressed EGF receptor and none of these cells showed an extra copy of chromosome 7. The results indicate that a single extra dose of a gene (for EGF receptor) may provide a selective advantage to cells in the late stages of tumorigenesis.

Topics: Antibodies, Monoclonal; Cell Line; Chromosome Mapping; Chromosomes, Human, 6-12 and X; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Genes; Humans; Melanoma; Neoplasm Metastasis; Nevus, Pigmented; Receptors, Cell Surface

Transforming growth factors (TGFs) were purified from serum-free medium conditioned by retrovirus-transformed Fisher rat embryo fibroblasts, mouse 3T3 cells, and two human melanoma cell lines. The purification of each TGF was monitored in a radioreceptor assay based on receptor crossreactivity with mouse submaxillary gland epidermal growth factor (mEGF) and was achieved by gel permeation chromatography of the acid-soluble TGF-containing activity, followed by reverse-phase high-pressure liquid chromatography with sequential use of acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. The amino-terminal sequences of rat, mouse, and human TGFs were determined. Extensive sequence homology was found among TGF polypeptides from different species and cell types. Alignment of the amino acid sequences of rat TGF, mEGF, and human urogastrone (hEGF) reveals statistically significant sequence homology. The reported results suggest that TGFs that compete for binding to the cellular EGF receptor and EGF may have evolved from a common progenitor.

Topics: Amino Acid Sequence; Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Embryo, Mammalian; Epidermal Growth Factor; Fibroblasts; Genes; Humans; Melanoma; Mice; Moloney murine leukemia virus; Neoplasm Metastasis; Peptides; Rats; Rats, Inbred F344; Retroviridae; Sarcoma Viruses, Feline; Transforming Growth Factors

Topics: Animals; Cell Division; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Neoplasm Metastasis; Neoplasms, Experimental; Ovarian Neoplasms; Peptides; Rats; Transferrin

It is suggested that a common proliferative factor is required for the growth of clones of both antigen-triggered B lymphocytes and carcinogen-triggered epithelial cells, and that the common growth-promoting substance is secreted by macrophages which have originally differentiated in support of the immune response. This macrophage-derived factor seems to be antigenically distinct from granulocyte colony-stimulating factor. Identification of the macrophage-derived clonal proliferative factor could provide new understanding of the proliferation of clones of immunocytes and neoplastic cells.

Topics: Animals; Antibody-Producing Cells; B-Lymphocytes; Cell Division; Clone Cells; Epidermal Growth Factor; Growth Substances; Humans; In Vitro Techniques; Macrophages; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms