transforming-growth-factor-beta and Colonic-Neoplasms

Colorectal cancer (CRC) is a serious public health issue, and it has the leading incidence and mortality among malignant tumors worldwide. CRC patients with metastasis in the liver, lung or other distant sites always have poor prognosis. Thus, there is an urgent need to discover the underlying mechanisms of metastatic colorectal cancer (mCRC) and to develop optimal therapy for mCRC. Transforming growth factor-β (TGF-β) signaling plays a significant role in various physiologic and pathologic processes, and aberrant TGF-β signal transduction contributes to mCRC progression. In this review, we summarize the alterations of the TGF-β signaling pathway in mCRC patients, the functional mechanisms of TGF-β signaling, its promotion of epithelial-mesenchymal transition, its facilitation of angiogenesis, its suppression of anti-tumor activity of immune cells in the microenvironment and its contribution to stemness of CRC cells. We also discuss the possible applications of TGF-β signaling in mCRC diagnosis, prognosis and targeted therapies in clinical trials. Hopefully, these research advances in TGF-β signaling in mCRC will improve the development of new strategies that can be combined with molecular targeted therapy, immunotherapy and traditional therapies to achieve better efficacy and benefit mCRC patients in the near future.

Topics: Colonic Neoplasms; Epithelial-Mesenchymal Transition; Humans; Rectal Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

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

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

Transforming growth factor beta (TGF-β) is a multipurpose cytokine, which plays a role in many cellular functions such as proliferation, differentiation, migration, apoptosis, cell adhesion and regulation of epithelial to mesenchymal transition. Despite many studies having observed the effect that TGF-β plays in colorectal cancer, its role in the colorectal stem cell population has not been widely observed.. This systematic review will analyse the role of TGF-β in the stem cell population of colorectal cancer.. The effects on the stem cell phenotype are through the downstream proteins involved in activation of the TGF-β pathway. Its involvement in the initiation of the epithelial to mesenchymal transition (EMT), the effect of colorectal invasion and metastasis regulated through the Smad protein involvement in the EMT, initiation of angiogenesis, promotion of metastasis of colorectal cancer to the liver and its ability to cross-talk with other pathways.. TGF-β is a key player in angiogenesis, tumour growth and metastasis in colon cancer.

Topics: Colonic Neoplasms; Epithelial-Mesenchymal Transition; Humans; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

Colon cancer is one of the third most common cancer in man, the second most common cancer in women worldwide, and the second leading cause of mortality in the USA. There are a number of molecular pathways that have been implicated in colon carcinogenesis, including TGF-β/Smad signaling pathway. TGF-β (transforming growth factor-beta) signaling pathway has the potential to regulate various biological processes including cell growth, differentiation, apoptosis, extracellular matrix modeling, and immune response. TGF-β signaling pathway acts as a tumor suppressor, but alterations in TGF-β signaling pathway promotes colon cancer cell growth, migration, invasion, angiogenesis, and metastasis. Here we review the role of TGF-β signaling cascade in colon carcinogenesis and multiple molecular targets of curcumin in colon carcinogenesis. Elucidation of the molecular mechanism of curcumin on TGF-β signaling pathway-induced colon carcinogenesis may ultimately lead to novel and more effective treatments for colon cancer.

Topics: Carcinogenesis; Chemoprevention; Clinical Trials as Topic; Colonic Neoplasms; Curcumin; Humans; NF-kappa B; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Colorectal cancer remains the most common cancer and the second leading cause of cancer mortality in Europe. There are a number of pathways that have been implicated in colorectal carcinogenesis, including TGF-beta (TGF-β)/Smad signalling pathway. The TGF-β pathway is involved in several biological processes, including cell proliferation, differentiation, migration and apoptosis. Here we review the role of TGF-β signalling cascade in colorectal carcinogenesis and provide some new molecular insights that may aid efforts towards targeted antitumor therapies.

Topics: Animals; Cell Transformation, Neoplastic; Colonic Neoplasms; Humans; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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

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

It is sometimes difficult but finally possible to distinguish colitis-associated neoplasms from sporadic neoplasms. The frequency of detection of precursor lesions of carcinoma (e.g. dysplasia, intraepithelial neoplasia and adenoma) has increased in recent years, which is most probably due to better endoscopic detection and thus improved histological diagnosis. Carcinogenesis of colitis-associated neoplasms is different from carcinogenesis in sporadic neoplasms because mutations and epigenetic changes are different or may occur at a different point in time. In the present article, these differences will be described and placed in context with carcinogenesis in ulcerative colitis.

Topics: Adenoma; Carcinoma in Situ; Cell Transformation, Neoplastic; Chromosome Aberrations; Colitis, Ulcerative; Colonic Neoplasms; DNA Methylation; DNA Mutational Analysis; Epigenesis, Genetic; Humans; Intestinal Mucosa; Microsatellite Instability; Neoplasm Invasiveness; Neoplasm Staging; Oxidative Stress; Precancerous Conditions; Reactive Oxygen Species; Transforming Growth Factor beta

Colorectal cancer is the second most common cause of cancer-related death in the United States. Twin studies suggest that 35% of all colorectal cancer cases are inherited. High-penetrance tumor susceptibility genes account for at most 3-6% of all colorectal cancer cases and the remainder of the unexplained risk is likely due to a combination of low to moderate penetrance genes. Recent genome-wide association studies have identified several SNPs near genes belonging to the transforming growth factor beta (TGF-beta) superfamily such as GREM1 and SMAD7. Together with the recent discovery that constitutively decreased TGFBR1 expression is a potent modifier of colorectal cancer risk, these findings strongly suggest that germline variants of the TGF-beta superfamily may account for a sizeable proportion of colorectal cancer cases. The TGF-beta superfamily signaling pathways mediate many different biological processes during embryonic development, and in adult organisms they play a role in tissue homeostasis. TGF-beta has a central role in inhibiting cell proliferation and also modulates processes such as cell invasion, immune regulation, and microenvironment modification. Mutations in the TGF-beta type II receptor (TGFBR2) are estimated to occur in approximately 30% of colorectal carcinomas. Mutations in SMAD4 and BMPR1A are found in patients with familial juvenile polyposis, an autosomal dominant condition associated with an increased risk of colorectal cancer. This chapter provides an overview of the genetic basis of colorectal cancer and discusses recent discoveries related to alterations in the TGF-beta pathways and their role in the development of colorectal cancer.

Topics: Bone Morphogenetic Proteins; Carcinoma; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; Mutation; Polymorphism, Genetic; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Treatment Outcome

Transforming growth factor-β (TGF-β) plays a central role in a wide array of cellular functions including control of cell growth and differentiation, embryonic development, wound healing, angiogenesis, and immune regulation. In the gastrointestinal tract, TGF-β can either promote or suppress inflammation and cancer formation. This report reviews recent data on the role of TGF-β in the pathogenesis of inflammatory bowel disease and how TGF-β might contribute to the cancer risk associated with chronic inflammation of the gut.

Topics: Animals; Cell Proliferation; Cell Transformation, Neoplastic; Colitis; Colonic Neoplasms; Humans; Inflammatory Bowel Diseases; Lymphocyte Activation; Mice; Th17 Cells; Transforming Growth Factor beta

Colon cancer is the third most common cancer and third most common cause of cancer-related death in the USA according to 2008 American Cancer Society statistics. The carcinogenesis of colon cancer has been associated with both genetics and environmental factors. It has been found that several signal pathways, including K-ras, Src/PI3K/Akt, beta-catenin, TGFbeta and p53 play critical roles in its pathogenesis. The 5 y survival rate of metastatic colon cancer is below 10%. Thus, it is necessary to further understand its biology and search for effective therapy. Azoxymethane (AOM) is a common model for colon cancer. It can specifically induce colon cancer similar to the pathogenesis of human sporadic colon cancer. Thus, it has been extensively used in the study of the molecular biology, prevention and treatment of colon cancer. After administration, AOM is metabolised into methylazoxymethanol by CYP2E1, which causes DNA mutations. Mutation of K-ras activates this pathway and its downstream PI3K/Akt pathway and MAPK pathway. Mutation of beta-catenin also prevents it from being degraded by GSK-3 and accumulation of beta-catenin leads to cell proliferation. TGFbeta, a pro-apoptotic protein, is inhibited. All of these changes form the basis of AOM carcinogenesis. This model has been used in the study of the genetic deficiencies of colon cancer and in the prevention and treatment of the disease. For example, TGF-betaR2 and adiponectin knockout mice are more susceptible to AOM, while high amylose cornstarch, green tea and artemisia have protective effects.

Topics: Adenocarcinoma; Adenoma; Adiponectin; Animals; Anticarcinogenic Agents; Apoptosis; Azoxymethane; Carcinogens; Colonic Neoplasms; Cytochrome P-450 CYP2E1; Diet; DNA Damage; Genes, ras; Humans; MAP Kinase Signaling System; Methylazoxymethanol Acetate; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta2

IL-6 plays a pivotal role in immune responses and certain oncologic conditions. The intense investigation of its biological activity and function led to the discovery of two different IL-6-driven signalling pathways. Binding to the membrane-bound IL-6 receptor (mIL-6R, CD126) causes the recruitment of two gp130 co-receptor molecules (CD130) and the activation of intracellular signalling cascades via gp130. Although this classical pathway is mainly limited to hepatocytes, neutrophils, monocytes/macrophages and certain other leukocyte populations, which express IL-6R on their surface, an alternative mechanism has also been described. Proteolytic cleavage of the mIL-6R protein or translation from alternatively spliced mRNA leads to the generation of a soluble form of the IL-6R (sIL-6R), which is likewise able to bind to IL-6. The resulting IL-6/sIL-6R complex is also capable of binding to gp130 and inducing intracellular signalling. Through this so-called 'trans-signalling' mechanism, IL-6 is able to stimulate cells that lack an endogenous mIL-6R. High levels of IL-6 and sIL-6R have been reported in several chronic inflammatory and autoimmune diseases as well as in cancer. Preclinical animal disease models have provided strong evidence that specific blockade of IL-6-regulated signalling pathways represents a promising approach for the therapy of these diseases. An optimised variant of the recently described fusion protein sgp30Fc is now heading towards its clinical evaluation.

Topics: Alternative Splicing; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Arthritis; Arthritis, Rheumatoid; Asthma; Autoimmune Diseases; Cell Line; Clinical Trials as Topic; Colitis; Colonic Neoplasms; Cytokine Receptor gp130; Disease Models, Animal; Drug Delivery Systems; Drug Evaluation, Preclinical; Humans; Inflammation; Interleukin-6; Leukocytes; Male; Mice; Neoplasms; Receptors, Interleukin-6; Recombinant Fusion Proteins; Signal Transduction; Solubility; Transforming Growth Factor beta

Three major mucosal systems exist in the body, the oral-gastrointestinal, the respiratory and the genitourinary systems. In particular, the gastrointestinal (GI) tract contains the largest mucosal surface in the body and is the major port of entry for foreign antigens. Therefore, the gut immune system has to differentiate to tolerate dietary antigens and expel infectious and harmful pathogens. During the complex but well-orchestrated immune responses in the mucosal system, T cells play a pivotal role in both immunity and tolerance. Of many T cell subpopulations, CD4(+)CD25(+) T regulatory cells (Tregs) are instrumental in regulation of immune responses in mucosea. Among the multitude of cytokines and factors that are produced in the gut, Transforming Growth Factor-beta (TGF-beta) is probably the most important one in influencing mucosal T cell responses. The interaction and mutual regulation between TGF-beta and CD4(+)CD25(+) Tregs may be the key in maintaining the balance between T cell immunity and tolerance in mucosal system. In this article, we attempt to discuss both beneficial and detrimental effects of TGF-beta and Tregs on oral tolerance, mucosal inflammation and autoimmunity, colon cancer and HIV infection in the gut.

Topics: Autoimmune Diseases; CD4 Antigens; Colonic Neoplasms; Cytokines; HIV Infections; Humans; Immune Tolerance; Immunity, Mucosal; Inflammation Mediators; Interleukin-2 Receptor alpha Subunit; Mucous Membrane; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

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

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

Research over the past decade has established that the progression from normal colonic epithelium to colon cancer is in every case a step-wise process in which specific pathologic and molecular markers can be identified for study and clinical therapy. Genetic and epigenetic instability appears fundamentally important to this process. We have now determined that this neoplastic progression occurs along a limited set of pathways, in which specific tumor suppressors are inactivated or oncogenes activated in a defined order. Although incomplete, our new understanding of the process of carcinogenesis in the colon has already significantly impacted patient care and will continue to do so for the foreseeable future. Increasingly rapid research developments and technologic advances will transform the way we prevent, diagnose, and treat this common and deadly form of cancer.

Topics: Colonic Neoplasms; Genes, p53; Genes, ras; Humans; Mutation; Rectal Neoplasms; Transforming Growth Factor beta

Topics: Animals; Colonic Neoplasms; Disease Progression; Genes, APC; Genes, DCC; Genes, Tumor Suppressor; Humans; Mice; Signal Transduction; Transforming Growth Factor beta

TGF-beta is a member of a superfamily associated with development and regulation of cell growth. Recently, its two receptors and intracellular components of signal transduction, a Smad family, have been identified. The type II receptor gene has been found to be mutated specifically in hereditary colon cancers, HNPCC. Alterations of the Smad 4 gene were also detected in pancreatic and colorectal cancers.

Topics: Colonic Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; Humans; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The TGF-betas are a family of cytokines with antiproliferative activity on many cell types. Recent findings demonstrate that the TGF-beta receptor complex functions as a tumor suppressor gene in human malignancy. Somatic mutations of the type II subunit of the TGF-beta receptor (RII) have been demonstrated in several different tumor types. RII frameshift mutations within a short coding region polyadenine repeat are particularly characteristic of colon and gastric cancers that also demonstrate the phenotype of microsatellite instability (RER cancers). These and other mutations as in the type I receptor (RI) are associated with both loss of cell surface TGF beta receptors and with resistance of the cancer cells to TGF-beta-induced growth inhibition. Restoration of receptor expression by gene transfection reverses the transformed phenotype in cancer cells that lacked functional RII or RI. These receptors and, by implication, TGF-beta as well as its complete signalling pathway, are thus new and novel additions to the family of human tumor suppressor genes.

Topics: Cell Division; Colonic Neoplasms; Genes, Tumor Suppressor; Humans; Mutation; Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

There is now clear-cut evidence that polypeptide growth factors control the proliferation of the normal gastrointestinal mucosa. Epidermal growth factor (EGF) stimulates normal growth throughout the gastrointestinal tract, and accelerates the healing of ulcerated epithelium. While the effects of gastrin were at first thought to be similarly widespread, the gastrin target now appears to be restricted to the enterochromaffin-like cells in the stomach. Isolated reports suggest that several other hormones, including fibroblast growth factor and the insulin-like growth factors, have similar proliferative effects. In contrast, indirect evidence suggests that somatostatin and transforming growth factor-beta inhibit the growth of the gastrointestinal mucosa. The same growth factors profoundly affect the growth of some gastrointestinal carcinomas. Prolonged hypergastrinaemia increases the risk of development of gastric endocrine tumours, but has no effect on the incidence of gastric adenocarcinoma. Gastrin also stimulates the in vivo growth of 50% of gastric and colorectal carcinoma xenografts, but has no consistent effect on the growth of carcinoma cell lines in vitro. EGF, on the other hand, significantly stimulates proliferation of many gastrointestinal cell lines in culture. Interest has recently focused on autocrine stimulation of gastrointestinal carcinoma growth. Elevated levels of EGF receptor, and of EGF or related mRNAs, have been demonstrated in gastric carcinomas, and the growth of some gastrointestinal cell lines is inhibited by antibodies against EGF, and by antisense oligonucleotides based on EGF mRNA. Similarly gastrin/cholecystokinin antagonists inhibit the growth of several colon carcinoma cell lines, although the spectrum of antagonist potencies suggests that classical gastrin and cholecystokinin receptors are not necessarily involved. Continued research on antagonists may therefore lead to novel therapies for gastrointestinal cancers.

Topics: Adult; Animals; Colonic Neoplasms; Digestive System Physiological Phenomena; Epidermal Growth Factor; ErbB Receptors; Gastrins; Gastrointestinal Hormones; Gastrointestinal Neoplasms; Glucagon-Like Peptides; Humans; Peptide YY; Peptides; Somatostatin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor alpha and beta 1 (TGF alpha and TGF beta 1) are representative members of two distinct and expanding families of polypeptide growth factors. TGF alpha is an epithelial cell mitogen, whereas TGF beta 1 inhibits epithelial cell growth; the role of these factors in contributing to the transformed phenotype is uncertain. Steady state mRNA expression for these growth factors and their receptors in a panel of human colon cancers and adjacent normal mucosa is presented. Based in part on results from transgenic mice in which TGF alpha is selectively overproduced in the mammary gland, a possible role for TGF alpha as a tumor promoter in the process of transformation is discussed.

Topics: Animals; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Male; Mice; Mice, Transgenic; Multigene Family; Proto-Oncogenes; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor alpha; Transforming Growth Factor beta

Colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways such as Notch and TGF-β contribute to these phenomena. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-β signaling pathway. We hypothesized that Smad3, mediates the Notch-induced stemness and EMT observed in CRC cells. The human colorectal carcinoma cell line HCT-116, stably transduced with constitutively active Notch-1 (ICN) or a GFP-vector control was treated with different combinations of TGF-β1, DAPT (a Notch inhibitor), or SIS3 (a Smad3 inhibitor). Western blot analysis was performed to determine the effects of Smad3 stimulation and inhibition on Notch and potential downstream EMT-related targets, CD44, Slug and Snail. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression and effectively inhibited CD44, Slug, and Snail expression. Colosphere forming ability was also reduced in cells with inhibited Smad3. These results indicate a key role of TGF-β signaling in Notch1-induced tumorigenesis, and suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.

Topics: Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Humans; Phosphorylation; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

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

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

Mesenchymal stem cell (MSC) therapy has been shown to have some therapeutic effects in rodent models and patients with IBD; however, its role in colon tumor models is controversial. In this study, the potential role and mechanisms of bone marrow-derived MSCs (BM-MSCs) in colitis-associated colon cancer (CAC) were investigated.. The CAC mouse model was established with azoxymethane (AOM) and dextran sulfate sodium (DSS). The mice were administered an intraperitoneal injection of MSCs once weekly for different periods. The progression of CAC and the cytokine expression in tissues was assessed. Immunofluorescence staining was used to detect MSCs localization. Levels of immune cells in the spleen and lamina propria of the colon were detected using flow cytometry. A co-culture of MSCs and naïve T cells was performed to determine the effect of MSCs on naïve T cell differentiation.. Early administration of MSCs inhibited the occurrence of CAC, while late administration promoted the progression of CAC. The inhibitory effect of early injection in mice was characterized by the expression of inflammatory cytokines in colon tissue was decreased, and induction of T regulatory cells (Tregs) infiltration via TGF-β. The promotive effect of late injection was characterized by a shift of T helper (Th) 1/Th2 immune balance toward a Th2 phenotype through IL-4 secretion. IL-12 can reverse this shift to Th2 accumulation in mice.. MSCs can curb the progression of colon cancer by inducing Treg accumulation via TGF-β at the early stage of inflammatory transformation but promote the progression of colon cancer by inducing a shift in Th1/Th2 immune balance to Th2 through IL-4 secretion at the late stage. And the immune balance of Th1/Th2 influenced by MSCs could be reversed by IL-12.

Topics: Animals; Colitis; Colon; Colonic Neoplasms; Cytokines; Dextran Sulfate; Disease Models, Animal; Interleukin-12; Interleukin-4; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Liver metastasis is the leading cause of death in colorectal cancer. Immunotherapy using immune checkpoint blockade (ICB) is ineffective due to its immunological cold tumor nature. Herein, we prepared a nanodrug (NCG) encapsulating the transforming growth factor-β receptor inhibitor galunisertib (Gal) and the sonosensitizer chlorin e6 (Ce6), which was aimed to turn this type of cold tumor into a hot one to promote the ICB-based immunotherapy against it. After delivery to the tumor, NCG under ultrasonic irradiation generated reactive oxygen species causing tumor immunogenic cell death and releasing immunostimulatory signals such as calreticulin and HMGB1, which increased tumor immunogenicity and activated the innate T lymphocyte immune response. Moreover, NCG responded to the acidic microenvironment and released Gal, inhibiting phosphorylation and inducing immunosuppressive Smad2/3 signaling. Consequently, the differentiation of MDSCs was inhibited, M1-like polarization of tumor-associated macrophages was induced, and the immunosuppressive barrier of tumor-associated fibroblasts was destroyed to increase the infiltration of effector T cells, which reversed the immunosuppression of the tumor microenvironment and improved the therapeutic efficacy of anti-PD-L1 antibodies. Notably, in the liver metastasis mouse model, combination therapy using NCG (+) and aPD-L1 inhibited the growth of colon cancer liver metastasis, manifesting potential in treating this popular yet intractable malignancy. STATEMENT OF SIGNIFICANCE: Only a limited number of patients with colorectal cancer and liver metastasis can benefit from immune checkpoint blockade therapy, as most of them are microsatellite stable, immunologically cold tumors. Interestingly, there is compelling evidence that sonodynamic therapy (SDT) can convert immunosuppressed cold tumors into hot ones, trigger tumor immunogenic cell death non-invasively, and boost cytotoxic T cells infiltration. However, its therapeutic efficacy is constrained by the abundance of transforming growth factor-β (TGF-β) cytokines in the tumor microenvironment. Here, we reported a TGF-β-targeted inhibitory nanodrug that improved SDT in colon cancer and liver metastasis, reversed the immunosuppressive tumor microenvironment and boosted the immune response to anti-PD-L1 therapy in this cancer. It demonstrated the potential to cure this prevalent but incurable malignancy.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; Immune Checkpoint Inhibitors; Immunity; Immunotherapy; Liver Neoplasms; Mice; Nanoparticles; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Microenvironment

Lipid metabolic reprogramming in colon cancer shows a potential impact on tumor immune microenvironment and is associated with response to immunotherapy. Therefore, this study aimed to develop a lipid metabolism-related prognostic risk score (LMrisk) to provide new biomarkers and combination therapy strategies for colon cancer immunotherapy.. Differentially expressed lipid metabolism-related genes (LMGs) including cytochrome P450 (CYP) 19A1 were screened to construct LMrisk in TCGA colon cancer cohort. The LMrisk was then validated in three GEO datasets. The differences of immune cell infiltration and immunotherapy response between LMrisk subgroups were investigated via bioinformatic analysis. These results were comfirmed by in vitro coculture of colon cancer cells with peripheral blood mononuclear cells, human colon cancer tissue microarray analysis, multiplex immunofluorescence staining and mouse xenograft models of colon cancer.. Six LMGs including CYP19A1, ALOXE3, FABP4, LRP2, SLCO1A2 and PPARGC1A were selected to establish the LMrisk. The LMrisk was positively correlated with the abundance of macrophages, carcinoma-associated fibroblasts (CAFs), endothelial cells and the levels of biomarkers for immunotherapeutic response including programmed cell death ligand 1 (PD-L1) expression, tumor mutation burden and microsatellite instability, but negatively correlated with CD8. A risk model based on lipid metabolism-related genes may predict prognosis and immunotherapeutic response in colon cancer. CYP19A1-catalyzed estrogen biosynthesis promotes vascular abnormality and inhibits CD8

Topics: Animals; Aromatase; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Endothelial Cells; Humans; Immunotherapy; Interleukin-6; Leukocytes, Mononuclear; Lipid Metabolism; Mice; Prognosis; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta; Tumor Microenvironment

Patrinia villosa, a traditional medicinal herb commonly used for treating intestinal-related diseases, has been commonly prescribed by Chinese medicine practitioners as a key component herb to treat colon cancer, although its anti-tumor effect and mechanisms of action have not been fully elucidated.. This study aimed to investigate the anti-tumor and anti-metastatic effects of Patrinia villosa aqueous extract (PVW), and its underlying mechanisms.. The chemical profile of PVW was analysed by high-performance liquid chromatography with photodiode-array detection (HPLC-DAD) method. Cell-based functional assays MTT, BrdU, scratch, and transwell were conducted to evaluate the effects of PVW on human colon cancer HCT116 and murine colon26-luc cells, assessing cytotoxicity, cell proliferation, motility, and migration, respectively. Western blotting was performed to assess the effect of PVW on the expression of key intracellular signaling proteins. In vivo studies were conducted using zebrafish embryos and tumor-bearing mice to evaluate the anti-tumor, anti-angiogenesis, and anti-metastatic effects of PVW in colon cancer.. Five chemical markers were identified and quantified in PVW. PVW exhibited significant cytotoxicity and anti-proliferative activity, as well as inhibitory effects on cell motility and migration in both HCT116 and colon 26-luc cancer cells via modulating protein expressions of TGF-β R1, smad2/3, snail, E-cadherin, FAK, RhoA, and cofilin. PVW (0.01-0.1 mg/ml) could significantly decrease the length of subintestinal vessels of zebrafish embryos through decreasing mRNA expressions of FLT1, FLT4, KDRL, VEGFaa, VEGFc, and Tie1. PVW (> 0.05 mg/ml) also significantly suppressed colon cancer cells migration in the zebrafish embryos. Furthermore, oral administration of PVW (1.6 g/kg) significantly inhibited tumor growth by decreasing the expressions of tumor activation marker Ki-67 and CD 31 in tumor tissues of HCT116 tumor-bearing mice. PVW could also significantly inhibit lung metastasis in colon 26-luc tumor-bearing mice by modulating their tumor microenvironment, including immune cells populations (T cells and MDSCs), levels of cytokines (IL-2, IL-12, and IFN-γ), as well as increasing the relative abundance of gut microbiota.. This study revealed for the first time the anti-tumor and anti-metastatic effects of PVW through regulation of TGF-β-smad2/3-E-cadherin, and FAK-cofilin pathways in colon cancer. These findings provide scientific evidence to support the clinical use of P. villosa in patients with colon cancer.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Humans; Mice; Patrinia; Smad2 Protein; Transforming Growth Factor beta; Tumor Microenvironment; Zebrafish; Zebrafish Proteins

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

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

Colon cancers are among the top three causes of cancer-related deaths. This study is a continuation of previous research aiming to identify effective treatments.. This study investigated the effects of Tarantula cubensis alcoholic extract (TCAE) and Nerium oleander (NO) distillate on the levels of midkine, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), alpha-fetoprotein (AFP), cyclooxygenase (COX)-2, insulin-like growth factor (IGF) and caspase-3 in the liver and colon tissues of rats with experimentally induced colon cancer.. The liver and colon tissues of rats were homogeneously divided into control, colon cancer (azoxymethane, AZM), AZM + TCAE, and AZM + NO distillate groups. The levels of midkine, TGF-β, VEGF, AFP, COX-2, IGF, and caspase-3 in the colon and liver tissues were measured by ELISA.. The levels of all parameters in colon and liver tissues in the AZM group were higher (p<0.05) than those in the control group. TCAE and NO distillate prevented (p < 0.05) increases in midkine, TGF-β, VEGF, AFP, COX-2, IGF, and caspase-3 levels in the colon. NO distillate prevented the increase in all parameters except IGF, whereas TCAE prevented the increase in all values apart from COX-2 and IGF levels in the liver (p<0.05).. NO distillate and TCAE may prevent the studied markers from reaching specified levels observed in the colon in AZM-induced colon cancer. The increases in the levels of the parameters in the liver were not as severe as those in the colon; however, an 18-week study period may not be sufficient for liver metastasis formation. Future molecular studies should investigate the mechanisms and pathways of these treatments in greater detail.

Topics: alpha-Fetoproteins; Animals; Biological Products; Biomarkers, Tumor; Caspase 3; Colonic Neoplasms; Cyclooxygenase 2; Liver; Midkine; Nerium; Plant Extracts; Rats; Spiders; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Colon cancer (CC) and epithelial ovarian cancer (EOC) are common and severe neoplasms frequently sharing a massive inflammatory involvement of peritoneum. A detailed molecular characterization of such carcinomatosis has not been performed, so far.. Omental adipocytes were isolated from thirty-three adult women who underwent primary surgery for CC or EOC. Expression of several pro-inflammatory genes was determined by real-time PCR and immunofluorescence. Data were related to the clinical phenotype of the patients.. CD68, FGFR1, and IL-6 were significantly more expressed in adipocytes from CC patients and VEGF in adipocytes from EOC. TNFα, TGFβ, or MCP-1, as well as the pro-inflammatory platform P2X7R-NLRP3, did not differ between the 2 cancers. White blood cell count, mirroring systemic inflammation, was related to adipocyte P2X7R (R = 0.508, p = 0.003), NLRP3 (R = 0.405; p = 0.02), and MCP-1 (R = 0.448; p = 0.009). P2X7R and NLRP3 were the only inflammatory factors significantly more expressed in patients carrying both omental and peritoneal carcinosis, who were also characterized by a higher leukocytosis. None of the tested inflammatory markers was associated with tumor grading for both neoplasms; however, the presence of metastases was associated with a higher adipocyte expression of FGFR1 and TGFβ.. We show here that rarely measured molecules seem to specifically characterize omental carcinomatosis of CC or EOC, while more common inflammatory agents like TNFα, TGFβ, or MCP-1 do not; the P2X7R-NLRP3 complex marks omental and peritoneal carcinosis and is related to circulating white blood cells and MCP-1, involved in monocyte-macrophage tissue infiltration; increased TGFβ and FGFR1 characterize the tumoral dissemination.

Topics: Colon; Colonic Neoplasms; Female; Humans; Inflammasomes; Interleukin-1beta; NLR Family, Pyrin Domain-Containing 3 Protein; Ovarian Neoplasms; Peritoneal Neoplasms; Peritoneum; Receptors, Purinergic P2X7; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Prolyl 4-hydroxylase subunit alpha 3 (P4HA3) has been known to be associated with a variety of human cancers. However, the role of P4HA3 on colon cancer growth and metastasis is unclear. In this study, we investigated the effect of P4HA3 on the growth and metastasis of colon cancer and its possible molecular mechanism. First of all, we demonstrated that P4HA3 expression was greatly higher in cells and tissues of colon cancer than that in non-tumor tissues and cells, and the prognosis of patients who had higher P4HA3 was distinctively poorer than patients who had lower level of P4HA3. Second, it was shown that P4HA3 knockdown strongly inhibited the migration, proliferation and invasion ability of colon cancer cells. However, P4HA3 over-expression accelerated the abilities. Meanwhile, P4HA3 could promote subcutaneous tumorigenesis in nude mice in vivo. In addition, P4HA3 knockdown significantly decreased mesenchymal markers Vimentin, N-cadherin and Snail expression and increased epithelial marker E-cadherin expression. And conversely, over-expression of P4HA3 produced the opposite effects. In the current study, there was further evidence that down-regulating P4HA3 significantly reduced both TGF-β and its following molecules including p-Smad2 as well as p-Smad3. However, overexpression of P4HA3 showed the opposite effect. In conclusion, this study shows that P4HA3 promotes the human colon cancer growth and metastasis by affecting TGF-β/Smad signaling pathway. P4HA3 may become a new target for early diagnosis, treatment and prognosis assessment of colon cancer.

Topics: Animals; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Procollagen-Proline Dioxygenase; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcriptome; Transforming Growth Factor beta

MicroRNAs (miRNAs) can affect the progression of colon cancer cells. A variety of miRNAs, especially miR-766-3p, are proved to be abnormally expressed in colon cancer, but the molecular mechanism of miR-766-3p in this cancer has not yet been fully defined.. Differentially expressed genes in the TCGA-COAD dataset were searched through bioinformatics analysis. MiR-766-3p and TGFBI mRNA levels were measured by qRT-PCR. TGFBI protein expression was measured via Western blot. Targeting relation between miR-766-3p and TGFBI was investigated by dual-luciferase reporter gene assay. Cell proliferation, invasion migration, and apoptosis were detected by cell functional assays.. MiR-766-3p was less expressed, while TGFBI was conspicuously highly expressed in colon cancer. MiR-766-3p high expression suppressed cell malignant behaviors and induced cell apoptosis in colon cancer. MiR-766-3p had a targeting relation with TGFBI verified by dual-luciferase assay. The cancer-suppressive impact of miR-766-3p overexpression was attenuated by overexpressing TGFBI.. MiR-766-3p/TGFBI axis suppressed malignant behaviors and facilitated apoptosis of colon cancer cells. MiR-766-3p may be an underlying target for colon cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a key process, which can promote the transition of tumor cells into other organs by weakening the cell-cell junctions. Tumor cell invasion and metastasis arising because of EMT can determine the prognosis of cancer. EMT can be induced by several growth factors including transforming growth factor-β (TGF-β), which can exert their effects by affecting several cell-signaling pathways. Fangchinoline (FCN), a kind of bisbenzylisoquinoline, belongs to the family Menispermaceae. FCN can display substantial antitumor effects against various malignant cell lines but its possible impact on EMT has not been explored. We examined the potential impact of FCN in affecting the activation of EMT in human colon cancer cells. We evaluated the influence of FCN on EMT in colon cancer cells by using Western blot analysis and reverse transcription-polymerase chain reaction assays. The cellular invasion and migration were observed by Boyden chamber and wound healing assays. Thereafter, the effect of the drug on proliferation and invasion was also evaluated by real-time cell analysis. FCN suppressed the levels of TGF-β-induced mesenchymal markers, such as fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail. However, FCN markedly enhanced the expression of epithelial markers such as occludin and E-cadherin. These results imply that FCN can potentially inhibit tumor metastasis through abrogating EMT. In addition, FCN downregulated c-Met/PI3K/Akt/mTOR and Wnt/β-catenin cell signaling pathways and mitigated tumor migration as well as invasion. Overall, our study suggests a potential novel role of FCN as an antimetastatic agent against human colon cancer cells.

Topics: Benzylisoquinolines; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Humans; Phosphatidylinositol 3-Kinases; Signal Transduction; Transforming Growth Factor beta

The interactions between platelets and tumor cells are well-known to play important roles in the progression of malignant tumors. Danshensu, a main water-soluble component of. The effects of Danshensu on the platelet activity and epithelial-mesenchymal transformation (EMT)-like invasive phenotype of SW620 colon cancer cells were assessed by stimulating with the supernatants from co-cultured platelets and SW620 cells with direct contact (SCP). The expression and secretion of proteins were determined by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Hematoxylin and eosin (H&E) staining was performed to analyzed the histopathology of tumor tissues and immunohistochemical staining was conducted to examine the protein expression in tumors.. Danshensu attenuated EMT-like characteristics and chemoresistance by inhibiting secretion capability of platelets and activation of the TGF-β/Smad signaling pathway, suggesting that it may be optimized to be a therapeutic agent for fighting against colon cancer.

Topics: Blood Platelets; Colonic Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Lactates; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

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

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

Epithelial-mesenchymal transition (EMT) is a crucial process in which the polarized epithelial cells acquire the properties of mesenchymal cells and gain invasive properties. We have previously demonstrated that manganese superoxide dismutase (MnSOD) can regulate the EMT phenotype by modulating the intracellular reactive oxygen species. In this report, we have demonstrated the EMT-suppressive effects of 2,3,5,6-Tetramethylpyrazine (TMP, an alkaloid isolated from Chuanxiong) in colon cancer cells. TMP suppressed the expression of MnSOD, fibronectin, vimentin, MMP-9, and N-cadherin with a parallel elevation of occludin and E-cadherin in unstimulated and TGFβ-stimulated cells. Functionally, TMP treatment reduced the proliferation, migration, and invasion of colon cancer cells. TMP treatment also modulated constitutive activated as well as TGFβ-stimulated PI3K/Akt/mTOR, Wnt/GSK3/β-catenin, and MAPK signaling pathways. TMP also inhibited the EMT program in the colon cancer cells-transfected with pcDNA3-MnSOD through modulation of MnSOD, EMT-related proteins, and oncogenic pathways. Overall, these data indicated that TMP may inhibit the EMT program through MnSOD-mediated abrogation of multiple signaling events in colon cancer cells.

Topics: Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3; Humans; Phosphatidylinositol 3-Kinases; Pyrazines; Superoxide Dismutase; Transforming Growth Factor beta

Immune checkpoint blockade (ICB) therapy, while groundbreaking, must be improved to promote enhanced durable responses and to prevent the development of treatment-refractory disease. Cancer therapies that engage, enable, and expand the antitumor immune response will likely require rationally designed combination strategies. Targeting multiple immunosuppressive pathways simultaneously may provide additional therapeutic benefit over singular targeting. We therefore hypothesized that the use of two molecules which inhibit three independent, but overlapping, pathways (TIGIT:CD155, PD-1/PD-L1, and TGFβ) would provide significant antitumor efficacy in the syngeneic ICB resistant colorectal tumor model MC38 expressing human carcinoembryonic antigen (CEA) in CEA transgenic mice. This novel combination treatment strategy has significant antitumor activity and survival benefit in two models of murine carcinomas, MC38-CEA (CRC) and TC1 (HPV

Topics: Animals; B7-H1 Antigen; Carcinoembryonic Antigen; Carcinoma; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Humans; Immune Checkpoint Inhibitors; Mice; Papillomavirus Infections; Programmed Cell Death 1 Receptor; Receptors, Immunologic; Transforming Growth Factor beta

Histone deacetylase 6 (HDAC6), a member of the HDAC family, has been identified as a potential therapeutic target for tumor therapy, but the function and underlying mechanisms of HDAC6 in colon cancer are incompletely characterized. Our study showed that the infiltration ratio of M2 macrophages was increased in colon cancer tissues with high HDAC6 expression. Similarly, the knockdown of HDAC6 in colon cancer cells inhibited cocultured macrophage M2 polarization in vitro. Analysis of the antibody chip revealed that HDAC6 promoted sIL-6R release to enhance macrophage M2 polarization. Mass spectrometry and immunoprecipitation demonstrated that, mechanistically, HDAC6 interacted with transforming growth factor β-activated kinase 1 (TAK1), deacetylated TAK1 at T178 and promoted TAK1 phosphorylation. TAK1-p38 MAPK signaling could further increase the phosphorylation and activity of ADAM17, which is responsible for shedding of IL-6R. Notably, the expression of phosphorylated TAK1 was positively correlated with HDAC6 expression and macrophage M2 polarization in human colon cancer tissues. Our study revealed a new HDAC6-TAK1-ADAM17 regulatory axis that mediates sIL-6R release and macrophage polarization in colon cancer.

Topics: Colonic Neoplasms; Histone Deacetylase 6; Humans; Macrophages; p38 Mitogen-Activated Protein Kinases; Transforming Growth Factor beta

The TGF-β receptor kinase inhibitors (TRKI) have been reported to inhibit tumorigenicity in colon cancer. However, there is no direct evidence showing that these inhibitors function through inhibiting the TGF-β- mediated tumor-promoting effects in vivo. We established a TGF-β inducible reporter system by inserting a luciferase reporter gene to the vector downstream of TGF-β-inducible promoter elements, and transfected it into colon cancer cell lines. TRKIs SB431542 and LY2109761 were used to treat TGF-β inducible cells in vitro and in vivo. The luciferase activity was induced 5.24-fold by TGF-β in CT26 inducible cells, while it was marginally changed in MC38 inducible cells lacking Smad4 expression. Temporary treatment of mice with SB431542 inhibited the TGF-β pathway and TGF-β induced bioluminescence activity in vivo. Long-term treatment with LY2109761 inhibited tumorigenicity and liver metastasis in vivo in concomitant with reduced luciferase activity in the tumor. In this study, we established a model to monitor the TGF-β pathway in vivo and to compare the antitumor effects of TRKIs. Based on this novel experimental tool, we provided direct evidences that LY2109761 inhibits tumorigenicity and liver metastasis by blocking the pro-oncogenic functions of TGF-β in vivo.

Topics: Animals; Benzamides; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Dioxoles; Disease Models, Animal; Humans; Mice; Protein Kinase Inhibitors; Pyrazoles; Pyrroles; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The paradoxical roles of transforming growth factor-β (TGFβ) signaling and nuclear receptor Nur77 in colon cancer development are known but the underlying mechanisms remain obscure. Inhibitor of differentiation 1 (ID1) is a target gene of TGFβ and a key promoter for colon cancer progression. Here, we show that Nur77 enhances TGFβ/Smad3-induced ID1 mRNA expression through hindering Smurf2-mediated Smad3 mono-ubiquitylation, resulting in ID1 upregulation. In the absence of TGFβ, however, Nur77 destabilizes ID1 protein by promoting Smurf2-mediated ID1 poly-ubiquitylation, resulting in ID1 downregulation. Interestingly, TGFβ stabilizes ID1 protein by switching Nur77 interaction partners to inhibit ID1 ubiquitylation. This also endows TGFβ with an active pro-tumorigenic action in Smad4-deficient colon cancers. Thus, TGFβ converts Nur77's role from destabilizing ID1 protein and cancer inhibition to inducing ID1 mRNA expression and cancer promotion, which is highly relevant to colon cancer stemness, metastasis and oxaliplatin resistance. Our data therefore define the integrated duality of Nur77 and TGFβ signaling in regulating ID1 expression and provide mechanistic insights into the paradoxical roles of TGFβ and Nur77 in colon cancer progression.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Inhibitor of Differentiation Protein 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Models, Biological; Nuclear Receptor Subfamily 4, Group A, Member 1; Protein Stability; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Ubiquitination

Colorectal cancer (CRC) is one of the most common malignant tumours in the world. Recent reports have revealed natural products displayed inhibition on colon cancer potential by suppressing transforming growth factor-β/Smads induced epidermal-mesenchymal transition (EMT). In this article, 12 kinds of natural berberine analogues were screened for their effects on the inhibition of the colon cancer cells, the results showed that demethyleneberberine (DM-BBR) exhibited an interesting and potential effect on inducing the apoptosis of HCT-116 cells with drug concentrations of 6, 12 and 18 μM. Particularly, DM-BBR reversed the EMT process by inhibiting the expression of p-Smad2 and p-Smad3 in the transforming growth factor-β/Smads signal pathway, up-regulated pro-apoptotic protein cleaved caspase-9, and blocked cell cycle at the S phase and increasing the expression of cyclin proteins P27 and P21. Taken together, these findings suggested that DM-BBR could promote apoptosis and suppress TGF-β/Smads induced EMT in the colon cancer cells HCT-116.

Topics: Antineoplastic Agents; Apoptosis; Berberine; Cell Proliferation; Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epithelial-Mesenchymal Transition; HCT116 Cells; Humans; Molecular Structure; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1

Topics: Adaptor Proteins, Signal Transducing; Animals; Carcinogenesis; Cell Differentiation; Cell Survival; Colon; Colonic Neoplasms; Epithelial Cells; Fetus; Inflammation; Kaplan-Meier Estimate; MAP Kinase Signaling System; Mice, Inbred C57BL; Mutation; Prognosis; Proto-Oncogene Proteins B-raf; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Spheroids, Cellular; Transcription Factors; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway; YAP-Signaling Proteins

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) are a family of enzymes. However, the clinical and functional roles of PLOD3 in colon adenocarcinoma (COAD) have not been investigated. The present study found that PLOD3 was highly upregulated in COAD, which may be resulted from its aberrant DNA methylation. The upregulation of both PLOD3 mRNA and protein was confirmed in our tissue samples. Moreover, high PLOD3 was identified to be associated with unfavorable prognosis in COAD. As genome instability is a hallmark of cancer, PLOD3 was expressed higher in COAD samples with high chromosomal instability (CIN-high) than those with low CIN (CIN-low) and higher in those with low MSI than high MSI, indicating that PLOD3 expression was associated with tumor genomic instability. Furthermore, immune cells showed significantly different infiltrating levels between the high and low PLOD3 expression groups, and the immune score was negatively correlated with PLOD3 expression and higher in samples with low PLOD3 expression, suggesting that high PLOD3 expression was associated with reduced immune cell infiltrating levels in COAD. To further uncover the underlying mechanism of PLOD3 in PLOD3, we compared the COAD samples of high PLOD3 expression with those of low PLOD3 expression and found that high expression of PLOD3 was associated with reduced expression of immune regulators and enhanced activities of two tumor-promoting pathways, including gluconeogenesis and TGF-beta signaling in epithelial-mesenchymal transition (EMT), suggesting that high expression of PLOD3 causes poor prognosis in COAD by weakening the immune cell infiltration and enhancing activities of tumor-promoting pathways. In summary, the present study highlights the importance of PLOD3 and provides the evidence about the functional role of PLOD3 in COAD.

Topics: Adenocarcinoma; Colonic Neoplasms; Databases, Factual; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genomic Instability; Gluconeogenesis; Humans; Immune System; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Prognosis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Up-Regulation

Inflammatory microenvironment is an important factor for promoting cancer invasion and metastasis, but the underlying molecular mechanisms remain unclear. Here, we mimicked an inflammatory microenvironment both in vitro and in vivo and investigated its effects on the invasion and metastasis of colon cancer. Moreover, colon cancer patient samples were also analyzed statistically. Conditioned medium from the differentiated macrophages induced invasion and migration of colon cancer cells in vitro, which could be reversed by the treatment of a neutralizing anti-growth differentiation factor 15 (GDF15) antibody, indicating GDF15 involvement in inflammation-induced invasiveness. Also, we observed similar effects of human recombinant GDF15 on colon cancer cells. Mechanistically, GDF15 activated c-Fos by separating it from Lamin A/C, increasing transcriptional activity of c-Fos and regulating EMT gene expressions. However, c-Fos knockdown using lentivirus shRNA plasmid inhibited GDF15-triggered invasion and migration in vitro. In vivo, inflammation caused by lipopolysaccharides obviously increased GDF15 secretion, and c-Fos knockdown reduced the lung metastasis of colon cancer cells in mice model. In addition, c-Fos expressions in patient samples were found to be associated with colon cancer metastasis and TNM stages. Taken together, GDF15 in inflammatory microenvironment induces colon cancer invasion and metastasis by regulating EMT genes by activating c-Fos, which might be a potential therapeutic target for metastatic colon cancer.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers, Tumor; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 15; Humans; Inflammation; Lamin Type A; Macrophages; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasms, Experimental; Proto-Oncogene Proteins c-fos; Transforming Growth Factor beta; Tumor Microenvironment

High T-cell infiltration in colorectal cancer (CRC) correlates with a favorable disease outcome and immunotherapy response. This, however, is only observed in a small subset of CRC patients. A better understanding of the factors influencing tumor T-cell responses in CRC could inspire novel therapeutic approaches to achieve broader immunotherapy responsiveness. Here, we investigated T cell-suppressive properties of different myeloid cell types in an inducible colon tumor mouse model. The most potent inhibitors of T-cell activity were tumor-infiltrating neutrophils. Gene expression analysis and combined in vitro and in vivo tests indicated that T-cell suppression is mediated by neutrophil-secreted metalloproteinase activation of latent TGFβ. CRC patient neutrophils similarly suppressed T cells via TGFβ in vitro, and public gene expression datasets suggested that T-cell activity is lowest in CRCs with combined neutrophil infiltration and TGFβ activation. Thus, the interaction of neutrophils with a TGFβ-rich tumor microenvironment may represent a conserved immunosuppressive mechanism in CRC.

Topics: Animals; Colonic Neoplasms; Humans; Lymphocytes, Tumor-Infiltrating; Matrix Metalloproteinases; Mice; Neutrophils; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment

MTA1, SOX4, EZH2, and TGF-β are all potent inducers of epithelial-mesenchymal transition (EMT) in cancer; however, the signaling relationship among these molecules in EMT is poorly understood. Here, we investigated the function of MTA1 in cancer cells and demonstrated that MTA1 overexpression efficiently activates EMT. This activation resulted in a significant increase in the migratory and invasive properties of three different cancer cell lines through a common mechanism involving SOX4 activation, screened from a gene expression profiling analysis. We showed that both SOX4 and MTA1 are induced by TGF-β and both are indispensable for TGF-β-mediated EMT. Further investigation identified that MTA1 acts upstream of SOX4 in the TGF-β pathway, emphasizing a TGF-β-MTA1-SOX4 signaling axis in EMT induction. The histone methyltransferase EZH2, a component of the polycomb (PcG) repressive complex 2 (PRC2), was identified as a critical responsive gene of the TGF-β-MTA1-SOX4 signaling in three different epithelial cancer cell lines, suggesting that this signaling acts broadly in cancer cells in vitro. The MTA1-SOX4-EZH2 signaling cascade was further verified in TCGA pan-cancer patient samples and in a colon cancer cDNA microarray, and activation of genes in this signaling pathway predicted an unfavorable prognosis in colon cancer patients. Collectively, our data uncover a SOX4-dependent EMT-inducing mechanism underlying MTA1-driven cancer metastasis and suggest a widespread TGF-β-MTA1-SOX4-EZH2 signaling axis that drives EMT in various cancers. We propose that this signaling may be used as a common therapeutic target to control epithelial cancer metastasis.

Topics: Cell Line, Tumor; Colonic Neoplasms; Enhancer of Zeste Homolog 2 Protein; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasms; Prognosis; Repressor Proteins; Signal Transduction; SOXC Transcription Factors; Trans-Activators; Transforming Growth Factor beta

Imbalance of the gut microbial community promotes inflammation and colorectal cancer (CRC). Previously, we demonstrated that freeze-dried Parabacteroides distasonis (Pd) suppressed obesity-driven colorectal tumorigenesis in mice. Here, we investigated if Pd could suppress the development of colon tumors in mice independent of obesity. Six-week-old male A/J mice were assigned to receive: (i) chow diet (CTR); (ii) chow with 0.04% wt/wt freeze-dried Pd (Pd-Early) or (iii) chow diet before switching to 0.04% Pd diet (Pd-Late). Mice remained on diet for 25 weeks with the switch for Pd-Late mice occurring after 19 weeks. All mice received 6 weekly injections of the colon carcinogen azoxymethane (AOM; 10 mg/kg I.P.) starting after 1 week on diet. Colon tumors were observed in 77, 55 and 40% in CTR, Pd-Early and Pd-Late mice, respectively (X2 = 0.047). Colonic expression of toll-like receptor 4, IL-4 and TNF-α was 40% (P < 0.01), 58% (P = 0.05) and 55% (P < 0.001) lower, respectively, in Pd-Early compared with CTR mice. Pd-Late mice displayed a 217% (P = 0.05) and 185% (P < 0.001) increase in colonic IL-10 and TGF-β expression, respectively, compared with CTR mice and similar increases in protein abundances were detected (47-145%; P < 0.05). Pd-Early and Pd-Late mice both demonstrated increased colonic expression of the tight junction proteins Zonula occludens-1 (P < 0.001) and occludin (P < 0.001) at the transcript (2-3-fold; P < 0.01) and protein level (30-50%; P < 0.05) relative to CTR. Our results support a protective role for Pd in colonic tumorigenesis and maintenance of intestinal epithelial barrier in AOM-treated mice.

Topics: Animals; Azoxymethane; Bacteroidetes; Carcinogenesis; Colon; Colonic Neoplasms; Humans; Inflammation; Interleukin-4; Intestinal Mucosa; Mice; Obesity; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta

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

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

Interaction of cancer cells with cancer-associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3-D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin-α5β1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E-cadherin-expressing adenocarcinoma cell lines, DLD-1 (colon) and MCF-7 (breast). When hybrid spheroids of DLD-1 cells with CAFs were embedded into collagen gel, DLD-1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor-α promoted the collective invasion, possibly by reducing the E-cadherin junction, as did the transforming growth factor-β inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor-β itself inhibited the cancer cell invasion. Efficient collective invasion of DLD-1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function-blocking Abs and siRNAs confirmed that DLD-1 cells adhered to fibronectin fibrils on CAFs mainly through integrin-α5β1. Anti-E-cadherin Ab promoted the single cell invasion of DLD-1 cells by dissociating the E-cadherin junction. Although the binding affinity of MCF-7 cells to CAFs was lower than DLD-1, they also collectively invaded the collagen matrix in a similar fashion to DLD-1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers.

Topics: A549 Cells; Adenocarcinoma; Amides; Benzamides; Cancer-Associated Fibroblasts; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chromones; Collagen; Colonic Neoplasms; Connective Tissue; Dioxoles; Epidermal Growth Factor; Fibroblasts; Fibronectins; Humans; Immunohistochemistry; Integrin alpha5beta1; MCF-7 Cells; Morpholines; Neoplasm Invasiveness; Pyridines; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Corilagin (CLG), a major component of several medicinal plants, can exhibit diverse pharmacological properties including those of anti-cancer, anti-inflammatory, and hepatoprotective qualities. However, there are no prior studies on its potential impact on the epithelial-to-mesenchymal transition (EMT) process. EMT can lead to dissemination of tumor cells into other organs and promote cancer progression. Hence, we aimed to investigate the effect of CLG on EMT and its mechanism(s) of action in tumor cells. We noted that CLG reduced the expression of various epithelial markers and up-regulated the expression of Occludin and E-cadherin in both basal and TGFβ-stimulated tumor cells. CLG treatment also abrogated cellular invasion and migration in colon and prostate carcinoma cells. In addition, CLG effectively attenuated the Wnt/β-catenin signaling cascade in TGFβ-stimulated cells. Overall, our study suggests that CLG may function as and effective modulator of EMT and metastasis in neoplastic cells.

Topics: Cadherins; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Glucosides; Humans; Hydrolyzable Tannins; Male; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Occludin; Prostatic Neoplasms; Transforming Growth Factor beta; Wnt Signaling Pathway

Mucinous adenocarcinoma is a distinct subtype of colorectal cancer (CRC) with a worse prognosis when compared with non-mucinous adenocarcinoma. The aim of this study was to compare somatic mutations and copy number alteration (CNA) between mucinous and non-mucinous CRC.. Data from The Cancer Genome Atlas-colon adenocarcinoma and rectum adenocarcinoma projects were utilized. Mucinous and non-mucinous CRC were compared with regard to microsatellite status, overall mutation rate, the most frequently mutated genes, mutations in genes coding for mismatch repair (MMR) proteins and genes coding for mucin glycoproteins. CNA analysis and pathway analysis was undertaken.. Mucinous CRC was more likely to be microsatellite instability-high (MSI-H) and hypermutated. When corrected for microsatellite status the single-nucleotide variation and insertion-deletion rate was similar between the two cohorts. Mucinous adenocarcinoma was more likely to have mutations in genes coding for MMR proteins and mucin glycoproteins. Pathway analysis revealed further differences between the two histological subtypes in the cell cycle, RTK-RAS, transforming growth factor-β, and TP53 pathways.. Mucinous CRC has some distinct genomic aberrations when compared with non-mucinous adenocarcinoma, many of which are driven by the increased frequency of MSI-H tumors. These genomic aberrations may play an important part in the difference seen in response to treatment and prognosis in mucinous adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Cohort Studies; Colonic Neoplasms; Datasets as Topic; DNA Copy Number Variations; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genomics; Humans; INDEL Mutation; Microsatellite Instability; Mucins; Mutation; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Rectal Neoplasms; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Tumor microenvironment (TME) crucially functions in tumor initiation and progression. Stroma-tumor interactions and cellular transdifferentiation are the prerequisite for tumor formation. Transforming growth factor-β (TGF-β), a major cytokine secreted by tumor-associated fibroblasts (TAFs) and cancer cells, is a crucial player involving cell transdifferentiation. Therefore, we hypothesized that these TAFs and cancer cells also affect normal colon epithelium. In our study, we found for the first time that colon cancer cells HCT116 and TAF-like CCD-18Co cells induced epithelial-mesenchymal transition (EMT)-like transdifferentiation in colon epithelial cells HCoEpiCs, with enhanced migratio. Dysfunction of TGF-β/Smads signal was also observed in the EMT-transformed HCoEpiCs. We wondered whether these phenomena were regulated by TGF-β/Smads signaling pathway. A TGFβ receptor kinase I (TβRI) inhibitor LY364947 was used. We found that the EMT induced by the HCT116- and CCD-18Co-derived CM was suppressed by the LY364947. Besides, different expression profiles for the components of TGF-β/Smads pathway were found in the EMT-like HCoEpiCs, but high expression of p-Smad2/3 and Smad4 was the common feature. Our observations suggest that the mechanisms of phenotypic transition of colon epithelial cells are cellular environment-dependent, which maybe a basis of potential therapy targeting TME.

Topics: Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Transdifferentiation; Colon; Colonic Neoplasms; Epithelial Cells; Epithelial-Mesenchymal Transition; HCT116 Cells; Humans; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

Programmed cell death protein 1(PD-1) blocking antibodies have been used to enhance immunity in solid tumors and achieve durable clinical responses with an acceptable safety profile in multiple types of cancer. However, only a subset of patients could benefit from PD-1 blockade therapy. Prognostic information including PD-1 ligand (PD-L1) expression, IFN-γ expression signature, tumor mutational burden, and microsatellite instability (MSI) have been evaluated for patients who are selected to receive immune checkpoint therapeutic treatment. Yet the relationship of those biomarkers in determining immune checkpoint therapy is largely unknown.. Immune-profiles of MSI subtype colon cancer were identified from integrating published MSI associated gene expression data. The enriched pathways and transcription factors were analyzed by GSEA assay. The infiltrations of immune cell types into MSI subtype colon cancer tissues were determined by CIBESORT assay.. In the MSI subtype colon cancer patients, PD-L1, IFN-γ and IFN-γ associated genes are highly expressed. And all those genes are favorable effects in colon cancer progress. In addition, we find that Wnt-β-catenin and TGFβ signaling pathways which are two important factors inhibiting PD-1 checkpoint blockade therapy are negatively related with MSI status. We also identify that the immune-profiles in MSI subtype colon cancer are contributed by M1 macrophage infiltration in the tumor environment.. Our results provide the detailed underlying mechanisms of MSI subtype cancer patients are sensitive to PD-1 checkpoint blockade.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Colonic Neoplasms; Gene Expression; Humans; Immunotherapy; Interferon Regulatory Factor-1; Interferon-gamma; Macrophages; Microsatellite Instability; MutL Protein Homolog 1; Phthalazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prognosis; Programmed Cell Death 1 Receptor; Transcriptome; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway

Recurrence and chemoresistance in colorectal cancer remain important issues for patients treated with conventional therapeutics. Metformin and phenformin, previously used in the treatment of diabetes, have been shown to have anticancer effects in various cancers, including breast, lung and prostate cancers. However, their molecular mechanisms are still unclear. In this study, we examined the effects of these drugs in chemoresistant rectal cancer cell lines. We found that SW837 and SW1463 rectal cancer cells were more resistant to ionizing radiation and 5-fluorouracil than HCT116 and LS513 colon cancer cells. In addition, metformin and phenformin increased the sensitivity of these cell lines by inhibiting cell proliferation, suppressing clonogenic ability and increasing apoptotic cell death in rectal cancer cells. Signal transducer and activator of transcription 3 and transforming growth factor-β/Smad signaling pathways were more activated in rectal cancer cells, and inhibition of signal transducer and activator of transcription 3 expression using an inhibitor or siRNA sensitized rectal cancer cells to chemoresistant by inhibition of the expression of antiapoptotic proteins, such as X-linked inhibitor of apoptosis, survivin and cellular inhibitor of apoptosis protein 1. Moreover, metformin and phenformin inhibited cell migration and invasion by suppression of transforming growth factor β receptor 2-mediated Snail and Twist expression in rectal cancer cells. Therefore, metformin and phenformin may represent a novel strategy for the treatment of chemoresistant rectal cancer by targeting signal transducer and activator of transcription 3 and transforming growth factor-β/Smad signaling.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemoradiotherapy; Colon; Colonic Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Fluorouracil; Humans; Male; Metformin; Mice; Mice, Nude; Neoplasm Recurrence, Local; Phenformin; Rectal Neoplasms; Signal Transduction; Smad Proteins; STAT3 Transcription Factor; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Tumor-associated fibroblasts (TAFs) contribute to transdifferentiation of stromal cells in tumor microenvironment. Epithelial-mesenchymal transition (EMT) is a procedure of phenotypic remodeling of epithelial cells and extensively exists in local tumoral stroma. Histone deacetylase (HDAC) inhibitor Tricostatin A (TSA) and sodium butyrate (SB) are reported to play important roles in the regulation of biological behaviour of cancer cells. However, whether TSA or SB is involved in control of EMT in colon epithelial cells induced by TAFs remains unidentified. In present study, we used conditioned medium (CM) form TAF-like CCD-18Co cells to stimulate 2D- and 3D-cultured colon epithelial HCoEpiC cells for 24 h and 4 d. We found that the CCD-18Co CM triggered multiple morphological changes in HCoEpiCs including prolonged cell diameters, down-regulation of E-cadherin and up-regulation of vimentin and α-SMA. Besides, ZEB1 and Snail expression and migration were also promoted by the CM. These phenomena were abolised by 5 μg/ml LY364947, a TGF-β receptor inhibitor. CCD-18Co induced up-regulation of HDAC1 and HDAC2 in the 2D and 3D models, while no change of HDAC4 exprerssion was found. Treatment of 2 μg/ml TSA reversed the CCD-18Co-induced morphological changes and migration of the HCoEpiCs, and suppressed the downregulation of E-cadherin and upregulation of vimentin, α-SMA, ZEB1 and Snail. However, the suppressive effect of 4 mg/ml SB on the EMT was not observed. TSA down-regulated the expressions of Smad2/3, p-Smad2/3 amd HDAC4. Besides, TSA promoted the apoptosis rate (36.84 ± 6.52%) comparing with the CCD-18Co-treated HCoEpiCs (3.52 ± 0.85%, P < 0.05), with promotion of Bax (0.5893±0.0498 in 2D and 0.8867±0.0916 in 3D) and reduction of Bcl-2 (0.0476±0.0053 in 2D and 0.0294±0.0075 in 3D). TSA stimulated expression of phosphorylated-p38 MAPK in 2D (0.3472±0.0249) and 3D (0.3188±0.0248). After pre-treatment with p38 MAPK inhibitor VX-702 (0.5 mg/ml), the apoptosis rate of TSA was decreased in 2D (10.32%) and 3D (5.26%). Our observations demonstrate that epigenetic treatment with HDAC inhibitor TSA may be a useful therapeutic tool for the reversion of TAF-induced EMT in colon epithelium through mediating canonical Smads pathway and non-canonical p38 MAPK signalling.

Topics: Apoptosis; Cancer-Associated Fibroblasts; Cell Line; Colon; Colonic Neoplasms; Epithelial Cells; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Neoplasm Proteins; Transforming Growth Factor beta

The formation of new blood vessels is essential for normal development, tissue repair and tumor growth. Here we show that inhibition of the kinase p38α enhances angiogenesis in human and mouse colon tumors. Mesenchymal cells can contribute to tumor angiogenesis by regulating proliferation and migration of endothelial cells. We show that p38α negatively regulates an angiogenic program in mesenchymal stem/stromal cells (MSCs), multipotent progenitors found in perivascular locations. This program includes the acquisition of an endothelial phenotype by MSCs mediated by both TGF-β and JNK, and negatively regulated by p38α. Abrogation of p38α in mesenchymal cells increases tumorigenesis, which correlates with enhanced angiogenesis. Using genetic models, we show that p38α regulates the acquisition of an endothelial-like phenotype by mesenchymal cells in colon tumors and damage tissue. Taken together, our results indicate that p38α in mesenchymal cells restrains a TGF-β-induced angiogenesis program including their ability to transdifferentiate into endothelial cells.

Topics: Animals; Azoxymethane; Carcinogens; Cell Proliferation; Cell Transdifferentiation; Colonic Neoplasms; Endothelial Cells; Endothelium, Vascular; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; HT29 Cells; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 14; Neoplasms, Experimental; Neovascularization, Pathologic; RNA, Small Interfering; Transforming Growth Factor beta

Natural killer cells harnessed from healthy individuals can be expanded ex vivo using various platforms to produce large doses for adoptive transfer into cancer patients. During such expansion, NK cells are increasingly activated and more efficient at killing cancer cells. Adoptive transfer however introduces these activated cells into a highly immunosuppressive tumor microenvironment mediated in part by excessive transforming growth factor beta (TGF-beta) from both cancer cells and their surrounding stroma. This microenvironment ultimately limits the clinical efficacy of NK cell therapy. In this study, we examined the use of a TGF-beta receptor kinase inhibitor, LY2157299, in preserving the cytotoxic function of ex vivo expanded, highly activated NK cells following sustained exposure to pathologic levels of TGF-beta in vitro and in a liver metastases model of colon cancer. Using myeloid leukemia and colon cancer cell lines, we show that the TGF-beta driven impairment of NK cell cytotoxicity is mitigated by LY2157299. We demonstrate this effect using quantitative cytotoxicity assays as well as by showing a preserved activated phenotype with high NKG2D/CD16 expression and enhanced cytokine production. In a mouse liver metastases model of colon cancer, we observed significantly improved eradication of liver metastases in mice treated with adoptive NK cells combined with LY2157299 compared with mice receiving NK cells or TGF beta inhibition alone. We propose that the therapeutic efficacy of adoptive NK cell therapy clinically will be markedly enhanced by complementary approaches targeting TGF-beta signaling in vivo.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; Cytotoxicity, Immunologic; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Liver Neoplasms; Mice; Models, Biological; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cancer cells alter their metabolism to support their malignant properties. In this study, we report that the glucose-transforming polyol pathway (PP) gene aldo-keto-reductase-1-member-B1 (

Topics: A549 Cells; Aldehyde Reductase; Animals; Cell Line, Tumor; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Glucose; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; L-Iditol 2-Dehydrogenase; Lung Neoplasms; MCF-7 Cells; Mice; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta

Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (T

Topics: Alleles; Animals; Cell Differentiation; Colonic Neoplasms; Disease Models, Animal; Drug Synergism; Female; Humans; Immune Evasion; Immunotherapy; Intestinal Mucosa; Intestines; Liver Neoplasms; Male; Mice; Mutation; Neoplasm Metastasis; Programmed Cell Death 1 Receptor; Stem Cells; T-Lymphocytes, Cytotoxic; Th1 Cells; Transforming Growth Factor beta; Tumor Microenvironment

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

Tumors that elude infiltration by CD8

Topics: CD8-Positive T-Lymphocytes; Colonic Neoplasms; Humans; Immune Evasion; Transforming Growth Factor beta; Transforming Growth Factors

Aberrant cell survival plays a critical role in cancer progression and metastasis. We have previously shown that ezrin, a cAMP-dependent protein kinase A-anchoring protein (AKAP), is up-regulated in colorectal cancer (CRC) liver metastasis. Phosphorylation of ezrin at Thr-567 activates ezrin and plays an important role in CRC cell survival associated with XIAP and survivin up-regulation. In this study, we demonstrate that in FET and GEO colon cancer cells, knockdown of ezrin expression or inhibition of ezrin phosphorylation at Thr-567 increases apoptosis through protein kinase A (PKA) activation in a cAMP-independent manner. Transforming growth factor (TGF) β signaling inhibits ezrin phosphorylation in a Smad3-dependent and Smad2-independent manner and regulates pro-apoptotic function through ezrin-mediated PKA activation. On the other hand, ezrin phosphorylation at Thr-567 by insulin-like growth factor 1 receptor (IGF1R) signaling leads to cAMP-dependent PKA activation and enhances cell survival. Further studies indicate that phosphorylated ezrin forms a complex with PKA RII, and dephosphorylated ezrin dissociates from the complex and facilitates the association of PKA RII with AKAP149, both of which activate PKA yet lead to either cell survival or apoptosis. Thus, our studies reveal a novel mechanism of differential PKA activation mediated by TGFβ and IGF1R signaling through regulation of ezrin phosphorylation in CRC, resulting in different cell fates. This is of significance because TGFβ and IGF1R signaling pathways are well-characterized tumor suppressor and oncogenic pathways, respectively, with important roles in CRC tumorigenesis and metastasis. Our studies indicate that they cross-talk and antagonize each other's function through regulation of ezrin activation. Therefore, ezrin may be a potential therapeutic target in CRC.

Topics: Apoptosis; Cell Proliferation; Colonic Neoplasms; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Humans; Phosphorylation; Receptor, IGF Type 1; Receptors, Somatomedin; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

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

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

T cell exclusion from cancers is a major problem for a proportion of patients suffering poor outcomes. Two papers recently published in Nature highlight the negative role of cancer associated fibroblast TGFβ. These studies indicate that TGFβ enables T cell exclusion in a proportion of colorectal and urothelial cancers, and in the latter disease may reduce their response to anti-PD-L1 immunotherapy.

Topics: Colonic Neoplasms; Humans; Immune Evasion; Immunotherapy; T-Lymphocytes; Transforming Growth Factor beta

Tumor microenvironment (TME) is the cellular environment in which tumor exists, and it contributes to tumor formation and progression. The TME is composed of tumor cells, stromal cells, cytokines, and chemotactic factors of which fibroblasts are the main cellular components. In our present study, we found that colorectal cancer (CRC) cells expressing integrin αvβ6 clearly could induce morphological changes in inactive fibroblasts and increased the expression of activated fibroblast markers such as α-smooth muscle actin (α-SMA) and fibroblast-activating protein (FAP). Those activated fibroblasts in the TME are called cancer-associated fibroblasts (CAFs). In order to investigate the mechanism by which CRC cells expressing integrin αvβ6 activated CAFs, a series of assays have been carried out in the follow-up. We found that CRC cells could secrete inactive transforming growth factor β (TGF-β); however, integrin αvβ6 activated TGF-β, which subsequently activated fibroblasts. This process was disrupted by knockdown of integrin αvβ6. In contrast, activated fibroblasts could promote CRC cell invasion. In particular, the strengthening effect on expression of integrin αvβ6 in colon cancer cells was obvious. Additionally, we found that CAFs could secrete stromal cell-derived factor-1 (SDF-1) and promote CRC cell metastasis in distant organs via the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) axis. Taken together, we assumed that CRC cells and CAFs activated one another and worked together to promote cancer progression, with integrin αvβ6 playing a role in the bi-directional regulation of these cells. Hence, integrin αvβ6 may serve as a therapeutic target for the future CRC treatment.

Topics: Antigens, Neoplasm; Cancer-Associated Fibroblasts; Cell Line, Tumor; Colonic Neoplasms; Humans; Integrins; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Microenvironment

Colon cancer, the second leading cause of cancer-related deaths in the world, is usually diagnosed in invasive stages. The interactions between cancer cells and cells located in their niche remain the crucial mechanism inducing tumor metastasis. The most important among those cells are cancer-associated fibroblasts (CAFs), the heterogeneous group of myofibroblasts transdifferentiated from numerous cells of different origin, including endothelium. The endothelial-to-mesenchymal transition (EndMT) is associated with modulation of cellular morphology, polarization and migration ability as a result of microtubule cytoskeleton reorganization. Here we reveal, for the first time, that invasive colon cancer cells regulate EndMT of endothelium via tubulin-β3 upregulation and its phosphorylation. Thus, we concluded that therapies based on inhibition of tubulin-β3 expression or phosphorylation, or blocking tubulin-β3's recruitment to the microtubules, together with anti-inflammatory chemotherapeutics, are promising means to treat advanced stages of colon cancer.

Topics: Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Transdifferentiation; Colonic Neoplasms; Endothelium; Enzyme Inhibitors; Humans; Microtubules; Neoplasm Invasiveness; Phosphorylation; Transforming Growth Factor beta; Tubulin; Up-Regulation; Wortmannin

In this study, we have uncovered a novel crosstalk between TGFβ and IGF-1R signaling pathways. We show for the first time that expression and activation of IRS-1, an IGF-1R adaptor protein, is decreased by TGFβ/Smad3 signaling. Loss or attenuation of TGFβ activation leads to elevated expression and phosphorylation of IRS-1 in colon cancer cells, resulting in enhanced cell proliferation, decreased apoptosis and increased tumor growth in vitro and in vivo. Downregulation of IRS-1 expression reversed Smad3 knockdown-mediated oncogenic phenotypes, indicating that TGFβ/Smad3 signaling inhibits cell proliferation and increases apoptosis at least partially through the inhibition of IRS-1 expression and activation. Additionally, the TGFβ/Smad3/IRS-1 signaling axis regulates expression of cyclin D1 and XIAP, which may contribute to TGFβ/Smad3/IRS-1-mediated cell cycle progression and survival. Given that loss of TGFβ signaling occurs frequently in colon cancer, an important implication of our study is that IRS-1 could be a potential therapeutic target for colon cancer treatment.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colon; Colonic Neoplasms; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Insulin Receptor Substrate Proteins; Phosphorylation; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; X-Linked Inhibitor of Apoptosis Protein

Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P=.005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer. Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial-mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Regulatory Networks; HCT116 Cells; Humans; Kallikreins; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor-β (TGF-β) induction of epithelial-mesenchymal transition (EMT) in SW480 was established as a system for studies of colon cancer metastasis. However, the epigenetic mechanisms underlying this process remain unknown. In mammal, polycomb repressive complex-2 (PRC2) is a highly conserved histone methyltransferase involved in epigenetic regulations. Enhancer of zeste Homolog 2 (EZH2) is the catalytic subunit of PRC2, which catalyzes methylation of lysine 27 of histone H3 (H3K27).. An inducible EMT system in colorectal cancer was utilized to study its mechanistic and phenotypic changes. Particularly, gene expression analysis was studied after immunoprecipitation.. In this study, we reported that EZH2 is significantly enriched in the promoter region of WNT5A after TGF-β induction in SW480 colon cancer cell line, which in turn silenced the expression of WNT5A. Furthermore, EZH2 inhibitor antagonized the TGF-β-induced morphological conversion associated with epithelial-mesenchymal transition (EMT). Conversely, inhibition of histone H3K27me3 reader CBX does not affect the WNT5A expression level during TGF-β-induced EMT.. Our results indicate that EZH2 was essential for the silencing of WNT5A during TGF-β-induced epithelial-mesenchymal transition of colon cancer cells.

Topics: Apoptosis; Cell Proliferation; Colonic Neoplasms; DNA Methylation; Enhancer of Zeste Homolog 2 Protein; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Histones; Humans; MicroRNAs; Promoter Regions, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Wnt-5a Protein

TGF-β is an important target for many cancer therapies under development. In addition to suppressing anti-tumor immunity, it has pleiotropic direct pro- and anti- tumor effects. The actions of increased endogenous TGF-β production remain unclear, and may affect the outcomes of anti-TGF-β cancer therapy. We hypothesize that tumor-derived TGF-β (td-TGF-β) plays an important role in maintaining tumor remission by controlling tumor proliferation in vivo, and that decreasing td-TGF-β in the tumor microenvironment will result in tumor progression. The aim of this study was to examine the effect of TGF-β in the tumor microenvironment on the balance between its anti-proliferative and immunosuppressive effects.. A murine BALB/c spontaneous colon adenocarcinoma cell line (CT26) was genetically engineered to produce increased active TGF-β (CT26-TGF-β), a dominant-negative soluble TGF-β receptor (CT26-TGF-β-R), or the empty neomycin cassette as control (CT26-neo). In vitro proliferation rates were measured. For in vivo studies, the three cell lines were injected into syngeneic BALB/c mice, and tumor growth was measured over time. Immunodeficient BALB/c nude mice were used to investigate the role of T and B cells.. In vitro, CT26-TGF-β-R and CT26-TGF-β cells showed increased and suppressed proliferation, respectively, compared to control (CT26-neo), confirming TGF-β has direct anti-tumor effects. In vivo, we found that CT26-TGF-β-R cells displayed slower growth compared to control, likely secondary to reduced suppression of anti-tumor immunity, as this effect was ablated in immunodeficient BALB/c nude mice. However, CT26-TGF-β cells (excess TGF-β) exhibited rapid early growth compared to control, but later failed to progress. The same pattern was shown in immunodeficient BALB/c nude mice, suggesting the effect on tumor growth is direct, with minimal immune system involvement. There was minimal effect on systemic antitumor immunity as determined by peripheral antigen-specific splenocyte type 1 cytokine production and tumor growth rate of CT26-neo on the contralateral flank of the same mice.. Although TGF-β has opposing effects on tumor growth, this study showed that excessive td-TGF-β in the tumor microenvironment renders the tumor non-proliferative. Depleting excess td-TGF-β may release this endogenous tumor suppressive mechanism, thus triggering the progression of the tumor. Therefore, our findings support cautions against using anti-TGF-β strategies in treating cancer, as this may tip the balance of anti-immunity vs. anti-tumor effects of TGF-β, leading to tumor progression instead of remission.

Topics: Adenocarcinoma; Animals; B-Lymphocytes; Carcinogenesis; Cell Growth Processes; Cell Line, Tumor; Colonic Neoplasms; Cytokines; Genetic Engineering; Immunity; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment

Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, is known to exhibit tumor suppressor activity in colon cancer, the mechanism of which is not understood. Here we show that R-spondin 1 (RSPO1)/LGR5 directly activates TGFβ signaling cooperatively with TGFβ type II receptor in colon cancer cells, enhancing TGFβ-mediated growth inhibition and stress-induced apoptosis. Knockdown of LGR5 attenuated downstream TGFβ signaling and increased cell proliferation, survival, and metastasis in an orthotopic model of colon cancer

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; HCT116 Cells; Humans; Neoplasm Metastasis; Receptors, G-Protein-Coupled; Receptors, Transforming Growth Factor beta; Thrombospondins; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Signaling Pathway

Metastasis of tumor cells occurs through lymphatic vessels, blood vessels and transcoelomic spreading. Growing evidence from in vivo and in vitro studies has indicated that tumor lymphangiogenesis facilitates metastasis. However, the regulation of lymphangiogenesis in colon cancer remains unclear. The aims of this study were to identify key miRNAs in colon cancer lymphangiogenesis and to investigate its target and mechanism.. miRNA microarray analysis was conducted to identify miRNAs in human lymphatic endothelial cells (HLECs) that were regulated by co-cultured human colon cancer cells. Gain- and loss-of-function studies were performed to determine the function of miR-27a, a top hint, on lymphangiogenesis and migration in HLECs. Furthermore, bioinformatics prediction and experimental validation were performed to identify miR-27a target genes in lymphangiogenesis.. We found that expression of miR-27a in HLECs was induced by co-culturing with colon cancer cells. Over-expression of miR-27a in HLECs enhanced lymphatic tube formation and migration, whereas inhibition of miR-27a reduced lymphatic tube formation and migration. Luciferase reporter assays showed that miR-27a directly targeted SMAD4, a pivotal component of the TGF-β pathway. In addition, gain-of-function and loss-of-function experiments showed that SMAD4 negatively regulated the length of lymphatic vessels formed by HLECs and migration.. Our data indicated that colon cancer cell induced the expression of miR-27a in HLECs, which promoted lymphangiogenesis by targeting SMAD4. Our finding implicated miR-27a as a potential target for new anticancer therapies in colon cancer.

Topics: Cells, Cultured; Coculture Techniques; Colonic Neoplasms; Humans; Lymphangiogenesis; Lymphatic Vessels; MicroRNAs; Signal Transduction; Transforming Growth Factor beta

TGFβ has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFβ can affect the extent and composition of inflammatory cells present in tumors, contextually promoting and inhibiting inflammation. While colon tumors display intratumoral inflammation, the contributions of TGFβ to this process are poorly understood. In human patients, we found that epithelial loss of TGFβ signaling was associated with increased inflammatory burden; yet overexpression of TGFβ was also associated with increased inflammation. These findings were recapitulated in mutant APC models of murine tumorigenesis, where epithelial truncation of TGFBR2 led to lethal inflammatory disease and invasive colon cancer, mediated by IL8 and TGFβ1. Interestingly, mutant APC mice with global suppression of TGFβ signals displayed an intermediate phenotype, presenting with an overall increase in IL8-mediated inflammation and accelerated tumor formation, yet with a longer latency to the onset of disease observed in mice with epithelial TGFBR-deficiency. These results suggest that the loss of TGFβ signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFβ inhibitors may result in a worse outcome by enhancing inflammatory responses.

Topics: Adenomatous Polyposis Coli Protein; Animals; Colonic Neoplasms; Cytokines; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasms, Experimental; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Cholera toxin B subunit (CTB) is a component of a licensed oral cholera vaccine. However, CTB has pleiotropic immunomodulatory effects whose impacts on the gut are not fully understood. Here, we found that oral administration in mice of a plant-made recombinant CTB (CTBp) significantly increased several immune cell populations in the colon lamina propria. Global gene expression analysis revealed that CTBp had more pronounced impacts on the colon than the small intestine, with significant activation of TGFβ-mediated pathways in the colon epithelium. The clinical relevance of CTBp-induced impacts on colonic mucosa was examined. In a human colon epithelial model using Caco2 cells, CTBp, but not the non-GM1-binding mutant G33D-CTBp, induced TGFβ-mediated wound healing. In a dextran sodium sulfate (DSS) acute colitis mouse model, oral administration of CTBp protected against colon mucosal damage as manifested by mitigated body weight loss, decreased histopathological scores, and blunted escalation of inflammatory cytokine levels while inducing wound healing-related genes. Furthermore, biweekly oral administration of CTBp significantly reduced disease severity and tumorigenesis in the azoxymethane/DSS model of ulcerative colitis and colon cancer. Altogether, these results demonstrate CTBp's ability to enhance mucosal healing in the colon, highlighting its potential application in ulcerative colitis therapy besides cholera vaccination.

Topics: Administration, Oral; Animals; Azoxymethane; Caco-2 Cells; Cholera; Cholera Toxin; Cholera Vaccines; Colitis, Ulcerative; Colon; Colonic Neoplasms; Dextran Sulfate; Disease Models, Animal; Female; Humans; Mice; Mice, Inbred C57BL; Mucous Membrane; Signal Transduction; Transforming Growth Factor beta; Wound Healing

Molecular targeting is an import strategy to treat advanced colon cancer. The current study demonstrates that expression of GRM3, a metabotropic glutamate receptor mainly expressed in mammalian central nervous system, is significantly upregulated in majority of human colonic adenocarcinomas tested and colon cancer cell lines. Knockdown of GRM3 expression or inhibition of GRM3 activation in colon cancer cells reduces cell survival and anchorage-independent growth in vitro and inhibits tumor growth in vivo. Mechanistically, GRM3 antagonizes TGFβ-mediated activation of protein kinase A and inhibition of Protein kinase B (AKT). In addition, TGFβ signaling increases GRM3 protein stability and knockdown of GRM3 enhances TGFβ-mediated tumor suppressor function. Further studies indicate that miR-487b-3p directly targets GRM3. Overexpression of miR-487b-3p mimics the effects of GRM3 knockdown and suppresses the tumorigenicity of colon cancer cells in vivo. Expression of miR-487b-3p is decreased in colon adenocarcinomas and inversely correlates with GRM3 expression. Taken together, these studies indicate that upregulation of GRM3 expression is a functionally important molecular event in colon cancer, and that GRM3 is a promising molecular target for colon cancer treatment. This is particularly interesting and important from a therapeutic standpoint because numerous metabotropic glutamate receptor antagonists are available, many of which have been found unsuitable for treatment of neuropsychiatric disorders for reasons such as inability to readily penetrate blood brain barriers. As GRM3 is upregulated in colon cancer, but rarely expressed in normal peripheral tissues, targeting GRM3 with such agents would not likely cause adverse neurological or peripheral side effects, making GRM3 an attractive and specific molecular target for colon cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice; MicroRNAs; Neoplasm Transplantation; Receptors, Metabotropic Glutamate; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Long non-coding RNA-activated by TGF-β (lncRNA-ATB) promotes the invasion-metastasis cascade in hepatocellular carcinoma via downregulating E-cadherin (E-cad) and inducing epithelial-to- mesenchymal transition (EMT) and is clinically significant in human colon cancer. However, its molecular mechanisms in colon cancer progression remain unclear. This study aimed to elucidate the role of lncRNA-ATB and its clinical value in colon cancer.. Expression levels of lncRNA-ATB in colon cancer tissues and colon cancer cell lines were evaluated using quantitative real-time polymerase chain reaction. The clinicopathological significance and prognostic value of lncRNA-ATB were investigated, and roles of lncRNA-ATB in regulating E-cad and other EMT-related markers expression and colon cancer progression were evaluated in vitro. Expression levels of lncRNA-ATB and E-cad in human plasma were evaluated.. Long non-coding RNA-activated by TGF-β was upregulated in colon cancer tissues compared with adjacent mucosa (P < 0.001). LncRNA-ATB levels were also higher in metastatic cancer tissues (P < 0.001). Among the three highly invasive colon cancer cell lines, lncRNA-ATB levels were relatively higher with concurrent low levels of E-cad compared with levels in the three low-invasive cell lines. LncRNA-ATB expression correlated with pN stage (P < 0.01) and American Joint Committee on Cancer stage (P < 0.01). Striking differences were observed in overall survival and disease-free survival in cases with both high lncRNA-ATB expression and low E-cad expression. Reduction of lncRNA-ATB increased expression of epithelial markers E-cad, ZO-1, and decreased expression of mesenchymal markers ZEB1 and N-cadherin (N-cad), and significantly influenced colon cancer cell progression. Plasma lncRNA-ATB was upregulated in colon cancer patients one month after surgery (P < 0.05).. Long non-coding RNA-activated by TGF-β may act on colon tumorigenesis by suppressing E-cad expression and promoting EMT process, and lncRNA-ATB inhibition may provide a promising therapeutic option for suppressing colon cancer progression.

Topics: Cadherins; Carcinogenesis; Colonic Neoplasms; Disease Progression; Down-Regulation; Female; Gene Expression; Humans; Male; Molecular Targeted Therapy; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; RNA, Long Noncoding; Survival Rate; Transforming Growth Factor beta; Up-Regulation

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa-miR-142-5p was up-regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR-142-5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR-142-5p. Functional studies of hsa-miR-142-5p identified its role in transforming growth factor beta (TGFβ) signalling as TGFβ receptor 2 and SMAD3 were degraded during both hsa-miR-142-5p overexpression and rotavirus infection. TGFβ is induced during rotavirus infection, which may promote apoptosis by activation of non-canonical pathways in HT29 cells. However, up-regulated miR-142-5p resulted in the inhibition of TGFβ-induced apoptosis suggesting its anti-apoptotic function. Rotavirus NSP5 was identified as a regulator of miR-142-5p expression. Concurrently, NSP5-HT29 cells showed inhibition of TGFβ-induced apoptosis and epithelial to mesenchymal transition by blocking non-canonical pathways. Overall, the study identified proviral function of hsa-miR-142-5p during rotavirus infection. In addition, modulation of TGFβ-induced non-canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.

Topics: Apoptosis; Colonic Neoplasms; Gene Expression Regulation, Viral; HT29 Cells; Humans; MicroRNAs; Protein Serine-Threonine Kinases; Proviruses; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Rotavirus; Rotavirus Infections; Smad3 Protein; Transforming Growth Factor beta

Colorectal cancer (CRC) is a notable cause of cancer‑associated mortality worldwide, making it a pertinent topic for the study of cancer and its treatment. Staphylococcal enterotoxin B (SEB), an enterotoxin produced by Staphylococcus aureus, has been demonstrated to exert anticancer and antimetastatic effects due to its ability to modify cell immunity and cellular signaling pathways. In the current study, SEB was investigated, including whether it exerts its growth inhibitory effects on colon adenocarcinoma cells. This may occur through the manipulation of a key tumor growth factor, termed transforming growth factor‑β (TGF‑β), and its signaling pathway transducer, Smad2/3. The human colon adenocarcinoma HCT116 cell line was treated with different concentrations of SEB, and cell number was measured using MTT assay at different treatment times. Smad2/3 RNA expression level was analyzed in untreated or SEB‑treated cells using quantitative polymerase chain reaction, which indicated significant differences between cell viability and Smad2/3 expression levels. SEB effectively downregulated Smad2/3 expression in the HCT116 cells at concentrations of 1 and 2 µg/ml (P=0.0021 and P=0.0017, respectively). SEB concentrations that were effective at inhibiting Smad2/3 expression were correlated with those able to inhibit the proliferation of the cancer cells. SEB inhibited Smad2/3 expression at the mRNA level in a concentration‑ and time‑dependent manner. The present study thus proposed SEB as an agent able to significantly reduce Smad2/3 expression in colon cancer cells, provoking moderate TGF‑β growth signaling and the reduction of tumor cell proliferation.

Topics: Adenocarcinoma; Cell Proliferation; Colonic Neoplasms; Enterotoxins; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Signal Transduction; Smad2 Protein; Staphylococcus aureus; Transforming Growth Factor beta

Clinical studies have linked microRNA-155 (miR-155) expression in the tumor microenvironment to poor prognosis. However, whether miR-155 upregulation is predictive of a pro- or antitumorigenic response is unclear, as the limited preclinical data available remain controversial. We examined miR-155 expression in tumor tissue from colon cancer patients. Furthermore, we investigated the role of this microRNA in proliferation and apoptosis, inflammatory processes, immune cell populations, and transforming growth factor-β/SMAD signaling in a chemically induced (azoxymethane-dextran sulfate sodium) mouse model of colitis-associated colon cancer. We found a higher expression of miR-155 in the tumor region than in nontumor colon tissue of patients with colon cancer. Deletion of miR-155 in mice resulted in a greater number of polyps/adenomas, an increased symptom severity score, a higher grade of epithelial dysplasia, and a decrease in survival. Surprisingly, these findings were associated with an increase in apoptosis in the normal mucosa, but there was no change in proliferation. The protumorigenic effects of miR-155 deletion do not appear to be driven solely by dysregulation of inflammation, as both genotypes had relatively similar levels of inflammatory mediators. The enhanced tumorigenic response in miR-155(-/-) mice was associated with alterations in macrophages and neutrophils, as markers for these populations were decreased and increased, respectively. Furthermore, we demonstrated a greater activation of the transforming growth factor-β/SMAD pathway in miR-155(-/-) mice, which was correlated with the increased tumorigenesis. Given the multiple targets of miR-155, careful evaluation of its role in tumorigenesis is necessary prior to any consideration of its potential as a biomarker and/or therapeutic target in colon cancer.

Topics: Animals; Apoptosis; Azoxymethane; Biomarkers; Carcinogens; Cell Proliferation; Colonic Neoplasms; Dextran Sulfate; Gene Deletion; Humans; Intestinal Mucosa; Mice; Mice, Inbred C57BL; MicroRNAs; Signal Transduction; Smad Proteins; Transfection; Transforming Growth Factor beta

Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remain controversial. In this study, we demonstrate that DPEP1 functions as a positive regulator for colon cancer cell metastasis. The expression of DPEP1 mRNA and proteins were upregulated in colon cancer tissues compared to normal mucosa. Gain-of-function and loss-of-function approaches were used to examine the malignant phenotype of DPEP1-expressing or DPEP1-depleted cells. DPEP1 expression caused a significant increase in colon cancer cell adhesion and invasion in vitro, and metastasis in vivo. In contrast, DPEP1 depletion induced opposite effects. Furthermore, cilastatin, a DPEP1 inhibitor, suppressed the invasion and metastasis of DPEP1-expressing cells. DPEP1 inhibited the leukotriene D4 signaling pathway and increased the expression of E-cadherin. We also show that DPEP1 mediates TGF-β-induced EMT. TGF-β transcriptionally repressed DPEP1 expression. TGF-β treatment decreased E-cadherin expression and promoted cell invasion in DPEP1-expressing colon cancer cell lines, whereas it did not affect these parameters in DPEP1-depleted cell lines. These results suggest that DPEP1 promotes cancer metastasis by regulating E-cadherin plasticity and that it might be a potential therapeutic target for preventing the progression of colon cancer.

Topics: Animals; Antigens, CD; Cadherins; Cell Adhesion; Cell Line, Tumor; Cilastatin; Colonic Neoplasms; Dipeptidases; Epithelial-Mesenchymal Transition; Female; GPI-Linked Proteins; HCT116 Cells; HT29 Cells; Humans; Leukotriene D4; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta; Transplantation, Heterologous

Metastasis is the main cause of mortality in cancer patients. Although there are many anti-cancer drugs targeting tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression; in particular, lymphangiogenesis is pivotal for metastasis in cancer. Here we report that lithium inhibits colon cancer metastasis by blocking lymphangiogenesis. Lithium reduces the expression of transforming growth factor-β-induced protein (TGFBIp) in colon cancer cells by inhibiting Smad3 phosphorylation via GSK3β inactivation. Moreover, lithium inhibits lymphatic endothelial cell migration, which is increased upon TGFBIp expression in tumor cells. Lithium had no significant effect on SW620 tumor growth in vitro and in vivo; however, it inhibited lymphangiogenesis in tumors. In tumor xenografts model, lithium was found to prevent metastasis to the lungs, liver, and lymph nodes by inhibiting TGFBIp-induced tumor lymphangiogenesis. Collectively, our findings demonstrate a novel role of lithium in the inhibition of colon cancer metastasis by blocking TGFBIp expression, and thereby TGFBIp-induced lymphangiogenesis, in primary tumors.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Lithium; Lymphangiogenesis; Mice; Neoplasm Metastasis; Neoplasm Proteins; Phosphorylation; Smad3 Protein; Transforming Growth Factor beta

The association of colitis with colorectal cancer has become increasingly clear with mast cells being identified as important inflammatory cells in the process. In view of the relationship between mast cells and cancer, we studied the effect and mechanisms of mast cells in the development of colon cancer. Functional and mechanistic insights were gained from ex vivo and in vivo studies of cell interactions between mast cells and CT26 cells. Further evidence was reversely obtained in studies of mast cell targeted Fcε-PE40 chimeric toxin. Experiments revealed mast cells could induce colon tumor cell proliferation and invasion. Cancer progression was found to be related to the density of mast cells in colonic submucosa. The activation of MAPK, Rho-GTPase, and STAT pathways in colon cancer cells was triggered by mast cells during cell-to-cell interaction. Lastly, using an Fcε-PE40 chimeric toxin we constructed, we confirmed the promoting effect of mast cells in development of colon cancer. Mast cells are a promoting factor of colon cancer and thus also a potential therapeutic target. The Fcε-PE40 chimeric toxin targeting mast cells could effectively prevent colon cancer in vitro and in vivo. Consequently, these data may demonstrate a novel immunotherapeutic approach for the treatment of tumors.

Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Exotoxins; Immunotoxins; Mast Cells; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Pseudomonas aeruginosa Exotoxin A; Receptors, IgE; rho GTP-Binding Proteins; STAT Transcription Factors; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Virulence Factors

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Topics: Adenoma; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoembryonic Antigen; Cell Movement; Cell Proliferation; Cells, Cultured; Colon; Colonic Neoplasms; Disease Progression; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Immunoenzyme Techniques; Immunoprecipitation; Mutation; Signal Transduction; Transforming Growth Factor beta

To develop prognostic biomarkers that can discriminate stage II-III colorectal cancer patients with high risk of postoperative recurrence, we conducted a genome-wide screening of relapse-related genes utilizing multiple microarray cohorts. Among differentially expressed genes between tumor and nontumor, we identified eight candidate genes associated with relapse in two datasets of stage II-III patients (n = 94 and 145, respectively, P < 0.05). Using datasets of laser-microdissected samples and FACS-purified cell populations, the localization of candidate genes, including COL4A2, COL4A1, VCAN and SERPINE1, were found predominantly in cancer stroma rather than epithelial components. Among those relapse-related stromal genes, VCAN mRNA, specifically expressed in cancer-associated fibroblasts, was further validated to be a prognostic factor in two additional independent datasets, consisting of 453 (P = 0.0334) and 89 (P = 0.0041) stage II-III patients. Furthermore, in our large set of formalin-fixed paraffin-embedded cohort (n = 338), VCAN protein was detected exclusively in cancer stroma by immunohistochemistry, demonstrating a stepwise increase of stromal VCAN from normal tissues through stage 0 to stage IV tumors. Stromal VCAN protein was associated with shorter relapse-free survival (RFS) in stage II-III colon cancer, independent of other clinical factors by multivariate analysis (P = 0.004). Stratified analyses revealed that stromal VCAN was a strong prognostic indicator particularly in stage II colon cancer (P = 0.0029). In all five analyzed cohorts, the expression of VCAN, in transcript or protein levels, was associated with poor RFS in stage II-III patients. We conclude that VCAN is a promising biomarker to identify stage II-III patients at high risk of relapse who may benefit from intensive postoperative management.

Topics: Aged; Biomarkers, Tumor; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Prognosis; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Versicans

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

Topics: Animals; Caco-2 Cells; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Cytokines; Exocytosis; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Inflammation; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; NF-kappa B; Promoter Regions, Genetic; rab27 GTP-Binding Proteins; RNA Interference; Stem Cells; Transcription Factor RelA; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Recently, the immunostimulatory roles of chemotherapeutics have been increasingly revealed, although bone marrow suppression is still a common toxicity of chemotherapy. While the numbers and ratios of different immune subpopulations are analyzed after chemotherapy, changes to immune status after each cycle of treatment are less studied and remain unclear.. To determine the tumor-specific immune status and functions after different cycles of chemotherapy, we treated CT26 tumor-bearing mice with one to four cycles of 5-fluorouracil (5-FU). Overall survival was not improved when more than one cycle of 5-FU was administered. Here we present data concerning the immune statuses after one and three cycles of chemotherapy. We analyzed the amount of spleen cells from mice treated with one and three cycles of 5-FU as well as assayed their proliferation and cytotoxicity against the CT26 tumor cell line. We found that the absolute numbers of CD8 T-cells and NK cells were not influenced significantly after either one or three cycles of chemotherapy. However, after three cycles of 5-FU, proliferated CD8 T-cells were decreased, and CT26-specific cytotoxicity and IFN-γ secretion of spleen cells were impaired in vitro. After one cycle of 5-FU, there was a greater percentage of tumor infiltrating CD8 T-cells. In addition, more proliferated CD8 T-cells, enhanced tumor-specific cytotoxicity as well as IFN-γ secretion of spleen cells against CT26 in vitro were observed. Given the increased expression of immunosuppressive factors, such as PD-L1 and TGF-β, we assessed the effect of early introduction of immunotherapy in combination with chemotherapy. We found that mice treated with cytokine induced killer cells and PD-L1 monoclonal antibodies after one cycle of 5-FU had a better anti-tumor performance than those treated with chemotherapy or immunotherapy alone.. These data suggest that a single cycle of 5-FU treatment promoted an anti-tumor immune response, whereas repeated chemotherapy cycles impaired anti-tumor immune functions. Though the amount of immune cells could recover after chemotherapy suspension, their anti-tumor functions were damaged by multiple rounds of chemotherapy. These findings also point towards early implementation of immunotherapy to improve the anti-tumor effect.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; B7-H1 Antigen; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Combined Modality Therapy; Cytokine-Induced Killer Cells; Cytotoxicity, Immunologic; Fluorouracil; Humans; Immunotherapy, Adoptive; Interferon-gamma; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-β (TGFβ) pathway activation were induced in CAFs. The putative TGFβ-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Proteins; Male; Neoplasm Invasiveness; Neoplasm Proteins; Paracrine Communication; Sarcoma; Transcription, Genetic; Transforming Growth Factor beta; Wnt Signaling Pathway

Topics: Adenocarcinoma; Aged; Colonic Neoplasms; Dermatomyositis; Female; Humans; Lymphatic Metastasis; Lymphocytes; Paraneoplastic Syndromes; Signal Transduction; Smad4 Protein; Transcription Factors; Transforming Growth Factor beta

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.

Topics: Adenocarcinoma; Ascitic Fluid; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cell Movement; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Interleukin-4; Interleukin-6; Leukocytes, Mononuclear; Lipopolysaccharides; Macrophage Activation; Macrophage Colony-Stimulating Factor; Macrophages; Muscle Proteins; Nerve Tissue Proteins; Primary Cell Culture; Signal Transduction; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Metastasis is the leading cause of cancer-related mortality in almost all types of cancers, including colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a critical process during the metastatic cascade. This process may be a potential target for the diagnosis and treatment of CRC. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in treating various types of human malignancies, including CRC. Our published data suggest that PZH can induce apoptosis, as well as inhibit cell proliferation and tumor angiogenesis, thus suppressing CRC growth in vitro and in vivo. We evaluated the therapeutic efficacy of PZH against CRC metastasis using a CRC liver metastasis mouse model to further explore the mechanisms underlying the antitumor action of PZH. MTT, migration, and Matrigel invasion assays were used to assess the effect of PZH on cell viability, migration and invasion. We then established an orthotopic liver metastasis model of colon cancer using microsurgical techniques. Mice were intragastrically administered 234 mg/kg/day dose of either PZH or saline for 14 days. The body and tumor weights of the mice were measured after they were sacrificed. Moreover, we examined the effect of PZH inhibition on liver metastasis. Finally, EMT-related proteins and the TGF-β signaling pathway were assessed using immunohistochemical staining (IHS). The present data revealed that PZH significantly inhibited the migration and invasion of CT-26 cells in a dose-dependent manner, which affirmed the inhibitory effect of PZH on CRC cell metastasis. No significant change was observed between the in vivo primary tumor growth and body weight. However, the control group had five cases of liver metastasis (5/6), whereas one case was found in the PZH group (1/6). Thus, PZH exhibited therapeutic efficacy against CRC metastasis without apparent toxicity. The inhibitory effect of PZH on EMT resulted in an increase in E-cadherin expression, as well as a decrease in N-cadherin expression. In addition, PZH significantly inhibited TGF-β, as well as the phosphorylation of Smad2/3 and Smad4 in the tumor tissues, indicating its suppressive action on TGF-β signaling. These molecular effects ultimately resulted in the inhibition of cancer cell EMT and tumor metastasis.

Topics: Animals; Antineoplastic Agents; Cadherins; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Genetic alterations in the TGFβ signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFβ signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined Apc(Δ716) Tgfbr2(ΔIEC) compound mutant mice that carry mutations in Apc and Tgfbr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple Tgfbr2(ΔIEC) mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these Tgfbr2(ΔIEC) mice developed invasive colon cancer associated with chronic inflammation. We also found that TGFβ signaling is suppressed in human colitis-associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causing MMP2 activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFβ signaling-repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in Tgfbr2(ΔIEC) mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated Tgfbr2(ΔIEC) mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFβ signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans.

Topics: Adenomatous Polyposis Coli Protein; Animals; Cell Proliferation; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Intestinal Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Transgenic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The anticancer effect of (1S,2S,3E,7E,11E)-3,7,11,15-cembratetraen-17,2-olide (LS-1) from Lobophytum sp. has been already reported in HT-29 human colorectal cancer cells. In this study, we examined the effect of LS-1 on the apoptosis induction of SNU-C5/5-FU, fluorouracil-resistant human colon cancer cells. Furthermore, we investigated whether the apoptosis-induction effect of LS-1 could arise from the activation of the TGF-β pathway. In SNU-C5/5-FU treated with LS-1 of 7.1 μM (IC50), we could observe the various apoptotic characteristics, such as the increase of apoptotic bodies, the increase of the sub-G1 hypodiploid cell population, the decrease of the Bcl-2 level, the increase of procaspase-9 cleavage, the increase of procaspase-3 cleavage and the increase of poly(ADP-ribose) polymerase cleavage. Interestingly, the apoptosis-induction effect of LS-1 was also accompanied by the increase of Smad-3 phosphorylation and the downregulation of c-Myc in SNU-C5/5-FU. LS-1 also increased the nuclear localization of phospho-Smad-3 and Smad-4. We examined whether LS-1 could downregulate the expression of carcinoembryonic antigen (CEA), a direct inhibitor of TGF-β signaling. LS-1 decreased the CEA level, as well as the direct interaction between CEA and TGF-βR1 in the apoptosis-induction condition of SNU-C5/5-FU. To examine whether LS-1 can induce apoptosis via the activation of TGF-β signaling, the SNU-C5/5-FU cells were treated with LS-1 in the presence or absence of SB525334, a TGF-βRI kinase inhibitor. SB525334 inhibited the effect of LS-1 on the apoptosis induction. These findings provide evidence demonstrating that the apoptosis-induction effect of LS-1 results from the activation of the TGF-β pathway via the downregulation of CEA in SNU-C5/5-FU.

Topics: Animals; Anthozoa; Antineoplastic Agents; Apoptosis; Colonic Neoplasms; Diterpenes; Down-Regulation; Drug Resistance, Neoplasm; Fluorouracil; HT29 Cells; Humans; Inhibitory Concentration 50; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) promotes tumor growth, invasion and metastasis in established tumors. In this study, we analyzed the effect of overexpressing an anti-TGF-β peptide fused to apolipoprotein A-I (ApoA-I) as a scaffold molecule. We generated and characterized stable MC38 colon carcinoma clones expressing ApoA-I fused to the anti-TGF-β peptide P144 and ApoA-I as control cells. We evaluated in vitro the gene expression profile, cell cycle and anchorage-independent growth. The in vivo tumorigenic potential and immunogenicity were analyzed inoculating the MC38 clones into C57BL/6 mice, recombination-activating gene 1 knockout mice or mice deficient in NK cells either subcutaneously or intrasplenically to generate hepatic metastases. While overexpression of ApoA-I had no effect on the parameters analyzed, ApoA-I fused to P144 markedly diminished the tumorigenic capacity and metastatic potential of MC38 in vitro and in vivo, thus generating a highly immunogenic cell line. MC38 cells transfected with ApoA-I fused to P144 triggered memory T cell responses able to eliminate the parental cell line upon re-challenge. In summary, expression of ApoA-I fused to P144 is a novel strategy to modulate TGF-β in tumor cells. These results highlight the potential of TGF-β as a target in the development of new antitumor treatments.

Topics: Animals; Apolipoprotein A-I; Cell Line, Tumor; Colonic Neoplasms; Female; Genetic Therapy; Immunotherapy; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptide Fragments; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Colorectal cancer is strongly associated with lipid metabolism. NPC1L1, a sterol transporter, plays a key role in modulating lipid homeostasis in vivo. Its inhibitor, ezetimibe, began to be used clinically to lower cholesterol and this caused the great debate on its role in causing carcinogenesis. Here we explored the role of NPC1L1 in colorectal tumorigenesis.. Wild-type mice and NPC1L1(-/-) (NPC1L1 knockout) mice were treated with azoxymethane (AOM)-dextran sodium sulfate (DSS) to induce colitis-associated colorectal tumorigenesis. Mice were sacrificed 10, 15, 18 or 20 weeks after AOM treatment, respectively. Colorectal tumors were counted and analyzed. Plasma lipid concentrations were measured using enzymatic reagent kit. Protein expression level was assayed by western blot.. NPC1L1(-/-) mice significantly had fewer tumors than wild-type. The ratio of malignant/tumor in NPC1L1(-/-) mice was significantly lower than in wild-type 20 weeks after AOM-DSS treatment. NPC1L1 was highly expressed in the small intestine of wild-type mice but its expression was undetectable in colorectal mucous membranes or tumors in either group. NPC1L1 knockout decreased plasma total cholesterol and phospholipid. NPC1L1(-/-) mice had significant lower intestinal inflammation scores and expressed inflammatory markers p-c-Jun, p-ERK and Caspase-1 p20 lower than wild-type. NPC1L1 knockout also reduced lymphadenectasis what may be caused by inflammation. NPC1L1 knockout in mice decreased β-catenin in tumors and regulated TGF-β and p-gp in adjacent colons or tumors. There was not detectable change of p53 by NPC1L1 knockout.. Our results provide the first evidence that NPC1L1 knockout protects against colitis-associated tumorigenesis. NPC1L1 knockout decreasing plasma lipid, especially cholesterol, to reduce inflammation and decreasing β-catenin, p-c-Jun and p-ERK may be involved in the mechanism.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azoxymethane; beta Catenin; Cell Transformation, Neoplastic; Colitis; Colonic Neoplasms; Dextran Sulfate; Disease Models, Animal; Homozygote; Intestinal Mucosa; Lipids; Membrane Transport Proteins; Mice; Mice, Knockout; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFβ. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFβ (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFβ stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFβ induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ-Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP-Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFβ signaling pathways.

Topics: Acyltransferases; Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Colon; Colonic Neoplasms; Hippo Signaling Pathway; Humans; Phosphoproteins; Protein Interaction Maps; Protein Serine-Threonine Kinases; Signal Transduction; Smad Proteins, Receptor-Regulated; Smad2 Protein; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins

Loss of response to TGF-β is a central event in the genesis of colorectal cancer (CRC), a disease that, in the majority cases, is refractory to growth inhibition induced by this cytokine. However, inactivating mutations at receptors and transducers from the TGF-β cascade occur only in approximately half of CRCs, suggesting the involvement of additional mechanisms altering the response to the cytokine. We have recently described the amplification of the 13q31 locus, where the miR-17-92 cluster maps, associated with overexpression of its members. In this study, we address the potential role of miR-20a, from the miR-17-92 cluster, in the suppression of TGF-β cytostatic response in CRC. Using the poorly tumorigenic and TGF-β-sensitive FET cell line that expresses low miR-20a levels, we first confirmed that miR-20a downmodulated CDKN1A expression, both at mRNA and protein level, through direct binding to its 3'-UTR. We demonstrated that miR-20a significantly diminished cell response to TGF-β by preventing its delay of G1/S transition and promoting progression into cell cycle. Moreover, besides modulating CDKN1A, miR-20a blocked TGF-β-induced transactivation of its promoter without affecting the post-receptor activation of Smad3/4 effectors directly. Finally, miR-20a abrogated the TGF-β-mediated c-Myc repression, a direct inhibitor of the CDKN1A promoter activation, most likely by reducing the expression of specific MYC-regulating genes from the Smad/E2F-based core repressor complex. Our experiments indicate that miR-20a interferes with the colonic epithelium homeostasis by disrupting the regulation of Myc/p21 by TGF-β, which is essential for its malignant transformation.

Topics: 3' Untranslated Regions; Binding Sites; Caco-2 Cells; Carcinoma; Cell Proliferation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; MicroRNAs; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; RNA Interference; Signal Transduction; Transcriptional Activation; Transfection; Transforming Growth Factor beta

The increased mRNA expression of chemotaxis- and angiogenesis-related factors in human monocytes following interaction with colon cancer cells has been shown to be suppressed by pre-treatment with low-dose lipopolysaccharide (LPS) (100 pg/ml). It has been demonstrated that low-dose LPS reduced the expression of RelB, a member of the nuclear factor (NF)-κB transcription factor family, in mouse macrophages and the NF-κB signaling pathway was important for tumor initiation and growth in tumor-associated macrophages. In addition, the signal transducer and activator of transcription 3 (STAT3) regulated innate immunity via Toll-like receptor (TLR)4 signaling. In the present study, the mRNA expression of signaling pathway- and suppression-related genes in human monocytes following a low-dose LPS treatment and subsequent interaction with colon cancer cells was investigated, in order to assess the molecular response.. The human monocyte cell line THP-1 was treated with LPS and, subsequently, co-cultured with the human colon cancer cell line DLD-1. The mRNA expression of various genes was then analyzed using quantitative real-time polymerase chain reaction (PCR).. The mRNA expression of RelB, STAT3, interleukin (IL)-10 and transforming growth factor (TGF)-β in THP-1 cells following interaction with DLD-1 cells was suppressed by pre-treatment with low-dose LPS (100 pg/ml).. Treating human monocytes with low-dose LPS may be useful for suppressing tumor progression and may be valuable for maintaining homeostasis.

Topics: Cell Line, Tumor; Coculture Techniques; Colonic Neoplasms; Disease Progression; Gene Expression Profiling; Humans; Interleukin-10; Lipopolysaccharides; Macrophages; Monocytes; RNA, Messenger; STAT3 Transcription Factor; Toll-Like Receptor 4; Transcription Factor RelB; Transforming Growth Factor beta

Vitamin D3 and its analogues have recently been shown to enhance the anti-tumour effects of 5- Fluorouracil (5-FU) both in vitro and in xenograft mouse model of colon cancer. This study measured the potential mechanism(s) by which vitamin D3 could synergise the tumouricidal activities of 5-FU in azoxymethane (AOM) rat model of colon cancer.. Seventy-five male Wistar rats were divided equally into 5 groups: Control, AOM, AOM-treated by 5-FU (5-FU), AOM-treated by vitamin D3 (VitD3), and AOM-treated by 5-FU + vitamin D3 (5-FU/D). The study duration was 15 weeks. AOM was injected subcutaneously for 2 weeks (15 mg/kg/week). 5-FU was injected intraperitoneally in the 9th and 10th weeks post AOM (8 total injections were given: 12 mg/kg/day for 4 successive days, then 6 mg/kg every other day for another 4 doses) and oral vitamin D3 (500 IU/rat/day; 3 days/week) was given from week 7 post AOM till the last week of the study. The colons were collected following euthanasia for gross and histopathological examination. The expression of β-catenin, transforming growth factor-β1 (TGF-β1), TGF-β type 2 receptor (TGF-βR2), smad4, inducible nitric oxide synthase (iNOS), and heat shock protein-90 (HSP-90) proteins was measured by immunohistochemistry. In colonic tissue homogenates, quantitative RT-PCR was used to measure the mRNA expression of Wnt, β-catenin, Dickkopf-1 (DKK-1) and cyclooxygenase-2 (COX-2) genes, while ELISA was used to measure the concentrations of TGF-β1, HSP-90 and COX-2 proteins.. Monotherapy with 5-FU or vitamin D3 significantly decreased the number of grown tumours induced by AOM (P < 0.05); however, their combination resulted in more significant tumouricidal effects (P < 0.05) compared with monotherapy groups. Mechanistically, vitamin D3/5-FU co-therapy significantly decreased the expression of Wnt, β-catenin, iNOS, COX-2 and HSP-90 and significantly increased the expression of DKK-1, TGF-β1, TGF-βR2, smad4 (P < 0.05), in comparison with their corresponding monotherapy groups.. Vitamin D3 and 5-FU synergise together and exhibit better anticancer effects by modulating Wnt/β-catenin pathway, TGF-β1 signals, iNOS, COX-2 and HSP-90. Further studies are required to illustrate the clinical value of vitamin D supplementation during the treatment of colon cancer with 5-FU in human patients.

Topics: Animals; beta Catenin; Cholecalciferol; Colonic Neoplasms; Disease Models, Animal; Fluorouracil; Humans; Male; Mice; Rats; Rats, Wistar; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cancer-associated fibroblasts (CAF) are major mediators in tumor microenvironment. We investigated the changes in protein expression in colon cancer-associated fibroblasts compared with normal fibroblasts (NF) in the context of searching for prognostic biomarkers, particularly for stage II patients.. CAFs and NFs isolated from colon cancer patients were used to identify differentially expressed proteins using quantitative proteomics. Stromal expression of deregulated proteins was analyzed by IHC. Prognostic impact was studied using external gene-expression datasets for training, then quantitative PCR and IHC for validation in different cohorts of patients. Combined datasets were used for prediction of risk assessment at stages II and III.. A desmoplastic signature composed of 32 proteins, highly specific for stromal components in colon cancer, was identified. These proteins were enriched for extracellular matrix organization components, TGFβ signaling pathway, fibrosis, and wound-healing proteins. The expression in CAFs of 11 upregulated proteins and four downregulated proteins, selected for biomarker validation, was verified by orthogonal techniques. LOXL2 displayed a high prognostic impact by using external independent datasets and further validation in two different cohorts of patients. High expression of LOXL2 was associated with higher recurrence P = 0.001 HR, 5.38 [95% confidence interval (CI), 1.70-17.01] and overall survival P = 0.001 HR, 8.52 (95% CI, 1.90-38.29). IHC analysis revealed a prognostic value for LOXL2 in stage II patients.. We identified LOXL2 to be associated with the outcome of colon cancer patients. Furthermore, it can be used to stratify patients at stages II and III for further therapeutic decisions.

Topics: Amino Acid Oxidoreductases; Biomarkers; Cell Line, Tumor; Cluster Analysis; Colonic Neoplasms; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Microfilament Proteins; Muscle Proteins; Neoplasm Staging; Prognosis; Protein Interaction Mapping; Protein Interaction Maps; Proteomics; Reproducibility of Results; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-β) signaling pathway, which is often inactivated by mutations of TGF-β type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in ∼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2(IEKO)) alone is not sufficient for intestinal tumor formation and lack of Pten (Pten(IEKO)) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (Pten(IEKO);Tgfbr2(IEKO)) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a β-catenin-independent mechanism. Inactivation of TGF-β signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-β signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.

Topics: Animals; Carcinogenesis; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Humans; Intestinal Mucosa; Mice; Mutation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

It has been suggested that cancer-associated fibroblasts (CAFs) positioned at the desmoplastic areas of various types of cancer are capable of executing a migratory program, characterized by accelerated motility and collective configuration. Since CAFs are reprogrammed derivatives of normal progenitors, including quiescent fibroblasts, we hypothesized that such migratory program could be context-dependent, thus being regulated by specific paracrine signals from the adjacent cancer population. Using the traditional scratch assay setup, we showed that only specific colon cancer cell lines (i.e. HT29) were able to induce collective CAF migration. By performing quantitative proteomics (SILAC), we identified a 2.7-fold increase of claudin-11, a member of the tight junction apparatus, in CAFs that exerted such collectivity in their migratory pattern. Further proteomic investigations of cancer cell line secretomes revealed a specific signature, involving TGF-β, as potential mediator of this effect. Normal colonic fibroblasts stimulated with TGF-β exerted myofibroblastic differentiation, occludin (OCLN) and claudin-11 (CLDN11) overexpression and cohort formation. Subsequently, inhibition of TGF-β attenuated all the previous effects. Immunohistochemistry of the universal tight junction marker occludin in a cohort of 30 colorectal adenocarcinoma patients defined a CAF subpopulation expressing tight junctions. Overall, these data suggest that cancer cells may induce CLDN11 overexpression and subsequent collective migration of peritumoral CAFs via TGF-β secretion.

Topics: Cell Line, Tumor; Cell Movement; Claudins; Colonic Neoplasms; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Tight Junctions; Transforming Growth Factor beta

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O(6)-methylguanine (O(6) MeG) adducts through O(6) MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-β signaling because such properties were completely abrogated by absorption of TGF-β under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-β-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease.

Topics: Animals; Azoxymethane; Carcinogens; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; G1 Phase Cell Cycle Checkpoints; Humans; Mesenchymal Stem Cells; Neoplasms, Experimental; Rats; Rats, Inbred Lew; Transforming Growth Factor beta; Wnt Signaling Pathway

Transforming growth factor-β (TGF-β) pathway is involved in primary tumor progression and in promoting metastasis in a considerable proportion of human cancers such as colorectal cancer (CRC). Therefore, blockage of TGF-β pathway signaling via an inhibitor could be a valuable tool in CRC treatment.. To evaluate the efficacy of systemic targeting of the TGF-β pathway for therapeutic effects on CRC, we investigated the effects of a TGβRI (TGF-β receptor 1) or TβRI kinase inhibitor, SD-208, on SW-48, colon adenocarcinoma cells. In this work, in vitro cell proliferation was studied by methyl thiazolyl tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays. Also, the histopathological and immunohistochemical evaluations were conducted by hematoxylin and eosin, and Ki-67 and CD34 markers were stained, respectively.. Our results showed no significant reduction in cell proliferation and vessel formation (170 ± 70 and 165 ± 70, P > 0.05) in treated SW-48 cells with SD-208 compared to controls.. Our data suggested that SD-208 could not significantly reduce tumor growth and angiogenesis in human colorectal cancer model at least using SW-48 cells.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Female; Humans; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Pteridines; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Tumor-stroma interactions are referred to as essential events in tumor progression. There has been growing attention that bone marrow-derived mesenchymal stem cells (BMSCs) can travel to tumor stroma, where they differentiate into tumor-associated fibroblast (TAF)-like cells, a predominant tumor-promoting stromal cell. However, little is definitively known about the contributors for this transition. Here, using an in vitro direct co-culture model of colon cancer cells and BMSCs, we identify that colon cancer cells can induce adjoining BMSCs to exhibit the typical characteristic of TAFs, with increased expression of α-smooth muscle actin (α-SMA). Importantly, the present data also reveals that activated Notch signaling mediates transformation of BMSCs to TAFs through the downstream TGF-β/Smad signaling pathway.

Topics: Actins; Bone Marrow Cells; Calcium-Binding Proteins; Cell Communication; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Colonic Neoplasms; Fibroblasts; Humans; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Mesenchymal Stem Cells; Receptors, Notch; Serrate-Jagged Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Microenvironment

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.. Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.. Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.. Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.

Topics: Coculture Techniques; Colonic Neoplasms; Curcumin; Epithelial-Mesenchymal Transition; Fibroblasts; Fluorouracil; HCT116 Cells; Humans; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

Activin is a multifunctional growth and differentiation factor of the growth factor-beta (TGF-β) superfamily, which inhibits the proliferation of colon cancer cells. It induces phosphorylation of intracellular signaling molecules (Smads) by interacting with its type I and type II receptors. Previous studies showed that human activin receptor-interacting protein 2 (hARIP2) can reduce activin signaling by interacting with activin type II receptors; however, the activity of hARIP2 in colon cancer has yet to be detailed. In vitro, overexpression of hARIP2 reduced activin-induced transcriptional activity and enhanced cell proliferation and colony formation in human colon cancer HCT8 cells and SW620 cells. Also, hARIP2 promoted colon cancer cell apoptosis, suggesting that a vital role in the initial stage of colon carcinogenesis. In vivo, immunohistochemistry revealed that hARIP2 was expressed more frequently and much more intensely in malignant colon tissues than in controls. These results indicate that hARIP2 is involved in human colon tumorigenesis and could be a predictive maker for colon carcinoma aggressiveness.

Topics: Activins; Apoptosis; Blotting, Western; Carcinogenesis; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptor-Interacting Protein Serine-Threonine Kinase 2; RNA, Neoplasm; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Histone methylation plays a crucial role in various biological and pathological processes including cancer development. In this study, we discovered that JARID2, an interacting component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in Transforming Growth Factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 lung cancer cell line and HT29 colon cancer cell line. The expression of JARID2 was increased during TGF-ß-induced EMT of these cell lines and knockdown of JARID2 inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. JARID2 knockdown itself had no effect in the expression of EMT-related genes but antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB family and microRNA-200 family. Chromatin immunoprecipitation assays showed that JARID2 was implicated in TGF-ß-induced transcriptional repression of CDH1 and microRNA-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of JARID2 protein, which may control PRC2 recruitment and histone methylation during TGF-ß-induced EMT of lung and colon cancer cell lines.

Topics: Antigens, CD; Cadherins; Colonic Neoplasms; Enhancer of Zeste Homolog 2 Protein; Epithelial-Mesenchymal Transition; Histones; HT29 Cells; Humans; Lung Neoplasms; MicroRNAs; Polycomb Repressive Complex 2; Transforming Growth Factor beta

The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients.

Topics: Adenocarcinoma; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; RNA Processing, Post-Transcriptional; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Selenium (Se) is an essential micronutrient that exerts its functions via selenoproteins. Little is known about the role of Se in inflammatory bowel disease (IBD). Epidemiological studies have inversely correlated nutritional Se status with IBD severity and colon cancer risk. Moreover, molecular studies have revealed that Se deficiency activates WNT signaling, a pathway essential to intestinal stem cell programs and pivotal to injury recovery processes in IBD that is also activated in inflammatory neoplastic transformation. In order to better understand the role of Se in epithelial injury and tumorigenesis resulting from inflammatory stimuli, we examined colonic phenotypes in Se-deficient or -sufficient mice in response to dextran sodium sulfate (DSS)-induced colitis, and azoxymethane (AOM) followed by cyclical administration of DSS, respectively. In response to DSS alone, Se-deficient mice demonstrated increased morbidity, weight loss, stool scores, and colonic injury with a concomitant increase in DNA damage and increases in inflammation-related cytokines. As there was an increase in DNA damage as well as expression of several EGF and TGF-β pathway genes in response to inflammatory injury, we sought to determine if tumorigenesis was altered in the setting of inflammatory carcinogenesis. Se-deficient mice subjected to AOM/DSS treatment to model colitis-associated cancer (CAC) had increased tumor number, though not size, as well as increased incidence of high grade dysplasia. This increase in tumor initiation was likely due to a general increase in colonic DNA damage, as increased 8-OHdG staining was seen in Se-deficient tumors and adjacent, non-tumor mucosa. Taken together, our results indicate that Se deficiency worsens experimental colitis and promotes tumor development and progression in inflammatory carcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Azoxymethane; Carcinogenesis; Colitis; Colonic Neoplasms; Deoxyguanosine; Dextran Sulfate; Diet; DNA Damage; Epidermal Growth Factor; Gene Expression Regulation; Inflammation; Mice; Mice, Inbred C57BL; Selenium; Signal Transduction; Transforming Growth Factor beta; Weight Loss

Interactions between the epithelium and surrounding mesenchyme/stroma play an important role in normal gut morphogenesis, the epithelial response to injury, and epithelial carcinogenesis. The tumor microenvironment, composed of stromal cells including myofibroblasts and immune cells, regulates tumor growth and the cancer stem cell niche. Deletion of epimorphin (Epim), a syntaxin family member expressed in myofibroblasts and macrophages, results in partial protection from colitis and from inflammation-induced colon cancer in mice. We sought to determine whether epimorphin deletion protects from polyposis in the Apcmin/+ mouse model of intestinal carcinogenesis. Epim-/- mice were crossed to Apcmin/+ mice; Apcmin/+ and Apcmin/+/Epim-/- mice were killed at 3 mo of age. Polyp numbers and sizes were quantified in small intestine and colon, and gene expression analyses for pathways relevant to epithelial carcinogenesis were performed. Primary myofibroblast cultures were isolated, and expression and secretion of selected growth factors from Apcmin/+ and Apcmin/+/Epim-/- myofibroblasts were examined by ELISA. Small bowel polyposis was significantly inhibited in Apcmin/+/Epim-/- compared with Apcmin/+ mice. Apcmin/+/Epim-/- compared with Apcmin/+ polyps and adjacent uninvolved intestinal mucosa had increased transforming growth factor-β (TGF-β) expression and signaling with increased P-Smad2/3 expression. Myofibroblasts isolated from Apcmin/+/Epim-/- vs. Apcmin/+ mice had markedly decreased hepatocyte growth factor (HGF) expression and secretion. We concluded that Epim deletion inhibits polyposis in Apcmin/+ mice, associated with increased mucosal TGF-β signaling and decreased myofibroblast HGF expression and secretion. Our data suggest that Epim deletion reduces tumorigenicity of the stromal microenvironment.

Topics: Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Colonic Neoplasms; Disease Models, Animal; Female; Gene Expression Regulation; Hepatocyte Growth Factor; Intestinal Mucosa; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Signal Transduction; Transforming Growth Factor beta

Photodynamic therapy (PDT) can lead to development of antigen-specific immune response and PDT-mediated immunity can be potentiated by T regulatory cell (Treg) depletion. We investigated whether the combination of PDT with cyclophosphamide (CY) could foster immunity against wild-type tumours expressing self-antigen (gp70).. Mice with CT26 tumours were treated with PDT alone or in combination with low-dose CY. T regulatory cell numbers and transforming growth factor-β (TGF-β) levels were measured at several time points after treatment. Mice cured by PDT+CY were rechallenged with CT26 and monitored for long-term survival.. Photodynamic therapy+CY led to complete tumour regression and long-term survival in 90% of treated mice while the absolute numbers of Treg decreased after PDT+CY and the TGF-β levels were reduced to a level comparable to naïve mice. Sixty-five percent of the mice treated with PDT+CY that survived over 90 days tumour free rejected the rechallenge with the same tumour when a second dose of CY was administered before rechallenge but not without.. Administration of CY before PDT led to depletion of Treg and potentiated PDT-mediated immunity, leading to long-term survival and development of memory immunity that was only uncovered by second Treg depletion.

Topics: Animals; Antigens, Neoplasm; Autoantigens; Colonic Neoplasms; Combined Modality Therapy; Cyclophosphamide; Immunologic Memory; Immunosuppressive Agents; Lymph Nodes; Mice; Mice, Inbred BALB C; Photochemotherapy; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway.. Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is aubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-b, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor.. Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

Topics: Animals; Cell Proliferation; Cell Survival; Colonic Neoplasms; Epigenesis, Genetic; Histones; Humans; Mice; Mitogen-Activated Protein Kinases; NIH 3T3 Cells; Polycomb Repressive Complex 2; RNA, Long Noncoding; RNA, Small Interfering; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Bone morphogenetic proteins (BMP) are part of the TGF-β-signaling pathway; genetic variation in these genes may be involved in colorectal cancer. In this study, we evaluated the association between genetic variation in BMP1 (11 tagSNPs), BMP2 (5 tagSNPs), BMP4 (3 tagSNPs), BMPR1A (9 tagSNPs), BMPR1B (21 tagSNPs), BMPR2 (11 tagSNPs) and GDF10 (7 tagSNPs) with risk of colon and rectal cancer and tumor molecular phenotype. We used data from population-based case-control studies (colon cancer n = 1,574 cases, 1,970 controls; rectal cancer n = 791 cases, 999 controls). We observed that genetic variation in BMPR1A, BMPR1B, BMPR2, BMP2 and BMP4 was associated with risk of developing colon cancer, with 20 to 30% increased risk for most high-risk genotypes. A summary of high-risk genotypes showed over a twofold increase in colon cancer risk at the upper risk category (OR = 2.49 95% CI = 1.95, 3.18). BMPR2, BMPR1B, BMP2 and GDF10 were associated with rectal cancer. BMPR2 rs2228545 was associated with an almost twofold increased risk of rectal cancer. The risk associated with the highest category of the summary score for rectal cancer was 2.97 (95% CI = 1.87, 4.72). Genes in the BMP-signaling pathway were consistently associated with CIMP+ status in combination with both KRAS-mutated and MSI tumors. BMP genes interacted statistically significantly with other genes in the TGF-β-signaling pathway, including TGFβ1, TGFβR1, Smad 3, Smad 4 and Smad 7. Our data support a role for genetic variation in BMP-related genes in the etiology of colon and rectal cancer. One possible mechanism is via the TGF-β-signaling pathway.

Topics: Adult; Aged; Bone Morphogenetic Proteins; Case-Control Studies; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; Genotype; Humans; Male; Middle Aged; Phenotype; Rectal Neoplasms; Signal Transduction; Transforming Growth Factor beta

We previously reported that oral ingestion of Lentinula edodes mycelia (L.E.M.) extract can inhibit the growth of a subcutaneously established melanoma in a T cell-dependent manner via mitigation of regulatory T cell (Treg)-mediated immunosuppression. In this study, we tested the antitumor effect and mechanism of oral ingestion of L.E.M. extract following inoculation of murine colon carcinoma colon-26 (C26) cells into the subserosal space of the cecum (i.c.) of syngeneic mice. In this model, the primary site of the immune response was gut-associated lymphoid tissue (GALT), which is known to be an immunological tolerance-inducing site for numerous dietary antigens. Oral ingestion of the L.E.M. extract suppressed the growth of i.c.-inoculated C26 cells in a T cell-dependent manner and restored the T cell response of the mesenteric lymph nodes and the spleen, not only to a tumor antigen-derived peptide, presented on H-2Ld molecules, but also to C26 cells. I.c. inoculation of C26 cells increased the potential of CD4+ T cells of the mesenteric lymph nodes to produce transforming growth factor (TGF)-β, but ingestion of the L.E.M. extract decreased the ability of both CD4+ and CD8+ T cells in the mesenteric lymph nodes to produce this immunosuppressive cytokine. Although ingestion of L.E.M. showed only a marginal effect on Tregs in this model, this treatment significantly reduced the plasma levels of TGF-β and IL-6, both of which were increased in the i.c. C26-inoculated mice. In summary, our results indicate that oral ingestion of L.E.M. extract can restore antitumor T cell responses of mice even when the primary antitumor immune response is elicited in GALT, and provide important implications for anticancer immunotherapy of human colon cancer.

Topics: Amino Acid Sequence; Animals; Biological Products; Cecum; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Eating; Female; Humans; Interleukin-6; Lymph Nodes; Mesentery; Mice; Mice, Inbred BALB C; Mice, Nude; Peptides; Peyer's Patches; Shiitake Mushrooms; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (T(reg)). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T(reg) infiltration and CD8(+) T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T(reg) against CD8(+) T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T(reg) cell infiltration and CD8(+) T-cell apoptosis was noted. TGF-β signaling blockade diminished apoptosis of CD8(+) T cells, implicating TGF-β as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-β by CCR5-deficient T(reg) or to enhance their cytotoxic effects against CD8(+) T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of T(reg) in inhibiting the antitumor responses of CD8(+) T cells, in the same way as CCL5 signaling blockade. Together, our findings establish that CCL5/CCR5 signaling recruits T(reg) to tumors and enhances their ability to kill antitumor CD8(+) T cells, thereby defining a novel mechanism of immune escape in colorectal cancer.

Topics: Animals; Apoptosis; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemokine CCL5; Colonic Neoplasms; Cytotoxicity, Immunologic; Gene Knockdown Techniques; Humans; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred BALB C; Mice, Nude; Receptors, CCR5; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Escape

Transforming growth factor (TGF)-β signaling, which is down-regulated by the E3 ubiquitin ligase Smad ubiquitin regulating factor 2 (Smurf2), promotes development of cancer. We identified a splice variant of Smurf2 (ΔE2Smurf2) and investigated its role in colon carcinogenesis in mice.. Colitis-associated colon cancer was induced in mice by administration of azoxymethane, followed by 3 cycles of oral administration of dextran sodium sulfate. Messenger RNA levels of Smurf2 in colon tumors and control tissue were measured by quantitative polymerase chain reaction; lymphocyte and cytokine levels were measured in tumor and tissue samples.. Tumor-infiltrating CD4(+) cells expressed higher levels of ΔE2Smurf2 than CD4(+) cells from nontumor tissues of wild-type mice. T cell-specific overexpression of ΔE2Smurf2 increased TGF-β signaling by suppressing protein levels of Smurf2, accompanied by an increase in levels of TGF-β receptor type II. Transgenic mice that overexpress ΔE2Smurf2 were protected against development of colitis-associated tumors and down-regulated proinflammatory cytokines such as interleukin-6. Patients with chronic inflammatory bowel disease had a significantly lower ratio of Smurf2/ΔE2Smurf2 than control individuals.. T cell-specific ΔE2Smurf2 degrades wild-type Smurf2 and controls intestinal tumor growth in mice by up-regulating TGF-β receptor type II, reducing proliferation and production of proinflammatory cytokines.

Topics: Animals; Cells, Cultured; Colitis; Colonic Neoplasms; Gene Expression Profiling; Hyaluronan Receptors; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-kit; Receptors, G-Protein-Coupled; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

TGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFβ signaling.. To test the importance of TGFβ signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFβ response (FET), or tumorigenic with TGFβ response (FETα) or tumorigenic with abrogated TGFβ response via introduction of dominant negative TGFβRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging.. Abrogation of TGFβ signaling through introduction of a dominant negative TGFβ receptor II (TGFβRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFβ signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFβ signaling is a metastasis suppressor, we rescued TGFβ signaling in CBS metastatic colon cancer cells that had lost TGFβ receptor expression due to epigenetic repression. Restoration of TGFβ signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFβ signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma.. The observations presented here indicate a metastasis suppressor role for TGFβ signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFβ signaling may be imprudent in some patient populations with residual TGFβ tumor suppressor activity.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; HEK293 Cells; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Microscopy, Video; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Experimental; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transplantation, Heterologous; X-Linked Inhibitor of Apoptosis Protein

Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21(cip1/waf1)). Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.

Topics: Activins; Apoptosis; Colon; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Humans; Phosphorylation; Proteasome Endopeptidase Complex; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitination

This study investigated the expression and biological role of TLR4 in human colon cancer cells' growth and survival, and its potential as a target for colon cancer therapy. Reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry (FCM) were used to detect the expression level of TLR4. MTT analysis was performed to evaluate cell proliferation and enzyme-linked immunosorbent assay (ELISA) to test the production of IL-8, VEGF, and TGF-beta. MAPKs and NF-kappaB were analyzed by Western blotting. Apoptosis was analyzed by flow cytometry with Annexin V and propidium iodide staining. The results showed that the human colon cancer cells HT-29, SW480, and Lovo all expressed TLR4 at both mRNA and protein levels, and TLR4 ligand LPS could not affect the expression of TLR4 and the proliferation of colon cancer cells. LPS increased phosphorylation of ERK1/2 and p38 and activated NF-kappaB. LPS promoted cytokine production, such as IL-8, VEGF, and TGF-beta. In addition, LPS induced resistance of human colon cancer cells to TRAIL-induced apoptosis and NF-kappaB activation was necessary for apoptosis resistance. The study identified the expression level of TLR4 in human colon cancer cells and TLR4 was functionally active. TLR4 may play important roles in promoting immune escape of human colon cancer cells by inducing immunosuppressive factors and apoptosis resistance.

Topics: Analysis of Variance; Apoptosis; Cell Proliferation; Colonic Neoplasms; Cytokines; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Lipopolysaccharides; TNF-Related Apoptosis-Inducing Ligand; Toll-Like Receptor 4; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Escape; Vascular Endothelial Growth Factor A

Transforming growth factor-β1 (TGF-β1) induces stromal fibroblast-to-myofibroblast transdifferentiation in the tumor-stroma interactive microenvironment via modulation of multiple phenotypic and functional genes, which plays a critical role in tumor progression. Up to now, the involvement of micro-RNAs (miRNAs) and their roles in TGF-β1-induced myofibroblast differentiation in tumor-stroma interaction are unclear. Using quantitative real-time RT-PCR, we demonstrated that the expression of micro-RNA-21 (miR-21) was upregulated in activated fibroblasts after treatment with TGF-β1 or conditioned medium from cancer cells. To determine the potential roles of miR-21 in TGF-β1-mediated gene regulation during myofibroblast conversion, we showed that miR-21 expression was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic. Interestingly, downregulation of miR-21 with the inhibitor effectively inhibited TGF-β1-induced myofibroblast differentiation while upregulation of miR-21 with a mimic significantly promoted myofibroblast differentiation. We further demonstrated that MiR-21 directly targeted and downregulated programmed cell death 4 (PDCD4) gene, which in turn acted as a negative regulator of several phenotypic and functional genes of myofibroblasts. Taken together, these results suggested that miR-21 participated in TGF-β1-induced myofibroblast transdifferentiation in cancer stroma by targeting PDCD4.

Topics: Apoptosis Regulatory Proteins; Blotting, Western; Cell Differentiation; Cells, Cultured; Colonic Neoplasms; Culture Media, Conditioned; Female; Fetus; Fibroblasts; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Lung; MicroRNAs; Myofibroblasts; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

The TGF-β signaling pathway is an essential regulator of many cellular process involved in carcinogenesis. Smad proteins are central to the function of TGF-β signaling. In this study, we evaluated genetic variation in TGFβ1, TGFβR1, Smad1, Smad2, Smad3, and Smad4 and risk of colon and rectal cancer.. Data are from a large case-control study of colon (n = 1,444 cases, 1,841 controls) and rectal (n = 754 cases, 856 controls) cancer participants with DNA.. Both TGFβ1 rs1800469 and rs4803455 were associated with colon cancer [odds ratio (OR) = 0.65 and 1.43, 95% CI = 0.51-0.84 and 1.18-1.73, respectively) but not rectal cancer. Likewise, 1 of 3 tagSNPs for TGFβR1, 2 of the 4 tagSNPs for Smad2, and 4 of 37 Smad3 tagSNPs were associated with colon cancer. Fewer significant associations were observed for rectal cancer, with only 1 tagSNP in Smad2 and 3 tagSNP in Smad3 having 95% CIs excluding 1.0. Several Smad3 tagSNPs were only associated with CpG island methylator phenotype. We observed several statistically significant interactions between genetic variation in the TGF-β signaling pathway and NFκB1, further illustrating its involvement in proposed mechanisms. In addition, we observed statistically significant interaction between TGFβ1, TGFβR1, and Smad3 and cigarette smoking, aspirin use, and estrogen status for both colon and rectal cancers. Variation in TGFβ1, TGFβR1, and Smad3 seemed to influence survival after diagnosis of colon and rectal cancer.. These findings provide further support for genetic variation in the TGF-β signaling pathway and risk of developing both colon and rectal cancers.. Insight into biological pathways is provided.

Topics: Adult; Aged; Case-Control Studies; Cell Growth Processes; Colonic Neoplasms; Female; Genetic Predisposition to Disease; Genetic Variation; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Rectal Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Metastasis causes most deaths from cancer yet mechanistic understanding and therapeutic options remain limited. Overexpression of the phosphatase PRL-3 (phosphatase of regenerating liver) is associated with metastasis of colon cancer. Here, we show that PRL-3 is a direct target of signaling by TGFβ, which is broadly implicated in progression and metastasis. We found that suppression of PRL-3 expression by TGFβ was mediated by Smad-dependent inhibition of PRL-3 transcription at the level of promoter activity. PRL-3 activation stimulated PI3K/AKT signaling that caused resistance to stress-induced apoptosis. PRL-3 overexpression promoted metastatic colonization in an orthotopic mouse model of colon cancer, whereas PRL-3 knockdown reduced metastatic potential. Altered metastatic phenotypes were not derivative of primary tumor development or local invasion but could be attributed to PRL-3-mediated cell survival. Our findings suggest that inhibiting PRL-3 expression might be an important mechanism through which TGFβ suppresses metastasis in colon cancer. In addition, our findings suggest that loss of TGFβ signaling, which occurs commonly during colon cancer progression, is sufficient to activate a PRL-3-mediated cell survival pathway that can selectively promote metastasis. Therefore, a major implication of our findings is that PRL-3 antagonists may offer significant value for antimetastatic therapy in patients with colon cancer.

Topics: Animals; Cell Line, Tumor; Chromatin Immunoprecipitation; Colonic Neoplasms; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Mice; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The Runt-related transcription factors (RUNX), mitogen-activated protein kinase (MAPK) 1 and eukaryotic translation initiation factor 4E (eIF4E) are potentially involved in tumorigenesis. We evaluated genetic variation in RUNX1 (40 tagSNPs), RUNX2 (19 tagSNPs), RUNX3 (9 tagSNPs), MAPK1 (6 tagSNPs), eIF4E (3 tagSNPs), eIF4EBP2 (2 tagSNP) and eIF4EBP3 (2 tagSNPs) to determine associations with colorectal cancer (CRC). We used data from population-based studies (colon cancer n = 1555 cases, 1956 controls; rectal cancer n = 754 cases, 959 controls with complete genotype data). Four statistically significant tagSNPs were identified with colon cancer and three tagSNPs were identified with rectal cancer. Whereas the independent risk estimates for each of the tagSNPs ranged from 1.21 to 1.52, the combined risk was greater than additive for any of the three combined high-risk genotypes {combined risk range 1.98 [95% confidence interval (CI) 1.45, 2.70] for eIF4E, RUNX1 and RUNX3 to 3.32 [95% CI 1.34, 8.23] for eIF43, RUNX2 and RUNX3}. For rectal cancer, the strongest association was detected for the combined genotype of RUNX1 and RUNX3 (odds ratio 1.87 95% CI 1.22, 2.87). Associations with specific molecular tumor phenotypes showed consistent and strong associations for CIMP+/MSI+ tumors where the risk estimates were consistently >10-fold and lower confidence bounds were over 3.00 for high-risk genotypes defined by RUNX1, RUNX2 and RUNX3. For CIMP+/KRAS2-mutated colon tumors, the combined risk for high-risk genotypes of RUNX2, eIF4E and RUNX1 was 7.47 (95% CI 1.58, 35.3). Although the associations need confirmation, the findings and their internal consistency underline the importance of genetic variation in these genes for the etiology of CRC.

Topics: Adult; Aged; Case-Control Studies; Colonic Neoplasms; Core Binding Factor Alpha 1 Subunit; Core Binding Factor Alpha 2 Subunit; Core Binding Factor Alpha 3 Subunit; Eukaryotic Initiation Factor-4E; Female; Genetic Variation; Genotype; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mutation; Polymorphism, Single Nucleotide; Rectal Neoplasms; Risk Factors; Signal Transduction; Transforming Growth Factor beta

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFβ signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFβ and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of function of Smad4 and retention of intact TGFβ receptors could synergistically increase the levels of VEGF, a major proangiogenic factor. Pharmacologic inhibition studies suggest that overactivation of the TGFβ-induced MEK-Erk and p38-MAPK (mitogen-activated protein kinase) auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in matrix metalloprotease 9 enhanced hypoxia-induced GLUT1 expression, increased aerobic glycolysis, and resistance to 5'-fluoruracil-mediated apoptosis. Interestingly, Smad4 specifically interacts with hypoxia-inducible factor (HIF) 1α under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFβ-induced auxiliary pathways, such as MEK-ERK, and p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention.

Topics: Cell Hypoxia; Cell Movement; Cell Transformation, Neoplastic; Colonic Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; Gene Silencing; HCT116 Cells; Humans; Matrix Metalloproteinase 9; Neoplasm Metastasis; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad4 Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The transforming growth factor-β (TGF-β) signaling pathway is involved in many aspects of tumorigenesis, including angiogenesis and metastasis. The authors evaluated this pathway in association with survival after a diagnosis of colon or rectal cancer.. The study included 1553 patients with colon cancer and 754 patients with rectal cancer who had incident first primary disease and were followed for a minimum of 7 years after diagnosis. Genetic variations were evaluated in the genes TGF-β1 (2 single nucleotide polymorphisms [SNPs]), TGF-β receptor 1 (TGF-βR1) (3 SNPs), smooth muscle actin/mothers against decapentaplegic homolog 1 (Smad1) (5 SNPs), Smad2 (4 SNPs), Smad3 (37 SNPs), Smad4 (2 SNPs), Smad7 (11 SNPs), bone morphogenetic protein 1 (BMP1) (11 SNPs), BMP2 (5 SNPs), BMP4 (3 SNPs), bone morphogenetic protein receptor 1A (BMPR1A) (9 SNPs), BMPR1B (21 SNPs), BMPR2 (11 SNPs), growth differentiation factor 10 (GDF10) (7 SNPs), Runt-related transcription factor 1 (RUNX1) (40 SNPs), RUNX2 (19 SNPs), RUNX3 (9 SNPs), eukaryotic translation initiation factor 4E (eiF4E) (3 SNPs), eukaryotic translation initiation factor 4E-binding protein 3 (eiF4EBP2) (2 SNPs), eiF4EBP3 (2 SNPs), and mitogen-activated protein kinase 1 (MAPK1) (6 SNPs).. After adjusting for American Joint Committee on Cancer stage and tumor molecular phenotype, 12 genes and 18 SNPs were associated with survival in patients with colon cancer, and 7 genes and 15 tagSNPs were associated with survival after a diagnosis of rectal cancer. A summary score based on "at-risk" genotypes revealed a hazard rate ratio of 5.10 (95% confidence interval, 2.56-10.15) for the group with the greatest number of "at-risk" genotypes; for rectal cancer, the hazard rate ratio was 6.03 (95% confidence interval, 2.83-12.75).. The current findings suggest that the presence of several higher risk alleles in the TGF-β signaling pathway increase the likelihood of dying after a diagnosis of colon or rectal cancer.

Topics: Aged; Colonic Neoplasms; Female; Genetic Variation; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Rectal Neoplasms; Signal Transduction; Transforming Growth Factor beta

To study the influence of tumor-associated macrophages (TAMs) on the biological function of SW620 cell.. Macrophage was induced into M2-type macrophage form with interleukin (IL)-4. CD68, macrophage mannose receptor (MMR), and inducible nitric oxide synthase (iNOS) were analyzed with Western blot. SW620 was co-cultured with TAMs in the Transwell. Cytokines including IL-10, IL-12, IL-23, and tramsforming growth factor-β (TGF-β) were detected with enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) in SW620 was analyzed with electrophoretic mobility shift assay (EMSA). The proliferation and apoptosis of SW620 cells after co-cultured with TAM were determined with tetrazolium four nitrogen (XTT) assay and fluorescence activated cell sorting (FACS), respectively. RESULTS IL-4 induced M2 type macrophage expressed CD68 and MMR instead of iNOS. After co-cultured with SW620 for 24 hours and 48 hours, M2 type macrophage secreted higher levels of IL-10 and TGF-β than the pre-culture level (P 0.05). The activity of NF-κB in SW620 decreased by 72% and 75% after 24 hours and 48 hours compared with the pre-culture level, respectively (both P<0.01). The activity of proliferation decreased by 48% and 59% and the apoptotic rates increased by 6.37% and 7.68% and 0.37% after 24 hours and 48 hours (all P<0.01) compared with the pre-culture levels.. TAM may inhibit the proliferation and promote the apoptosis of SW620 by suppressing the activity of NF-κB.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Colonic Neoplasms; Humans; Interleukins; Macrophages; NF-kappa B; Nitric Oxide Synthase Type II; Transforming Growth Factor beta

Regulatory T (Treg) cells (CD4+ CD25high FoxP3+) regulate mucosal tolerance; their adoptive transfer prevents or reduces symptoms of colitis in mouse models of inflammatory bowel disease. Colonic CD90+ mesenchymal myofibroblasts and fibroblasts (CMFs) are abundant, nonprofessional antigen-presenting cells in the normal human colonic mucosa that suppress proliferation of activated CD4+ effector T cells. We studied CMF suppressive capacity and evaluated the ability of CMF to induce Treg cells.. Allogeneic cocultures of CD4+ T cells and CMFs, derived from normal mucosa of patients undergoing colectomy for colon cancer or inflamed colonic tissues from patients with ulcerative colitis or Crohn's disease, were used to assess activation of the Treg cells.. Coculture of normal CMF with resting or naïve CD4+ T cells led to development of cells with a Treg phenotype; it also induced proliferation of a CD25+ CD127- FoxP3+ T cells, which expressed CTLA-4, interleukin-10, and transforming growth factor-β and had suppressive activities. In contrast to dendritic cells, normal CMFs required exogenous interleukin-2 to induce proliferation of naturally occurring Treg cells. Induction of Treg cells by normal CMFs required major histocompatibility complex class II and prostaglandin E2. CMFs from patients with inflammatory bowel diseases had reduced capacity to induce active Treg cells and increased capacity to transiently generate CD4+CD25+/- CD127+ T cells that express low levels of FoxP3.. CMFs suppress the immune response in normal colon tissue and might therefore help maintain colonic mucosal tolerance. Alterations in CMF-mediated induction of Treg cells might promote pathogenesis of inflammatory bowel diseases.

Topics: Cell Proliferation; Cells, Cultured; Coculture Techniques; Colon; Colonic Neoplasms; Dinoprostone; Forkhead Transcription Factors; Histocompatibility Antigens Class II; Humans; Immune Tolerance; Immunity, Mucosal; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Leukocyte Common Antigens; Lymphocyte Activation; Myofibroblasts; Paracrine Communication; Phenotype; Signal Transduction; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta

Microsatellite instability (MSI), which occurs in 15% of colorectal cancer, has been shown to have a lower incidence of metastasis and better patient survival rates compared with microsatellite stable colorectal cancer. However, a mechanistic understanding of the basis for this difference is very limited. Here, we show that restoration of TGFβ signaling by re-expression of TGFβ receptor II in MSI colon cancer cells increased PI3K/AKT activation, conferred resistance to growth factor deprivation stress-induced apoptosis, and promoted cell motility in vitro. Treatment with a potent PI3K inhibitor (LY294002) blocked the prosurvival and promotility effects of TGFβ, indicating that TGFβ-mediated promotion of cell survival and motility is dependent upon activation of the PI3K/AKT pathway. Analysis of apoptotic effectors that are affected by TGFβ signaling indicated that Bim is an effector of TGFβ-mediated survival. In addition, TGFβ-induced down-regulation of E-cadherin contributed to the prosurvival effect of TGFβ, and restoration of TGFβ signaling in MSI colon cancer cells increased liver metastasis in an orthotopic model in vivo. Taken together, our results demonstrate that restoration of TGFβ signaling promotes cell survival, motility, and metastatic progression in MSI colon cancer cells and indicate that TGFβ receptor II mutations contribute to the favorable outcomes in colon cancer patients with MSI.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Survival; Chromones; Colonic Neoplasms; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microsatellite Repeats; Morpholines; Neoplasm Metastasis; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.

Topics: Activins; Apoptosis; Blotting, Western; Butyrates; Calcium-Binding Proteins; Cell Nucleus; Cell Proliferation; Cells, Cultured; Chromatin Immunoprecipitation; Colonic Neoplasms; Connective Tissue Growth Factor; Humans; Immunoprecipitation; Intercellular Signaling Peptides and Proteins; Luciferases; Membrane Proteins; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Wnt Proteins

The transforming growth factor-β (TGF-β) signaling pathway progresses through a series of protein phosphorylation regulated steps. Smad4 is a key mediator of the classical TGF-β signaling pathway; however, reports suggest that TGF-β can activate other cellular pathways independent of Smad4. By investigating the TGF-β-regulated phosphoproteome, we aimed to uncover new functions controlled by TGF-β. We applied titanium dioxide to enrich phosphopeptides from stable isotope labeling with amino acids in cell culture (SILAC)-labeled SW480 cells stably expressing Smad4 and profiled them by mass spectrometry. TGF-β stimulation for 30 min resulted in the induction of 17 phosphopeptides and the repression of 8 from a total of 149 unique phosphopeptides. Proteins previously not known to be phosphorylated by TGF-β including programmed cell death protein 4, nuclear ubiquitous casein and cyclin-dependent kinases substrate, hepatoma-derived growth factor and cell division kinases amongst others were induced following TGF-β stimulation, while the phosphorylation of TRAF2 and NCK-interacting protein kinase are examples of proteins whose phosphorylation status was repressed. This phosphoproteomic screen has identified new TGF-β-modulated phosphorylation responses in colon carcinoma cells.

Topics: Amino Acid Sequence; Cell Line, Tumor; Colonic Neoplasms; Germinal Center Kinases; Humans; Isotope Labeling; Mass Spectrometry; Molecular Sequence Data; Peptide Fragments; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proteome; Proteomics; Signal Transduction; Smad4 Protein; Titanium; TNF Receptor-Associated Factor 2; Transforming Growth Factor beta

We examined the effects of adoptive T-cell transfer (ACT) on the population of regulatory T cells (Tregs) in a mouse colorectal cancer transplant model. In an in vivo study, Treg populations in Balb/c mice colon26 transplant model after ACT were analyzed in peripheral blood, local lymph node, and tumor. In an in vitro study CD4+ cells were cultured in medium containing TGF-beta to induce Tregs. LAK cells were added or not in this Treg induction system. Treg induction after coculture with LAK was investigated. We also studied the role of IFN-gamma in the mechanism of Treg induction. Tregs in the draining lymph nodes and tumor were significantly suppressed by ACT. The induction of Tregs in vitro was inhibited by coculture with LAK cells. Furthermore, Tregs in the cultured cells were significantly inhibited by addition of exogenous IFN-gamma. Moreover, Tregs were increased by addition of IFN-gamma mAb. ACT may decrease Tregs in tumor-bearing hosts. One of the mechanisms is considered to be IFN-gamma inhibiting the induction of Tregs.

Topics: Adenocarcinoma; Adoptive Transfer; Animals; Blotting, Western; CD4-Positive T-Lymphocytes; Colonic Neoplasms; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Forkhead Transcription Factors; Immunoenzyme Techniques; Immunotherapy; Interferon-gamma; Killer Cells, Lymphokine-Activated; Lymph Nodes; Male; Mice; Mice, Inbred BALB C; Real-Time Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Cells, Cultured

Though TGF-beta inhibition enhances antitumor immunity mediated by CD8(+) T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-beta blockade, we tested the effect of anti-TGF-beta combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-beta antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-beta antibodies compared to vaccinated mice without anti-TGF-beta, suggesting that anti-TGF-beta synergistically enhanced irradiated tumor vaccine efficacy. CD8(+) T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8(+) T cells. Depletion of CD25(+) T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-beta did not change the number of CD25(+) T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-beta production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-beta enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-beta levels found in patients with cancer and that the effect is not dependent on TGF-beta solely from CD4(+)CD25(+) T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.

Topics: Animals; Antibodies, Monoclonal; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Colonic Neoplasms; Female; Flow Cytometry; Interleukin-13; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Mice; Mice, Inbred BALB C; Mice, Knockout; Natural Killer T-Cells; Receptors, Interleukin-4; Signal Transduction; STAT6 Transcription Factor; Survival Rate; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vaccination

Inactivation of the tumour suppressor gene SMAD4 is a genetically late event in gastrointestinal carcinogenesis. SMAD4 is a transmitter of growth-inhibitory effects of transforming growth factor-beta (TGF-beta), an important tumour promoter capable of inducing an epithelial to mesenchymal transition (EMT). The role of SMAD proteins in late, tumour-promoting effects of TGF-beta is not well understood.. The change of molecular differentiation markers typical for EMT upon SMAD4 re-expression in SW480 cells was determined using Western blotting, immunohistochemistry and confocal laser microscopy. The influence of SMAD4 on the migration of SW480 cells was assessed in wound healing and pore migration assays.. SMAD4 suppresses invasiveness and mediates reversion of SW480 cells from a mesenchymal-like to a polarized epithelial phenotype, with features of enterocyte-like differentiation. Moreover, SMAD4 reconstitution was associated with down-regulation of endogenous TGF-beta cytokines, suggesting that autocrine TGF-beta signaling may be involved in the EMT.. These results provide further evidence for a role of SMAD4 as a regulator of invasion, a process of prime importance in carcinogenesis but hitherto poorly understood in molecular terms.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Epithelial Cells; Humans; Mesoderm; Mice; Neoplasm Invasiveness; Signal Transduction; Smad4 Protein; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Wound Healing

The role of transforming growth factor-beta (TGF-beta) in the development of hepatic metastasis from colon cancer is not clearly elucidated. The aim of this study was to determine the role of TGF-beta in the development of such metastasis.. Two human colon cancer cell lines were utilized: FET-alpha cells (intact TGF-beta inhibitory response), and CBS cells (defects in TGF-beta inhibitory response caused by a deficiency in type II receptor activity). The ability of these cell lines to metastasize was analysed in an orthotopic colon cancer mouse model.. FET-alpha cells did not metastasize to the liver, but showed lung metastasis in 10% of the animals, whereas CBS cells gave rise to metastasis in 65%. Following the elimination of TGF-beta activity by transfection and overexpression of dominant negative type II receptor, FET-alpha cells demonstrated liver and lung metastasis in 70% of the animals. Similarly, after the restoration of type II receptor activity by ectopic expression, CBS cells formed metastasis in fewer (10%) animals.. The results of our study demonstrate for the first time that TGF-beta displays selective metastasis suppressor activity. These abnormal pathways can serve as selective targets for future development of targeted therapies.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Burden

TGF-β promotes cell migration and invasion, an attribute that is linked to the pro-metastasis function of this cytokine in late stage cancers. The LIM 1863 colon carcinoma organoid undergoes epithelial-mesenchymal transition (EMT) in response to TGF-β. This process is markedly accelerated by TNF-α, and we found that the levels of miR-21 and miR-31 were prominently elevated under the synergistic actions of TGF-β/TNF-α. Consistent with this, overexpression of either miR-21 or miR-31 significantly enhanced the effect of TGF-β alone on LIM 1863 morphological changes. More importantly, transwell assays demonstrated the positive effects of both miR-21 and miR-31 in TGF-β regulation of LIM 1863 motility and invasiveness. Elevated levels of miR-21 and miR-31 also enhanced motility and invasiveness of other colon carcinoma cell lines. We present compelling evidence that TIAM1, a guanidine exchange factor of the Rac GTPase, is a direct target of both miR-21 and miR-31. Indeed in LIM 1863 cells, suppression of TIAM1 is required for miR-21/miR-31 to enhance cell migration and invasion. Therefore, we have uncovered miR-21 and miR-31 as downstream effectors of TGF-β in facilitating invasion and metastasis of colon carcinoma cells.

Topics: Blotting, Northern; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; MicroRNAs; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

TGF-β signalling can play a paradoxical cell type specific role in cancer progression. Smad4 is a key mediator of the TGF-β pathway, and is mutated and/or deleted in many cancers. To investigate Smad4 regulated TGF-β signalling in colon cancer we conducted an iTRAQ mass spectrometry quantitative screen using wild type SW480 (Smad4 negative) colon carcinoma cells and stably restored Smad4 positive SW480 cells. In cells possessing a restored canonical TGF-β signalling pathway, 48 h TGF-β stimulation induced the expression of 15 proteins and repressed 1 protein, while in Smad4 wild type cells, TGF-β induced 7 proteins and repressed 2 proteins. The expression of several S100 protein family members (A2, A4, A10, and A11), transgelin-2 and AKAP12, amongst others, were shown to be regulated by TGF-β in a Smad4 dependent manner. We observed that S100 A4 could be repressed by TGF-β, independently of Smad4 expression, while other Smad4 independent TGF-β responses were restricted to induction of ribosomes and cytoskeletal proteins. Our proteomic screen has identified new Smad4 dependent and independent TGF-β responses in colon carcinoma cells.

Topics: Cell Line, Tumor; Colonic Neoplasms; Humans; Immunoblotting; Isotope Labeling; Mass Spectrometry; Models, Biological; Neoplasm Proteins; Proteomics; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-β (TGFβ) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFβ receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFβ-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HG(low) cell lines (e.g., glioblastoma) results in impaired gene activation by TGFβ. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFβ signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

Topics: Angiogenesis Inhibitors; Animals; Base Sequence; Clusterin; Colonic Neoplasms; Down-Regulation; Genes, Reporter; Humans; Luciferases; Mice; Mice, Inbred C57BL; MicroRNAs; Molecular Sequence Data; Mutation; Proto-Oncogene Proteins c-myc; Receptors, Transforming Growth Factor beta; Ribonuclease III; Transforming Growth Factor beta; Untranslated Regions

Expression of matrix metalloproteinase 7 (MMP7) is increased in the human colorectal carcinomas, and correlates with malignant progression. However, its contribution to colon cancer pathogenesis is not understood thoroughly. To investigate the roles of MMP7 in colon cancer progression, we introduced an Mmp7 knockout mutation into the cis-Apc/Smad4 mutant mouse, a model of invasive colon cancer in which SMAD4-dependent TGF-beta family signaling is inactivated. We demonstrate here that lack of MMP7 reduces the number and size of tumors in the cis-Apc/Smad4 mice. On the other hand, MMP7-deficiency does not affect the depth of tumor invasion, number of stromal fibroblasts or levels of extracellular matrix components in the tumors. These results indicate that MMP7 is required for tumor formation, but not for the invasion or fibrosis of the colon cancer whose SMAD4-dependent TGF-beta family signaling is blocked.

Topics: Adenocarcinoma; Animals; Cell Count; Collagen Type I; Colonic Neoplasms; Disease Progression; Female; Fibroblasts; Fibrosis; Genes, APC; Male; Matrix Metalloproteinase 7; Mice; Mice, Knockout; Neoplasm Invasiveness; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) are IMiDs proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-alpha is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed. These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependent adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-beta or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Colonic Neoplasms; Female; Forkhead Transcription Factors; Glucocorticoid-Induced TNFR-Related Protein; Humans; Immunosuppressive Agents; Interleukin-10; Lenalidomide; Mice; Mice, Inbred BALB C; Receptors, Nerve Growth Factor; Receptors, OX40; Receptors, Transforming Growth Factor beta; Receptors, Tumor Necrosis Factor; T-Lymphocytes, Regulatory; Thalidomide; Transforming Growth Factor beta

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

Topics: Adenocarcinoma; Animals; Blotting, Western; Colonic Neoplasms; Disease Models, Animal; DNA-Binding Proteins; Flow Cytometry; Helicobacter Infections; Immunohistochemistry; Inflammation; Mice; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; Smad3 Protein; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Despite a primary tumor suppressor role, there is compelling evidence suggesting that TGF-beta can promote tumor growth, invasion and metastasis in advanced stages of colorectal cancer. Blocking these tumor-promoting effects of TGF-beta provides a potentially important therapeutic strategy for the treatment of colorectal cancer. However, little is known about how the inhibitors of TGF-beta receptor kinases affect colorectal carcinogenesis in vivo. Here, we have observed that a novel dual kinase inhibitor of TGF-beta type I and type II receptors, LY2109761, inhibits TGF-beta-mediated activation of Smad and non-Smad pathways in CT26 colon adenocarcinoma cells having K-Ras mutation. The inhibitor attenuates the oncogenic effects of TGF-beta on cell migration, invasion and tumorigenicity of CT26 cells. Furthermore, LY2109761 decreases liver metastases and prolongs survival in an experimental metastasis model. These findings suggest that the dual kinase inhibitor LY2109761 has potential therapeutic value for metastatic colorectal cancer.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Genes, ras; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mutation; Neoplasm Invasiveness; Pyrazoles; Pyrroles; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

TGF-beta is a pluripotent cytokine that mediates its effects through a receptor composed of TGF-beta receptor type II (TGFBR2) and type I (TGFBR1). The TGF-beta receptor can regulate Smad and nonSmad signaling pathways, which then ultimately dictate TGF-beta's biological effects. We postulated that control of the level of TGFBR2 is a mechanism for regulating the specificity of TGF-beta signaling pathway activation and TGF-beta's biological effects. We used a precisely regulatable TGFBR2 expression system to assess the effects of TGFBR2 expression levels on signaling and TGF-beta mediated apoptosis. We found Smad signaling and MAPK-ERK signaling activation levels correlate directly with TGFBR2 expression levels. Furthermore, p21 levels and TGF-beta induced apoptosis appear to depend on relatively high TGFBR2 expression and on the activation of the MAPK-ERK and Smad pathways. Thus, control of TGFBR2 expression and the differential activation of TGF-beta signaling pathways appears to be a mechanism for regulating the specificity of the biological effects of TGF-beta.

Topics: Apoptosis; Blotting, Western; Cells, Cultured; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Humans; Luciferases; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Colorectal cancer is the second leading cause of cancer related mortality, with a majority of deaths resulting from metastases. Few in vivo models allow for the study of the complex process of metastasis. The purpose of this study was to determine the effects of epidermal growth factor receptor activation and TGFbeta pathway attenuation in FET, a weakly tumorigenic human colon cancer cell line, in an orthotopic model.. Using FET, FETalpha, FETalphaDNRII, and FETDNRII cells were constructed. Tumors were orthotopically implanted onto the colons of BALB/c nude mice. After 7 wk, the mice were euthanized and organs extracted for examination. All cell lines demonstrated primary invasion. FETalpha was weakly metastatic compared with FETalphaDNRII and FETDNRII, which demonstrated metastases to the lung and liver, respectively.. Epidermal growth factor receptor (EGFR) activation transforms a nontumorigenic cell line into a tumorigenic but not metastatic one. The tumorigenic line becomes metastatic with the attenuation of TGFbeta signaling. Loss of EGFR activation in the TGFbeta inhibited line results in a decreased metastatic burden, but importantly, changes the organotropic homing from lung to liver. Thus, these in vivo studies demonstrate that EGFR activation and TGFbeta signaling pathways play a role in tumorigenicity and in pattern of metastases.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; ErbB Receptors; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Genes involved in the TGF-beta signaling pathway are often altered in several types of cancers. The TGF-beta-resistant human colon cancer cell line HT-29 has inactivated TbetaRII and deficient expression of RUNX3 and Smad4, which are involved in the TGF-beta signaling pathway.. Western blot and immunocytochemistry were performed to confirm gene expression, the MTT assay to detect cell growth, flow cytometry to investigate the cell cycle and the TUNEL to detect cell apoptosis.. In the absence of TGF-beta, Bim was upregulated, cell growth was inhibited and apoptosis was induced. TGF-beta treatment did not affect RUNX3 expression; however, the increase in Bim expression was significant and time dependent. Interestingly, Smad4 but not Smad2/3 was also upregulated upon exposure to TGF-beta. This was not the case after TGF-beta treatment of parent HT-29 cells. As expected, TGF-beta further inhibited cell growth and induced apoptosis in HT-29/RUNX3+ cells.. Our data demonstrate that RUNX3 is involved in TGF-beta-dependent and -independent cell growth inhibition and apoptosis induction pathways.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Core Binding Factor Alpha 3 Subunit; Gene Expression; Humans; Membrane Proteins; Proto-Oncogene Proteins; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

To investigate the regulation of Toll-like receptors (TLRs) on immune suppressive cytokines in situ colon cancer cells.. The mRNA and protein expression spectrum of TLRs in HT-29 cells were determined by RT-PCR and Western blot respectively. The cytokines and chemokines levels of supernant of HT-29 stimulated by lipoplysaccharide(LPS) were detected with ELISA.. TLR1-9 were expressed in HT-29 cells on mRNA level. After LPS stimulation, TLR4 mRNA and protein expressions were up-regulated in HT-29 cells, and TGF-beta, VEGF, IL-8, CCL20 and IL-6 levels increased significantly(all P<0.01). Except IL-6, up-regulation of the other cytokines was not suppressed by NF-kappa B inhibitor.. TLRs expressed on colon cancer cells may elevate the immune suppressive cytokines and chemokines, which promote the immune escape of cancer cells.

Topics: Chemokine CCL20; Colonic Neoplasms; Cytokines; HT29 Cells; Humans; Interleukin-6; NF-kappa B; RNA, Messenger; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta; Up-Regulation

Aminoacyl-transfer ribonucleic acid (tRNA) synthetases-interacting multifunctional protein (AIMP) 2 is a factor associated with the macromolecular protein synthesis machinery consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors. However, it was shown to work as a multifaceted regulator through the versatile interactions with diverse signal mediators. For instance, it can mediate pro-apoptotic response to DNA damage and tumor necrosis factor-alpha (TNF-alpha) stimulus and growth-arresting signal by transforming growth factor (TGF)-beta. Considering that these pathways are critically implicated in the control of tumorigenesis, AIMP2 is expected to work as a potent tumor suppressor with broad coverage against different cancer types. Here we investigated whether AIMP2 would give gene dosage effect on its pro-apoptotic and anti-proliferative activities using the wild-type, hetero- and homozygous AIMP2 cells and whether AIMP2 would be critical in preventing tumorigenesis using different in vivo tumor models. Both the apoptotic responses to DNA damage and TNF-alpha and sensitivity to growth arresting TGF-beta signal were reduced in AIMP2 hetero- and homozygous cells compared with the wild-type cells in dose-dependent manner. In all the in vivo carcinogenesis experiments, reduction of AIMP2 level in heterozygous AIMP2 mice provided higher susceptibility to tumor formation. Thus, this work proves the functional significance of AIMP2 in determination of cell proliferation and death, and as a haploinsufficient tumor suppressor.

Topics: Amino Acyl-tRNA Synthetases; Animals; Apoptosis; Cells, Cultured; Colonic Neoplasms; Disease Susceptibility; DNA Damage; Heterozygote; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasms; NF-kappa B; Papilloma; Skin Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins

More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. When the dynamics of phosphorylated ERK response to TNFalpha were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-alpha. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor-alpha autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNFalpha treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence.

Topics: Apoptosis; Autocrine Communication; Blotting, Western; Chemokines; Colonic Neoplasms; Computational Biology; Dual-Specificity Phosphatases; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Flow Cytometry; Fluorescent Antibody Technique; Genes, ras; Humans; Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Functional expression of KAL1 gene is critical in the migration of GnRH neurons from the olfactory placode to the hypothalamus in embryogenesis. This gene thus far has not been shown to play a functional role in any other physiological or pathological process either in the developed brain or in peripheral tissues. We show here that KAL1 gene expression is decreased in early stage and increased in later stages of cancers. Screening of colon, lung and ovarian cancer cDNA panels indicated significant decrease in KAL1 expression in comparison to corresponding uninvolved tissues. However, KAL1 expression increased with the progression of cancer from early (I and II) stages to later (III and IV) stages of the cancer. There was a direct correlation between the TGFbeta and KAL1 expression in colon cancer cDNA. Using colon cancer cell lines, we showed that TGFbeta induces KAL1 gene expression and secretion of anosmin-1 protein (KAL1 coded protein). We further report that hypoxia induces anosmin-1 expression; anosmin-1 protects cancer cells from apoptosis activated by hypoxia and increases cancer cell mobility. Using siRNA technique we found that KAL1 expression following hypoxia is hypoxia-inducible factor (HIF-1)alpha dependent. Our results suggest that KAL1 gene expression plays an important role in cancer metastasis and protection from apoptosis.

Topics: Base Sequence; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Disease Progression; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Sequence Data; Nerve Tissue Proteins; Neurons; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sequence Analysis, DNA; Transforming Growth Factor beta

Signaling pathways enabling transforming growth factor-beta (TGFbeta)'s conversion from a tumor suppressor to a tumor promoter are not well characterized. TGFbeta utilizes intracellular SMADs to mediate growth suppression; however, TGFbeta-induced proliferative pathways may become more apparent when SMAD signaling is abrogated. Here, we determined regulation of the tumor suppressor PTEN by TGFbeta utilizing SMAD4-null colon cancer cells. TGFbeta downregulated PTEN mRNA and simultaneously induced growth proliferation. TGFbeta also induced both SMAD2 and SMAD3 nuclear translocation, but only triggered SMAD2-specific transcriptional activity in the absence of SMAD4. Interference of SMAD2 with DN-SMAD2 enhanced TGFbeta-induced cell proliferation, but downregulation of PTEN expression by TGFbeta was unaffected. TGFbeta increased PI3K tyrosine phosphorylation, and inhibition of PI3K pharmacologically or by DN-p85 transfection reversed both TGFbeta-induced PTEN suppression and TGFbeta-induced cell proliferation. Thus, TGFbeta activates PI3K to downregulate PTEN for enhancement of cell proliferation that is independent of SMAD proteins.

Topics: Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Enzyme Activation; Gene Expression Regulation, Neoplastic; Genes, Dominant; Humans; Phosphatidylinositol 3-Kinases; PTEN Phosphohydrolase; RNA; RNA, Messenger; Signal Transduction; Smad1 Protein; Smad4 Protein; Transforming Growth Factor beta

TGFbeta exerts a potent tumor-suppressive effect in the human colon carcinoma CBS and Moser cells. However, TGFbeta can also function as a tumor promoter. The mechanisms underlying the tumor promoting effect of TGFbeta are not understood. Both the CBS and Moser cells were found to express mutant (truncated) APC. Expression of this form of APC did not interfere with the tumor-suppressive function of TGFbeta. However, when APC expression was knocked down in these cells, TGFbeta function switched from that of tumor suppression to that of tumor promotion. TGFbeta stimulated cellular invasion and anchorage-independent growth in APC knocked-down cells. Knocking down APC expression abrogated the ability of TGFbeta to induce the expression of the tumor suppressor E-cadherin and the cyclin dependent kinase inhibitor p21/Waf1. TGFbeta now stimulated the constitutive TCF transcriptional activation activity associated with the beta-catenin/Wnt pathway in the APC knocked-down cells. Thus, the level of APC expression determined the type of TGFbeta function in these human colon carcinoma cells.

Topics: Base Sequence; Cell Line, Tumor; Colonic Neoplasms; Gene Knockdown Techniques; Genes, APC; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transcriptional Activation; Transforming Growth Factor beta

In the majority of human tumors, expression of the c-MYC oncogene becomes constitutive. Here, we report that c-MYC directly regulates the expression of AP4 via CACGTG motifs in the first intron of the AP4 gene. Induction of AP4 was required for c-MYC-mediated cell cycle reentry of anti-estrogen arrested breast cancer cells and mitogen-mediated repression of the CDK inhibitor p21. AP4 directly repressed p21 by occupying four CAGCTG motifs in the p21 promoter via its basic region. AP4 levels declined after DNA damage, and ectopic AP4 interfered with p53-mediated cell cycle arrest and sensitized cells to apoptosis induced by DNA damaging agents. AP4 expression blocked induction of p21 by TGF-beta in human keratinocytes and interfered with up-regulation of p21 and cell cycle arrest during monoblast differentiation. Notably, AP4 is specifically expressed in colonic progenitor and colorectal carcinoma cells. In conclusion, our results indicate that c-MYC employs AP4 to maintain cells in a proliferative, progenitor-like state.

Topics: Base Sequence; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Repressor Proteins; Transcription Factors; Transforming Growth Factor beta

The mutational inactivation of transforming growth factor beta receptor type II (TGFBR2) occurs in approximately 30% of colon cancers and promotes the formation of colon cancer by inhibiting the tumor suppressor activity of the TGFB signaling pathway. TGFBR2 mutations occur in >90% of microsatellite unstable (MSI) colon cancers and affect a polyadenine tract in exon 3 of TGFBR2, called BAT-RII, which is vulnerable to mutation in the setting of DNA mismatch repair (MMR) system deficiency. In light of the vulnerable nature of the BAT-RII tract in the setting of MMR inactivation and the favorable effects of TGFBR2 inactivation in colon cancer, analysis of TGFBR2 inactivation provides an opportunity to assess the roles of genomic instability vs. clonal selection in cells acquiring TGFBR2 BAT-RII tract mutations in MSI colon cancer formation. The contribution of genomic instability and/or clonal evolution to the mutational inactivation of TGBFR2 in MSI colon cancers has not been studied in a systematic way that would allow a determination of the relative contribution of these two mechanisms in the formation of MSI colon cancer. It has not been demonstrated whether the BAT-RII tract mutations are strictly a consequence of the BAT-RII region being hypermutable in the setting of MMR deficiency or if the mutations are rather a consequence of clonal selection pressure against the TGFB receptor. Through the use of defined cell line systems, we show that both genomic instability and clonal selection of TGFB resistant cells contribute to the high frequency of TGFBR2 mutations in MSI colon cancer.

Topics: Amino Acid Substitution; Cell Line; Cell Line, Tumor; Colonic Neoplasms; DNA Mutational Analysis; Drug Resistance, Neoplasm; Gene Frequency; Gene Silencing; Growth Inhibitors; HCT116 Cells; Humans; Microsatellite Instability; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Metastasis, the major cause of cancer death, is a multistep process that requires interactions between cancer cells and stromal cells and between cancer cells and extracellular matrix. Molecular alterations of the extracellular matrix in the tumor microenvironment have a considerable impact on the metastatic process during tumorigenesis. Here we report that elevated expression of betaig-h3/TGFBI (transforming growth factor, beta-induced), an extracellular matrix protein secreted by colon cancer cells, is associated with high-grade human colon cancers. Ectopic expression of the betaig-h3 protein enhanced the aggressiveness and altered the metastatic properties of colon cancer cells in vivo. Inhibition of betaig-h3 expression dramatically reduced metastasis. Mechanistically, betaig-h3 appears to promote extravasation, a critical step in the metastatic dissemination of cancer cells, by inducing the dissociation of VE-cadherin junctions between endothelial cells via activation of the integrin alphavbeta5-Src signaling pathway. Thus, cancers associated with overexpression of betaig-h3 may have an increased metastatic potential, leading to poor prognosis in cancer patients.

Topics: Amino Acid Sequence; Animals; Antigens, CD; Cadherins; Cell Line, Tumor; Colonic Neoplasms; Endothelial Cells; Endothelium, Vascular; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Integrins; Intercellular Junctions; Lung Neoplasms; Mice; Mice, SCID; Molecular Sequence Data; Neoplasm Invasiveness; Neovascularization, Pathologic; Oligopeptides; Receptors, Vitronectin; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta) suppresses the malignant phenotype of human colon carcinoma cells through diverse cellular pathways. Activation of beta-catenin/Wnt signal pathway underlies the malignant phenotype of human colon carcinomas. The Smad family of signal transducing, sequence-specific transcription activators are mediators of TGFbeta signaling. In this report, we showed that TGFbeta suppressed the beta-catenin/Wnt signal pathway in human colon carcinoma cells and stimulated an adhesion response in these cells in a Smad4/DPC4 independent manner. Smad/DCP4, however, was found to be linked to the growth-inhibitory action of TGFbeta.

Topics: beta Catenin; Cell Adhesion; Cell Line, Tumor; Colonic Neoplasms; Humans; Microscopy, Confocal; Signal Transduction; Smad4 Protein; Transfection; Transforming Growth Factor beta; Wnt Proteins

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.

Topics: Actin Cytoskeleton; Actins; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cells, Cultured; Coculture Techniques; Collagen Type I; Colonic Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Humans; Neoplasm Invasiveness; rho GTP-Binding Proteins; Transforming Growth Factor beta

FET cells, derived from an early-stage colon carcinoma, are nontumorigenic in athymic mice. Stable transfection of a dominant-negative transforming growth factor beta (TGFbeta) type II receptor (DNRII) into FET cells that express autocrine TGFbeta shows loss of TGFbeta signaling and increased tumorigenicity in vivo indicating tumor suppressor activity of TGFbeta signaling in this model. The ability of tumorigenic cells to withstand growth factor and nutrient deprivation stress (GFDS) is widely regarded as a key attribute for tumor formation and progression. We hypothesized that increased tumorigenicity of FET/DNRII cells was due to loss of participation of autocrine TGFbeta in a "fail-safe" mechanism to generate cell death in response to this stress. Here, we document that loss of autocrine TGFbeta in FET/DNRII cells resulted in greater endogenous cell survival in response to GFDS due to activation of the phosphoinositide 3-kinase (PI3K)/Akt/survivin pathway. Treatment of FET DNRII cells with a PI3K inhibitor (LY294002) inhibited Akt phosphorylation and reduced survivin expression resulting in increased apoptosis in FET/DNRII cells. We also show that exogenous TGFbeta increased apoptosis in FET cells through repression of the PI3K/Akt/survivin pathway during GFDS. These results indicate that the PI3K/Akt/survivin pathway is blocked by TGFbeta signaling and that loss of autocrine TGFbeta leads to increased cell survival during GFDS through the novel linkage of TGFbeta-mediated repression of survivin expression. Inhibition of survivin function by dominant-negative approaches showed that this inhibitor of apoptosis family member is critical to cell survival in the FET/DNRII cells, thus indicating the importance of this target for TGFbeta-mediated apoptosis.

Topics: Apoptosis; Carcinoma; Caspases; Cell Survival; Colonic Neoplasms; Culture Media, Serum-Free; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Survivin; Transforming Growth Factor beta; Tumor Cells, Cultured

Clinical evidence suggests that estradiol replacement therapy reduces colon cancer risk in 'post'menopausal women. In colon epithelial cells, the estrogen receptor beta (ERbeta) is the predominant ER subtype and is thought to mediate the genomic effect of estrogens. The first aim of this study was to investigate the consequence of ERbeta deficiency on intestinal tumorigenesis in the Apc(Min/+) mouse model. Furthermore, to explore the biological mechanisms by which estrogens may influence the pathogenesis of colorectal cancer, we performed gene expression profiles in colonocytes from ovariectomized wild-type (WT) vs. ERbeta(-/-) mice, treated with estradiol (E(2)) or vehicle. Specifically in female, ERbeta deficiency was found to be associated with higher adenoma multiplicity in the small intestine, but not in the colon. Furthermore, tumors from ERbeta(-/-)Apc(Min/+) female mice were on average significantly larger than those from control Apc(Min/+) mice. Higher steady-state proliferation in epithelial cells of the jejunum and colon from ERbeta(-/-)Apc(Min/+) vs. Apc(Min/+) female mice was confirmed by BrdU incorporation assay. Interestingly, functional categorization of microarray results revealed the TGFbeta signaling pathway to be modulated in colonocytes, especially for the WT + E(2) vs. WT + Vehicle and the ERbeta(-/-) + E(2) vs. WT + E(2) comparisons. Using quantitative PCR analysis, we observed transcripts from ligands of the TGFbeta pathway to be upregulated in colonocytes from E(2)-treated WT and ERbeta(-/-) mice and downregulated in ERbeta-deficient mice, mostly in an E(2)-independent manner. Therefore, our results demonstrate that ERbeta deficiency enhances small intestinal tumorigenesis and suggest that modulation of the TGFbeta signaling pathway could contribute to the protective role of estrogens on intestinal tumorigenesis.

Topics: Adenoma; Animals; Bromodeoxyuridine; Colonic Neoplasms; Disease Models, Animal; Estrogen Receptor beta; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Intestinal Neoplasms; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Oligonucleotide Array Sequence Analysis; Ovariectomy; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

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

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

The extracellular matrix and the interactive signalling between its components are thought to play a pivotal role for tumour development and metastasis formation. An altered matrix composition as potential underlying pathology for the development of colorectal cancer was hypothesized.. In a retrospective study of patients with colon cancer, the extracellular matrix in tumour-free bowel specimen was investigated in comparison with non-infected bowel specimen from patients operated on for colonic diverticulosis. The following matrix parameters with known associations to tumour formation, cell proliferation, invasion and metastasis were analysed by immunohistochemistry and quantified by a scoring system: VEGF, TGF-beta, ESDN, CD117, c-erb-2, cyclin D1, p53, p27, COX-2, YB-1, collagen I/III, MMP-13, PAI and uPAR. Expression profiles and correlations were calculated.. The comparison of the two groups revealed a significantly decreased immunostaining for CD117 and TGF-beta in the cancer group (8.5+/-2.6 vs 10.3+/-2,1 and 4.9+/-1.5 vs 8.1+/-3, respectively), whereas PAI scores were significantly higher than in patients with diverticular disease (8.1+/-1.6 vs 6.2+/-0.9). Overall correlation patterns of matrix parameters indicated pronounced differences between tumour-free tissue in cancer patients compared with patients with diverticular disease.. Our results indicate distinct differences in the colonic tissue architecture between cancer patients and patients with diverticulitis that support the notion of an altered matrix composition predisposing to the development of colon cancer.

Topics: Collagen Type I; Collagen Type III; Colon; Colon, Sigmoid; Colonic Neoplasms; Cyclin D1; Cyclooxygenase 2; Diverticulosis, Colonic; DNA-Binding Proteins; Extracellular Matrix; Female; Humans; Male; Matrix Metalloproteinase 13; Membrane Proteins; Middle Aged; Nuclear Proteins; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins c-kit; Receptor, ErbB-2; Retrospective Studies; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Y-Box-Binding Protein 1

Bone morphogenetic proteins (BMPs) regulate cell differentiation, proliferation, and apoptosis through a canonical SMAD signaling cascade. Absence of BMP signaling causes the formation of intestinal juvenile polyps in the colon cancer-prone syndrome familial juvenile polyposis. As sporadic colon cancers appear to have intact BMP signaling, we evaluated if K-RAS, driving a mitogenic pathway frequently activated in colon cancer, negatively affects BMP growth suppression. We treated non-tumorigenic but activated RAS/ERK FET cells with BMP2, and in combination with pharmacological or genetic inhibition of RAS/ERK, examined BMP-SMAD signaling, transcriptional activity, and cell growth, and also assessed p21(WAF1) mRNA, transcriptional activation, and protein levels. BMP2 increased nuclear phospho-SMAD1 2-fold, which increased another 2-3 fold when RAS/ERK was inhibited. BMP2 increased BMP-specific SMAD transcriptional activity 2-fold over control and decreased cell growth, but inhibition of RAS/ERK further enhanced BMP-specific transcriptional activity by an additional 1.5-2 fold and enhanced growth suppression by 20%. BMP-induced growth suppression is mediated in part by p21(WAF1), not by transcriptional upregulation but by improved p21 protein stability, which is inhibited by RAS/ERK. In colon cancer cells, BMP-SMAD signaling and growth suppression is facilitated by p21(WAF1) but modulated by oncogenic K-RAS to reduce the growth suppression directed by this pathway.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Nucleus; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Protein Kinase Inhibitors; PTEN Phosphohydrolase; ras Proteins; RNA, Messenger; Signal Transduction; Smad Proteins; Thermodynamics; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Recent data indicate that the Bone Morphogenetic Protein BMP-7 exhibits mucosal protection against experimental colitis in rats, suggesting that this cytokine exerts direct actions in intestinal epithelial cells during inflammatory bowel diseases and other precancerous lesions of the colon. In this study, we investigated the functional expression of BMP-7 and its receptors in normal human colon crypts, aberrant crypt foci (ACF) in sigmoiditis and colorectal tumors, and their derived cancer cell lines. Transcripts encoding BMP-7 receptors type II (BMPRII, ActRII, ActRIIB) and type I (ALK-2) were clearly detected by RT-PCR in several premalignant and carcinoma cell lines. The cytokine was identified by immunohistochemistry in surface epithelial cells and crypts in the normal colon mucosa, ACF in sigmoiditis, sporadic high grade dysplastic adenoma, and in 9 of 16 colon carcinomas (56.2%). In addition, the conditioned medium collected from the adenoma PC/AA/C1 and carcinoma HCT8/S11 and SW48 cell lines in culture contained significant levels of BMP-7 ranging from 0.17 to 0.38 ng/ml. We found that BMP-7 induced scattering and proinvasive responses (EC50=1 ng/ml) in kidney and colon cancer cell lines through SMAD4 and src -independent pathways and signaling cascades using FAK phosphorylation at Y925 and activation of ERK1/2, Rac1 and JNK. This phosphorylation of FAK on Y925 was also induced by the proinvasive agent EGF. Taken together, our findings suggest that BMP-7 exerts divergent effects in the colon mucosa, one counteracting transient inflammatory situations and the other linked to pejorative functions during chronic ulcerative diseases and the neoplastic progression.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Line, Tumor; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; HT29 Cells; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; rac GTP-Binding Proteins; Retrospective Studies; Smad4 Protein; Transforming Growth Factor beta

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; PTEN Phosphohydrolase; ras Proteins; Smad4 Protein; Transcription, Genetic; Transforming Growth Factor beta

Approximately 15% of human colon cancers have microsatellite instability (MSI) and carry frameshift mutations in a polyadenine tract (BAT-RII) in the type II transforming growth factor beta (TGF-beta) receptor (TGFBR2), a required component of the TGF-beta receptor. The BAT-RII mutations in MSI colon cancers make the tumors resistant to the effects of TGF-beta. In cultured epithelial cells, TGF-beta can inhibit cell proliferation and induce apoptosis, and in vitro it can regulate the expression of a variety of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors. These effects are context- and tissue type-dependent, raising questions about which of these in vitro effects of TGF-beta signaling inactivation contribute to the formation of primary colon cancer. Thus, this study sought to determine the pathogenetically relevant effects of TGFBR2 inactivation in primary MSI colon cancers with mutant BAT-RII. Colon cancers with mutant BAT-RII were found to have increased proliferation compared to cancers with wild-type BAT-RII. Assessment of cdk4, cyclin D1 and p27(kip1) expression revealed that only cdk4 expression was increased in the cancers with mutant BAT-RII. In order to determine if TGFBR2 inactivation was the cause of these changes, TGFBR2 was reconstituted in an MSI colon cancer cell line, resulting in decreased proliferation and decreased cdk4 expression and kinase activity. These results suggest that TGFBR2 mutations in primary colon cancers may be responsible for the increased proliferation and cdk4 expression in these tumors and provide evidence that deregulation of cdk4 is a pathogenic in vivo consequence of TGFBR2 inactivation in primary colon cancer.

Topics: Adult; Aged; Aged, 80 and over; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Humans; Intracellular Signaling Peptides and Proteins; Microsatellite Repeats; Middle Aged; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retrospective Studies; Transforming Growth Factor beta; Tumor Cells, Cultured

Nuclear factor kappa B (NF-kappaB) has been implicated in cancer cell survival. We explored the role of the TGF-beta pathway in the regulation of NF-kappaB in colon cancer cells. TGF-beta-1 treatment of the colon adenocarcinoma cell line FET-1, results in an early increase in IkappaB-alpha phosphorylation that precedes NF-kappaB nuclear translocation and DNA binding activity. Activation of the TGF-beta type I receptor is required for the TGF-beta-mediated activation of NF-kappaB. No activation of NF-kappaB is observed in a Smad4 null cell line, SW480, even though TGF-beta does result in IkappaB-alpha phosphorylation in these cells. Smad4 restores the TGF-beta-1-mediated NF-kappaB activation in SW480 cells. TGF-beta-1 treatment fails to activate NF-kappaB or phosphorylate IkappaB-alpha in FET-1 cells expressing the inhibitory Smad, Smad7. Taken together, these results suggest a role for Smad4 in the transcriptional activation of NF-kappaB, and a direct effect of Smad 7 inhibiting IkappaB-alpha phosphorylation rather than through the well-established inhibition of Smad2/3 phosphorylation with subsequent inhibition of the TGF-beta pathway.

Topics: Active Transport, Cell Nucleus; Activin Receptors, Type I; Animals; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; DNA; Enzyme Activation; Epithelial Cells; Humans; I-kappa B Kinase; NF-kappa B; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Smad7 Protein; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1

Recently, it was reported that exogenous bone morphogenetic protein (BMP)-2 acted as an antiproliferative agent in a variety of cell lines, including normal and cancerous gastric cell lines, indicating that BMP-2 plays an important role during cell growth. However, despite the loss of BMP-2 expression in several cancers, the underlying mechanism remains unknown. Epigenetic silencing through DNA methylation is one of the key steps during carcinogenesis. In this study, we found, through analysis by the methylation-specific polymerase chain reaction technique, CpG island methylation of the BMP-2 promoter region in gastric and colon cancer cell lines. BMP-2 mRNA was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. Moreover, 24 of the 56 (42.9%) gastric cancer tissues exhibited promoter methylation. Immunohistochemical staining revealed that 18 of the 24 (75%) gastric cancer tissues without methylation signals exhibited BMP-2 expression, whereas among 20 cancer tissues with strong methylation signals only four (20%) expressed BMP-2 (P = 0.0003). These findings indicate that BMP-2 methylation is strongly associated with the loss of BMP-2 protein expression in the primary gastric carcinomas. BMP-2 methylation was more often observed in diffuse type (60.7%) than in intestinal type (25%) gastric carcinomas (P = 0.007). Thus, aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be related to gastric carcinogenesis, particularly in the diffuse type.

Topics: Aged; Base Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Colonic Neoplasms; CpG Islands; Diet; DNA Methylation; Epigenesis, Genetic; Female; Gene Silencing; Humans; Immunoenzyme Techniques; Male; Middle Aged; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-beta (TGF-beta) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-beta signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3(-/-) mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2-positive cells, CD4(+) T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IFN-gamma, and tumor necrosis factor-alpha, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-beta signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans.

Topics: Animals; Cecum; Cell Proliferation; Colonic Neoplasms; Cytokines; DNA, Bacterial; Female; Genetic Predisposition to Disease; Helicobacter Infections; Inflammation; Male; Mice; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc; Risk Factors; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

In hematopoietic cells the transforming potential of the ecotropic viral integration site 1 (Evi1) oncogene is thought to be dependent upon the ability to inhibit TGFbeta signaling. Although Evi1 has recently been implicated in certain epithelial cancers, the effects of Evi1 on transformation and TGFbeta signaling in epithelial cells are not completely understood. Herein, we have determined the effects of Evi1 on TGFbeta signaling in intestinal epithelial cells. Stable expression of Evi1 in non-transformed intestinal epithelial cells inhibited induction of some Smad3-dependent TGFbeta target genes, such as PAI1. However, TGFbeta-mediated induction of cellular adhesion signaling components such as integrin1 and paxillin was not inhibited by Evi1; nor did Evi1 inhibit TGFbeta-mediated epithelial to mesenchymal transition. Likewise, Evi1 did not inhibit TGFbeta-mediated downregulation of cyclin D1 or block TGFbeta-mediated growth inhibition. However, Evi1 did inhibit TGFbeta-mediated apoptosis by a process that involves phosphoinositide-3-kinase (PI3K) and its downstream effector AKT. The ability of Evi1 to suppress apoptosis is not restricted to TGFbeta-mediated cell death, since Evi1 also protects intestinal epithelial cells from taxol-mediated apoptosis. Evi1 is overexpressed in some human colon cancer cell lines, and overexpression is associated with amplification of the Evi1 gene. Knockdown of Evi1 by siRNA inhibited AKT phosphorylation in HT-29 human colon cancer cells and increased their sensitivity to taxol-mediated apoptosis. These data indicate that Evi1 functions as a survival gene in intestinal epithelial cells and colon cancer cells, activating PI3K/AKT and conveying resistance to both physiological and therapeutic apoptotic stimuli.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; DNA-Binding Proteins; Drug Resistance, Neoplasm; Humans; Intestinal Mucosa; Oncogene Protein v-akt; Paclitaxel; Phosphatidylinositol 3-Kinases; Proto-Oncogenes; Rats; Signal Transduction; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta

Platelets are rich sources of growth factors and enzymes that are implicated in a number of diseases including obesity, atherosclerosis, heart disease, syndrome X, liver and kidney diseases and certain types of cancers. In this research we investigated, if platelets in Zucker obese rats differ from their lean counterparts with respect to the levels of TGF-beta and COX isoforms, implicated in the pathogenesis of chronic diseases. In addition, we investigated if energy intake of the animals affects the platelet physiology. Platelets were isolated from obese and lean rats bearing preneoplastic lesions in their colon. Prior to platelet isolation these rats were fed either ad libitum (Ob or Ln) or energy restricted (Ob-ER or Ln-ER) diets for 8 weeks (n = 8/group). The levels of TGF-beta1/-beta2 and COX-1/-2 proteins in platelets were analyzed by Western blot. The platelets of the Ob rats had significantly higher levels of TGF-beta1, COX-1/-2 (p < 0.001) than did the platelets of the Ln rats and were not affected by moderate energy restriction. There were no significant differences in the protein expression of platelet TGF-beta2 among any of the groups. These results demonstrate that cytokines and candidates playing a role in the pathogenesis of chronic diseases, such as TGF-beta1 and COX-1/-2, are over-expressed in platelets of Zucker obese rats by comparison to their lean counterparts. These findings also demonstrate that the genotype of the animals exerts a significant effect on the biochemical composition of the platelets and could contribute to the pathogenesis of colon cancer and other metabolic abnormalities associated with obesity.

Topics: Animals; Blood Platelets; Colonic Neoplasms; Cyclooxygenase 1; Cyclooxygenase 2; Diet, Carbohydrate-Restricted; Energy Intake; Female; Isoenzymes; Membrane Proteins; Obesity; Precancerous Conditions; Rats; Rats, Zucker; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

To determine whether sodium butyrate (NaB), a major short-chain fatty acid produced in the human gut by bacterial fermentation of dietary fiber, enhances transforming growth factor (TGF)-beta signaling and potentiates its tumor suppressor activity in the gut.. The molecular mechanisms by which dietary fiber decreases the risk of colon cancers are poorly characterized. TGF-beta is an important tumor suppressor in the gut and has many similar biologic activities as NaB. Therefore, we hypothesized that the chemo-preventive effects of NaB are mediated in part by enhancing TGF-beta signaling and its tumor suppressor function in the gut.. The effects of NaB on Smad3 expression in rat intestinal epithelial (RIE-1) cells and 6 human colon cancer cell lines were examined. The effects of NaB on TGF-beta-induced Smad3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2) gene expression were also examined in RIE-1 cells. Finally, the effects of NaB and TGF-beta on anchorage-independent growth were examined in Akt-transformed RIE-1 cells.. NaB induced Smad3 in RIE-1 cells and in 4 human colon cancer cell lines. NaB enhanced TGF-beta-induced Smad3 phosphorylation and potentiated TGF-beta-induced PAI-1 expression. NaB and TGF-beta synergistically inhibited anchorage-independent growth of Akt-transformed RIE-1 cells.. These results demonstrate that NaB induces Smad3 and potentiates TGF-beta signaling and its tumor suppressor activity in gut epithelial cells. Our data reveal a novel molecular mechanism that may explain in part the beneficial effects of dietary fiber in decreasing the risk of colon cancers.

Topics: Animals; Butyric Acid; Cells, Cultured; Colonic Neoplasms; Dietary Fiber; Epithelial Cells; Humans; Intestinal Mucosa; Rats; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

In tumor cells that have lost responsiveness to the growth inhibitory effects of transforming growth factor beta (TGFbeta), increased TGFbeta production by the tumor cells often contributes to cancer progression, primarily through paracrine mechanisms. Here we investigated the major components of the activator protein-1 (AP-1) complex in the TGFbeta1 promoter of human colon carcinoma cells (HCCCs). In contrast to untransformed epithelial cells (UECs), HCCCs displayed constitutive activation of AP-1 at the proximal AP-1 site in the human TGFbeta1 promoter. Further, in contrast to the JunD and Fra-2 components present in the AP-1 complex at this AP-1 site in UECs, c-Fos was the major detectable AP-1 component in HCCCs. Thus, transcriptional factor switching had occurred in HCCCs relative to the UECs, with regard to the proximal AP-1 site of the human TGFbeta1 promoter. Small interfering RNAs (siRNAs) against c-Fos significantly suppressed AP-1 activity at the relevant AP-1 site, and led to a decrease in TGFbeta1 secretion by the HCCCs. Our results indicate for the first time that c-Fos binding at the TGFbeta1 promoter proximal AP-1 site in HCCCs is required for TGFbeta1 production by the tumor cells. Further, we demonstrated that blockade of TGFbeta1 secretion by c-Fos siRNA led to a suppression of the cellular migration and mitogenesis of NIH 3T3 fibroblasts in a paracrine fashion. Thus, c-Fos may have utility as a target for blocking tumor cell-secreted TGFbeta1, thereby suppressing the migratory behavior associated with the malignant phenotype of HCCCs.

Topics: Animals; Carcinoma; Cell Movement; Coculture Techniques; Colonic Neoplasms; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Mice; NIH 3T3 Cells; Paracrine Communication; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; RNA, Small Interfering; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Since modern treatment of liver metastases includes serial hepatectomies and portal vein obstruction, we investigated the effects of portal vein ligation (PVL) and partial hepatectomy (PH) on tumor growth.. The effects of 70% PH and PVL on liver and lung metastases were evaluated in mice. Tumor growth and liver regeneration were assessed by morphometry and immunohistochemistry for PCNA and BrdU. The effect of growth factors of liver regeneration on CT-26 cells was tested in vitro, and TGF-beta secretion of CT-26 cells was measured by ELISA. RNA synthesis of TGF-beta and activin A was measured by RT-PCR.. Liver regeneration after PH and PVL was similar in tumor-free mice. Intrahepatic tumor growth was lower after PH than after PVL (p=0.016). Extrahepatic tumor growth was not different. In contrast to PVL, liver regeneration was delayed after PH in metastatic livers (p=0.001). Tested growth factors of liver regeneration stimulated CT-26 cells in vitro, and CT-26 secreted significant amounts of TGF-beta in vitro and in vivo.. Although similar in tumor-free mice, liver regeneration significantly differed between PVL and PH in metastatic livers. In addition, PH and PVL differently affected intrahepatic tumor growth.

Topics: Animals; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Hepatectomy; Liver Neoplasms; Liver Regeneration; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Portal Vein; Transforming Growth Factor beta

Immunosuppressed individuals undergoing organ transplantation are at increased risk of recurrences of initial cancers, although how immunosuppressive therapy increases early cancer metastasis remains unclear.. The metastatic fate of luciferase-expressing rat metastatic colon cancer cells (luc-RCN-H4) injected intravenously into the liver of syngeneic and allogeneic rats was examined in the presence of the immunosuppressant cyclosporin A (CsA) by in vivo luminescent technique. With respect to potential tumor-progressing factors, contribution of chemokine receptors and transforming growth factor (TGF)-beta1 to early metastasis was evaluated using their specific signaling inhibitors.. F344 rats injected in the liver with luc-RCN-H4 cells did not always exhibit the formation of tumors and showed a dormant state as long as 60 days after inoculation without CsA. However, CsA released early luc-RCN-H4 cells from dormancy within 2 weeks at nearly 100% in liver and preferentially promoted metastasis to the lymph nodes (approximately 40%). A similar dissemination occurred even in minor histocompatibility complex-disparate hosts. As a tumor-progressing factor, RCN-H4 cells aberrantly expressed chemokine receptors CXCR4 and CCR7. The chemokine receptor (CXC) R4-specific antagonist AMD3100 decreased early metastasis of luc-RCN-H4 cells in rats with ischemic liver conditions (P<0.05), but CsA treatment did not enhance early adhesion. Use of CsA was able to facilitate TGF-beta1 expression and the subsequent TGF-beta-mediated random migration was blocked by the use of the specific signaling inhibitor SB431542 in vitro.. Whereas the chemokine receptor expression by cancer cells is implicated with early organotropic dissemination even under CsA-mediated immune suppression, rather, CsA enhances the late-phase progression after tumor adhesion through TGF-beta1 expression.

Topics: Adenocarcinoma; Animals; Benzamides; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclosporine; Dioxoles; Disease Progression; Gene Expression Regulation, Neoplastic; Image Processing, Computer-Assisted; Killer Cells, Natural; Liver Neoplasms; Luminescence; Lymphatic Metastasis; Male; Neoplasm Metastasis; Rats; Rats, Inbred F344; Receptors, Chemokine; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

We examined the hypothesis that a failure of the immune system to eradicate tumors is due to the immunosuppressive environment created by the growing tumor, which is influenced by the site of tumor growth. We demonstrated that T cell responses to a bystander Ag in mice were suppressed by a growing CT26 tumor. T cells purified from the growing tumor expressed mRNA for IL-10, TGF-beta, and Foxp3. Intracellular cytokine staining revealed a high frequency of IL-10-secreting macrophages, dendritic cells, and CD4+ and CD8+ T cells infiltrating the tumor. In contrast, T cell IFN-gamma production was weak and CD8+ CTL responses were undetectable in mice with CT26 lung metastases and weak and transient following s.c. injection of CT26 cells, but were enhanced in the presence of anti-IL-10 and anti-TGF-beta. Consistent with this, removal of CD8+ T cells abrogated CTL responses and promoted progression of the s.c. tumor. However, in the lung model, depletion of CD8+ T cells significantly reduced the tumor burden. Furthermore, depletion of CD4+ or CD25+ T cells in vivo reduced tumor burden in s.c. and lung models, and this was associated with significantly enhanced IFN-gamma production by CD8+ T cells. These findings suggest that tumor growth facilitates the induction or recruitment of CD4+ regulatory T cells that secrete IL-10 and TGF-beta and suppress effector CD8+ T cell responses. However, CD8+ T regulatory cells expressing IL-10 and TGF-beta are also recruited or activated by the immunosuppressive environment of the lung, where they may suppress the induction of antitumor immunity.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Forkhead Transcription Factors; Immunity, Innate; Immunosuppression Therapy; Interleukin-10; Lung Neoplasms; Lymphocyte Count; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred BALB C; Mice, Transgenic; RNA, Messenger; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

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

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

We have recently shown that quinoxaline 1,4-dioxide (QdNO) derivatives, namely 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ), 2-benzoyl-3-phenyl-quinoxaline 1,4-dioxide (BPQ) and 2-acetyl-3-methyl-quinoxaline 1,4-dioxide (AMQ), suppress the growth of T-84 human colon cancer cells. Here we show that the growth-suppressive effects of QdNOs are due to their ability to induce cell cycle arrest and/or apoptosis. While AMQ blocked more than 60% of cells at the G2/M phase without inducing apoptosis, DCQ caused a significant increase in apoptotic cells with no noticeable effects on the cycling of cells. Treatment with BPQ resulted in G2/M cell cycle arrest and induction of apoptosis. With regard to the effects of QdNOs on molecules that regulate apoptosis and the G2 to M transition, both BPQ and AMQ inhibited the expression of cyclin B, while DCQ significantly decreased the levels of Bcl-2 and increased Bax expression. Next, we investigated whether transforming growth factor-beta1 (TGF-beta1) and/or extracellular signal-regulated kinase (ERK) mediate the antiproliferative and apoptotic effects of QdNOs in colon cancer cells. Interestingly, the above QdNOs increased differentially total TGF beta1 mRNA expression and decreased TGF alpha mRNA and ERK phosphorylation. None of these QdNOs induced changes in TGF beta-2 mRNA expression. The addition of a specific inhibitor of MEK greatly enhanced apoptosis in cells treated with DCQ, suggesting that the inhibition of ERK phosphorylation may explain, to an extent, the apoptogenic effects of this compound. Taken together, these findings provide insights into possible molecular mechanisms of growth inhibition by QdNOs that could aid in their evaluation for anticancer therapy.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Division; Cells, Cultured; Colonic Neoplasms; Cyclin B; Extracellular Signal-Regulated MAP Kinases; Humans; Proto-Oncogene Proteins c-bcl-2; Quinoxalines; Transforming Growth Factor beta; Transforming Growth Factor beta1

We used a spheroid model of colon carcinoma to analyze integrin dynamics as a function of the epithelial-mesenchymal transition (EMT), a process that provides a paradigm for understanding how carcinoma cells acquire a more aggressive phenotype. This EMT involves transcriptional activation of the beta6 integrin subunit and a consequent induction of alphavbeta6 expression. This integrin enhances the tumorigenic properties of colon carcinoma, including activation of autocrine TGF-beta and migration on interstitial fibronectin. Importantly, this study validates the clinical relevance of the EMT. Kaplan-Meier analysis of beta6 expression in 488 colorectal carcinomas revealed a striking reduction in median survival time of patients with high beta6 expression. Elevated receptor expression did not simply reflect increasing tumor stage, since log-rank analysis showed a more significant impact on the survival of patients with early-stage, as opposed to late-stage, disease. Cox regression analysis confirmed that this integrin is an independent variable for these tumors. These findings define the alphavbeta6 integrin as an important risk factor for early-stage disease and a novel therapeutic candidate for colorectal cancer.

Topics: Animals; Autocrine Communication; Biomarkers, Tumor; Carcinoma; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha5; Integrin beta Chains; Intestinal Mucosa; Mesoderm; Mice; Mice, Nude; Neoplasm Invasiveness; Prognosis; Transcription, Genetic; Transforming Growth Factor beta

Transforming growth factor-betas (TGF-betas), cytokines expressed in the colon, play important roles as tumor suppressors and tumor promoters during colorectal carcinogenesis. TGF-beta signaling pathway involves activation of Smad2 and Smad3 by the type I receptor and formation of Smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. Most human colorectal cancers are resistant to the tumor suppressor effects of TGF-beta, and a subset of human colorectal cancers have mutations in Smad2 and Smad4. The purpose of this study was to determine whether Smads are required for TGF-beta signaling in colon cancer cells. First, we selected a colon cancer cell line (MC-26) that has a functional TGF-beta signaling pathway. We found that MC-26 cells expressed Smad2, Smad3, and Smad4 mRNAs by reverse transeription-polymerase chain reaction and confirmed that the TGF-beta signaling pathway is functional using a transient transfection assay with 3TP-Lux reporter plasmid. TGF-beta also inhibited cell growth and induced apoptosis in MC-26 cells. When MC-26 cells were transiently transfected with dominant-negative carboxyl-terminal truncation mutants of Smad2, Smad3, and Smad4, TGF-beta-induced 3TP-Lux reporter activity was significantly reduced, suggesting that Smad2, Smad3, and Smad4 are attractive novel therapeutic targets for regulating TGF-beta signaling in colorectal cancers. Because MC-26 cells express TGF-beta activated Smads, have a functional TGF-beta signaling pathway, and are sensitive to the growth inhibitory and apoptotic effects of TGF-beta, they can serve as an excellent model to examine TGF-beta signaling in colorectal cancers.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; DNA-Binding Proteins; Mice; RNA, Messenger; Signal Transduction; Smad Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

TGF-beta signaling is essential for development and proliferative homeostasis. During embryogenesis, maternal determinants act in concert with TGF-beta signals to form mesoderm and endoderm. In contrast, ectoderm specification requires the TGF-beta response to be attenuated, although the mechanisms by which this is achieved remain unknown. In a functional screen for ectoderm determinants, we have identified Ectodermin (Ecto). In Xenopus embryos, Ecto is essential for the specification of the ectoderm and acts by restricting the mesoderm-inducing activity of TGF-beta signals to the mesoderm and favoring neural induction. Ecto is a RING-type ubiquitin ligase for Smad4, a TGF-beta signal transducer. Depletion of Ecto in human cells enforces TGF-beta-induced cytostasis and, moreover, plays a causal role in limiting the antimitogenic effects of Smad4 in tumor cells. We propose that Ectodermin is a key switch in the control of TGF-beta gene responses during early embryonic development and cell proliferation.

Topics: Animals; Base Sequence; Blastula; Bone Morphogenetic Protein Receptors; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cells, Cultured; Colon; Colonic Neoplasms; DNA-Binding Proteins; Gene Library; Germ Layers; Humans; Molecular Sequence Data; Receptors, Growth Factor; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Xenopus; Xenopus Proteins

Although transforming growth factor-beta (TGF-beta) is both a suppressor and promoter of tumorigenesis, its contribution to early tumor suppression and staging remains largely unknown. In search of the mechanism of early tumor suppression, we identified the adaptor protein ELF, a beta-spectrin from stem/progenitor cells committed to foregut lineage. ELF activates and modulates Smad4 activation of TGF-beta to confer cell polarity, to maintain cell architecture, and to inhibit epithelial-to-mesenchymal transition. Analysis of development of colon cancer in (adult) elf+/-/Smad4+/-, elf+/-, Smad4+/-, and gut epithelial cells from elf-/- mutant mouse embryos pinpoints the defect to hyperplasia/adenoma transition. Further analysis of the role of ELF in human colorectal cancer confirms reduced expression of ELF in Dukes' B1 stage tissues (P < 0.05) and of Smad4 in advanced colon cancers (P < 0.05). This study indicates that by modulating Smad 4, ELF has a key role in TGF-beta signaling in the suppression of early colon cancer.

Topics: Animals; Apoptosis; Cell Growth Processes; Colonic Neoplasms; DNA-Binding Proteins; Humans; Intestinal Mucosa; Mice; Mice, Knockout; Smad4 Protein; Spectrin; Trans-Activators; Transforming Growth Factor beta

TGF-beta1/signaling has been shown to be associated with proapoptotic and antimitotic activities in epithelial tissues. Genistein, a major component of soybean isoflavone, has multiple functions resulting in anticancer proliferation. We herein showed that genistein dose-dependently increased TGF-beta1 mRNA expression in mouse colon cancer MC-26 cells. A mouse monoclonal anti-TGF-beta1 neutralizing antibody partially, but not completely, blocked the growth inhibition by genistein. By using adenoviral vector, we demonstrated that Smad7 overexpression attenuated genistein-induced growth inhibition and apoptosis as determined by MTT and apoptosis ELISA. Smad7 overexpression also inhibited upregulation of p21 and caspase-3 activity by geinistein. To further confirm inhibitory effect of genistein in MC-26 cells require TGF-beta1/Smad signaling, we employed Western blot and electrophoretic mobility shift assay to detect formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, respectively. Data revealed that genistein induced an evident formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, indicating increased TGF-beta1 signaling. Taken together, these findings first provided insights into possible molecular mechanisms of growth inhibition by genistein that required Smad signaling, which could aid in its evaluation for colon tumor prevention.

Topics: Animals; Cell Division; Cell Line, Tumor; Colonic Neoplasms; DNA-Binding Proteins; Genistein; Mice; Phosphorylation; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Dendritic cell (DC)-based tumor vaccine is a promising therapy for malignancies. Recent studies showed greater potency with DC/tumor fusion vaccines against acute myeloid leukemia and melanoma compared with lysate-pulsed DC vaccines. We compared these two vaccine strategies against murine colon cancer and investigated whether DC/tumor fusion cells continue to produce tumor antigens following fusion as a possible explanation for their increased potency. Using a mouse colon cancer model, CT26, we first showed that the DC/CT26 fusion vaccine is more effective in preventing tumor implantation than CT26 lysate-pulsed DC vaccine. Next, CT26 made to constitutively produce bioactive TGF-beta, a surrogate of tumor-derived products, was fused to DCs and found to produce bioactive TGF-beta 72 h after fusion. Our results suggest the DC/tumor fusion vaccine is more potent against colon cancer than the lysate-pulsed DC vaccine. These fusion cells have the distinct advantage of prolonged interaction with tumor antigens in vivo.

Topics: Animals; Antigens, Neoplasm; Biomarkers; Cancer Vaccines; Cell Differentiation; Cell Extracts; Cell Fusion; Cells, Cultured; Colonic Neoplasms; Dendritic Cells; Female; Gene Expression Regulation; Immunotherapy; Mice; Mice, Inbred BALB C; Survival Rate; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 microg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 microg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-beta2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.

Topics: Catechin; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Receptor, IGF Type 1; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta2

It is recognised that stromal cells determine cancer progression. We have previously shown that active TGFbeta produced by rat colon carcinoma cells modulated NO production in rat endothelial cells. To elucidate the role of TGFbeta and NO in the mechanisms of interaction of colon carcinoma cells with stromal cells and in cancer progression, we transfected REGb cells, a regressive colon carcinoma clone secreting latent TGFbeta, with a cDNA encoding for a constitutively-secreted active TGFbeta. Out of 20 injected rats only one tumour progressed, which was resected and sub-cultured (ReBeta cells). ReBeta cells secreted high levels of active TGFbeta. The adhesive properties of REGb and Rebeta cells to endothelial cells were similar, showing that the secretion of active TGFbeta is not involved in tumour cell adhesion to endothelial cells. ReBeta, but not REGb, cell culture supernatants inhibited cytokine-dependent NO secretion by endothelial cells, but inhibition of NO production was similar in co-cultures of REGb or ReBeta cells with endothelial cells. Therefore, secretion of active TGFbeta regulated endothelial NO synthase activity when tumour cells were distant from, but not in direct contact with, endothelial cells. However, only ReBeta cells inhibited cytokine-dependent secretion of NO in coculture with macrophages, indicating that the active-TGFbeta-NO axis confers an advantage for tumour cells in their interaction with macrophages rather than endothelial cells in cancer progression.

Topics: Animals; Carcinoma; Cell Adhesion; Coculture Techniques; Colonic Neoplasms; Disease Progression; Endothelial Cells; Macrophages; Nitric Oxide; Rats; Rats, Inbred Strains; Transforming Growth Factor beta; Tumor Cells, Cultured

The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon.. We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients.. BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients.. These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.

Topics: Adenomatous Polyposis Coli; Animals; Apoptosis; beta Catenin; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Carrier Proteins; Cell Differentiation; Cell Division; Cell Line, Tumor; Colon; Colonic Neoplasms; Cytoskeletal Proteins; DNA-Binding Proteins; Humans; Intestinal Mucosa; Mice; Protein Serine-Threonine Kinases; Proteins; Receptors, Growth Factor; Smad Proteins; Smad1 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

We investigated the mechanism by which cancers evade the growth inhibitory effects of TGF-beta. Using two p21-/- somatically deleted human epithelial cell lines, we find that TGF-beta serves as a growth stimulator rather than a growth suppressor to cells lacking p21. In addition, TGF-beta stimulated p21-/- cells exhibited a mesenchymal phenotype, demonstrated by an upregulation of vimentin and decreased expression of E-cadherin. Analysis of primary human breast cancers by immunohistochemical labeling confirmed a correlation between p21 loss and positive vimentin expression. These data provide a molecular mechanism explaining how nongastrointestinal cancers can escape the anti-proliferative effects of this cytokine and simultaneously use this pathway for growth advantage.

Topics: Breast Neoplasms; Cadherins; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin

Topics: Breast Neoplasms; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Humans; Transforming Growth Factor beta

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)-beta stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF-beta-upregulated extracellular matrix glycoprotein tenascin-C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast-stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)-like repeats of TNC through EGF-receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c-Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA-Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Lineage; Collagen; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Activation; Fibroblasts; Hepatocyte Growth Factor; Humans; Intracellular Signaling Peptides and Proteins; Laminin; Muscle Cells; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Proteoglycans; rac GTP-Binding Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein; Solubility; Tenascin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF) beta is a potent regulator of cell-matrix and cell-cell adhesions (collectively termed cellular adhesions). Cellular adhesions play crucial roles in controlling the differentiation of epithelial cells and in maintaining the integrity of the epithelium. Loss of TGF beta-responsiveness is thought to be an important early initiating event in the malignant progression of epithelial cancer. In the TGFbeta-responsive human colon adenocarcinoma Moser cells, TGFbeta promotes cellular adhesions and suppresses their malignant phenotype. TGFbeta promotes cell-matrix adhesion by inducing the synthesis of extracellular matrix (ECM) adhesion molecules and the expression of integrin receptors for these molecules (termed ECM remodeling). TGFbeta promotes cell-cell adhesion through the induction of E-cadherin expression, an epithelial associated homotypic cell-cell adhesion molecule, which also functions as a tumor suppressor in colon cancer. How TGFbeta regulates E-cadherin expression is not known. In this study, we showed that the induction of E-cadherin by TGFbeta was mediated through the activation of focal adhesion kinase (FAK), a major signaling molecule in focal adhesion contacts and that the activation of FAK was due to ECM remodeling and increased cell-matrix interactions. Thus, TGFbeta regulates cell-cell adhesion through its ability to remodel the ECM and to activate FAK through ECM remodeling.

Topics: Adenocarcinoma; Cadherins; Cell Adhesion; Colonic Neoplasms; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Protein-Tyrosine Kinases; Transforming Growth Factor beta

Transforming growth factor (TGF) beta mediates a tumor suppressor pathway in human colon epithelial cells. We were interested in identifying and characterizing novel genes regulated by the TGF beta pathway in the colon. We employed expression microarrays to identify transcripts induced by TGF beta in Vaco 330, a colon adenoma cell line. We then used expression microarrays to determine which of these TGF beta induced transcripts are down-regulated in metastatic colon cancer. Northern analysis and real-time reverse transcription PCR confirmed and quantified our findings from the microarrays. These analyses highlighted C8orf4 as induced by TGF beta in colon cells. Moreover, C8orf4 is expressed in most normal colon mucosa samples, and is not expressed in most colon cancer metastases or colon cancer cell lines. Colon cancer primary tumors showed reduced expression of C8orf4 relative to normal mucosa, possibly reflecting contributions of C8orf4 expression in stromal cells. C8orf4 is a gene regulated by TGF beta signaling and loss in advanced colon cancer suggests C8orf4 may play a role in colon cell differentiation or growth regulation.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Gene Expression Profiling; Humans; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stromal Cells; Transcription, Genetic; Transforming Growth Factor beta

Apoptosis is a physiologic process in normal development, tissue remodeling and cell turnover. This cell death is noninflammatory and nonimmunogenic, but when associated with a danger signal, it can activate the immune system. However, the capacity of apoptotic cells to activate the immune system is not clearly established, although dead tumor cells have been largely exploited as a source of TAA in cellular therapy against cancer. From these cellular preparations, contradictory results have been reported on the effect of apoptotic cells as an effective source of TAA and their immunologic properties. These conflicting data strongly suggest that the optimal preparation of apoptotic cells derived from tumor cells remains to be determined. In this work, we studied and compared the efficacy of antitumor immune responses derived from repeated injections using different preparations of apoptotic cells. We investigated the importance of HSP70 and TGF-beta expression in apoptotic cells used in the treatment of an established and nonimmunogenic rat carcinoma. UVB-mediated apoptosis did not affect TGF-beta expression in tumor cells, whereas HS treatment sharply downregulated it. Thus, downregulation of TGF-beta permits normal DC activation and maturation and the induction of tumor immunity. We conclude that HS followed by UVB irradiation is a superior source of tumor antigen for the treatment of established tumors. Future work will determine whether HS independently upregulates HSP70, thereby suppressing expression of active TGF-beta, or whether the 2 are linked via a still undefined mechanism.

Topics: Animals; Apoptosis; Cancer Vaccines; Carcinoma; Colonic Neoplasms; Dendritic Cells; Down-Regulation; HSP70 Heat-Shock Proteins; Humans; Immunotherapy; Rats; Transforming Growth Factor beta; Ultraviolet Rays; Up-Regulation

Current therapies for gliomas often fail to address their infiltrative nature. Conventional treatments leave behind small clusters of neoplastic cells, resulting in eventual tumor recurrence. In the present study, we have evaluated the antitumor activity of neural progenitor cells against gliomas when stereotactically injected into nucleus Caudatus of Fisher rats. We show that the rat neural progenitor cell lines HiB5 and ST14A, from embryonic hippocampus and striatum primordium, respectively, are able to prolong animal survival and, in 25% of the cases, completely inhibit the outgrowth of N29 glioma compared with control animals. Delayed tumor outgrowth was also seen when HiB5 cells were inoculated at the site of tumor growth 1 week after tumor inoculation or when a mixture of tumor cells and HiB5 cells were injected s.c. into Fisher rats. HiB5 cells were additionally coinoculated together with two alternative rat gliomas, N32 and N25. N32 was growth inhibited, but rats inoculated with N25 cells did not show a prolonged survival. To evaluate the possibility of the involvement of the immune system in the tumor outgrowth inhibition, we show that HiB5 cells do not evoke an immune response when injected into Fisher rats. Furthermore, the rat neural progenitor cells produce all transforming growth factor beta isotypes, which could explain the observed immunosuppressive nature of these cells. Hence, some neural progenitor cells have the ability to inhibit tumor outgrowth when implanted into rats. These results indicate the usefulness of neural stem cells as therapeutically effective cells for the treatment of intracranial tumors.

Topics: Animals; Brain Neoplasms; Colonic Neoplasms; Corpus Striatum; Glioma; Hippocampus; Humans; Immunosuppressive Agents; Male; Neurons; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Stem Cells; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel trade mark when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-beta (TGF-beta) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-beta. Functional N-cadherin activity is implicated in TGF-beta stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-beta1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-beta stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

Topics: Animals; Antigens, CD; Cadherins; Cell Adhesion Molecules; Cell Line; Cell Movement; Cell Polarity; Coculture Techniques; Colonic Neoplasms; Culture Media, Conditioned; Fibroblasts; Humans; Mitogen-Activated Protein Kinases; Muscle Cells; Neoplasm Invasiveness; Phenotype; Rats; Spheroids, Cellular; Transforming Growth Factor beta; Transforming Growth Factor beta1

The epithelial-mesenchymal transition (EMT) is an essential component of embryonic development, tissue remodeling, and wound repair. In addition, many epithelial tumors, including colorectal carcinomas, appear to undergo this transition that may facilitate their invasion. Using a novel model of EMT in colon carcinoma in which the inflammatory cytokine TNF-alpha accelerates this TGF-beta directed process, we report that TNF-alpha stimulation upregulates expression of the chemokine IL-8, and that this upregulation is dependent on the transcription factor NF-kappaB. Significantly, this effect is not merely an inflammatory response by these colon carcinoma cells because IL-8 expression is induced in cells undergoing a TGF-beta-driven EMT in the absence of exogenous TNF-alpha. During the EMT, a concomitant increase in the chemokine receptor CXCR-1, but not CXCR-2, also occurs. Moreover, both IL-8 and CXCR-1 function in the chemokinetic and chemotactic migration of colon carcinoma cells as assessed by antibody inhibition studies. These studies establish that the regulated expression of a specific chemokine and its receptor are linked to the EMT and they provide a biochemical framework for understanding the mechanisms by which the EMT promotes migration.

Topics: Cell Movement; Chemotaxis; Colonic Neoplasms; Epithelium; Humans; Interleukin-8; Mesoderm; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; NF-kappa B; Receptors, Interleukin-8A; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Alterations of TGF-beta signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-beta or a dominant-negative TGF-betaRII, we demonstrate that TGF-beta signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-dependent IL-6 trans-signaling prevented tumor progression in vivo. Taken together, our data provide novel insights into TGF-beta signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on inhibition of TGF-beta-dependent IL-6 trans-signaling.

Topics: Animals; Blotting, Western; Colonic Neoplasms; Disease Models, Animal; Disease Progression; DNA-Binding Proteins; Endoscopy, Digestive System; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Interleukin-6; Intestinal Mucosa; Mice; Mice, Knockout; Mice, Transgenic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Interleukin-6; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes; Trans-Activators; Transforming Growth Factor beta

The activin type II receptor (ACVR2) gene is a putative tumor suppressor gene that is frequently mutated in microsatellite-unstable colon cancers (MSI-H colon cancers). ACVR2 is a member of the transforming growth factor (TGF)-beta type II receptor (TGFBR2) family and controls cell growth and differentiation. SMAD proteins are major intracellular effectors shared by ACVR2 and TGFBR2 signaling; however, additional shared effector mechanisms remain to be explored. To discover novel mechanisms transmitting the ACVR2 signal, we restored ACVR2 function by transfecting wild-type ACVR2 (wt-ACVR2) into a MSI-H colon cancer cell line carrying an ACVR2 frameshift mutation. The effect of ACVR2 restoration on cell growth, SMAD phosphorylation, and global molecular phenotype was then evaluated. Decreased cell growth was observed in wt-ACVR2 transfectants relative to ACVR2-deficient vector-transfected controls. Western blotting revealed higher expression of phosphorylated SMAD2 in wt-ACVR2 transfectants versus controls, suggesting cells deficient in ACVR2 had impaired SMAD signaling. Microarray-based differential expression analysis revealed substantial ACVR2-induced overexpression of genes implicated in the control of cell growth and tumorigenesis, including the activator protein (AP)-1 complex genes JUND, JUN, and FOSB, as well as the small GTPase signal transduction family members, RHOB, ARHE, and ARHGDIA. Overexpression of these genes is shared with TGFBR2 activation. This observed similarity between the activin and TGF-beta signaling systems suggests that activin may serve as an alternative activator of TGF-beta effectors, including SMADs, and that frameshift mutation of ACVR2 may contribute to MSI-H colon tumorigenesis via disruption of alternate TGF-beta effector pathways.

Topics: Activin Receptors, Type II; Cell Division; Cell Line, Tumor; Colonic Neoplasms; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta

Marked increased expression of cyclooxygenase 2 (COX-2), a prostaglandin-synthesizing enzyme that is pharmacologically inhibited by nonsteroid anti-inflammatory-type drugs, is a major early oncogenic event in the genesis of human colon neoplasia. We report that, in addition to inducing expression of COX-2, colon cancers further target the prostaglandin biogenesis pathway by ubiquitously abrogating expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme that physiologically antagonizes COX-2. We find that 15-PGDH transcript and protein are both highly expressed by normal colonic epithelia but are nearly undetectable in colon cancers. Using gene transfection to restore 15-PGDH expression in colon cancer cells strongly inhibits the ability of these cells to form tumors in immune-deficient mice and demonstrates 15-PGDH to have functional colon cancer tumor suppressor activity. In interrogating the mechanism for 15-PGDH expression loss in colon cancer, we determined that colonic 15-PGDH expression is directly controlled and strongly induced by activation of the TGF-beta tumor suppressor pathway. These findings thus delineate an enzymatic pathway that induces colon cancer suppression, a pathway that is activated by TGF-beta and mediated by 15-PGDH.

Topics: Cell Proliferation; Cells, Cultured; Colonic Neoplasms; Cyclooxygenase 2; Humans; Hydroxyprostaglandin Dehydrogenases; Immunohistochemistry; Intestinal Mucosa; Isoenzymes; Kinetics; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins

The trimeric extracellular matrix molecule laminin-5 and its constituent chains (alpha 3, beta 3, gamma 2) are normally not detectable intracellularly in intestinal epithelial cells but the laminin gamma 2 chain can be detected in cancer cells at the invasive front of a subset of colon carcinomas. These cells are subjected to cytokines such as transforming growth factor beta 1 (TGF-beta 1) and hepatocyte growth factor (HGF), produced by the tumour cells or by the surrounding stromal cells. The purpose of the present work was to investigate whether TGF-beta 1 and HGF, known to stimulate the LAMC2 gene encoding the laminin gamma 2 chain, might synergize to activate the LAMC2 promoter, and to identify the promoter elements involved. We find evidence for synergy between TGF-beta and HGF with respect to laminin gamma 2 chain expression and promoter activation and demonstrate that this requires the 5' activator protein-1 (AP-1) element of the promoter and an additional upstream element which is also responsive to co-expression of the Smad3 protein from the TGF-beta signalling pathway. The transcripts encoding the other laminin-5 chains are not synergistically activated by HGF and TGF-beta. Thus the synergistic activation of the LAMC2 gene is mediated via different cis-elements and results in an overproduction of the laminin gamma 2 chain relative to the other laminin-5 constituent chains. This difference may explain why laminin gamma 2 chains accumulate in the cells at the invasive front of colon carcinomas.

Topics: Carcinoma; Colonic Neoplasms; DNA-Binding Proteins; Drug Synergism; Hepatocyte Growth Factor; Humans; Laminin; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Response Elements; Signal Transduction; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcription Factor AP-1; Transforming Growth Factor beta; Tumor Cells, Cultured

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

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

To identify potential effectors of transforming growth factor (TGF)-beta-mediated suppression of colon cancer, we used GeneChip expression microarrays to identify TGF-beta-induced genes in VACO 330, a nontransformed TGF-beta-sensitive cell line derived from a human adenomatous colon polyp. PMEPA1 was identified as a gene highly up-regulated by TGF-beta treatment of VACO 330. Northern blot analysis confirmed TGF-beta induction of PMEPA1 in VACO 330, as well as a panel of three other TGF-beta-sensitive colon cell lines. PMEPA1 induction could be detected as early as 2 h after TGF-beta treatment and was not inhibited by pretreatment of cells with cycloheximide, suggesting that PMEPA1 is a direct target of TGF-beta signaling. Wild-type PMEPA1 and an alternative splice variant lacking the putative transmembrane domain were encoded by the PMEPA1 locus and were shown by epitope tagging to encode proteins with differing subcellular localization. Both variants were found to be expressed in normal colonic epithelium, and both were shown to be induced by TGF-beta. Consistent with TGF-beta playing a role in terminal differentiation of colonocytes, in situ hybridization of normal colonic epithelium localized PMEPA1 expression to nonproliferating, terminally differentiated epithelium located at the top of colonic crypts. Intriguingly, in situ hybridization and Northern blot analysis showed that the expression of PMEPA1 was well maintained both in colon cancer primary tumors and in colon cancer liver metastases. PMEPA1 is thus a novel TGF-beta-induced marker of a differentiated crypt cell population. Moreover, as PMEPA1 expression is maintained, presumptively in a TGF-beta-independent manner after malignant transformation and metastasis, it demonstrates that even late colon cancers retain a strong capacity to execute many steps of the normal colonic differentiation program.

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Membrane Proteins; Molecular Sequence Data; Protein Isoforms; Signal Transduction; Subcellular Fractions; Transfection; Transforming Growth Factor beta; Up-Regulation

High levels of the cell growth inhibitor transforming growth factor-beta1 (TGF-beta1) are often found in a variety of human cancers. However, the physiological significance of this overexpression depends on the availability of the biologically active form of TGF-beta1 within the extracellular matrix of the tumor microenvironment. To determine the expression and activation status of TGF-beta1 in chemically induced tumors, 6-wk-old A/J mice were injected intraperitoneally with either azoxymethane (AOM) (10 mg/kg body weight, once a week for 6 wk) or normal saline solution, and colon tumors were isolated 24 wk following the last injection. An enzyme-linked immunosorbent assay for TGF-beta1 revealed a significant increase (1.7-fold, P < 0.05) in total TGF-beta1 protein in tumors. Interestingly, while 80% of the total TGF-beta1 in the control colon tissues was in the active form, only 50% was found to be active in tumors. Together with our earlier observations that TGF-beta1 mRNA levels are unchanged in A/J tumors, these data further support a mechanism whereby elevated TGF-beta1 levels result from a defective activation and turnover of this protein. Because plasmin is known to be a major activator of TGF-beta1 in vivo, we hypothesized that reduced plasmin activity may be responsible for the observed dysregulation of TGF-beta1 processing in these behaviorally benign tumors. With a fluorogenic peptide substrate for serine proteases, a deficiency in plasmin activity was found in the tumors. Furthermore, semiquantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis of a panel of genes involved in the plasminogen activation system, including plasminogen activator inhibitor-1 (PAI-1), urokinase-plasminogen activator (u-PA), and urokinase-receptor (u-PAR-1), demonstrated a significant upregulation (approximately fourfold to sixfold, P < 0.05) in the expression of each of these genes in the tumor tissue. In addition, no significant changes were observed in the expression levels of thrombospondin-1 (TSP-1) and insulin-like growth factor type II receptor (IGF-IIR), which also mediate the activation of latent TGF-beta1. To gain further insight into the functionality of the TGF-beta1 pathway, cDNA microarrays were performed and the expression levels of a panel of 21 TGF-beta1-specific target genes were determined in AOM-induced tumors that overexpress the ligand. A significant dysregulation in the expression of each of these targets was observed, p

Topics: Animals; Azo Compounds; Colonic Neoplasms; Fibrinolysin; Gene Expression Regulation; Male; Mice; Oligonucleotide Array Sequence Analysis; Plasminogen Activator Inhibitor 1; Protein Processing, Post-Translational; Receptor, IGF Type 2; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombospondin 1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator

We examined transforming growth factor-beta 1 (TGF-beta 1) effects on cell cycle progression of human colon carcinoma FET cells. TGF-beta 1 inhibited DNA synthesis and cyclin-dependent kinase (CDK) activity after release from growth arrest in association with induction of the p21 CDK inhibitor, whereas cyclins, CDKs, and p27 protein levels remained relatively unchanged. The decrease in CDK2 kinase activity was the result of increased p21 association with cyclin A-CDK2 and cyclin E-CDK2. TGF-beta 1 treatment in late G(1) showed reduced induction of p21 protein levels in association with increased DNA synthesis. Consequently, p21 induction in early G(1) is critical for TGF-beta 1 inhibition of CDK2 kinase activity. Although TGF-beta 1 treatments in late G(1) failed to induce p21 protein, p21 mRNA induction was observed in late G(1) and in S phase. Further analysis showed that TGF-beta 1 treatment in early G(1) increases p21 protein stability throughout the G(1) and S phases of the cell cycle. Our results demonstrate that TGF-beta 1 stimulation of p21 is regulated at the posttranscriptional and transcriptional levels. This is a novel mechanism of TGF-beta 1 inhibition requiring early G(1) induction and stabilization of p21 protein, which binds to and inhibits cyclin E-CDK2 and cyclin A-CDK2 kinase activity rather than direct modulation of cyclin or CDK protein levels as seen in other systems.

Topics: Adenocarcinoma; CDC2-CDC28 Kinases; Colonic Neoplasms; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Kinetics; Luciferases; Macromolecular Substances; Neoplasm Proteins; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; RNA, Messenger; S Phase; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-beta1 (TGF-beta1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-beta1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-beta1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8-33-fold, n=16, P<0.001), together with a two-fold increase in TIL apoptosis (range 1.6-2.5-fold, n=14, P<0.001), relative to FasL-negative nests within the same tumours. The overall level of neutrophils present in all tumours examined was low (mean 0.3%, n=16), with FasL expression by tumour nests associated with a mean two-fold decrease in neutrophils, irrespective of TGF-beta1 expression. Together, our results suggest that tumour-expressed FasL is inhibitory rather than stimulatory towards antitumour immune responses.

Topics: Colonic Neoplasms; Fas Ligand Protein; Humans; Immunity, Cellular; Immunoenzyme Techniques; In Situ Nick-End Labeling; Lactoferrin; Leukocyte Common Antigens; Ligands; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Membrane Glycoproteins; Neutrophils; Paraffin Embedding; Transforming Growth Factor beta; Transforming Growth Factor beta1

Several epidemiological studies have supported the concept that high energy intake, obesity, and/or hyperinsulinemia are risk factors for colon cancer. Previously, it was shown that Zucker obese rats are more sensitive to chemically induced colon cancer than their lean counterparts. The present study investigated whether moderate (20-25%) dietary energy restriction (ER) would attenuate colon carcinogenesis in the Zucker obese rat model. Six-week-old Zucker obese (fa/fa) rats and lean (Fa/Fa) rats received s.c. injections of azoxymethane at a dose of 10 mg/kg body weight once weekly for 2 weeks. A week later, obese rats (n = 16) were assigned to an ER diet (Ob-ER group), based on a low-fat AIN-93G semisynthetic diet. The remaining obese and lean rats (n = 16 rats/group) were fed the low-fat diet ad libitum (Ob group and Ln group, respectively). All rats were euthanized after 8 weeks, and their colons were assessed for aberrant crypt foci (ACF; n = 8/group) or for the expression of transforming growth factor (TGF)-beta and cyclooxygenase (COX) isoforms at the protein and mRNA transcript levels (n = 8/group). Ob rats had a higher number of advanced ACF (crypt multiplicity >or=7) than Ln rats. Dietary ER significantly reduced the appearance of advanced ACF in Ob-ER rats without significantly affecting the blood insulin level or body weights. TGF-beta and COX isoforms were differentially expressed in the colonic mucosae of Ob and Ln rats. Dietary ER significantly reduced TGF-beta1/beta2 and COX-1/2 protein expression in obese rats. This study is the first to demonstrate that moderate ER attenuated TGF-beta and COX protein expression and the carcinogenic process in Zucker obese rats. These findings provide insights leading to the proposal that the mechanism(s) underlying the early events of colon carcinogenesis in Zucker obese rats may extend beyond the role of excessive body weight and hyperinsulinemia per se.

Topics: Animals; Azoxymethane; Blood Glucose; Body Weight; Caloric Restriction; Carcinogens; Colonic Neoplasms; Cyclooxygenase 1; Cyclooxygenase 2; Eating; Female; Insulin; Isoenzymes; Lactic Acid; Membrane Proteins; Obesity; Precancerous Conditions; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Zucker; RNA, Messenger; Transforming Growth Factor beta; Triglycerides

Smad4 is an intracellular transmitter of TGF-beta signals and its tumor suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition. However, there is accumulating evidence that this hypothesis may be too simple. The roles of TGF-beta in carcinogenesis are complex and also comprise tumor promoting functions particularly in late stage carcinogenesis. Importantly, functional inactivation of Smad4 in colon carcinomas frequently occurs at late stages when tumors acquire invasive and metastatic capabilities. We have previously reported that stable re-expression of Smad4 in SW480 human colon carcinoma cells was adequate to suppress tumor growth in nude mice. However, it did not affect cell growth in vitro nor did it restore TGF-beta responsiveness. Here, we report that Smad4 transcriptionally induced classical cadherins including the invasion suppressor E-cadherin, presumably re-establishing epithelial morphology. Smad4-induced cadherins were able to recruit catenins to the plasma membrane and were functionally active in cell-cell adhesion. These results indicate a novel pathway of Smad4-mediated tumor suppression and suggest that Smad4 in colon cells may be involved in the maintenance of epithelial traits.

Topics: alpha Catenin; Animals; beta Catenin; beta-Galactosidase; Blotting, Northern; Blotting, Western; Cadherins; Colonic Neoplasms; Cytoskeletal Proteins; DNA-Binding Proteins; Genes, Tumor Suppressor; Humans; Immunoenzyme Techniques; Mice; Mice, Nude; Ribonuclease, Pancreatic; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Colonic tumors of human origin produce abundant transforming growth factor (TGF)-beta suggesting that TGF-beta is critical to their growth. Dietary lipids regulate a number of growth factors including TGF-beta. Whether elevated TGF-beta levels are consistently expressed in colonic tumors irrespective of the environmental milieu in an in vivo model is not known and forms the main objective of the present study. Male F344 rats were injected with azoxymethane, 10 weeks later, rats bearing preneoplastic lesions were fed a low fat (5% corn oil) diet and 3 high fat (5% corn oil with 18% corn oil, fish oil or beef tallow) diets for 16 weeks. Colonic tumors and mucosae were processed and assessed for TGF-beta status. TGF-beta1 and -beta2 mRNA levels were upregulated in colonic tumors more than in mucosae of all diet groups. Dietary lipids modulated TGF-beta mRNA in both tumors and mucosae, high corn and fish oil diets upregulated TGF-beta1 significantly more than the low fat corn oil or high fat beef tallow diets. Immunohistochemical assessments of tissues with different biological features revealed that TGF-beta1 and -beta2 were elevated in tumors and in selected microscopic preneoplastic lesions compared to normal mucosae. This is the first in vivo study, documenting that developing colonic tumors acquire upregulated TGF-beta phenotype even in the presence of lipid environments capable of differentially regulating TGF-beta in normal mucosae. Elevated expression of TGF-beta in a selected subset of microscopic preneoplastic lesions suggests that TGF-beta plays an important role on both early and late stages of colon carcinogenesis.

Topics: Animals; Azoxymethane; Body Weight; Carcinogens; Cattle; Colonic Neoplasms; Epithelial Cells; Fish Oils; Immunohistochemistry; Lipid Metabolism; Male; Meat; Models, Biological; Phenotype; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Up-Regulation; Zea mays

Growth of Caco-2 and many cancer cells is inhibited by 1,25(OH)(2)D(3). Whereas TGF-beta 1 inhibits normal colonic epithelial cell growth, most human colon cancer-derived cells, including Caco-2 and SW480 cells, are resistant to it. The mechanisms underlying these antiproliferative actions and resistance to TGF-beta growth inhibition are largely unknown. We observed that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] sensitized Caco-2 and SW480 cells to TGF-beta 1 growth inhibitory effects. Versus 1,25(OH)(2)D(3) alone, the combination of 1,25(OH)(2)D(3) and TGF-beta 1 significantly reduced cell numbers. Also, the amount of active TGF-beta 1 was increased (~4-fold) by this secosteroid in conditioned media from Caco-2 cells. The 1,25(OH)(2)D(3) increased the expression of IGF-II receptors (IGF-IIR), which facilitated activation of latent TGF-beta 1, and was found to activate TGF-beta signaling in Caco-2 cells. By using neutralizing antibodies to human TGF-beta 1, we showed that this cytokine contributes to secosteroid-induced inhibition of Caco-2 cell growth. Also, 1,25(OH)(2)D(3) was found to enhance the type I TGF-beta receptor mRNA and protein abundance in Caco-2 cells. Whereas the 1,25(OH)(2)D(3)-induced sensitization of Caco-2 cells to TGF-beta 1 was IGF-IIR independent, the type I TGF-beta 1 receptor was required for this sensitization. Thus 1,25(OH)(2)D(3) treatment of Caco-2 cells results in activation of latent TGF-beta 1, facilitated by the enhanced expression of IGF-IIR by this secosteroid. Also, 1,25(OH)(2)D(3) sensitized Caco-2 cells to growth inhibitory effects of TGF-beta 1, contributing to the inhibition of Caco-2 cell growth by this secosteroid.

Topics: Activin Receptors, Type I; Caco-2 Cells; Calcitriol; Cell Count; Cell Division; Colonic Neoplasms; Culture Media, Conditioned; Gene Expression; Humans; Protein Serine-Threonine Kinases; Receptor, IGF Type 2; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Activation of the tumour suppressor p53 by DNA damage induces either cell cycle arrest or apoptotic cell death. The cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1), whereas the apoptotic effect is mediated by transcriptional activation of mediators including PUMA and PIG3 (ref. 2). What determines the choice between cytostasis and apoptosis is not clear. Here we show that the transcription factor Myc is a principal determinant of this choice. Myc is directly recruited to the p21(Cip1) promoter by the DNA-binding protein Miz-1. This interaction blocks p21(Cip1) induction by p53 and other activators. As a result Myc switches, from cytostatic to apoptotic, the p53-dependent response of colon cancer cells to DNA damage. Myc does not modify the ability of p53 to bind to the p21(Cip1) or PUMA promoters, but selectively inhibits bound p53 from activating p21(Cip1) transcription. By inhibiting p21(Cip1) expression Myc favours the initiation of apoptosis, thereby influencing the outcome of a p53 response in favour of cell death.

Topics: Animals; Apoptosis; Cell Cycle; Cell Cycle Proteins; Colonic Neoplasms; COS Cells; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA; DNA Damage; DNA Footprinting; DNA-Binding Proteins; Flow Cytometry; Gene Deletion; Gene Expression Regulation, Neoplastic; Helminth Proteins; Humans; Kruppel-Like Transcription Factors; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Patients with ulcerative colitis are at risk for colon cancer and frequently have microsatellite instability,which, in turn, is usually associated with inactivation of transforming growth factor (TGF) beta signaling. TGF-beta1 deficiency in mice can lead to colon cancer that is preceded by precancerous lesions having submucosal inflammation and hyperplastic crypts. Germ-free TGF-beta1-deficient mice are free of inflammation, hyperplasia, and cancer, but when reintroduced into a Helicobacter hepaticus-containing specific pathogen-free room, these lesions reappear. Because adenoma/carcinoma but not inflammation/hyperplasia is dependent on the genetic backgrounds tested, colitis is required, but not sufficient, for carcinogenesis. This animal model should provide insight into the protective role of TGF-beta1 in early stages of ulcerative colitis-associated human colon cancer.

Topics: Animals; Colitis, Ulcerative; Colonic Neoplasms; Disease Models, Animal; Female; Genetic Predisposition to Disease; Germ-Free Life; Humans; Male; Mice; Mice, Inbred C3H; Mice, Knockout; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor beta1 (TGFbeta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. We recently demonstrated that colon carcinoma cells transfected with oncogenic cellular K-rasV12, but not oncogenic cellular H-rasV12, switched from TGFbeta1-insensitive to TGFbeta1-growth-stimulated and also became more invasive (Yan, Z., Deng, X., and Friedman, E. (2001) J. Biol. Chem. 276, 1555-1563). We now demonstrate that TGFbeta1 growth stimulation of colon carcinoma cells is Ras-dependent and smad-independent. In U9 colon carcinoma cells, which are responsive to TGFbeta1 by growth stimulation, a truncating mutation at Gln-311 was found in the smad4 gene. Very little smad4 protein was detected in these cells. Loss of smad4 protein was confirmed by functional studies. In U9 cells co-transfected wild-type smad4, but not mutant smad4, mediated response of the 3TP-lux and pSBE promoter reporter constructs to TGFbeta1. Proliferation initiated by TGFbeta1 in U9 cells required Ras-mediated down-regulation of p21cip1 protein. Less p21cip1 was associated with cdk2 small middle dotcyclin complexes in TGFbeta1-treated U9 cells, and the cdk2 complexes had increased kinase activity. Elevation of p21cip1 levels diminished proliferative response to TGFbeta1. U9 cells expressing DN-N17ras neither proliferated in response to TGFbeta1 nor down-regulated the cdk inhibitor p21cip1, and TGFbeta1 activation of 3TP-lux in U9 cells was inhibited by DN-N17ras in a dose-dependent manner. TGFbeta1 also decreased p21cip1 levels and stimulated proliferation in SW480 cells, which express mutant K-Ras but no smad4 protein. TGFbeta1 did not activate or inhibit the p21cip1 promoter construct in U9 cells even in the presence of co-transfected smad4, or alter p21cip1 mRNA levels. Thus the decrease in p21cip1 levels was mediated by a TGFbeta-initiated Ras-dependent, but smad-independent post-transcriptional mechanism.

Topics: Blotting, Northern; Blotting, Western; CDC2-CDC28 Kinases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mutation; Plasmids; Protein Binding; Protein Serine-Threonine Kinases; ras Proteins; RNA Processing, Post-Transcriptional; Signal Transduction; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

During neoplastic progression, alterations in transforming growth factor beta1 (TGF-beta1) dependent control of cell growth may be an important mechanism of selective proliferation of transformed cellular clones. Defective regulation of TGF-beta1 receptors has been reported to occur in a number of human malignant tumours while little is known of the actual levels of this growth inhibitory cytokine in cancer. On the basis of the demonstrated ability of major lipid peroxidation products such as 4-hydroxynonenal to modulate TGF-beta1 expression and synthesis, we speculated that decreased lipid oxidation, as frequently observed in neoplastic tissues, would contribute to the selective promotion of tumour growth through decreased expression of the cytokine within the tumour mass.. To seek a possible association between steady state levels of major aldehydic end products of lipid peroxidation and TGF-beta1 content in human colon cancer at different stages of growth.. Tissue biopsies from 15 adult patients with colon adenocarcinoma of different TNM and G stagings were compared with regard to lipid peroxidation aldehydes and net TGF-beta1 levels. For a more comprehensive analysis, cytokine type I and II receptors were measured in tumour biopsies. In one set of experiments, to support the conclusions, the apoptotic effect of TGF-beta1 was evaluated in a human colon cancer cell line, CaCo-2, retaining receptor changes consistent with those observed in cancer patients.. With the exception of two extremely advanced cases (T4/G3) in which tissue levels of lipid peroxidation were within the normal range, 4-hydroxynonenal was significantly decreased in all other cancer specimens. Consistent with lipid peroxidation levels, TGF-beta1 protein was markedly decreased or even negligible compared with the corresponding normal tissue surrounding the tumour in all tested biopsies except for the two T4/G3 colon cancers in which cytokine content was again within the normal range. As regards TGF-beta1 receptors, both in tumour sections and CaCo-2 cells, downregulation was greater for TGF-beta1 receptor I than for receptor II. Of note, in CaCo-2 cells, incubation with appropriate doses of TGF-beta1 led to marked nuclear fragmentation and apoptosis.. Evasion of human colon cancer cells from TGF-beta1 mediated growth inhibition appears to be due not only to downregulation of TGF-beta1 receptors, which is inconsistent and unrelated to cancer development, but also to the constant low concentration of this cytokine in the tumour mass. The associated levels of lipid peroxidation aldehydes, much lower than in control tissue, probably represent a lower stimulus for TGF-beta1 production in the neoplastic area and thus a favourable condition for neoplastic progression.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Apoptosis; Caco-2 Cells; Colonic Neoplasms; Disease Progression; Female; Humans; Intestinal Mucosa; Lipid Peroxidation; Male; Malondialdehyde; Middle Aged; Neoplasm Proteins; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1

Decorin, a small proteoglycan containing a dermatan sulfate (DS) chain, is expressed abnormally in human colon cancer stroma. The aim of this study was to determine neoplastic changes in DS chains from human colon cancer and normal colonic mucosa. Proteoglycans were extracted from human colon cancer and normal colonic mucosa and successively digested with enzymes. The glycosaminoglycan obtained was fluoro-labeled with 2-aminopyridine at reducing terminals and fractionated by HPLC. Fluoro-labeled DS chains were collected and digested with bovine testicular hyaluronidase, followed by HPLC. The repeating disaccharide connected to the linkage region [glucuronosyl-galactosyl-galactosyl-xylosyl(2-aminopyridine)] of pyridylaminated DS chains from both types of tissue was glucuronosyl-N-acetylgalactosamine. The other glucuronic acid of the pyridylaminated DS chain was located 12 saccharides from the reducing terminal in colon cancer, and 18 saccharides from the reducing terminal in normal colon. The saccharide sequence of DS chains from human colon cancer is altered from that in normal colon.

Topics: Aminopyridines; Animals; Cattle; Chromatography, High Pressure Liquid; Colon; Colonic Neoplasms; Decorin; Dermatan Sulfate; Electrophoresis; Extracellular Matrix Proteins; Fluorescent Dyes; Humans; Hyaluronoglucosaminidase; Intestinal Mucosa; Proteoglycans; Sequence Analysis; Transforming Growth Factor beta

A deficient leukocyte recruitment has been suggested in tumor vasculature, but little is known about the underlying molecular mechanism. To characterize leukocyte-endothelium interaction in experimental colon cancer, quantify the main endothelial cell adhesion molecules (CAMs), and evaluate the effect of tumor-derived products.. Leukocyte recruitment was assessed by intravital videomicroscopy in mice bearing HT29-derived tumors. Endothelial CAMs were measured using the dual-radiolabeled antibody technique. The role of molecules mediating leukocyte rolling (P-, E-, and L-selectin) or adhesion (intercellular adhesion molecule 1 [ICAM-1] and vascular cell adhesion molecule 1 [VCAM-1]) carcinoembryonic antigen (CEA), and transforming growth factor (TGF) beta1 was assessed through immunoblockade, whereas participation of nitric oxide (NO) and cyclooxygenase (COX) metabolites were evaluated by means of nonselective and selective inhibition.. Basal and lipopolysaccharide-stimulated leukocyte rolling and adhesion were markedly reduced in tumor vasculature. ICAM-1 immunoblockade prevented leukocyte adhesion in both tumor and nontumor microvessels. Neither baseline nor LPS-induced endothelial ICAM-1, P-, and E-selectin expression in tumors were reduced with respect to nontumor vasculature. Although VCAM-1 expression was reduced in tumor endothelium, immunoneutralization of VCAM-1 failed to reverse LPS-induced leukocyte recruitment in this setting. CEA immunoblockade and COX inhibition did not modify the deficient leukocyte rolling. Nonselective NO inhibition partially reversed the defective adhesion response in tumor microvessels. Finally, TGF-beta1 immunoblockade partially and selectively restored impaired leukocyte rolling and adhesion in tumor microvessels.. Impaired leukocyte recruitment in tumor vasculature cannot be attributed to a depressed expression of the main CAMs. Selective restoration after NO inhibition and TGF-beta1 immunoblockade suggests involvement of both molecules in this phenomenon.

Topics: Animals; Antibodies, Monoclonal; Carcinoembryonic Antigen; Cell Adhesion; Cell Communication; Chemotaxis, Leukocyte; Colonic Neoplasms; E-Selectin; Endothelium, Vascular; HT29 Cells; Humans; Intercellular Adhesion Molecule-1; L-Selectin; Leukocytes; Mice; Mice, Nude; Nitric Oxide; P-Selectin; Prostaglandin-Endoperoxide Synthases; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Cell Adhesion Molecule-1

Downregulation of TGF-beta receptors is implicated in colon cancer development. Inactivation of either of the two transmembrane serine/threonine kinases, TGF-beta1 types I/II receptors, is now implicated in carcinogenesis, especially gastrointestinal carcinogenesis.. We generated transgenic mice, called pS2-dnRII or ITF-dnRII, of which the dominant negative mutant of the TGF-beta type II receptor was expressed under the control of tissue-specific promoters, the pS2 promoter for stomach and ITF for intestine. They were either infected with H.pylori (ATCC 43504 strain, CagA+ and VacA+) or administered with azoxymethane to determine the significance of loss of TGF-beta signalling in gastrointestinal carcinogenesis.. Gastric adenocarcinoma developed in pS2-dnRII mice, whereas only chronic active gastritis was noted in wild-type littermates after 36 weeks of H.pylori infection. Mice lacking in TGF-beta signalling specifically in the stomach showed a significantly higher proliferation cell nuclear antigen-labelling index when infected with H.pylori than wild-type littermates (P < 0.01). Development of colonic aberrant crypt foci was provoked in mice by intraperitoneal injections of azoxymethane, and ITF-dnRII mice showed significantly higher incidences of ACF and colon cancers than wild-type littermates.. Maintaining normal TGF-beta signalling in the gastrointestinal tract seems to be important either for preventing abnormal mucosal proliferation, or for suppressing or retarding carcinogenesis.

Topics: Animals; Azoxymethane; Carcinogens; Carcinoma; Colonic Neoplasms; Disease Susceptibility; Gastritis; Helicobacter Infections; Helicobacter pylori; Mice; Mice, Transgenic; Receptors, Transforming Growth Factor beta; Signal Transduction; Stomach Neoplasms; Transforming Growth Factor beta

We report the immunological characterization of three colon carcinoma cell lines, COLO 205, SW620 and SW403, which we selected to combine with cytokine-secreting fibroblasts for the development of an allogeneic tumour cell vaccine. The cell lines expressed HLA-A2 as well as shared tumour-associated antigens (TAAs) representative of colon carcinomas: CEA, Ep-CAM, MUC1, HER2/neu and MAGE antigens. They did not secrete high levels of the immunosuppressive factors TGF-beta, IL-10 or prostaglandins. The lines presented TAAs in a manner recognized by immune effector cells, which was demonstrated by the lysis of SW620 by HLA-A2-restricted anti-p53 cytotoxic T lymphocytes (CTL). COLO 205 and SW620 were genetically modified to express the co-stimulatory molecule CD80 (B7.1), which increased the ability of the cells to stimulate CTL in vitro. CTL clones derived from HLA-A2+ peripheral blood mononuclear cells stimulated with the CD80-expressing lines lysed the stimulator cell and an HLA-A2+ colon cancer cell line, but did not lyse an isogeneic fibroblast line or an HLA-A2- colon cancer cell line. CTL clones derived from colon carcinoma patients immunized with an allogeneic vaccine containing these lines demonstrated killing of autologous tumour cells, the vaccine cell lines and other HLA-A2+ colon cancer cell lines, but not fibroblasts isogeneic to certain of the target cell lines. Our studies demonstrate that these colon carcinoma cell lines express shared TAAs that can induce CTLs which recognize and lyse other colon carcinoma cells, and support the continued clinical evaluation of the CD80 gene modified allogeneic colon cell/cytokine-secreting fibroblast carcinoma vaccine.

Topics: Antigen Presentation; Antigens, Neoplasm; B7-1 Antigen; Cancer Vaccines; Carcinoembryonic Antigen; Cell Adhesion Molecules; Cells, Cultured; Colonic Neoplasms; Cytokines; Cytotoxicity, Immunologic; Epithelial Cell Adhesion Molecule; Fibroblasts; HLA-A2 Antigen; Humans; Isoantigens; Lymphocyte Activation; Mucin-1; Neoplasm Proteins; Receptor, ErbB-2; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras(G12V) transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivo than controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928-30936). We now show that stable expression of oncogenic Ki-ras(G12V) converts the HD6-4 colon cancer cell line from insensitive to TGF-beta1 to growth-promoted by TGF-beta1. Each of three Ki-ras(G12V) transfectants responded to TGF-beta1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-beta1. The wild-type Ki-ras transfectants lack functional TGF-beta receptors, whereas all three Ki-ras(G12V) transfectants expressed functional TGF-beta receptors that bound (125)I-TGF-beta1. The previous studies showed that in cells with wild-type Ki-ras, TGF-beta receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-beta receptor III (TbetaRIII) was incomplete. We now show that the betaglycan form of TbetaRIII is highly modified following translation when transiently expressed in Ki-ras(G12V) cells, whereas no such post-translational modification of TbetaRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TbetaRIII post-translational modification in Ki-ras(G12V) cells and TGF-beta1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TbetaRIII post-translational modification.

Topics: Activin Receptors, Type I; Animals; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Genes, ras; Humans; Neoplasm Invasiveness; Phenotype; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteoglycans; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta1 (TGFbeta) simultaneously induces the expression of fibronectin, fibronectin receptor, laminin, and laminin receptor (alpha6beta1 integrin) in the human colon cancer cell line Moser (Int J Cancer, 57:742, 1994). Induction of fibronectin and induction of fibronectin receptor by TGFB are tightly coupled, and disrupting fibronectin induction disrupts the induction of fibronectin receptor and cellular adhesion to fibronectin (J Cellular Physiol, 170:138, 1997). We recently demonstrated the efficacy of using antisense chain-specific laminin RNA expression vectors to disrupt the induction by TGFP of the multichain laminin molecule (J Cellular Physiol, 178:296, 1999). We now show in this report that Moser cells used alpha6 and beta1 integrins to adhere to laminin, and, as is the fibronectin and fibronectin receptor system, disrupting the induction by TGFbeta of the ligand laminin by the expression of antisense laminin A chain RNA disrupted the induction of 125I-laminin binding and cellular adhesion to laminin. Disrupting laminin induction also blocked the induction of alpha6 and beta1 integrin laminin receptor by TGFbeta. We conclude that disrupting the induction of the ligand laminin by TGFbeta disrupts TGFbeta-regulated laminin receptor function by suppressing the induction of alpha6 and beta1 integrins. Therefore, targeted disruption of the ligand laminin may be an effective means in disrupting the function of both the ligand and its receptor in cells that utilize the laminin and laminin receptor system in malignant cell behavior.

Topics: Colonic Neoplasms; Extracellular Matrix; Humans; Integrins; Laminin; Receptors, Laminin; RNA, Antisense; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor (TGF)-beta is a protein family which affects multiple cellular functions including survival, proliferation, differentiation and adhesion. Among the three known isoforms, TGF-beta1 is commonly overexpressed in solid malignancies. Recent studies in knock-out mice demonstrated non-redundant roles of different TGF-beta isoforms in development. The present study was performed to assess tumour-associated expression of the three TGF-beta isoforms in colon carcinoma. We report that colon carcinoma progression is associated with gradual and significant increases in expression of TGF-beta1 and TGF-beta2 mRNA and proteins. By contrast, TGF-beta3 expression was detected in normal colonic mucosa and, at slightly higher levels, in tumour tissues. In addition, plasma levels of both TGF-beta1 and TGF-beta2 were significantly higher in cancer patients when compared with unaffected individuals. Taken together, our results indicate distinct expression patterns of the three TGF-beta isoforms in colon carcinoma cells and possible systemic effects of TGF-beta1 and TGF-beta2 in tumour patients.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma in Situ; Colonic Neoplasms; Disease Progression; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3

Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.

Topics: Adenoma; Cell Division; Colonic Neoplasms; DNA-Binding Proteins; Genes, Reporter; Growth Inhibitors; Humans; Luciferases; Mutation; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Because autocrine transforming growth factor beta (TGF-beta) can suppress carcinogenesis, which is often associated with telomerase activation, we studied whether autocrine TGF-beta inhibits telomerase activity. Restoration of autocrine TGF-beta activity in human colon carcinoma HCT116 cells after reexpression of its type II receptor (RII) led to a significant reduction of telomerase activity and the mRNA level of telomerase reverse transcriptase (hTERT), whereas suppression of the autocrine TGF-beta activity with a dominant negative RII without the cytoplasmic domain (deltaRII) in human breast cancer MCF-7 cells led to a significant increase of telomerase activity and hTERT mRNA level. This appears to be due to repression of hTERT mRNA transcription because exogenous TGF-beta treatment of MCF-7 cells transiently transfected with a hTERT promoter-reporter construct significantly repressed the hTERT promoter activity in a dose-dependent manner. Furthermore, the hTERT promoter activity was significantly decreased in HCT116 RII cells and increased in MCF-7 deltaRII cells when compared with their respective controls. Therefore, autocrine TGF-beta appears to target hTERT promoter to inhibit telomerase activity.

Topics: Autocrine Communication; Breast Neoplasms; Colonic Neoplasms; DNA-Binding Proteins; Enzyme Activation; Female; Humans; RNA; RNA, Messenger; Telomerase; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

A hallmark of transforming growth factorbeta (TGFbeta) action is the induction of the synthesis and secretion of extracellular-matrix adhesion molecules and induction of the cell-surface expression of integrin receptors for these molecules (termed extracellular-matrix remodeling). The signal pathways leading to extracellular-matrix remodeling and the significance of extracellular-matrix remodeling in TGFbeta function is not well-understood. In the epithelium-derived human colon cancer cell line Moser, TGFbeta induces extracellular-matrix remodeling in a protein kinase Calpha-dependent manner. In this study we showed that TGFbeta was a potent inducer of the homotypic cell-cell adhesion molecule E-cadherin and its undercoat-associated proteins, the catenins and dramatically increased the amount of E-cadherin/gamma-catenin complex formation. We found that the induction of E-cadherin and alpha- and beta-catenin by TGFbeta was also dependent on protein kinase Calpha, whereas the induction of gamma-catenin was independent of protein kinase Calpha but dependent on other protein kinase C isoforms. We also found that protein kinase Calpha-dependent induction of extracellular-matrix remodeling and subsequent cell-matrix interaction requiring both fibronectin and laminin were a prerequisite for the induction of E-cadherin (and alpha- and beta-catenin but not gamma-catenin) by TGFbeta. We therefore concluded that two signal pathways exist in TGFbeta-regulated expression of E-cadherin and the catenins. We also concluded that a functional significance of TGFbeta-induced extracellular matrix remodeling is the activation of signal transduction mechanisms through increased interaction between extracellular matrix fibronectin and laminin and their cell-surface integrin receptors, which lead to the induction of E-cadherin (and alpha- and beta-catenin).

Topics: alpha Catenin; beta Catenin; Cadherins; Cell Adhesion; Colonic Neoplasms; Cytoskeletal Proteins; Desmoplakins; Extracellular Matrix; gamma Catenin; Gene Expression; Humans; Isoenzymes; Protein Kinase C; Protein Kinase C-alpha; Signal Transduction; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

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

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

Adjuvant chemotherapy improves survival among patients with stage III colon cancer, but no reliable molecular predictors of outcome have been identified.. We evaluated loss of chromosomal material (also called loss of heterozygosity or allelic loss) from chromosomes 18q, 17p, and 8p; cellular levels of p53 and p21(WAF1/CIP1) proteins; and microsatellite instability as molecular markers. We analyzed tumor tissue from 460 patients with stage III and high-risk stage II colon cancer who had been treated with various combinations of adjuvant fluorouracil, leucovorin, and levamisole to determine the ability of these markers to predict survival.. Loss of heterozygosity at 18q was present in 155 of 319 cancers (49 percent). High levels of microsatellite instability were found in 62 of 298 tumors (21 percent), and 38 of these 62 tumors (61 percent) had a mutation of the gene for the type II receptor for transforming growth factor beta1 (TGF-beta1). Among patients with microsatellite-stable stage III cancer, five-year overall survival after fluorouracil-based chemotherapy was 74 percent in those whose cancer retained 18q alleles and 50 percent in those with loss of 18q alleles (relative risk of death with loss at 18q, 2.75; 95 percent confidence interval, 1.34 to 5.65; P=0.006). The five-year survival rate among patients whose cancer had high levels of microsatellite instability was 74 percent in the presence of a mutated gene for the type II receptor for TGF-beta1 and 46 percent if the tumor did not have this mutation (relative risk of death, 2.90; 95 percent confidence interval, 1.14 to 7.35; P=0.03).. Retention of 18q alleles in microsatellite-stable cancers and mutation of the gene for the type II receptor for TGF-beta1 in cancers with high levels of microsatellite instability point to a favorable outcome after adjuvant chemotherapy with fluorouracil-based regimens for stage III colon cancer.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Chemotherapy, Adjuvant; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 8; Colonic Neoplasms; Disease-Free Survival; Female; Fluorouracil; Genetic Markers; Humans; Loss of Heterozygosity; Male; Microsatellite Repeats; Middle Aged; Mutation; Neoplasm Staging; Proportional Hazards Models; Randomized Controlled Trials as Topic; Risk; Survival Analysis; Transforming Growth Factor beta

Tributyrin has been shown to be cytostatic to tumor cells by inducing differentiation and apoptosis. On the other hand, immunological NK cells can kill tumor cells, particularly when stimulated with interleukin-2 (IL-2) and/or interleukin-12(IL-12). However, little is known about whether and how both antitumor mechanisms act together, although in vivo such an interaction must exist. Here we demonstrate in vitro, that pretreatment of human LS 174T colon cancer cells with nontoxic concentrations of tributyrin augments the sensitivity to spontaneous NK cell activity two-fold. However, when NK cells have been activated with an optimized combination of IL-2 and IL-12, the immunocytotoxicity increases up to five-fold (from 14% to 70%), versus a 3.8-fold increase against untreated cancer cells. These effects are accompanied by increased IFN-gamma secretion and decreased TGF-beta1 secretion. Tributyrin is found to be a potent inducer of ICAM-1, LFA-3 and Fas on target cells corresponding to an increase of the FasL expression by IL-2/IL-12 on the effector cells. Our data suggest a synergistic link between induction of tumor cell differentiation and immunological defense mechanisms that may provide a rational basis for the improvement of clinical protocols, especially for colon cancer.

Topics: Colonic Neoplasms; Cytotoxicity, Immunologic; Drug Synergism; Humans; Interferon-gamma; Interleukin-12; Interleukin-2; Killer Cells, Natural; Transforming Growth Factor beta; Trialkyltin Compounds; Tumor Cells, Cultured

Deoxycholic acid and other secondary bile acids have long been considered tumour promoters in the colon. However, their effect on cell migration, known to play an important role in colon carcinogenesis, has not been studied so far.. To investigate the possible effects of deoxycholic acid on colon cancer-cell migration in culture.. Human colon carcinoma cells (Caco-2) were seeded on basement membrane matrix. To evaluate replication-blocked cell migration, we wounded confluent monolayers of cells with a sterile scalpel, and inhibited cell replication with mitomycin C. Immediately after wounding, the cells were exposed to 0-100 micromol/l deoxycholic acid. Migration over 72 h was monitored using a phase contrast microscope.. Replication-blocked migration was stimulated by deoxycholic acid in a dose-dependent manner, with the maximum effect at 20 micromol/l deoxycholic acid. Enhancement of migration rate was unaffected by immunoneutralization of transforming growth factor beta (a known migration-promoting peptide). However, specific inhibition of protein kinase C markedly inhibited deoxycholic acid-induced Caco-2 cell migration.. In addition to its well-established role in the enhancement of proliferation, deoxycholic acid also stimulates colon cancer-cell migration along the basement membrane matrix. The mechanism of this stimulation is likely to involve protein kinase C. Deoxycholic acid-stimulated migration might additionally contribute to the tumour-promoting effects of secondary bile acids in the colon.

Topics: Caco-2 Cells; Cell Division; Cell Movement; Colonic Neoplasms; Deoxycholic Acid; Dose-Response Relationship, Drug; Humans; Mitomycin; Protein Kinase C; Transforming Growth Factor beta; Tumor Cells, Cultured

Alterations in the transforming growth factor-beta (TGF-beta) pathway are implicated in the pathogenesis of colorectal cancer. We hypothesize that alterations in the TGF-beta pathway contribute to differential sensitivity of mice to the colon carcinogen azoxymethane (AOM). A/J (sensitive) and AKR/J (resistant) mice were injected intraperitoneally with AOM (10 mg/kg of body weight once a week for 6 wk). Twenty-four weeks after AOM exposure, mutational analysis of TGF-beta type II receptor (TbetaR-II) from normal colons and from tumors showed no AOM-induced alterations. A significant decrease (1.5-fold, P < 0.05) in TbetaR-II mRNA levels, however, was found in A/J tumors with the RNase protection assay. Immunofluorescence of TbetaR-II showed marked loss of staining in A/J tumors. The RNase protection assay and sequence analysis of the downstream signaling molecule Smad3 revealed no carcinogen-induced alterations in either strain. To gain further insight into the functionality of the pathway, expression of TGF-beta, TGF-beta type I receptor (TbetaR-I), and several downstream targets of TGF-beta signaling, including Smad7, c-myc, and p15, was examined. Although no alterations in TGF-beta, TbetaR-I, or Smad7 were found in tumors, a significant increase in c-myc expression (2.5-fold, P < 0.05 ) and a significant decrease in p15 expression (4.5-fold, P < 0.05 ) were noted. Concomitant repression of TbetaR-II and overexpression of c-myc may render epithelial cells insensitive to TGF-beta-mediated growth arrest, a possibility that also is suggested by this model. The significant decrease in p15 expression in tumors provides additional evidence that TGF-beta signaling may be markedly attenuated during colon tumorigenesis.

Topics: Adenoma; Animals; Azoxymethane; Carcinogens; Cell Cycle Proteins; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; Male; Mice; Mice, Inbred A; Mice, Inbred AKR; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins

Rectal administration of trinitrobenzene sulfonic acid (TNBS) produces chronic colitis in experimental animals. However, the role of epithelial cellular protein(s) in this model is unknown. We examined whether oral tolerance can be induced in this model with colon epithelial cell proteins and whether it is organ specific. Rats were fed five times with extracts of LS-180 human colon cancer cells or HT 1080 human fibroblast cells. Syngeneic normal rat colon or small intestinal extracts were fed to separate groups of rats. After oral feedings, each rat received TNBS by enema. Rats were killed 15 days later, and the following were measured: gross and histologic disease score, weight, thickness, and myeloperoxidase values of colon and serum interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) levels. Rectal TNBS alone produced severe colitis with a 26% mortality rate. Rats fed LS-180 or rat colon extract before TNBS enema were protected, as evidenced by reductions in mortality rate, disease scores, and myeloperoxidase values. However, rats fed HT 1080 or small intestine extract lacked such protection. To examine the possible mechanism of the oral tolerance, T lymphocytes from mesenteric lymph nodes and spleen of LS-180 extract-fed rats were passively transferred to naive rats, and this was followed by TNBS enema. These rats showed clear protection. Protected animals had low IFN-gamma and high TGF-beta levels. This study demonstrates that cellular protein(s) from human colon epithelial cells, but not from human fibroblasts, can induce oral tolerance in experimental colitis. This oral tolerance is mediated by primed mesenteric and splenic T lymphocytes.

Topics: Administration, Oral; Animals; Cell Extracts; Colitis; Colon; Colonic Neoplasms; Epithelium; Female; Fibroblasts; Humans; Immune Tolerance; Immunization, Passive; Interferon-gamma; Intestine, Small; Lymph Nodes; Peroxidase; Proteins; Rats; Rats, Sprague-Dawley; Rectum; Spleen; Tissue Extracts; Transforming Growth Factor beta; Trinitrobenzenesulfonic Acid; Tumor Cells, Cultured

Transforming growth factor beta1 (TGFbeta) inhibits cellular proliferation, promotes differentiation, and stimulates the expression and secretion of the extracellular matrix adhesion molecules fibronectin and laminin and the colon-associated intercellular adhesion molecule carcinoembryonic antigen. This is collectively called the TGFbeta-mediated adhesion response and occurs in the human colon cancer cell line Moser while the cell line KM12SM is relatively unresponsive to TGFbeta. We have previously shown that TGFbeta rapidly stimulates protein kinase C (PKC) phosphotransferase activity in the Moser cells and that the induction of the adhesion response (but not antiproliferation) by TGFbeta is dependent on PKC. Because resistance to growth factors may be due to translational suppression and the translation initiation factor eIF-4E may alleviate translational suppression, we determined the effect of eIF-4E expression on the responses of Moser and KM12SM cells to TGFbeta. Ectopic expression of eIF-4E in the TGFbeta-responsive Moser cells enhanced the activation of PKC by TGFbeta and the induction of the adhesion response, especially the secretion of adhesion molecules, but not the antiproliferative response. Ectopic expression of eIF-4E in the TGFbeta-resistant KM12SM cells increased TGFbeta stimulation of PKC and the TGFbeta-mediated adhesion response (but not antiproliferation). The secretion of adhesion molecules was significantly increased by TGFbeta. These results showed in these cells that eIF-4E promotes TGFbeta-regulated adhesion but not antiproliferation in a PKC-dependent manner.

Topics: Carcinoembryonic Antigen; Cell Adhesion; Cell Division; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Eukaryotic Initiation Factor-4E; Fibronectins; Humans; Laminin; Peptide Initiation Factors; Protein Binding; Protein Biosynthesis; Protein Kinase C; RNA, Messenger; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Solid tumors evade the host immunologic responses they initiate by unknown mechanisms. The authors investigated patterns of cytokine content in human colon carcinomas, colon cancer cell lines in vitro, and nude mouse xenografts from those lines in order to clarify those mechanisms.. Epithelial tumor cell lines were developed from specimens of human colon adenocarcinoma. Aliquots of these cells were then xenografted into female heterozygous BALB/c nu/+ immunologically deficient mice and serially passaged. Original tumors, cell lines, and resultant xenografts were then analyzed for histology/cytology and for levels of TGF-beta and TNF-alpha by enzyme linked immunoassay.. Cytokine levels were elevated beyond baseline mucosal levels in original tumors and xenograft mouse tumors but not detectable in extracts from epithelial cultures.. While the precise source of cytokine production remains unclear, these data suggest tumor/host interactions not found in pure epithelial cancer cells in culture.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The cytokine transforming growth factor beta-1 (TGFbeta1), was transfected into a TGFbeta1-negative rat colon carcinoma. The growth of isografts of TGFbeta1-expressing tumors was compared to that of vector control transfectants. The TGFbeta1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFbeta1-transfected tumor tissue had significantly greater infiltration of both CD4+ and CD8+ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFbeta1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFalpha) than did TIL from the vector control tumor. The TGFbeta1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFbeta1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFalpha on tumor proliferation in vitro. These results suggest that TGFbeta1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth.

Topics: Animals; Carcinoma; Cell Movement; Chimera; Colonic Neoplasms; Cytokines; Growth Inhibitors; Interleukin-10; Lymphocytes, Tumor-Infiltrating; Mice; Mice, SCID; Rats; Rats, Inbred F344; Rats, Inbred WF; Recombinant Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Actins; Animals; Bone Marrow Cells; Cell Survival; Coculture Techniques; Colonic Neoplasms; Fibroblasts; Humans; Immunohistochemistry; Mesoderm; Mice; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Colonic Neoplasms; Fibroblasts; Rats; Transforming Growth Factor beta

At least two separate genetic pathways of carcinogenesis in sporadic colon cancer involving the accumulation of mutations at various genetic loci have been described. About 15% of sporadic colorectal carcinomas arise via a mechanism associated with microsatellite instability (MSI) and mutations in transforming growth factor beta receptor II (TGFbetaRII), insulin-like growth factor II receptor (IGFIIR) and BAX, whilst the remaining 85% are associated with aneuploidy and gross chromosomal rearrangements. An 81-year-old woman had a sigmoid colon carcinoma resected and 18 months later developed two additional carcinomas of the caecum and transverse colon. To investigate whether there was a common genetic mechanism of carcinogenesis for the three lesions, MSI status was assessed, TGFbetaRII, IGFIIR and BAX were analysed for mutations and protein expression of transforming growth factor beta1 (TGFbeta1) and p53 were studied using immunohistochemistry. The caecal and transverse colonic carcinomas were both MSI positive but different mutations were identified in each lesion. No genetic abnormalities were identified in the sigmoid colonic carcinoma. This suggests that each carcinoma arose via a separate genetic mechanism of carcinogenesis.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; bcl-2-Associated X Protein; Colon; Colonic Neoplasms; DNA Mutational Analysis; Female; Genes, p53; Growth Substances; Humans; Microsatellite Repeats; Mutation; Neoplasms, Multiple Primary; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 2; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Many human tumor cells are resistant to growth inhibition by TGF beta 1. Resistance may be caused by mutations in TGFbeta receptors or in other components of the TGF beta signal transduction systems, or by knockout of the retinoblastoma (Rb) gene, which in fibroblasts converts cellular response to TGF beta 1 from growth inhibition to growth stimulation. Our earlier studies showed such a switch in response to TGF beta 1 occurred in 45% of colon cancers but without deletion of Rb. We now show that insulin-like growth factor binding protein 3 (IGFBP-3) mediates the TGF beta 1-induced proliferation of 3 metastatic or highly aggressive colon carcinoma cell lines. TGF beta 1 increases IGFBP-3 abundance while phosphorothiolated antisense oligonucleotides to IGFBP-3 block the growth-promoting effect of TGF beta 1 in each of 3 lines.IGFBP-3 induces carcinoma cell growth in a dose-dependent and time-dependent manner in vitro. IGFBP-3 may confer a selective growth advantage on tumor cells in vivo because levels of mature IGFBP-3 were elevated at least 2-fold in 7 of 10 resected colon cancers compared with adjacent normal tissue.

Topics: Adenocarcinoma; Cell Division; Colonic Neoplasms; Humans; Insulin-Like Growth Factor Binding Protein 3; Neoplasm Invasiveness; Oligonucleotides, Antisense; Recombinant Proteins; Thionucleotides; Transforming Growth Factor beta; Tumor Cells, Cultured

The association of transforming growth factor-beta 1 (TGF-beta 1) with a matrix metalloproteinase (MMP) and a tissue inhibitor of metalloproteinase (TIMP), as well as myometrial invasion of endometrial cancer was studied.. The effects of TGF-beta 1 on cellular invasiveness, gelatinase activity, and expression of TIMP-1 were examined in 2 endometrial adenocarcinoma cell lines, KLE and Ishikawa. Plasma was obtained from 8 endometrial cancer patients with Stage-Ia disease, from 6 with Stage-Ib disease, and from 4 with Stage-Ic disease, and the levels of TGF-beta 1 were measured by enzyme immunoassays. The immunohistochemical expression of MMP-9, TIMP-1, TGF-beta 1, and TGF-beta receptor Type I in tumor tissue from the same patients also was detected.. Invasiveness, gelatinase activity, and the expression of TIMP-1 were higher in KLE cells than in Ishikawa cells, and they were increased by treatment with rTGF-beta 1. The expression of TGF-beta receptor Type I was higher in KLE cells than in Ishikawa cells, which were unresponsive to exogenous TGF-beta 1. The plasma levels of TGF-beta 1 were greater in Stage-Ib and Stage-Ic patients than in Stage-Ia patients. MMP-9 and TGF-beta receptor Type I were expressed mainly in tumor cells, while TIMP-1 and TGF-beta 1 were localized in both tumor epithelial cells and stromal cells. MMP-9 and TIMP-1 were expressed only in Stage-Ib and Stage-Ic patients, although TGF-beta 1 and TGF-beta receptor Type I were ubiquitous.. Myometrial invasion of endometrial cancers involves an increase in gelatinase activity, regulated to some extent by TGF-beta 1 in an autocrine or paracrine fashion.

Topics: Adenocarcinoma; Colonic Neoplasms; Endometrial Neoplasms; Female; Gelatinases; Humans; Immunohistochemistry; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Myometrium; Neoplasm Invasiveness; Neoplasm Staging; Receptors, Transforming Growth Factor beta; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Tumor Cells, Cultured

Alterations in transforming growth factor beta1 (TGF-beta1) and its type II receptor (TbetaR-II) have been implicated in the pathogenesis of a variety of human cancers and animal tumor models. We postulated that TGF-beta1 and TbetaR-II alterations may also be involved in mouse colon tumorigenesis induced by the chemical carcinogen, azoxymethane (AOM). In the present study, normal colon tissues and AOM-induced colon tumors from SWR/J mice were analyzed for mutational changes in the TbetaR-II gene, and the expression and localization of TGF-beta1 and TbetaR-II were examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemisty. Direct DNA sequencing of the coding sequence of the TbetaR-II gene revealed no mutational changes in tumors induced by AOM when compared with the sequence from normal colon tissue. However, TGF-beta1 and TbetaR-II mRNA levels in tumor samples were increased 1.8-fold (p<0.01) and 1.3-fold (p<0.01), respectively, when compared with control mouse colon tissue. The results of immunohistochemical analysis of TGF-beta1 and TbetaR-II were correlated with mRNA expression data. An increase in staining intensity of both TGF-beta and TbetaR-II were observed in colon tumors. These findings suggest that alterations in the expression of TGF-beta1 and TbetaR-II may be involved in the pathogenesis of colon tumors induced by AOM in mice.

Topics: Adenocarcinoma; Amino Acid Substitution; Animals; Azoxymethane; Carcinogens; Codon; Colonic Neoplasms; DNA Mutational Analysis; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Male; Mice; Neoplasm Proteins; Point Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Transforming Growth Factor beta1

Fibrosis in normal tissues is a common and dose-limiting late complication of radiotherapy at many cancer sites, but its pathogenesis is poorly understood. We undertook a controlled study of the effect of irradiation on the collagen production of fibroblasts cultured from skin biopsies taken from patients undergoing radiotherapy treatment. Eight weeks after a single 8 Gy fraction using 300 kV X-rays, five patients treated at the Royal Marsden Hospital underwent biopsy of the irradiated site and of the contralateral, unirradiated body site. Fibroblasts from irradiated and control, unirradiated sites were cultured in vitro, and collagen production rates were measured during a 48-hour incubation under standardized conditions and in the presence and absence of transforming growth factor beta(1)(TGF beta(1)), 1 ng/ml, using HPLC. Collagen production was elevated in cells cultured from irradiated skin; median collagen production rates 61.16 pmoles hydroxyproline/10(5)cells/hour in irradiated cells, 39.78 pmoles hydroxyproline/10(5)cells/hour in unirradiated cells, P = 0.016 (Mann-Whitney U-test). In fibroblasts from unirradiated sites, collagen production rates were increased by the addition of TGF beta(1); however, in three of the cell lines cultured from irradiated sites this effect of TGF beta(1)on collagen production was not observed.

Topics: Aged; Aged, 80 and over; Biopsy; Breast Neoplasms; Cells, Cultured; Chromatography, High Pressure Liquid; Collagen; Colonic Neoplasms; Female; Fibroblasts; Flow Cytometry; Humans; Lung Neoplasms; Male; Middle Aged; Prostatic Neoplasms; Skin; Time Factors; Transforming Growth Factor beta

Transforming Growth Factor-beta 1 (TGF-beta 1) regulates the proliferation of normal epithelial cells, and resistance to TGF-beta 1 growth inhibition is a common feature of human cancers including lung cancer. In order to understand the mechanism of resistance to growth inhibition by TGF-beta 1 and to reverse the regulation of proliferation in lung cancer, we determined the genomic structure of the genes involved in the signal transduction pathway of TGF-beta 1 and performed an initial mutation survey of the complete coding region of the genes in lung cancer and cell lines with the resistance to growth inhibition by TGF-beta 1. First, a mutation analysis of the TGF-beta type II receptor (TGF-beta RII) was performed. Point mutations of the gene were detected in several colon cancers and an adenocarcinoma of the lung in the poly-A sequence. No mutations of Smad 2, 3, 4, 5 and TGF-beta type I receptor (TGF-beta IR) genes were detected in a series of the tumors we tested, although several mutations of Smad 2 and 4 were previously reported. Frequent alterations of the p15 gene and reduced expression of p21 we already reported from our previous studies. We also determined the genomic structure of the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), which is involved in activating TGF-beta 1, and performed an initial mutation survey of the complete coding sequences of the gene. A point mutation at exon 40 was found in one lung adenocarcinoma cell line. In summary, alterations in the many genes involved in the signal transduction of TGF-beta 1 were found and may mediate the loss of TGF-beta 1 responsiveness in lung cancer. The molecular targets for the regulation of the proliferation of lung cancer are thought to be p15, p21 and the transcriptional regulators.

Topics: Cell Division; Colonic Neoplasms; Drug Resistance, Neoplasm; Gene Deletion; Humans; Lung Neoplasms; Point Mutation; Polymerase Chain Reaction; Receptor, IGF Type 2; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor (TGF) beta1 has an antitumorigenic role in the gastrointestinal tract and may be associated with the development of colon neoplasia. In the present study we investigated whether TGF-beta1 production in mucosa is lower in the distal colon, which is where clinical evidence shows that the incidence of colon neoplasia is higher, and whether TGF-beta1 levels were lower in the mucosa of patients with colon adenoma. Production of colon mucosa TGF-beta1 was investigated by means of a 24-hour organ culture with biopsy specimens taken from different segments of the colon of 58 normal subjects by using an enzyme immunoassay. TGF-beta1 production in colon mucosa from locations near the site of sporadic adenoma was also investigated in 46 patients. TGF-beta1 production gradually increased from the rectum to the ascending colon in a statistically significant manner in both normal (r = 0.77, P < .0001) and adenoma-bearing (r = 0.8, P < .0001) mucosa. When TGF-beta1 production was compared between normal and adenoma-bearing mucosa, levels were lower in the latter, although statistically significant results were seen only in the transverse colon (P < .05). TGF-beta1 production has clear site dependency, being lowest in the rectum and highest in the ascending colon. Furthermore, low levels of TGF-beta1 are associated with the development of adenoma. Our results suggest the possibility that this site dependency is associated with the higher epidemiologic incidence of colon neoplasia in the distal colon.

Topics: Actins; Adenoma; Adult; Aged; Biopsy; Colon; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Male; Middle Aged; Organ Culture Techniques; Rectum; RNA, Messenger; Tissue Distribution; Transforming Growth Factor beta

The present study investigated the pathogenesis of desmoplastic stroma formation, which is characteristic of most bile duct carcinomas and other scirrhous carcinomas. Using immunohistochemical analysis, the expression of collagen types I and IV, laminin and TGF-beta1 was examined in human extrahepatic bile duct carcinoma and compared with gastric and colon carcinoma. In addition to delineating the basement membranes of carcinoma nests and blood vessels, collagen type IV was present along the thick bundles of collagenous fibers in the stroma of extrahepatic bile duct carcinoma and scirrhous gastric carcinoma. The immunoreactivity of collagen type IV was strong in the adjacent or surrounding interstitium of tumor cell nests, but was absent or weak in older, more central portions of the tumor that contained sclerotic collagen. In situ hybridization demonstrated active expression of collagen alpha1(IV) mRNA in extrahepatic bile duct carcinoma and scirrhous gastric carcinoma cells. These results suggest that, although collagen type IV is typically a component of the basement membrane, it is expressed in the interstitial stroma of extrahepatic bile duct carcinoma and scirrhous gastric carcinoma where it may play a role in desmoplastic stroma formation.

Topics: Adenocarcinoma; Basement Membrane; Bile Duct Neoplasms; Bile Ducts, Extrahepatic; Collagen; Colonic Neoplasms; Humans; Immunoenzyme Techniques; In Situ Hybridization; Laminin; RNA, Messenger; Stomach Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1

Stromal reaction is important for the growth of cancer both in primary and metastatic sites. To demonstrate this reaction during the hepatic metastasis of human colon carcinoma, we histologically investigated alterations to the distribution and phenotype of hepatic stellate cells (HSCs), the only mesenchymal cells in the liver parenchyma, using a nude mouse model. Intrasplenically injected colon carcinoma LM-H3 cells migrated into the space of Disse and underwent proliferation, in close association with hepatocytes and HSCs, at 2 days. At 14 days, HSCs were accumulated around the tumor mass and expressed alpha-smooth muscle actin, a marker for HSC activation. We next investigated in vitro the growth factors involved in the interactions between LM-H3 cells and HSCs. Conditioned medium of rat HSCs which underwent culture-induced activation contained platelet-derived growth factor (PDGF)-AB, hepatocyte growth factor (HGF) and transforming growth factor (TGF)-beta, and could augment LM-H3-cell proliferation and migration. Neutralizing antibodies against PDGF-AA and PDGF-BB and those against PDGF-BB and HGF inhibited proliferation and migration, respectively, of LM-H3 cells, whereas antibody against TGF-beta had no effect. LM-H3 cells expressed PDGF receptors-alpha and -beta and c-met. Conditioned medium of LM-H3 cells contained PDGF-AB, and could enhance HSC proliferation and migration. This augmenting effect was suppressed by treatment with anti-PDGF-AB antibody. The present study has demonstrated that bidirectional interactions involving PDGF and HGF take place in vitro between colon carcinoma cells and HSCs, raising the possibility that similar interactions might be involved in the stromal reaction during hepatic metastasis.

Topics: Adenocarcinoma; Animals; Cell Communication; Cell Division; Cell Movement; Cells, Cultured; Colonic Neoplasms; Culture Media, Conditioned; Female; Growth Substances; Hepatocyte Growth Factor; Hepatocytes; Humans; Liver; Liver Neoplasms; Mice; Mice, Nude; Platelet-Derived Growth Factor; Rats; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

Topics: Cell Line; Colonic Neoplasms; Cycloheximide; Dexamethasone; Enzyme Induction; Epithelial Cells; Glucocorticoids; Humans; Immediate-Early Proteins; Lymphocytes; Macrophages; Nuclear Proteins; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

We previously demonstrated that mutational inactivation of transforming growth factor beta type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor beta signaling blockade distal to RII. The transforming growth factor beta pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.

Topics: Adult; Colonic Neoplasms; Female; Humans; Male; Microsatellite Repeats; Middle Aged; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factorbeta1 (TGFbeta1) elicits a multitude of cellular responses from the epithelial-derived human colon cancer Moser cells. TGFbeta1 induces the expression of laminin and fibronectin, and previous studies show that the induction of fibronectin is functionally associated with the regulation of carcinoembryonic antigen (CEA) expression by TGFbeta1 (Huang and Chakrabarty, 1994, J Biol Chem 269:28764-28768). In this study we constructed antisense laminin chain-specific expression vectors and determined their efficacy in blocking the expression and the induction of the large multichain laminin molecule by TGFbeta1. We also determined the functional role of laminin in several TGFbeta1-mediated responses: growth inhibition, downmodulation of anchorage-independent growth, and cellular invasion. Expression of either antisense laminin chain A, B1, or B2 RNA resulted in a downmodulation of endogenous laminin mRNA expression and blocked the induction of laminin protein by TGFbeta1 without affecting the induction of other adhesion molecules such as fibronectin or CEA. It is concluded that antisense RNA directed to only one of the laminin chains was sufficient to disrupt the induction of the complex laminin molecule in quite a specific manner. Expression of antisense laminin RNA downregulated cellular adhesion to extracellular matrix (ECM) laminin and blocked the ability of TGFbeta1 to upmodulate adhesion to ECM laminin. Expression of antisense laminin RNA, however, did not alter the downregulating effect of TGFbeta1 on cellular proliferation, anchorage-independent growth, or cellular invasion, suggesting that the induction of laminin did not play a significant functional role in these TGFbeta1-mediated cellular responses. It is likely that other adhesion pathways may be involved in mediating the action of TGFbeta1 in this cell line.

Topics: Carcinoembryonic Antigen; Cell Adhesion; Cell Division; Colonic Neoplasms; Epithelial Cells; Extracellular Matrix; Fibronectins; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Laminin; Neoplasm Invasiveness; RNA, Antisense; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Evidence is accumulating which indicates that cyclooxygenase-2 (COX-2) is involved in the pathogenesis of colorectal cancer. We evaluated the expression of COX-2 in replication error-positive (RER) colon cancers, colon cancers metastatic to liver and azoxymethane (AOM)-induced rat colonic tumors. Immunohistochemistry showed that COX-2 was low to undetectable in normal human mucosa, but abundant in the RER adenocarcinomas we examined. COX-2 immunoreactivity in metastatic colon cancers was less abundant, but clearly detectable. In the colon of AOM-treated rats, COX-2 protein was not detectable in normal mucosa, but present in most of the epithelial cells comprising the tumors. The TGF-beta1 staining pattern in these human and rat tumors was similar to that observed for COX-2. The role of TGF-beta in RER adenocarcinomas is complex because of the increased mutation rate of TGF-beta type II receptors. Northern analysis showed abundant TGF-beta1 mRNA in AOM-induced tumors, but not in paired mucosa. TGF-beta1 induced the expression of COX-2 mRNA and protein in intestinal epithelial cells (IEC-6). Chronic TGF-beta1 treatment caused a TGF-beta-dependent overexpression of COX-2 in rat intestinal epithelial cells (RIE-1). TGF-beta1 may regulate COX-2 expression during the colonic adenoma to carcinoma sequence.

Topics: Animals; Azoxymethane; Carcinogens; Colon; Colonic Neoplasms; Cyclooxygenase 1; Cyclooxygenase 2; Enzyme Induction; Epithelial Cells; Humans; Immunohistochemistry; Intestinal Mucosa; Isoenzymes; Liver Neoplasms; Male; Membrane Proteins; Neoplasm Proteins; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred F344; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) acts as a potent inhibitor of cell growth and tumor progression but loss of this negative regulation can contribute to tumor development. Some studies have reported an association between disease progression and TGF-beta1 expression in patients with colorectal carcinoma, but their results were not always consistent.. Serum levels of TGF-beta1 were measured using an enzyme-linked immunoadsorbent assay in 121 consecutive patients with colorectal carcinoma and compared with TGF-beta1 serum levels in 31 healthy volunteers. Serum levels of TGF-beta1 also were measured in 50 patients who underwent curative surgical resection (part of the 121 preoperative patients) to compare their levels with preoperative serum levels of TGF-beta1.. Serum levels of TGF-beta1 in patients with colorectal carcinoma (45+/-15 ng/mL) (mean+/-the standard deviation) were significantly higher than those in the healthy control group (32+/-4 ng/mL) (P = 0.001). Serum levels of TGF-beta1 increased with increasing tumor stage (P < 0.01). Serum levels of TGF-beta1 were correlated significantly with depth of tumor invasion, lymph node metastasis, distant metastasis, and serum levels of carcinoembryonic antigen (CEA). Serum levels of TGF-beta1 tended to increase with increasing CEA (correlation coefficient = 0.21; P < 0.05). The mean serum level of TGF-beta1 in patients with colorectal carcinoma before surgery (45+/-14 ng/mL) (n = 50) significantly decreased to 34+/-7 ng/mL, which was within the normal range (32+/-4 ng/mL), after curative surgical resection of the tumor (P = 0.0000). Serum levels of TGF-beta1 after tumor resection decreased more significantly in patients with higher preoperative levels of TGF-beta1 (from 53+/-12 ng/mL to 36+/-6 ng/mL) (n = 30).. The results of the current study suggest that serum levels of TGF-beta1 in colorectal carcinoma patients may be associated with disease progression and may be used as a biomarker in the management of colorectal carcinoma patients. The authors believe further studies with a large number of patients for a longer follow-up period are necessary to conclude whether serum levels of TGF-beta1 carry significant clinical relevance.

Topics: Aged; Analysis of Variance; Biomarkers, Tumor; Carcinoembryonic Antigen; Case-Control Studies; Colonic Neoplasms; Disease Progression; Female; Humans; Male; Middle Aged; Neoplasm Staging; Prognosis; Rectal Neoplasms; Transforming Growth Factor beta

TGFbeta can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFbeta antimitogenic responses. Here we report that oncogenic Ras inhibits TGFbeta signaling in mammary and lung epithelial cells by negatively regulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFbeta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFbeta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. Our results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFbeta and Ras signals in normal cells, and for the silencing of antimitogenic TGFbeta functions by hyperactive Ras in cancer cells.

Topics: Animals; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Colonic Neoplasms; COS Cells; DNA-Binding Proteins; Dose-Response Relationship, Drug; Humans; Luciferases; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Models, Genetic; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; ras Proteins; Signal Transduction; Smad2 Protein; Smad3 Protein; Time Factors; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

After injury and loss of epithelial cells, intestinal barrier function is reestablished by migration of viable epithelial cells from the wound edge (restitution). Myofibroblasts are located close to the basal surface of epithelial cells. This study aimed to investigate the role of human colonic subepithelial myofibroblasts in epithelial restitution. Primary cultures of subepithelial myofibroblasts were established. Monolayers of the epithelial cell lines IEC-6 and T84 were "wounded" in a standard manner to create an in vitro model of restitution. Migration of epithelial cells across the wound edge was assessed following culture in myofibroblast-conditioned medium. Myofibroblast expression of transforming growth factor (TGF)-beta isoforms was examined using RT-PCR, and TGF-beta isoform bioactivity was assessed using Mv 1 Lu bioassay. Myofibroblast-conditioned medium, via a TGF-beta-dependent pathway, significantly enhanced migration of epithelial cells across the wound edge and significantly inhibited cell proliferation in wounded monolayers. Messenger RNA for TGF-beta1, -beta2, and -beta3 was detected in the myofibroblasts, and Mv 1 Lu bioassay showed the presence of predominantly bioactive TGF-beta3. This study shows that human colonic subepithelial myofibroblasts secrete predominantly bioactive TGF-beta3 and enhance restitution in wounded epithelial monolayers via a TGF-beta-dependent pathway.

Topics: Animals; Cell Division; Cell Line; Cell Movement; Colon; Colonic Neoplasms; Culture Media, Conditioned; Epithelial Cells; Fibroblasts; Gene Expression; Humans; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

We recently identified DPC4/Smad4 as a candidate tumor suppressor gene mutated or lost in one half of pancreatic carcinomas and in a subset of colon and biliary tract carcinomas. DPC4 plays a key role in signal transduction of the TGF-beta superfamily of molecules and inactivation of TGF-beta mediated growth inhibition is supposed to be the driving force for DPC4 inactivation in human tumors. However, DPC4 mediated tumor suppression by reconstitution of defective cells has not yet been reported. Here we show suppression of tumorigenicity in nude mice by stable reexpression of DPC4 in SW480 colon carcinoma cells. In vitro growth of DPC4-transfected cells was not affected and resistance towards TGF-beta mediated growth inhibition was retained. Instead, cells exhibited morphological alterations and adhesion and spreading were accelerated. These phenotypic changes were associated with reduced expression levels of the endogenous urokinase-type plasminogen activator (uPA) and plasminogen-activator-inhibitor-1 (PAI-1) genes, the products of which are implicated in the control of cell adhesion and invasion. In patients, high expression levels of uPA and PAI-1 correlate with poor prognosis. Thus, reduced expression of uPA and PAI-1 is consistent with suppression of tumorigenicity in DPC4 reconstituted cells. These results demonstrate DPC4's tumor suppressive function and suggest a potential role for DPC4 as a modulator of cell adhesion and invasion.

Topics: Animals; Cell Adhesion; Cell Division; Cell Movement; Colonic Neoplasms; DNA-Binding Proteins; Humans; Mice; Mice, Nude; Phenotype; Plasminogen Activator Inhibitor 1; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate NO synthesis in a variety of normal cells. In the present study, we investigated the influence of TGF-beta1 upon NO production in tumor cells and its consequences for tumor development. During the growth of PROb colon carcinoma cells intraperitoneally injected in syngeneic BDIX rats, intratumoral concentration of TGF-beta1 increases while NO concentration stays very low. Tumor regression induced by intraperitoneal injections of a lipid A is associated with a decrease in TGF-beta1 and an increase in NO intratumoral concentration. In these tumors, PROb tumor cells are the NO- and TGF-beta1-secreting cells. Using PROb cells transfected with an expression vector coding for TGF-beta1 antisense mRNA, we demonstrate in vitro that there is an inverse correlation between the amount of TGF-beta1 secreted and the ability of PROb cells to secrete NO. As the same results were obtained in the presence of an anti-TGF-beta type II receptor neutralizing antibody, and as exogenous TGF-beta1 is without any effect on NO secretion by PROb cells, TGF-beta1 apparently down-regulates NO synthesis in PROb cells by an intracellular mechanism. These results suggest that endogenous TGF-beta1 constitutes a potential target in a search for new antitumoral agents.

Topics: Activin Receptors, Type I; Animals; Carcinoma; Colonic Neoplasms; Down-Regulation; Female; Immunotherapy; Intracellular Fluid; Lipid A; Male; Neoplasm Transplantation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Serine-Threonine Kinases; Rats; Rats, Inbred Strains; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Antisense; Second Messenger Systems; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The transforming growth factor beta (TGF-beta) pathway is known to play an important role in both human and urine colon cancer. However, the staging, ligand specificity, and mechanism underlying the tumor suppressive activity of this pathway are unknown. We developed a mouse model for colon cancer that identifies an early role for TGF-beta1 in tumor suppression and implicates TGF-beta2 or TGF-beta3 in the prevention of metastasis. Analysis of the development of colon cancer in TGF-beta1 knockout mice pinpoints the defect to the hyperplasty/adenoma transition and reveals that the mechanism involves an inability to maintain epithelial tissue organization and not a loss of growth control, increased inflammatory activity, or increased genetic instability. These mice provide a unique opportunity to investigate the specific role of TGF-beta1 at this critical transition in the development of colon cancer.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli Protein; Animals; Apoptosis; beta Catenin; Biomarkers; Cecum; Cell Division; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Crosses, Genetic; Cytoskeletal Proteins; Disease Progression; DNA; DNA-Binding Proteins; DNA, Neoplasm; Genes, APC; Genetic Predisposition to Disease; Humans; Hyperplasia; Inflammation; Intestinal Mucosa; Mice; Mice, Knockout; Microsatellite Repeats; Neoplasm Metastasis; Nuclear Proteins; Specific Pathogen-Free Organisms; Trans-Activators; Transforming Growth Factor beta

We established in vitro heterotypic co-cultures of a human colon carcinoma cell line, HT-29, and a human hepatocyte line, tPH5CH, using transwells to investigate possible interactions between colon carcinoma cells and hepatocytes during the metastatic process. Co-culture, but not HT-29 conditioned medium, inhibited tPH5CH cell proliferation, and this inhibition was blocked by an anti-TGF-beta1 antibody. Significantly more activated TGF-beta1 was released by co-culture than by each cell line cultured alone, whereas there were no significant differences between the total TGF-beta1 released by the individual cultured cell lines and that released by co-culture. These data indicate that an interaction between human colon carcinoma cells and hepatocytes activates TGF-beta1 in vitro.

Topics: Carcinoma; Cell Communication; Coculture Techniques; Colonic Neoplasms; Humans; Liver; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor betas (TGF-betas) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-beta receptors have been shown to be indispensable for TGF-beta-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-beta1 and transfection of antisense TGF-beta1 to reduce TGF-beta1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-beta modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-beta system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-deltaRII and FET6-deltaRII), or with a vector containing the TGF-beta1 cDNA (CBS4-beta1S and FET-beta1S). Expression of the truncated RII reduced TGF-beta sensitivity, whereas overexpression of RII increased TGF-beta sensitivity. TGF-beta overexpression did not affect TGF-beta response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-deltaRII and CBS4-beta1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-deltaRII cells formed rapidly growing tumors, and FET-beta1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-beta signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-beta expression or TGF-beta signaling capability affects malignancy in a contextual manner.

Topics: Animals; beta-Galactosidase; Cell Division; Colonic Neoplasms; Culture Media, Conditioned; Genes, Reporter; Humans; Luciferases; Mice; Mice, Nude; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Time Factors; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Smad4/DPC4 is a tumor suppressor gene frequently mutated or deleted in pancreatic and metastatic colon cancers. Smad4 acts as a cofactor that binds transforming growth factor-beta (TGF-beta) receptor-activated Smad2 and Smad3 generating transcriptional complexes. Using SW480.7 colon carcinoma cells, defective in Smad4 function, we have investigated whether this loss plays a role in the resistance of colon cancer cells to the antiproliferative effects of TGF-beta. SW480.7 cells contain only one Smad4 allele, which we found encodes a wild type protein that is not expressed. We generated SW480.7 cells conditionally expressing Smad4 via an ecdysone-inducible system. Smad4 expression in these cells failed to rescue TGF-beta antiproliferative and gene responses (c-myc down-regulation and induction of p21/Cip1 and plasminogen activator inhibitor-1). SW480.7 cells contain an activated Ki-ras oncogene. Hyperactivation of Ras can inhibit Smad nuclear accumulation by their phosphorylation at mitogen-activated protein kinase sites. Co-transfection into SW480.7 cells of Smad4 together with a Ras phosphorylation-resistant Smad3 (but not with wild type Smad2, Smad3, adenomatous polyposis coli (APC), or TGF-beta type II receptor) restored the TGF-beta antiproliferative response. These results suggest that loss of Smad4 function by both deletion and silencing and inhibition of Smad2/3 function by a hyperactive Ras pathway jointly prevent TGF-beta antiproliferative responses in SW480.7 colon cancer cells.

Topics: Alleles; Animals; Base Sequence; Cell Division; Cell Line; Colonic Neoplasms; DNA Primers; DNA-Binding Proteins; Gene Silencing; Humans; ras Proteins; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

We have previously described a type I transforming growth factor (TGF)-beta receptor (TbetaR-I) polymorphic allele, TbetaR-I(6A), that has a deletion of three alanines from a nine-alanine stretch. We observed a higher than expected number of TbetaR-I(6A) homozygotes among tumor and nontumor DNA from patients with a diagnosis of cancer. To test the hypothesis that TbetaR-I(6A) homozygosity is associated with cancer, we performed a case-control study in patients with a diagnosis of cancer and matched healthy individuals with no history of cancer and who were identical in their gender and their geographical and ethnic background to determine the relative germ-line frequencies of this allele. We found nine TbetaR-I(6A) homozygotes among 851 patients with cancer. In comparison, there were no TbetaR-I(6A) homozygotes among 735 healthy volunteers (P < 0.01). We also observed an excess of TbetaR-I(6A) heterozygotes in cancer cases compared to controls (14.6% versus 10.6%; P = 0.02, Fisher's exact test). A subset analysis revealed that 4 of 112 patients with colorectal cancer were TbetaR-I(6A) homozygotes (P < 0.01). Using mink lung epithelial cell lines devoid of TbetaR-I, we established stably transfected TbetaR-I and TbetaR-I(6A) cell lines. We found that, compared to TbetaR-I, TbetaR-I(6A) was impaired as a mediator of TGF-beta antiproliferative signals. We conclude that TbetaR-I(6A) acts as a tumor susceptibility allele that may contribute to the development of cancer, especially colon cancer, by means of reduced TGF-beta-mediated growth inhibition.

Topics: Activin Receptors, Type I; Alleles; Analysis of Variance; Breast Neoplasms; Case-Control Studies; Colonic Neoplasms; Female; Genetic Predisposition to Disease; Germinoma; Heterozygote; Homozygote; Humans; Male; Neoplasms; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta

Although some in vitro studies indicate that macrophages exert cytotoxic responses against tumour cells by production of reactive oxygen intermediates (ROI), no obvious impairment of tumour cell growth is visible in various human malignant tumours, which contain a large number of tumour-associated macrophages (TAM). We made use of an in vivo-like co-culture model of multicellular tumour spheroids of three colon carcinoma cell lines (HRT-18, HT-29, CX-2) and three functionally different phenotypes of human macrophages (27E10, RM3/1, 25F9) to investigate if tumour cells deactivate macrophage cytotoxicity. The production of ROI was measured by a lucigenin-amplified chemiluminescence assay in a 96-well-microplate luminometer. Different capabilities to produce ROI by different macrophage phenotypes were observed. However, independent of the macrophage phenotype and the tumour cell type a significant inhibition of ROI formation was found in co-cultures after 1 hr, 1 and 2 days. Macrophages were also suppressed by tumour cell supernatants, which contained anti-inflammatory cytokines transforming growth factor-beta1 (TGF-beta1) and negligible levels of interleukin-4 (IL-4) and IL-10 as shown by enzyme-linked immunosorbent assay (ELISA). Although recombinant human cytokines TGF-beta1, IL-10 and IL-4 inhibited the production of ROI in freshly isolated monocytes, these cytokines had no effect on differentiated macrophage phenotypes, indicating that these cytokines are not involved in mediating tumour-induced suppression of ROI production by human macrophages.

Topics: Adenocarcinoma; Coculture Techniques; Colonic Neoplasms; Cytokines; Glucocorticoids; Humans; Interferon-gamma; Interleukin-10; Interleukin-4; Luminescent Measurements; Macrophage Activation; Macrophages; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured

We have devised a new drug screening assay to discover anti-cancer drugs which inhibit Ras-mediated cellular signals, by utilizing a Ras-responsive element (RRE)-driven reporter gene system. We found that hypothemycin, an anti-bacterial, reduces RRE-dependent transcription. Treatment of tumor cells with hypothemycin resulted in reduced expression of Ras-inducible genes, including MMP (matrix metalloproteinase)-1, MMP-9, transforming growth factor-beta (TGF-beta), and vascular endothelial growth factor (VEGF), but not that of the constitutively expressed gene, MMP-2. The results of zymography demonstrated that hypothemycin reduced the production of MMP-9 and MMP-3, another Ras-inducible MMP, in the culture medium. Hypothemycin selectively inhibits anchorage-independent growth of Ras-transformed cells in comparison with anchorage-dependent growth. These findings suggest that hypothemycin inhibits Ras-mediated cellular signaling. Daily treatment of tumor-bearing mice with hypothemycin resulted in significant inhibition of tumor growth. Since MMP-1, MMP-3 and MMP-9 play important roles in tumor invasion and TGF-beta and VEGF are involved in tumor angiogenesis, hypothemycin is considered to be an example of a new class of antitumor drugs, whose antitumor efficacy can be at least partly attributed to inhibition of Ras-inducible genes.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Colonic Neoplasms; Endothelial Growth Factors; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, Reporter; Humans; Lymphokines; Matrix Metalloproteinases; Mice; Mice, Inbred BALB C; Mice, Nude; ras Proteins; Signal Transduction; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Zearalenone

Transforming growth factor beta1 (TGFbeta1) inhibits cellular proliferation and induces the expression of the matrix adhesion molecules fibronectin (FN) and laminin (LM) in a concurrent manner, followed by the induction of the intercellular adhesion molecule carcinoembryonic antigen (CEA) (collectively designated as adhesion responses) in TGFbeta1-responsive human colon carcinoma cells. Exactly how TGFbeta1 controls cellular adhesion and proliferation is poorly understood. In the present report, we showed that down-regulating protein kinase Calpha (PKCalpha) expression blocked the induction of these adhesion responses by TGFbeta1, showing that PKCalpha is a postreceptor focal point controlling the induction of these molecules. Down-regulating PKCalpha expression, however, had minimal effect on the antiproliferative response to TGFbeta1 or the induction of p21/WAF1, a marker associated with the antiproliferative effect of TGFbeta1, demonstrating that the adhesion signal pathway is distinct from that of antiproliferation. Blockade of FN induction blocked the induction of CEA but not the induction of LM. Blockade of LM induction, on the other hand, had no effect on the induction of FN and CEA. These results established the existence of two distinct and parallel postPKCalpha adhesion signal pathways, one leading to the induction of LM and the other leading to the induction of FN and CEA.

Topics: Carcinoembryonic Antigen; Cell Adhesion; Cell Division; Colonic Neoplasms; Fibronectins; Humans; Isoenzymes; Laminin; Protein Kinase C; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.

Topics: Adenocarcinoma; Adoptive Transfer; Animals; Bone Marrow Transplantation; Colonic Neoplasms; Graft vs Host Disease; Immunosuppressive Agents; Interleukin-2; Intestines; Killer Cells, Natural; Liver; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, SCID; Skin; Time Factors; Transforming Growth Factor beta; Transplantation, Homologous

Although transforming growth factor (TGF)-beta1 is a potent growth inhibitor of normal epithelial cells including colonocytes, TGF-beta1 has also been implicated as an enhancer of colon cancer metastasis. Decreasing TGF-beta1 protein levels in the metastatic U9 colon cancer cell line by antisense methodology decreased both U9 cell metastasis to the liver and s.c. tumor formation in a nude mouse system, and the tumors that did arise had regained TGF-beta1 expression (F. Huang et al, Cell Growth Differ., 6: 1635-1642, 1995). In addition, in a clinical immunohistochemistry study, colon cancers with elevated TGF-beta1 protein levels were found to be 18 times more likely to recur as distant metastases than colon cancers expressing low TGF-beta1 levels, after resection of the primary tumor (E. Friedman et al, Cancer Epidemiol. Biomark. Prev., 4:549-554, 1995). Because both studies implicated TGF-beta1 in colon cancer metastasis, we wished to know whether a selection bias for TGF-beta1 was maintained in metastatic cells or was only a property of the primary site tumors that were likely to metastasize. TGF-beta1 levels were measured using two different antibodies in paired primary site cancers and their metastases by immunohistochemistry and, in selected cases, by Western blot analysis. In 16 of 21 cases (76%) with antibody G and 23 of 31 cases (74%) with antibody P, higher expression of TGF-beta1 was found in colon cancer cells invading local lymph nodes compared with primary site colon cancer cells, or (2 and 6 cases, respectively) high TGF-beta1 expression in the primary site cancer was maintained in invasive cells. Analysis by Western blotting using both antibodies also demonstrated that higher levels of TGF-beta1 protein were found in metastases compared with the primary site tumor or normal tissue. Additional cases of paired primary site colon cancer, local lymph node metastases, and cancer cells metastasizing to distant sites were examined. In six of eight such cases (75%), TGF-beta1 levels were increased in both invasive cell populations compared with the primary site cancer (five cases), or high levels in the primary site cancer were maintained in the metastatic cells (one case). These data suggest that TGF-beta1 plays a role in promoting colon cancer metastasis throughout the metastatic process in roughly 75% of cases. TGF-beta1 may increase metastasis by paracrine mechanisms, such as suppression of local immune response or increased angiogenesis, as wa

Topics: Blotting, Western; Carcinoma; Colonic Neoplasms; Humans; Immunohistochemistry; Liver Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Cells, Cultured

The effect of phenylbutyrate (PB), a nontoxic differentiation inducer, in human colon carcinoma cell lines treated with 5-fluorodeoxyuridine (FUdR) was evaluated. Two HT-29 human colon carcinoma subclones (U4 well differentiated and U9 poorly differentiated) were equally growth inhibited by 16 h of FUdR (0.2 microM) treatment but recovered cell growth in 3-6 days after the removal of FUdR. PB as a single agent had minimal effect on cell growth, but after FUdR treatment, PB inhibited cell growth for 12 days. The inhibition of cell growth in FUdR-treated cells by PB was more sustained in U4 than U9 cells and was associated with an increased and sustained expression of p21waf1 protein, secretion of transforming growth factor beta1, mediators of p53-dependent or -independent G1 cell cycle arrest, and an increase in the alkaline phosphatase activity as well, considered a marker of differentiation in colon carcinoma cells. These effects of PB were seen only in FUdR-pretreated cells because PB alone had minimal effect on the expression of these genes. The sequential use of FUdR followed by PB in patients with colon carcinoma should be explored because two subclones of HT29, irrespective of their state of differentiation, respond to this clinically achievable regimen.

Topics: Alkaline Phosphatase; Antimetabolites, Antineoplastic; Cell Differentiation; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; Floxuridine; HT29 Cells; Humans; Phenylbutyrates; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta1 (TGF-beta1) is a cytokine known to play a key role in the control of cell growth. TGF-beta1 potently inhibits the proliferation of human and rodent-derived epithelial cells. Colonic precancerous and moderately differentiated cancer cells are responsive to TGF-beta1, whereas malignant colon cancer cells are resistant to the inhibitory action of the cytokine. These observations have been derived exclusively from in vitro studies. Therefore, the main aim of our study was to determine whether TGF-beta1 exerts a growth-restraining action on colon carcinogenesis in vivo. TGF-beta1 was sequestered into ethylene acetate copolymer matrices and "loaded" preparations were implanted intraperitoneally (i.p.) in rats. One week later, the animals were treated with dimethylhydrazine (DMH), a colon procarcinogen. Empty matrices devoid of TGF-beta1 but containing bovine serum albumin (BSA) carrier served as the appropriate control preparations. The number of aberrant crypt foci (ACF), considered to be preneoplastic lesions of the colon, was scored. Tumor formation and size were assessed at the appropriate times. TGF-beta1 released in a sustained manner from copolymer matrices: (i) markedly inhibited colonic ACF formation and the number of aberrant crypts and (ii) significantly reduced colonic tumor formation and size.

Topics: 1,2-Dimethylhydrazine; Animals; Colonic Neoplasms; Drug Implants; Male; Polymers; Precancerous Conditions; Rats; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Invasive growth of epithelial tumor cells, a major cause of death from cancer in humans, involves loss of epithelial polarity and dedifferentiation. Transforming growth factor beta (TGFbeta) is regarded as a major tumor suppressor during early tumor development because it inhibits cell-cycle progression and tumor growth. Many dedifferentiated, late-stage tumors are resistant to growth inhibition by TGFbeta, however, and even secrete TGFbeta. In line with this, TGFbeta is involved in angiogenesis, wound healing and epithelial-mesenchymal transition (EMT) during development. Ha-Ras-transformed mammary epithelial cells (EpRas) undergo TGFbeta-induced EMT maintained via a TGFbeta autocrine loop. Thus, we have analyzed whether signal transduction by the TGFbeta receptor (TGFbetaR) is required for local tumor cell invasion and metastasis.. A dominant-negative type II TGFbetaR (TGFbetaRII-dn) was expressed using retroviral vectors in EpRas cells and highly metastatic mesenchymal mouse colon carcinoma cells (CT26). In both cell types, TGFbetaRII-dn blocked TGFbetaR signaling and heavily delayed tumor formation. In EpRas cells, TGFbetaRII-dn prevented EMT. In the dedifferentiated mesenchymal CT26 cells, TGFbetaRII-dn caused mesenchymal-to-epithelial transition and inhibited their in vitro invasiveness in several assays. In addition, TGFbetaRII-dn completely abolished metastasis formation by CT26 cells. Furthermore, several human carcinoma lines lost in vitro invasiveness when treated with neutralizing TGFbeta antibodies or soluble receptor variants. Finally, human colon carcinoma cells (hnPCC) expressing a mutated, non-functional TGFbetaRII were non-invasive in vitro, a defect restored by re-expressing wild-type TGFbetaRII.. Cell-autonomous TGFbeta signaling is required for both induction and maintenance of in vitro invasiveness and metastasis during late-stage tumorigenesis. TGFbetaRII therefore represents a potential target for therapeutical intervention in human tumorigenesis.

Topics: Animals; Cell Cycle; Colonic Neoplasms; Epithelial Cells; Humans; Mesoderm; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Macrophage migration inhibitory factor (MIF) was recently rediscovered as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. MIF is constitutively expressed in various cells and enhances production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interferon gamma. Recently, it was reported that MIF mRNA was overexpressed in prostatic tumors, which suggests that MIF is a protein involved in tumor cell growth beyond inflammatory and immune responses.. We examined the expression of MIF in the murine colon carcinoma cell line colon 26 by Western and Northern blot analyses and immunohistochemistry. Next, we investigated the effects of transforming growth factor (TGF) beta, basic fibroblast growth factor (b-FGF), and platelet-derived growth factor (PDGF) on the expression of MIF mRNA. Furthermore, we examined whether MIF is involved in tumor cell proliferation, using an MIF anti-sense plasmid transfection technique.. We demonstrated that MIF protein and its mRNA were highly expressed in colon 26 cells, using Western and Northern blot analyses, respectively. By immunohistochemical analysis, we found that MIF was localized largely in the cytoplasm of the tumor cells. In response to TGF-beta, b-FGF, and PDGF, MIF mRNA expression was significantly up-regulated. Following this, we transfected the cells with an anti-sense MIF plasmid, which revealed that this treatment induced significant suppression of cell proliferation.. Although MIF plays multifunctional roles in a broad spectrum of pathophysiological states, little has been done to investigate the role of this protein in association with tumor growth. The current results suggest the possibility that MIF induces tumor cell growth in concert with other growth factors, which encouraged us to investigate a novel approach for tumor therapy using an anti-MIF antibody and an MIF anti-sense plasmid transfection technique.

Topics: Animals; Cell Division; Colonic Neoplasms; DNA, Antisense; Fibroblast Growth Factor 2; Gene Expression; Immunohistochemistry; Macrophage Migration-Inhibitory Factors; Mice; Plasmids; Platelet-Derived Growth Factor; RNA, Messenger; RNA, Neoplasm; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The results of our previous studies of mouse embryo fibroblasts showed that fibronectin expression and fibronectin receptor expression are tightly coregulated and that fibronectin modulates expression of its receptor in response to treatment with the differentiation-inducing agent N,N,-dimethylformamide (Varani and Chakrabarty, 1990, J. Cell. Physiol., 143:445-454; Huang et al., 1994, J. Cell. Physiol., 161:470-482). We also found that transforming growth factor beta1 (TGFbeta1) induces a more differentiated phenotype in the epithelium-derived human colon carcinoma cell line Moser and upregulates the expression of both fibronectin and its receptor (Huang and Chakrabarty, 1994, Int. J. Cancer, 57:742-746). By expressing antisense fibronectin RNA in Moser cells, we have downregulated fibronectin mRNA expression and thus blocked the ability of TGFbeta1 to induce fibronectin expression (Huang and Chakrabarty, 1994, J. Biol. Chem., 269:28764-28768). In this study, we examined the effect of antisense fibronectin RNA expression on the induction of fibronectin receptor by TGFbeta1 and tested the hypothesis that the induction of fibronectin expression by TGFbeta1 is required for the induction of fibronectin receptor expression. Blocking fibronectin induction by TGFbeta1 attenuated the ability of TGFbeta1 to upregulate the expression of cell-surface fibronectin receptors, alpha5beta1 integrin expression, and adhesion to extracellular matrix fibronectin. We therefore conclude that induction of fibronectin expression is required for optimal upregulation of fibronectin receptor expression by TGFbeta1.

Topics: Animals; Cell Membrane; Colonic Neoplasms; Down-Regulation; Fibronectins; Gene Expression Regulation; Humans; Kinetics; Mice; Receptors, Fibronectin; Recombinant Proteins; RNA, Messenger; Swine; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Up-Regulation

We investigated both the effect and the mechanism of oral (p.o.) administration of PSK, a protein-bound polysaccharide derived from Basidiomycetes, on the anti-tumor T-cell response in gut-associated lymphoid tissue (GALT). The p.o. administration of PSK significantly suppressed the growth of colon 26 carcinoma (C-26) inoculated into the subserosal space of the cecum (i.c.), and augmented the tumor-neutralizing activity of the draining mesenteric lymph node (LN) cells. PSK treatment also significantly decreased the levels of immunosuppressive factors such as plasma transforming growth factor (TGF)-beta in the i.c. C-26-inoculated mice. We also evaluated the improving effect of PSK on the anti-tumor T-cell response in GALT by utilizing B7-transfected P815 mastocytoma (B7/P815). The PSK treatment promoted the rejection of i.c.-inoculated B7/P815 and restored the CD4+ T-cell-dependent proliferative response of the draining mesenteric LN cells against in vitro restimulation. Furthermore, the treatment also decreased the TGF-beta production but increased the IFN-gamma production of these cells. The p.o. administration of PSK, however, showed no effect in the CD8+ T-cell-dependent cytolytic activity of the draining mesenteric LN cells after in vitro restimulation. Overall, these results indicate that the p.o. administration of PSK can improve the impaired anti-tumor CD4+ T-cell response in GALT, mainly through a suppression of TGF-beta production and a restoration of IFN-gamma production.

Topics: Administration, Oral; Animals; Antibiotics, Antineoplastic; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Female; Humans; Immunologic Factors; Immunosuppression Therapy; Interferon-gamma; Lymph Nodes; Mesentery; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Proteoglycans; Specific Pathogen-Free Organisms; Transforming Growth Factor beta

The host-tumor interaction may play an important role in determining tumor progress. Recent studies have shown that this interaction can be influenced by the release of soluble factors by tumor cells and tumor-infiltrating lymphocytes (TIL). The aim of our study is to characterize the nature of cytokines and growth factors and their relationship to the cellular infiltrates in 16 patients with ovarian cancer using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Total RNA from 20 malignant and 10 benign specimens were used to assay for expression of 12 cytokines. Additionally, monoclonal antibodies (MAbs) were used to detect T cells, CD4+ helper and CD8+ cytotoxic/suppressor T-cell subtypes, B cells, and macrophages. Our results showed the expression of transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in 19, 17, and 10 malignant specimens, P < .001, .001, and .05, respectively. Other cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), TNF-beta/LT, IL-2, and IL-6 were expressed in a few cases, and IL-1alpha and IL-4 expression were not detected. The benign samples did not express IL-10, but GM-CSF, TGF-beta1, and IL-8 were expressed in one, one, and four specimens, respectively. Interestingly, in four cases in which samples from the primary and relapse tumors were available for analysis, the tumors in relapse showed a significant increase for TGF-beta1 (P < .05) and a decreased trend in IL-10 mRNA levels. The source of these factors was tumor cells as detected immunohistochemically. This combined alteration of TGF-beta1 and IL-10 was associated with a significant reduction in number of TIL in general, and CD8+ and macrophages in particular (P = .036 and .049, respectively). Our findings suggest the important role of certain soluble factors in the complex process of tumor progression. Furthermore, understanding the tumor-host relationship and the factors influencing the interaction may be helpful in developing effective and innovative treatment methods.

Topics: Adenocarcinoma; Adult; Aged; Colonic Neoplasms; Cytokines; DNA Primers; Esophageal Neoplasms; Fallopian Tube Neoplasms; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunohistochemistry; Interferon-gamma; Interleukins; Lymphocytes, Tumor-Infiltrating; Middle Aged; Ovarian Neoplasms; RNA; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Many studies have been conducted to assess the potential preneoplastic nature of colonic aberrant crypt foci (ACF), but still the biological significance of these foci and their relationship to colon neoplasia remains to be elucidated. In the present paper a battery of variables suggested to be indicative for colon cancer development has been studied in relation to ACF in rats. These include: (i) the degree of dysplasia; (ii) the type of mucus production; (iii) the cellular immunohistochemical expression and distribution of transforming growth factors alpha and beta and their respective receptors, epidermal growth factor receptor and transforming growth factor beta receptors I and II and phosphorylated cellular tyrosine. The parameters have been investigated in ACF selected from a previous study where the foci were induced under different circumstances, leading to disparities in the number as well as the crypt multiplicity obtained. The present study showed that for all parameters investigated, apart from sialomucin production, the different experimental conditions had no effect on the individual ACF, irrespective of the number and distribution of the different categories of ACF among the various diets. However, it was shown that the degree of dysplasia correlated strongly with crypt multiplicity and that all the investigated ACF lacked expression of transforming growth factor alpha and expressed a reduced amount of transforming growth factor beta compared with normal crypts. These observations may indicate that ACF are preneoplastic lesions and supports the suggestion that they may, at least in the rat, have the potential to gradually progress to tumors, but no single ACF showed particular characteristics indicating specific proneness to tumor development. The study could not confirm the presence of sialomucin-producing ACF as a valid marker for tumor development.

Topics: Animals; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Dietary Fats; Dietary Fiber; Dietary Sucrose; ErbB Receptors; Gene Expression Regulation; Growth Substances; Intestinal Mucosa; Male; Mucins; Precancerous Conditions; Rats; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; Sialomucins; Transforming Growth Factor alpha; Transforming Growth Factor beta

The aim of this study was to evaluate a possible relationship between oxidative stress and transforming growth factor beta 1 (TGF beta 1) expression in human colon adenocarcinoma. Crohn's disease, an inflammatory pathology of the intestine often regarded to as precancerous, was also examined. Indices of impaired redox balance were monitored in blood and in bioptic samples from 10 adult patients with adenocarcinoma of the colon and from five patients with Crohn's disease. On tissue samples TGF beta 1 mRNA expression was also determined. Ten healthy adults provided normal reference values for plasma indices of oxidative stress, and normal tissue distant from the lesions was used for comparative analysis. Fluorescent adducts with plasma proteins of malonaldehyde (MDA) and 4-hydroxynonenal (HNE) were significantly lower than controls in the plasma from cancer patients and significantly higher in the plasma from Crohn's patients. In adenocarcinoma biopsies, susceptibility to lipid peroxidation processes and TGF beta 1 expression were below the relative control; in Crohn's disease, lipid peroxidation and cytokine expression were both above the relative control. The findings obtained suggest the existence of an association between oxidative damage and fibrogenic cytokine expression in the human intestine. Further studies are needed to conclusively prove the correlation between the two events.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Case-Control Studies; Colonic Neoplasms; Crohn Disease; Female; Gene Expression; Humans; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Precancerous Conditions; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-beta) binds the type II TGF-beta growth factor receptor (RII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the RII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of RII mutations, we selected 24 human cancer cell lines from various tissues which were previously reported to be resistant to the inhibitory effects of TGF-beta. We developed protocols for non-isotopic SSCP analysis of PCR products from genomic DNA samples, and we tested them for microsatellite instability. PCR-SSCP analysis followed by DNA sequencing identified deletion mutations in the exon 3 poly-adenine tract in three colon tumor cell lines: LS174T and SW48 had a single base deletion and LS411 had a two base deletion. Among the 24 previously unreported cell lines, only these three demonstrated microsatellite instability. These and other recent data indicate that RII mutations are essentially confined to colon and gastric cancers with microsatellite instability. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.

Topics: Cell Division; Colonic Neoplasms; DNA Mutational Analysis; Humans; Microsatellite Repeats; Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Stomach Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Repair of colonic epithelial erosions requires cell migration. This study aimed to examine the effects of physiologically relevant short-chain fatty acids on migration in colonic epithelial cell lines.. Butyrate, propionate, and acetate were added to confluent monolayers of LIM1215 colon cancer cells after wounding. Migration in circular wounds was assessed after 24 hours.. The migration of LIM1215 cells was stimulated in a concentration-dependent manner by all short-chain fatty acids. In four experiments, 2 mmol/L butyrate, 8 mmol/L propionate, and 16 mmol/L acetate induced 112.6% +/- 6.7%, 98.5% +/- 5.4%, and 63.4% +/- 7.2% (mean +/- SEM) stimulation above control migration, respectively. Their effects were additive at submaximal concentrations and reversible. Butyrate also stimulated migration in two other colon cancer cell lines, Caco-2 and LIM2405. However, butyrate failed to stimulate the migration of nongastrointestinal and nonepithelial cell lines. The stimulatory effect of butyrate required protein and RNA synthesis but was independent of cell proliferation, presence of serum, beta-oxidation, transforming growth factor beta, intracellular acidification, and substratum composition.. In wounded in vitro models of colonic epithelium, short-chain fatty acids promote cell migration. If such an effect occurs in vivo, it would have ramifications for the biology and pathobiology of the colonic mucosa.

Topics: Animals; Caco-2 Cells; Carbon Radioisotopes; Cell Division; Cell Movement; Colonic Neoplasms; Cycloheximide; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Epithelium; Fatty Acids, Volatile; Fibroblast Growth Factors; Fibrosarcoma; Humans; Hydrogen-Ion Concentration; L-Lactate Dehydrogenase; Leucine; Lung; Mink; RNA; Thymidine; Transforming Growth Factor beta; Tritium; Tumor Cells, Cultured

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Cytokines; DNA Primers; Ephrin-A1; Epidermal Growth Factor; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Growth Substances; Humans; Interferon-gamma; Interleukin-1; Interleukins; Intestinal Mucosa; Membrane Proteins; Polymerase Chain Reaction; Protein Biosynthesis; Protein-Tyrosine Kinases; Receptor, EphA2; Recombinant Proteins; RNA, Messenger; Swine; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Cytokines released from tumour cells may have function as signals to neighbouring immune and inflammatory cells. Several studies have shown that the immunoregulatory cytokines IL-10 and transforming growth factor-beta1 (TGF-beta1) as well as prostaglandin-E2 (PGE2) play an important role in tumour-induced immunosuppression. The aim of the study was to investigate the effect of colon carcinoma cell lines on IL-10 production in peripheral monocytes (PBMC) and lamina propria mononuclear cells (LPMC). We examined four colon carcinoma cell lines (HT-29, Caco-2, Colo-320 and HCT-116) and determined their production of TGF-beta1, IL-10 and PGE2. Peripheral monocytes were isolated by density gradient centrifugation and LPMC were isolated from surgical specimens using a collagenase digestion method. Monocytes and LPMC were cultured with colon carcinoma cell conditioned medium or in co-culture with colon carcinoma cells. Supernatants were then determined for the production of IL-10 by ELISA assays. All colon carcinoma cell lines stimulated peripheral monocytes as well as LPMC to produce markedly increased levels of IL-10. Colon cancer cells secreted negligible levels of IL-10, but high amounts of TGF-beta1 and PGE2. Neutralization of TGF-beta1 by administration of anti-TGF-beta as well as neutralization of PGE2 with anti-PGE2 antisera reduced the IL-10 production of monocytes markedly, indicating that tumour cell-derived TGF-beta1 and PGE2 are major factors for IL-10 stimulation. In vitro stimulation of monocytes with TGF-beta1 and PGE2 could confirm that TGF-beta1 as well as PGE2 at picogram concentrations were able to prime monocytes for enhanced IL-10 production. Our results demonstrate that colon carcinoma cell lines enhance the ability of monocytes and intestinal macrophages to produce IL-10. The stimulation of monocyte IL-10 by colon cancer cell-derived TGF-beta1 and PGE2 may act as a tumour-protecting mechanism by impairing the activation of anti-tumour cytokines.

Topics: Carcinoma; Colonic Neoplasms; Culture Media, Conditioned; Dinoprostone; Humans; Interleukin-10; Monocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta proteins are key regulators of cellular growth and differentiation. To understand the role of TGF-beta in colonic tumour progression, 47 paraffin embedded samples from colonic tumours (13 adenomas, and 34 adenocarcinomas) were studied. Gene mutations in the region coding for the active protein were studied by PCR SSCP analysis of exons 5, 6, and 7. TGF-beta1 mRNA expression and localization were studied by NISH using cDNA probes generated by RT-PCR. Protein distribution was investigated by immunohistochemistry using antibodies against both intracellular and extracellular forms. Three mutations were found: one in exon 5 (Dukes C) and two in exon 7 (Dukes C and A). TGF-beta1 mRNA was observed in 9 (69%) of 13 adenomas and in 30 (88%) of 34 adenocarcinomas. Levels of TGF-beta1 mRNA and proteins were higher in colorectal carcinomas than in colorectal adenomas. Tubular adenomas associated with dysplasia showed greater expression of TGF-beta1 than adenomas without dysplasia and than non-neoplastic colonic mucosa. In dysplasia and cancer, epithelial neoplastic cells and stromal cells were positive for TGF-beta1. In normal tissue endothelial cells and granulocytes sporadically showed immunoreactivity for TGF-beta1, whereas epithelial cells were all negative. The three mutations in TGF-beta1 exons 5, 6 and 7 found in colorectal adenocarcinomas suggest that TGF-beta1 mutation may play a role in colorectal carcinogenesis and that the presence of the mutant form contributes to the transformed state. Our two other findings, the loss of staining gradient in normal colonic mucosa and the higher levels of TGF-beta1 mRNA and proteins in colorectal carcinomas than in colorectal adenomas, indicate that the de-regulation of TGF-beta1 expression may occur as an early event in colorectal carcinogenesis. Although TGF-beta1 expression is generally a reflection of cellular differentiation, tumour grade is not associated with TGF-beta1 mRNA expression. Beside being present in the epithelial cells of the colonic tumours, TGF-beta1 mRNA also occurred in the stroma: its localization in the macrophages adjacent to tumour strongly suggests that TGF-beta1 could be secreted by macrophages. This hypothesis should lead to new therapeutic strategies for colorectal carcinoma.

Topics: Adenocarcinoma; Adenoma; Colonic Neoplasms; DNA Probes; DNA, Neoplasm; Exons; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Mutation; Paraffin Embedding; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; RNA, Messenger; Transforming Growth Factor beta

The high incidence of colorectal cancer in Western society is believed to be strongly related to diet. Mutation of the p53 gene is a late event in colorectal carcinogenesis, and thus, the majority of pre-malignant adenomas express wild-type p53. As loss of p53 protein function is an important step in colorectal carcinogenesis, we investigated whether naturally occurring lumenal factors can modulate the expression of p53 in non-tumorigenic human colonic adenoma cell lines. Levels of p53 protein and mRNA were measured in adherent cells which had been incubated with growth-inhibitory concentrations of sodium butyrate (a by-product of dietary fibre fermentation) or sodium deoxycholate (a bile acid) for up to 48 hr. We report that both butyrate and deoxycholate can down-regulate the expression of wild-type and mutant p53. In contrast, incubation for 48 hr with the endogenous inhibitory growth factor TGFbeta1 did not alter p53 protein expression. Thus, in addition to cellular mechanisms which regulate p53 function, such as post-translational stabilisation, nuclear exclusion, negative feedback inhibition of p53 mRNA translation or binding of p53 by cellular proteins, p53 protein levels also may be regulated by changes in the level of p53 gene transcription. Furthermore, we show that lumenal factors are able to affect directly the expression of p53 protein in colonic epithelial cells.

Topics: Adenoma; Butyrates; Butyric Acid; Cell Division; Cholagogues and Choleretics; Colonic Neoplasms; Deoxycholic Acid; Down-Regulation; Gene Expression Regulation, Neoplastic; Histamine Antagonists; Humans; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

This study used a co-culture system with Transwell tissue-culture inserts to investigate the role of primary cultures of rat peritoneal mesothelial cells on the proliferation of rat colon-carcinoma cells (CC531 cells). Mesothelial cells significantly inhibited the growth of CC531 cells, while, conversely, CC531 cells stimulated the growth of mesothelial cells. Receptor-binding studies demonstrated the presence of high-affinity IGF-I receptors on the mesothelial and CC531 cells. Both cell types also produced IGF-I, as measured by radioimmunoassay. IGF-I stimulated DNA synthesis in mesothelial cells, but had no effect on the growth of CC531 cells. In co-culture, it was found that IGF-I potentiated the inhibitory effect of mesothelial cells on CC531 cells. The effect of IGF-I on mesothelial-cell proliferation was additive to the stimulatory effect of CC531 cells. TGF-beta had no effect on the growth of the CC531 cells, suggesting that this growth (-inhibitory) factor is not involved in the inhibitory effect of mesothelial cells on CC531 cell growth. The study provides evidence for the existence of a paracrine loop between mesothelial and colon-carcinoma cells, giving more insight into the basic cellular mechanisms that may modulate the growth of intraperitoneal colon carcinoma. Inhibition of CC531-cell proliferation by rat mesothelial cells might explain the earlier finding that tumour cells grow poorly in a surgically uncompromised abdomen.

Topics: Animals; Cell Division; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; Culture Media, Conditioned; Disease Models, Animal; Dose-Response Relationship, Drug; Epithelial Cells; Immunohistochemistry; Insulin-Like Growth Factor I; Keratins; Male; Paracrine Communication; Rats; Rats, Inbred Strains; Receptor, IGF Type 1; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin; von Willebrand Factor

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.

Topics: Carcinoma; Cell Nucleus; Codon, Terminator; Colonic Neoplasms; Drug Resistance, Neoplasm; Frameshift Mutation; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombination, Genetic; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Serum transforming growth factor beta 1 (TGF beta 1) concentration was determined in 47 patients with primary hepatic carcinoma (PHC) and 77 patients with various liver diseases and gastrointestinal tumors by means of enzyme-linked immunosorbent assay system. The result revealed that serum TGF beta 1 level of PHC patients were significantly higher (257.6 +/- 126.0 micrograms/L) than those in normal subjects (81.5 +/- 43.5 micrograms/L), in patients with hepatitis (152.4 +/- 98.4 micrograms/L), cirrhosis (191.8 +/- 96.3 micrograms/L), hepatic benign tumor (91.9 +/- 37.9 micrograms/L), metastatic liver cancer (146.4 +/- 73.7 micrograms/L) and gastrointestinal tumor (128.7 +/- 56.4 micrograms/L) (P < 0.01 or 0.05). Serum TGF beta 1 level was elevated in 34 of 47 (72.3%) PHC patients, including 5 of 7 (91.4%) small (< 5 cm) PHC patients. The sensitivity and specificity of diagnosis of TGF beta 1 in PHC were 72.3% and 77.9%, respectively. The diagnostic positivity was 78.5% in AFP-negative PHC patients. The combined assay of serum TGF beta 1 and AFP could further raise the detection rate of PHC up to 93.6%. Above results suggest that serum TGF beta 1 might be a candidate for a novel tumor marker for diagnosis and monitoring of PHC.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Colonic Neoplasms; Humans; Liver Neoplasms; Sensitivity and Specificity; Stomach Neoplasms; Transforming Growth Factor beta

Topics: Adenoma; Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Biological Evolution; Colonic Neoplasms; Cytoskeletal Proteins; DNA Repair; DNA-Binding Proteins; DNA, Satellite; Fungal Proteins; Humans; Mice; Microsatellite Repeats; Models, Genetic; Mutation; MutS Homolog 2 Protein; Neoplasms; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta) type I (RI) and type II (RII) receptors are essential for TGFbeta signal transduction. A human colon carcinoma cell line, designated GEO, is marginally responsive to TGFbeta and expresses a low level of RI mRNA relative to colon carcinoma cells, which are highly responsive to TGFbeta. Hence, the role of RI as a limiting factor for TGFbeta sensitivity and the contribution of low RI levels to the malignant phenotype of GEO cells were examined. Stable transfection of a tetracycline-regulatable rat RI cDNA increased TGFbeta1 binding to RI and resulted in increased growth inhibition by exogenous TGFbeta1. In contrast, although stable transfection of an RII expression vector into the same GEO cells increased TGFbeta1 binding to RII, growth inhibition by exogenous TGFbeta1 was not altered. This indicated that the low level of RI is a limiting factor for the growth-inhibitory effects of TGFbeta in GEO cells. RI-transfected cells were growth-arrested at a lower saturation density than GEO control cells. They also showed reduced growth and clonogenicity in plating efficiency and soft agarose assays, whereas RII-transfected cells did not show any differences from the NEO control cells in these assays. Tetracycline repressed RI expression in transfected cells and reversed the reduction in plating efficiency of RI-transfected clones, confirming that growth effects were due to increased RI expression in transfected cells. TGFbeta1 neutralizing antibody stimulated the proliferation of RI-transfected cells but had little effect on GEO control cells, indicating that increased autocrine-negative TGFbeta activity also resulted from increased RI expression. Tumorigenicity in athymic nude mice was significantly delayed in RI-transfected cells. These results indicate that low RI expression can be a limiting factor for response to exogenous TGFbeta, as well as TGFbeta autocrine-negative activity, and that reduction of RI expression can contribute to malignant progression.

Topics: Activin Receptors, Type I; Animals; Antigens, CD; Cell Division; Cell Line; Cloning, Molecular; Colonic Neoplasms; DNA Replication; Fibronectins; Humans; Integrin alpha5; Mice; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Tetracycline; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Retinoids have shown great promise as chemopreventive against the development of squamous cell carcinomas of the upper aerodigestive tract. However, the exact mechanism by which they block new tumors from arising is unknown. Here, we report that 13-cis- and all-trans-retinoic acid, used at clinically achievable doses of 10(-6) mol/L or less, can directly and specifically affect cell lines cultured from oral squamous cell carcinomas, inducing them to switch from an angiogenic to an anti-angiogenic phenotype. Although retinoic-acid-treated and untreated tumor cells make the same amount of interleukin-8, the major inducer of neovascularization produced by such tumor lines, they vary in production of inhibitory activity. Only the retinoic-acid-treated cells produce a potent angio-inhibitory activity that is able to block in vitro migration of endothelial cells toward tumor cell conditioned media and to halt neovascularization induced by such media in the rat cornea. Anti-angiogenic activity is induced in the tumor cells by low doses of retinoids in the absence of toxicity with a kinetics that suggest that it could be contributing to the effectiveness of the retinoids as chemopreventive agents.

Topics: Animals; Breast Neoplasms; Carcinoma, Squamous Cell; Colonic Neoplasms; Cornea; Endothelium, Vascular; Female; Fibrosarcoma; Humans; Interleukin-8; Keratinocytes; Neovascularization, Pathologic; Neovascularization, Physiologic; Neutralization Tests; Phenotype; Rats; Rats, Inbred F344; Tongue Neoplasms; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Sodium butyrate (NaB) is known to induce the process of cell differentiation, particularly for epithelial colonic cells. We previously observed that treatment with NaB in association with interleukin 2 (IL2), cures 60% of peritoneal carcinomatosis induced by injection of DHDK12/TRb cells in syngenic rats [15]. In the present work, we evidenced in vitro metabolic alterations of the DHDK12/TRb cell line treated with NaB, followed by an apoptotic process. Flow cytometric analysis evidenced that the tumour cells were arrested in the G1 and G2 phases of the cell cycle for the adherent cells to the plastic. Biological analysis of cells and debris released in the culture medium were essentially apoptotic cells. Complementary, the NaB-induced apoptotic process was confirmed by the staining of the nucleus from releasing cells by Hoechst 33258 and the DNA fragmentation revealed by DNA electrophoresis. Mitochondrial activity and glucose consumption were significantly stimulated after NaB treatment, which reveal an alteration of the metabolic activity of the treated tumour cells. As a consequence, we measured a significant increase of the active TGF beta 1 production, a cytokine previously described to participate to the epithelial cell differentiation. These in vitro data were confirmed in vivo showing a significant expression of apoptotic tumour cells in NaB- or NaB/IL2-treated tumours. Thus, the present results in the rat peritoneal carcinomatosis treatment show that combination of apoptotic process induced by NaB with immunostimulation by IL2 has powerful therapeutic properties.

Topics: Animals; Apoptosis; Butyrates; Cell Cycle; Cell Line; Colonic Neoplasms; DNA, Neoplasm; Drug Therapy, Combination; Flow Cytometry; Immunohistochemistry; In Vitro Techniques; Interleukin-2; Nucleosomes; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

We have reported previously that highly metastatic LuM1 cells derived from colon carcinoma colon 26 secrete larger amounts of gelatinase B than NM11 cells with poor metastatic potential, and that an increase in this gelatinase B secretion can be induced by autocrine factors (Hyup et A, Cancer Res., 54: 3611-3616, 1994). In the present study, a partial characterization was achieved by comparison of the autocrine factor preparation (fraction G) from serum-free medium conditioned with metastatic LuM1 cells with soluble factors known to stimulate gelatinase B secretion. Secretion of gelatinase B by LuM1 cells was augmented by tumor necrosis factor alpha, transforming growth factor beta1 (TGF-beta1), interleukin 1beta, or epidermal growth factor, and specific neutralizing antibodies abolished the induced increases. Platelet-derived growth factor and insulin-like growth factor 1 had no effect on gelatinase B secretion by LuM1 cells. The enhancement of gelatinase B secretion by fraction G was partially inhibited by the antibody to TGF-beta1. TGF-beta1 was detected in both active and latent forms in serum-free medium conditioned with LuM1 or NM11 cells, with the amount of TGF-beta1 higher in the former case. Gelatinase B secretion by LuM1 cells was enhanced by the addition of TGF-beta1 to the culture medium, but that by NM11 cells was not seriously affected, although the latter bound more of the factor. These results indicate the involvement of this growth factor in the autocrine stimulation of gelatinase B secretion by LuM1 cells. However, the autocrine factor effect was not fully explained by TGF-beta1 in the medium, and the involvement of some other unknown factor(s) was thus indicated.

Topics: Animals; Carcinoma; Collagenases; Colonic Neoplasms; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Growth Substances; Interleukin-1; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Metalloendopeptidases; Mice; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The present study was performed to investigate processes involved in circumvention of the immune system by advanced stages of tumor growth in the liver. The efficacy of Kupffer cells and pit cells against cancer cells was tested in vivo in an experimental model of colon carcinoma metastasis in rat liver. Liver tumors were induced by administration of CC531 colon cancer cells into the vena portae. After 3 weeks, livers were obtained and partly fixed for electron microscopic procedures or frozen in liquid nitrogen for enzyme and immunohistochemistry at the light microscope level. The activation status of Kupffer cells was studied by expression of Ia-antigen (MHC class II) and by measurement of glucose-6-phosphate dehydrogenase (G6PDH) activity in the cells in situ as a measure of production of reactive oxygen species. Large numbers of Kupffer cells were found in liver parenchyma surrounding colon carcinomas when compared with levels in control livers, but these cells were not activated. Large numbers of activated monocytes and macrophages, cytotoxic T cells but only a few pit cells were found to be recruited to the boundary between liver parenchyma and tumors or their stroma. In those areas where cancer cells invaded liver parenchyma, only newly recruited macrophages and some Kupffer cells were present but few cytotoxic T cells or pit cells were found. The low activation status of Kupffer cells both in terms of production of reactive oxygen species and Ia-antigen expression and the absence of significant numbers of pit cells at tumor sites suggest that Kupffer cells and pit cells do not play a significant role in advanced stages of tumor growth. High levels of prostaglandin E2 were detected in the parenchyma of livers containing tumors and transforming growth factor beta was detected in the stroma of the tumors, therefore suggest that cytotoxicity of newly recruited monocytes, macrophages and cytotoxic T cells may be limited in these stages because of local production of these immunosuppressive factors.

Topics: Animals; Carcinoma; Colonic Neoplasms; Dinoprostone; Disease Models, Animal; Killer Cells, Natural; Kupffer Cells; Liver; Liver Neoplasms, Experimental; Macrophages; Male; Microscopy, Electron; Monocytes; Rats; Rats, Inbred Strains; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Adenocarcinoma; Colonic Neoplasms; DNA, Satellite; Gastrointestinal Neoplasms; Humans; Microsatellite Repeats; Mutation; Phenotype; Receptor, IGF Type 1; Transforming Growth Factor beta

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

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

Transforming growth factor-beta proteins are key regulators of cellular growth and differentiation. To understand the role of TGF-beta in colonic tumour progression, 47 paraffin-embedded samples from colonic tumours (13 adenomas, and 34 adenocarcinomas) were studied. Gene mutations in the region coding for the active protein were studied by PCRSSCP analysis of exons 5, 6, and 7. TGF-beta 1 mRNA expression and localization were studied by NISH using cDNA probes generated by RT-PCR. Protein distribution was investigated by immunohistochemistry using antibodies against both intracellular and extracellular forms. Three mutations were found: one in exon 5 (Dukes C) and two in exon 7 (Dukes C and A). mRNA TGF-beta 1 was observed in 9 (69%) of 13 adenomas and in 30 (88%) of 34 adenocarcinomas. Levels of TGF-beta 1 mRNA and proteins were higher in colorectal carcinomas than in colorectal adenomas. Tubular adenomas associated with dysplasia showed greater expression of TGF-beta 1 than adenomas without dysplasia and than non neoplastic colonic mucosa. In dysplasia and cancer, epithelial neoplastic cells and stromal cells were positive for TGF-beta 1. In normal tissue endothelial cells and granulocytes sporadically showed immunoreactivity for TGF-beta 1, whereas epithelial cells were all negative. The three mutations in TGF-beta 1 exons 5, 6 and 7 found in colorectal adenocarcinomas suggest that TGF-beta 1 mutation may play a role in colorectal carcinogenesis and that the presence of the mutant form contributes to the transformed state. Our two other findings, the loss of staining gradient in normal colonic mucosa and the higher levels of TGF-beta 1 mRNA and proteins in colorectal carcinomas than in colorectal adenomas, indicate that the deregulation of TGF-beta 1 expression may occur as an early event in colorectal carcinogenesis. Although TGF-beta 1 expression is generally a reflection of cellular differentiation, tumour grade is not associated with mRNA TGF-beta 1 expression. As well as being present in the epithelial cells of the colonic tumours, mRNA TGF-beta 1 also occurred in the stroma. Its localization in the macrophages adjacent to tumour strongly suggests that TGF-beta 1 could be secreted by macrophages. This hypothesis should lead to new therapeutic strategies for colorectal carcinoma.

Topics: Adenocarcinoma; Adenoma; Colonic Neoplasms; Exons; Humans; Immunohistochemistry; Mutation; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) has been extensively studied as an exogenous agent that stimulates the expression of extracellular matrix proteins and their cell-surface integrin receptors in a variety of cell types. However, the recent demonstration of autocrine TGF-beta growth effects in a number of cell types suggests that the steady-state expression of extracellular matrix and integrin proteins and their biological activity may also be under autocrine TGF-beta control. Previously, we reported that repression of autocrine TGF-beta 1 activity by constitutive expression of a full-length TGF-beta 1 antisense cDNA led to abrogation of autocrine negative TGF-beta and, as a result, increased tumorigenicity and anchorage-independent growth of a poorly tumorigenic, well-differentiated colon carcinoma cell line designated FET (Wu, S., Theodorescu, D., Kerbel, R. S., Willson, J. K. V., Mulder, K. M., Humphrey, L. E., and Brattain, M. G. (1992) J. Cell Biol. 116, 187-196). Consequently, we have used this model system to study the effects of repression of autocrine TGF-beta 1 activity on the expression of integrin alpha 5 beta 1 and integrin alpha 5 beta 1-mediated cell adhesion to fibronectin. The expression of the integrin alpha 5 subunit was reduced in TGF-beta 1 antisense transfected FET cells at both mRNA and protein levels as determined by RNase protection assays and immunoprecipitation, respectively. Autocrine TGF-beta 1 had no effect on the transcription of integrin alpha 5 and beta 1 subunits, indicating that autocrine TGF-beta 1 may regulate integrin alpha 5 beta 1 expression at the post-transcriptional level. The diminished expression of integrin alpha 5 beta 1 on the cell surface led to the reduced adhesion of TGF-beta 1 antisense transfected cells to fibronectin. This phenomenon could be reversed by treatment with exogenous TGF-beta 1.

Topics: Cell Adhesion; Colonic Neoplasms; Fibronectins; Gene Expression Regulation; Humans; Integrins; Receptors, Fibronectin; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.

Topics: Animals; Brain; Cell Communication; Cell Line; Colonic Neoplasms; Cytokines; Endothelium, Vascular; Enzyme Induction; Interferon-gamma; Mice; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Rats; Recombinant Proteins; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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

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

VACO-330, a nontransformed cell line established from a human colon adenoma, undergoes spontaneous apoptosis and shedding of cells into the culture medium. Shed cells were shown to be apoptotic, both by nuclear morphology and by generation of a typical "laddered" pattern of degraded DNA. Quantitation of DNA released into the medium, compared with the amount retained on the plate, demonstrated that 6.2 +/- 1.1% of the total cell mass underwent apoptotic death daily. The addition of transforming growth factor beta (20 ng/ml) accelerated this spontaneous apoptotic rate 3.2-fold. Moreover, apoptosis could be rapidly induced in up to 45% of the VACO-330 cells by using brief exposure to a calcium chelating medium to release the cells from the substratum. We suggest that transforming growth factor beta is a likely physiological regulator of apoptosis during maturation of the colonic epithelial cells. We additionally suggest the existence of an alternate pathway, which at the time of shedding from the crypt induces apoptosis in colonic epithelial cells that have escaped earlier apoptotic signals.

Topics: Adenoma; Apoptosis; Cell Adhesion; Cell Differentiation; Collagen; Colonic Neoplasms; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.

Topics: Carcinoma; Cell Adhesion; Cell Differentiation; Colonic Neoplasms; DNA; Humans; Integrins; Organ Culture Techniques; Phenotype; Transforming Growth Factor beta; Tumor Cells, Cultured

PDGF-B released from colon tumor cells regulated tumor growth in athymic mice in a paracrine manner by inducing blood vessel formation. A positive correlation was found between expression of PDGF B-chain in cells grown in vitro and the number of factor VIII-positive blood vessels in tumors induced by three classes of colon carcinoma cell lines. Elevated expression of PDGF-B was also correlated with tumor size. Each cell line had the same mutations in the colon cancer genes APC, DCC, and p53 and had wild type c-K-ras genes (Huang et al. [1994] Oncogene, 9:3701-3706.) eliminating the possibility that any differences in tumor blood vessel formation were due to mutations and/or deletions in these genes. Colon carcinoma cells released biologically active PDGF capable of stimulating the growth of NIH3T3 cells, which was inhibited by neutralizing antisera to PDGF-AB chains. An inverse correlation was found between induction of factor VIII-positive blood vessels and expression of vascular endothelial growth factor (VEGF), while no correlation was seen with expression of either TGF alpha or k-FGF. Basic fibroblast growth factor (FGF) expression was not detected in these tumor cells. TGF beta 1 was capable of inducing PDGF-B expression in the undifferentiated U9 colon carcinoma cell line, but this sensitivity was not seen in differentiated cells. In contrast, TGF beta 1 inhibited VEGF expression in both undifferentiated cells and differentiated colon cancer cells. Thus, TGF beta 1 has two roles in the growth of undifferentiated U9 colon carcinoma cells in vivo: direct stimulation of cell proliferation as we have showed in earlier studies, and an increase in angiogenesis by inducing PDGF-B.

Topics: Animals; Blood Vessels; Cell Division; Colonic Neoplasms; Endothelial Growth Factors; Growth Substances; Hormones; Humans; Lymphokines; Male; Mice; Mice, Inbred BALB C; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Spontaneously arising, TGF beta 1-resistant colonies were isolated directly from the soft agarose plates of MOSER human colon carcinoma cells grown in the presence of TGF beta 1 but in the absence of serum. The colonies were cloned by limiting dilution and screened in a monolayer proliferation assay for sensitivity to TGF beta 1 and TGF beta 2 isoforms. Cell clones selectively sensitive or resistant to these isoforms in the growth inhibition assay displayed similar differential sensitivities to TGF beta isoforms for production of the extracellular matrix proteins laminin and fibronectin, as well as for the expression of the colon cell differentiation marker carcinoembryonic antigen. Differential receptor binding profiles for TGF beta 1 and TGF beta 2 were observed among the clones. The isolation of cell clones selectively resistant or sensitive to TGF beta isoforms as well as the identification of differential receptor binding profiles among the clones indicate the heterogeneity of TGF beta responsiveness that exists naturally in human colon tumor cells and stress the importance of defining mechanisms underlying differential responsiveness to TGF beta isoforms.

Topics: Carcinoma; Cell Division; Colonic Neoplasms; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

Transforming growth factor beta (TGF-beta) is released by a variety of cells and known to be involved in many different processes including the immune response, wound healing and carcinogenesis. As most experimental investigations have been based on quantitative analysis of TGF-beta production using a bioassay, it seemed important to test the validity and limitations of this method. This paper analyses several parameters that may impair TGF-beta quantification by bioassay. Recommendations are made concerning the influence of technical parameters and the presence of other cytokines (EGF and bFGF) commonly released by cultured cells to which the Mv1Lu mink lung epithelial cell line (CCL64) is sensitive.

Topics: Animals; Biological Assay; Blood Physiological Phenomena; Carcinoma; Cattle; Cell Line; Colonic Neoplasms; Culture Media, Conditioned; Culture Media, Serum-Free; Cytokines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelium; Fibroblast Growth Factor 2; Growth Inhibitors; Humans; Lung; Mink; Preservation, Biological; Recombinant Proteins; Sensitivity and Specificity; Swine; Transforming Growth Factor beta

Xenografted tumours were produced in nude mice by injection of HCT-8 human colon tumour cells. At average volumes of about 750 mm3, animals were injected with fast green vital dye, and 20 min later, tumours were excised and dissected into viable (stained) and necrotic portions (unstained). Viable and necrotic regions were then examined for cell yields, colony forming efficiencies, and levels of basic fibroblast growth factor (FGF-2), transforming growth factors-beta 1 and -alpha (TGF-beta 1, TGF-alpha), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) using enzyme-linked immunoassay (ELISA) procedures. Levels in the viable and necrotic regions were compared to levels in unseparated tumours. The average extent of necrosis in HCT-8 tumours of this size was 64%. The data for cell yields, colony forming efficiencies FGF-2, VEGF, TGF-beta 1 and TGF-alpha indicated that values determined in the unseparated tumours could be understood on the basis of the weighted average between viable and necrotic tissue, with the higher values occurring in the viable tissue. Low levels of FGF-2 and VEGF were found in the necrotic portions of the tumour while no measurable levels of TGF-beta 1 and TGF-alpha could be determined. PDGF levels were, however, equivalent in both the viable and necrotic regions indicating that necrotic tissue could be an important reservoir for this growth factor.

Topics: Colonic Neoplasms; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Growth Substances; Humans; Lymphokines; Necrosis; Neoplasms, Experimental; Platelet-Derived Growth Factor; Tissue Distribution; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

As colon epithelial cells migrate up the cylindrical colonic crypt, they terminally differentiate and lose their ability to divide. Elevated levels of the epithelial cell mitogen TGF alpha have been found at the top of the crypt by other investigators, causing us to speculate that colon epithelial cells lose mitogenic response to TGF alpha as they differentiate. We tested this hypothesis by using the HT29 colon carcinoma sublines U4 and U4H as models of one colonocyte lineage, fluid-transporting enterocytes. TGF alpha was mitogenic for the U4 cells, but inhibited the growth of the more differentiated U4H cells. However, p44 MAP kinase was activated by TGF alpha in both U4 and U4H cells, as well as in two control undifferentiated HT29 sublines which showed no change in proliferation in response to TGF alpha. In addition, TGF alpha activated the EGF receptor in each line by increasing its tyrosine phosphorylation. No relationship was found in these four lines between response to TGF alpha and level of expression of either the EGF receptor or two EGF receptor ligands, TGF alpha and amphiregulin. Activated EGF receptors initiate both growth-inhibitory and mitogenic signals in these cells since blocking some of the EGF receptors on TGF alpha-growth-inhibited U4H cells and TGF alpha-unresponsive U9 cells overrode the inhibitory signals and made both U9 and U4H cells sensitive to mitogenesis by added TGF alpha. These data imply that upon reaching stages of greater maturation, colon enterocytes lose proliferative response to TGF alpha because of changes in signaling by their EGF receptors.

Topics: Amphiregulin; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Cell Line; Colon; Colonic Neoplasms; EGF Family of Proteins; Enzyme Activation; Epithelial Cells; Epithelium; ErbB Receptors; Gene Expression; Glycoproteins; Growth Substances; Humans; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Intestinal Absorption; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Escape from negative growth regulation by transforming growth factor beta (TGF-beta) as a result of the loss of TGF-beta type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-beta 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-beta 1-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-beta autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Topics: Animals; Base Sequence; Cell Line; Clone Cells; Colonic Neoplasms; DNA Primers; Gene Expression; Humans; Mice; Mice, Nude; Molecular Sequence Data; Mutation; Phenotype; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Matrilysin is a member of the matrix metalloproteinase gene family, which is believed to play an important role in tumor invasion and metastasis. We examined the effects of over- and under-expression of matrilysin on the ability of colon cancer cells to migrate across an artificial membrane in vitro. Introduction of matrilysin caused colon cancer cells to become more invasive as assessed by an in vitro invasion assay. In contrast, expression of matrilysin was down-regulated by all trans-retinoic acid or by introduction of anti-sense matrilysin in BM314 colon cancer cells. This down-regulation caused these cells to become less invasive. We demonstrated a correlation between matrilysin level and the invasive potential of human colon cancer cells, implying an important role for matrilysin in the control of tumor invasion in vitro.

Topics: Colonic Neoplasms; DNA, Complementary; Down-Regulation; Humans; Matrix Metalloproteinase 7; Metalloendopeptidases; Neoplasm Invasiveness; RNA, Antisense; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Here we describe human colon carcinoma cell clones, isolated from a transforming growth factor beta (TGF-beta)-responsive parental cell line, which display differential sensitivities to TGF-beta 1 and TGF-beta 2 isoforms. In a monolayer proliferation assay, some clones were sensitive to both isoforms (IC50 = 0.1-0.6 ng/ml; S1S2) while others were resistant to both isoforms (IC50 > 5 ng/ml; R1R2). Still other clones (R1S2) were sensitive to TGF-beta 2 (IC50 = 0.1-0.2 ng/ml), but were resistant to TGF-beta 1 (IC50 > or = 5 ng/ml). In S1S2 cells, both TGF-beta isoforms resulted in a repression of c-myc mRNA expression, a concentration-dependent increase in fibronectin levels, and an enhanced production of the colon cell differentiation marker carcinoembryonic antigen. In contrast, R1R2 cells did not display these responses, or did so only to a limited extent. In R1S2 cells, TGF-beta 2 elicited these responses, yet TGF-beta 1 was essentially without effect. Receptor cross-linking experiments indicated that TGF-beta resistance in this model system was not generally associated with a complete lack of expression of either type I or II receptors. Moreover, the R1S2 type clones were heterogeneous, although the majority of them displayed binding to type I receptors by TGF-beta 2 but not by TGF-beta 1. These data suggest that either the TGF-beta 1 and TGF-beta 2 isoforms differ with respect to their ability to interact with the type I and II classes of receptors, or the TGF-beta 1 and TGF-beta 2 isoforms can interact with distinct receptor proteins of the type I and II classes in this model system.

Topics: Carcinoembryonic Antigen; Carcinoma; Cell Differentiation; Colonic Neoplasms; Drug Resistance; Fibronectins; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Proto-Oncogene Proteins c-myc; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Cell Division; Colonic Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA Repair; Endometrial Neoplasms; Female; Genes, Tumor Suppressor; Humans; Mice; Mice, Nude; Mutation; Ovarian Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Human colon cancer cell lines with high rates of microsatellite instability were found to harbor mutations in the type II TGF-beta receptor (RII) gene. Eight such examples, due to three different mutations, were identified. The mutations were clustered within small repeated sequences in the RII gene, were accompanied by the absence of cell surface RII receptors, and were usually associated with small amounts of RII transcript. RII mutation, by inducing the escape of cells from TGF-beta-mediated growth control, links DNA repair defects with a specific pathway of tumor progression.

Topics: Amino Acid Sequence; Animals; Colonic Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; Disease Progression; DNA Repair; DNA, Neoplasm; DNA, Satellite; Frameshift Mutation; Humans; Mice; Molecular Sequence Data; Neoplasm Transplantation; Phenotype; Receptors, Transforming Growth Factor beta; Repetitive Sequences, Nucleic Acid; RNA, Messenger; Sequence Deletion; Transforming Growth Factor beta; Tumor Cells, Cultured

The authors determined the molecular mechanisms for the failure of transforming growth factor-beta (TGF-beta) to inhibit the growth of SW1116 and SW48 colon cancer cell lines.. Transforming growth factor-beta is a bifunctional regulator of cell growth that typically stimulates proliferation of mesenchymal cells, but inhibits proliferation of normal epithelial cells. In the colon, TGF-beta appears to arrest proliferation of enterocytes as they leave the intestinal crypt and move to the villus tip. Transforming growth factor-beta actions are mediated by binding to heteromeric complexes of type I and type II TGF-beta receptors. Loss of TGF-beta responsiveness may contribute to uncontrolled cell growth and cancer.. The effects of TGF-beta 1 on DNA synthesis were measured by incorporation of tritiated thymidine into DNA of cultures of moderately differentiated adenocarcinoma (SW48) and poorly differentiated adenocarcinoma (SW1116) colon cell lines and a mink lung epithelial cell line (CCL-64). The effects of TGF-beta on the expression of c-myc, TGF-alpha, and TGF-beta in SW48 cells, SW1116 cells, and CCL-64 cells (c-myc only) were measured by Northern blot analysis. Expression of TGF-beta receptors in the cell lines was measured using competitive binding assays, receptor affinity labelling techniques, and reverse transcriptase-polymerase chain reaction.. Incubation with TGF-beta 1 (50 ng/mL) did not decrease serum-stimulated uptake of [3H]-thymidine into actively growing cultures of SW48 or SW1116 cells, but suppressed DNA synthesis of actively growing CCL-64 cells by 90%. Similarly, incubation with TGF-beta 1 (12 ng/mL) for 4 hours did not substantially alter the mRNA levels of c-myc, TGF-alpha, and TGF-beta 1 in either colon tumor cell line, although levels of c-myc mRNA in CCL-64 cells were reduced by TGF-beta 1 treatment. Competitive displacement of [125I]-TGF-beta 1 binding detected high levels (16,500 TGF-beta receptors per cell) of specific, high-affinity (200 pmol/L half-displacement) TGF-beta receptors on CCL-64 cells. In marked contrast, very low levels of TGF-beta 1 binding to SW1116 cells (250 receptors per cell) and SW48 cells (260 receptors per cell) were detected. Autoradiograms of CCL-64 cells affinity labelled with [125I]TGF-beta 1 revealed the presence of type I, type II, and type III TGF-beta receptors. No TGF-beta receptors were identified on SW1116 cells, and only very low levels of the nonsignaling type III TGF-beta receptors were detected on SW48 cells. Reverse transcriptase-polymerase chain reaction amplification detected mRNAs for type I, type II, and type III TGF-beta receptors in CCL-64 cells. SW48 cells, and SW1116 cells.. These results suggest that the lack of growth inhibition by TGF-beta in SW48 and SW1116 colon cancer cells may be caused by a lack of expression of functional TGF-beta receptors.

Topics: Activin Receptors, Type I; Base Sequence; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Epithelium; Genes, myc; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression of many types of human cells by arresting them in the G1 phase of the cell cycle. The arrest is mediated through interactions of various cyclin-dependent protein kinases (CDKs) and their inhibitors. We demonstrate that treatment with TGF-beta induces increased levels of WAF1/Cip1/p21, a potent inhibitor of various cyclin-CDK kinase activities, in two colon cancer cell lines (LS1034 and LS513), which are sensitive to TGF-beta-induced growth arrest. The induction in at least one of these cell lines (LS1034,p53-) is p53-independent. No WAF1 induction was observed after TGF-beta treatment in a third cell line (HT-29), which is completely insensitive to the cytoinhibitory effect of TGF-beta. In both LS513 and LS1034, WAF1 induction correlated with reduced cyclin E-associated kinase activity in vitro and suppression of the retinoblastoma susceptibility gene (Rb) protein phosphorylation in vivo. In addition, WAF1 was physically associated with cyclin E in the two sensitive cell lines. These results suggest that WAF1/Cip1/p21 is a mediator of cellular sensitivity to TGF-beta.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Blotting, Western; Cell Cycle; Cell Division; Cell Line; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA Primers; Exons; Genes, p53; Humans; Kinetics; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Protamine Kinase; Retinoblastoma Protein; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

We studied the antitumor immune response in gut-associated lymphoid tissue (GALT), which is the tolerance-inducing site for numerous dietary antigens. The mice inoculated with colon 26 carcinoma (C-26) into the subserosal space of the cecum (i.c.) showed a more rapid tumor growth than did the mice inoculated s.c. with C-26 into the flank. In addition, the serum of the i.c. C-26-inoculated mice showed a more potent suppressive activity, and their plasma contained a higher level of transforming growth factor than the s.c. C-26-inoculated mice. We also evaluated the tumor-specific T-cell response in the GALT by utilizing B7-transfected P815 mastocytoma (B7/P815). The rejection of i.c. inoculated B7/P815 was delayed compared to that of the s.c. inoculated B7/P815. The draining axillary lymph node (LN) cells of the s.c. B7/P815-inoculated mice exhibited a CD4+ T-cell-dependent proliferative response to in vitro restimulation, whereas the draining mesenteric LN cells of the i.c. B7/P815-inoculated mice exhibited no apparent response even with the addition of interleukin 2. However, such draining mesenteric LN cells did produce higher levels of interleukin 2 and transforming growth factor beta than the draining axillary LN without any stimulation, and their production of such cytokines depend on the CD4+ and CD8+ cells, respectively. Collectively, our results suggest the possibility that the impaired antitumor T-cell response in the GALT may be attributed to "bystander suppression" by TGF-beta-producing CD8+ T cells.

Topics: Animals; Carcinoma; Colonic Neoplasms; Endotoxins; Female; Immune Tolerance; Immunity, Cellular; Immunity, Mucosal; Intestines; Lymph Nodes; Lymphocyte Activation; Mesentery; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Serum Albumin; Spleen; T-Lymphocytes; Transfection; Transforming Growth Factor beta

Topics: Adenocarcinoma; Antibodies, Neoplasm; Carcinoma, Squamous Cell; Cell Line; Colonic Neoplasms; Epithelium; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin A; Immunoglobulin A, Secretory; Interferon-gamma; Interleukin-6; Neoplasm Proteins; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Secretory Component; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

A transforming growth factor beta1 (TGF beta1) antisense expression plasmid under constitutive control of the Rous sarcoma virus promoter was introduced into the highly tumorigenic and invasive colon carcinoma U9A cell line, which uses its autocrine TGF beta1 as a growth-stimulating factor. Stable transfectants were infrequent, and only the K6 transfectant exhibited 39 and 33%, respectively, of the levels of TGF beta1 mRNA and active, secreted TGF beta1 protein of the parental line. K6 exhibited no change in TGF beta2 expression, and TGF beta3 expression was not detected in either parental or transfectant cells. Compared to the parental line, the K6 antisense transfectant exhibited a 3-fold increase in lag time in anchorage-dependent colony formation. The parental line was 44 times as invasive through a collagen l-coated polycarbonate membrane in vitro as K6 cells and, after s.c. injection at low-cell inocula, U9A cells induced tumors 75 times as large in vivo as did the K6 antisense transfectant. The decreases in in vitro invasion and anchorage-dependent colony formation seen in K6 cells were largely reversed by the addition of TGF beta1. Tumors that did arise from the K6 antisense transfectant cells had lost antisense TGF beta1 expression and expressed the same TGF beta1 mRNA levels as controls. U9A cells were more metastatic to the liver after intrasplenic injection than K6 cells. These findings demonstrate a role for autocrine TGE beta1 in colon cancer tumorigenicity and invasion. They also show that a relatively small decrease in TGF beta1 levels was enough to markedly decrease colon carcinoma cell aggressiveness. This is not unprecedented, as we had found in an earlier study that a small, 2-4-fold increase in TGF beta1 protein levels in human colon cancers correlated with disease progression to metastases (E. Friedman et al., Cancer Epidemiol, Biomarkers & Prev., 4:549-554, 1995).

Topics: Animals; Base Sequence; Cell Division; Cell Line; Cell Survival; Colonic Neoplasms; DNA Primers; DNA Probes; DNA, Antisense; Humans; Kinetics; Liver Neoplasms; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Plasmids; Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Various peptide growth factors have been found to exert functional effects among epithelial cell populations. This study assessed the role of certain fibroblast growth factors (FGFs) (acidic FGF, basic FGF, and keratinocyte growth factor) in the regulation of intestinal epithelial cell proliferation and restitution.. Recombinant growth factors were added to subconfluent cultures of IEC-6, Caco-2, and HT-29 cell lines with subsequent assessment of [3H]-thymidine incorporation. The effects on an in vitro model of restitution were assessed by quantitation of cells migrating into standard wounds established in confluent monolayers of IEC-6 cells. Transforming growth factor beta (TGF-beta) content of growth factor-treated wounded monolayers was assessed by Northern blot and bioassay.. Acidic FGF, basic FGF, and keratinocyte growth factor caused a modest increase in proliferation of IEC-6, Caco-2, and HT-29 cell lines. Acidic FGF and basic FGF promoted intestinal epithelial cell restitution in vitro up to 10-fold, in conjunction with the enhanced expression of TGF-beta messenger RNA and protein. Promotion of IEC-6 restitution by acidic and basic FGF could be blocked by addition of immunoneutralizing anti-TGF-beta antisera.. FGFs that exert effects on fibroblast cells also exert effects on intestinal epithelial cell populations and may help promote epithelial cell restitution, the initial step of intestinal wound healing through a TGF-beta-dependent pathway.

Topics: Animals; Blotting, Northern; Cell Division; Cell Line; Cell Movement; Colonic Neoplasms; Epithelial Cells; Epithelium; Fibroblast Growth Factor 1; Fibroblast Growth Factor 10; Fibroblast Growth Factor 2; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Growth Substances; Humans; Intestines; Rats; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

nm23 has properties of a metastasis suppressor gene and also has been implicated in the control of response to transforming growth factor beta 1 (TGF beta 1) by studies in melanoma cells. In this report, we have examined the role of nm23 in two HT29 colon carcinoma sublines at different stages in tumor progression with different responses to TGF beta 1: the HD3 subline, which shows TGF beta 1-induced growth arrest and differentiation; and the more invasive and tumorigenic U9 subline, which induces tumors 7-fold as large as those induced by HD3 cells with one-half the latency. Analysis by semiquantitative reverse transcription-polymerase chain reaction showed that antisense phosphorothiolated oligonucleotides to the nm23 initiation site (nm23 AS oligos) decreased nm23 mRNA levels 2-8-fold in HD3 and U9 cells when normalized to beta-actin mRNA levels. However, a role for nm23 in TGF beta 1-mediated responses could only be found in HD3 cells. nm23 AS oligos inhibited the differentiation property of cell adherence over 90% in HD3 cells, and this loss of adherence could be partially blocked by concurrent treatment with TGF beta 1. In contrast, U9 cell adherence was not detectably altered by nm23 AS oligos, whether added in the presence or absence of TGF beta 1. The TGF beta 1-induced inhibition of HD3 cell proliferation was blocked by nm23 AS oligos, whereas the TGF beta 1-induced proliferation of U9 cells was unaffected by nm23 AS oligos.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Base Sequence; Cell Adhesion; Cell Differentiation; Cell Division; Colonic Neoplasms; Humans; Molecular Sequence Data; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta 1 regulates the expression of extracellular matrix adhesion molecules and the carcinoembryonic antigen gene family of glycoproteins in the Moser colon carcinoma cell line. Expression of fibronectin antisense RNA in the Moser cells down-regulated fibronectin mRNA expression and blocked the ability of transforming growth factor beta 1 to stimulate fibronectin secretion. Cells expressing antisense fibronectin RNA exhibited a higher rate of proliferation, changed their morphology, and produced more laminin in response to treatment with transforming growth factor beta 1. The stimulation of carcinoembryonic antigen secretion by transforming growth factor beta 1 (which normally lagged 10-16 h behind that of fibronectin secretion stimulated by transforming growth factor beta 1) was blocked by the expression of antisense fibronectin RNA. Thus, the stimulation of fibronectin secretion by transforming growth factor beta 1 was a prerequisite for subsequent stimulation of carcinoembryonic antigen secretion. In addition, the stimulation of the cellular expression of carcinoembryonic antigen and three carcinoembryonic antigen cross-reactive species of glycoproteins by transforming growth factor beta 1 was down-modulated in cells expressing antisense fibronectin RNA. We therefore conclude that fibronectin may play an important role in the mechanisms of transforming growth factor beta 1 regulation of the expression of carcino-embryonic antigen gene family of glycoproteins.

Topics: Carcinoembryonic Antigen; Colonic Neoplasms; Down-Regulation; Fibronectins; Humans; RNA, Antisense; Transforming Growth Factor beta; Tumor Cells, Cultured

Human colon cancer development is associated with the accumulation of mutations and deletions in the suppressor genes DCC, APC and p53 and mutations in the dominant oncogene K-ras, with loss of wild type alleles. In earlier studies we had observed that about half of the resected human colon cancers placed into primary culture were growth stimulated by TGF beta 1. This group included the more advanced cancers which were either poorly differentiated primary-site cancers or metastases. In contract, the more differentiated colon cancers were inhibited or unaffected by TGF beta 1, indicating that a switch in response to TGF beta 1 occurs during colon cancer progression. Different sublines of the HT29 colon carcinoma cell line model the resected cancers, responding to TGF beta 1 by proliferation, inhibition or no growth modulation. The current study shows that while the poorly differentiated, TGF beta 1-stimulated sublines are most tumorigenic, all the sublines have the same spectrum of mutations: truncating mutations in both APC (adenomatous polyposis coli) alleles, no activated ras genes, mutated and thus overexpressed p53, and very low expression of DCC compared to normal colon cells. Genes other than the four already implicated in colon carcinoma evolution are responsible for the mitogenic response to TGF beta 1 found in the more advanced cancers.

Topics: Base Sequence; Cell Division; Colonic Neoplasms; Genes, APC; Genes, DCC; Genes, p53; Genes, ras; Genes, Tumor Suppressor; Humans; Molecular Sequence Data; Mutation; Transforming Growth Factor beta

Transforming growth factor beta 1 (TGF-beta 1) switches from an inhibitor of tumor cell growth to a stimulator of growth and invasion during human colon carcinoma progression. We originally observed that metastatic colon carcinoma cells in primary culture responded to TGF-beta 1 by proliferation, whereas moderate to well-differentiated primary site colon carcinomas were growth inhibited by TGF-beta 1 (P. Schroy et al., Cancer Res., 50: 261-265, 1990). We then cloned several colon carcinoma cell lines which modeled these responses to TGF-beta 1 and expressed TGF-beta 1 (M. M. Hafez et al., Cell Growth & Differ., 1: 617-626, 1990; 3: 753-762, 1992). Two of these colon carcinoma cell lines, U9 and HD3, which activate approximately equal amounts of TGF-beta 1 and express equal amounts of TGF-beta receptors, are now used to compare the effects of TGF-beta 1 in modulating invasive behavior. The U9 cell line exhibits autocrine-positive growth regulation in vitro by TGF-beta 1, whereas the HD3 cell line shows the opposite response, autocrine-negative regulation. Blocking endogenous TGF-beta 1 with isotype-specific antibody inhibited U9 cell growth because autocrine TGF-beta 1 acts as a mitogen for U9 cells. In contrast, antibody to TGF-beta 1 stimulated HD3 cell proliferation because autocrine TGF-beta 1 inhibits growth of these cells. U9 cells were 13-fold more invasive in vitro through a collagen I layer than HD3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Division; Collagen; Colonic Neoplasms; Humans; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Proto-Oncogene Proteins c-fos; Transforming Growth Factor beta; Tumor Cells, Cultured

Various peptide growth factors have been found to regulate epithelial cell function within the mucosal epithelium of the gastrointestinal tract. In this study hepatocyte growth factor/scatter factor (HGF/SF) was found to stimulate intestinal epithelial cell proliferation: 2.5-fold in the non-transformed rat small intestinal epithelial cell line IEC-6 and 1.9-fold in the human colon cancer-derived HT-29 cell line. In addition, HGF/SF enhanced epithelial cell restitution, the initial step involved in gastrointestinal wound healing, in an in vitro model. Migration of IEC-6 in wounded monolayers was enhanced up to 7-fold. Enhancement of restitution by HGF could be completely abrogated by addition of immunoneutralizing anti-TGF beta 1, indicating that this process is mediated through a TGF beta-dependent pathway. These findings suggest that HGF exerts functional effects on intestinal epithelial cell populations and may play a role in the morphogenesis of the gastrointestinal tract and its remodeling following injury.

Topics: Animals; Cell Division; Cell Line; Cell Movement; Colonic Neoplasms; Epithelial Cells; Hepatocyte Growth Factor; Humans; Intestines; Rats; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Adenocarcinoma; Animals; Basement Membrane; Cell Communication; Collagen; Colonic Neoplasms; Digestive System; Digestive System Physiological Phenomena; Fibroblasts; Gastrointestinal Neoplasms; Mutation; Neoplasm Invasiveness; Rats; Transforming Growth Factor beta

Although a great deal is known about the cellular effects of exogenous transforming growth factor-beta (TGF-beta) treatment and the effects of various exogenous agents (including TGF-beta's themselves) on TGF-beta expression, studies of cellular controls for autocrine TGF-beta expression and function have been rare. Since exogenous TGF-beta treatment blocks progression through the cell cycle, it seemed likely that autocrine TGF-beta activity would be induced by growth states in which there was little or no cell division such as confluency or quiescence. Specific TGF-beta 1 or beta 2 neutralizing antibody treatment of a colon carcinoma cell line designated CBS showed that autocrine TGF-beta activity could be demonstrated in quiescent cells but not in preconfluent cells. Studies of kinetics of TGF-beta 1 and beta 2 mRNA levels during the establishment of quiescence revealed a significant increase of both isoforms in quiescent cells. The quiescent cells also secreted three- to fourfold and four-to fivefold higher levels of total (latent plus active) TGF-beta 1 and beta 2 protein in the conditioned media than the confluent cells and preconfluent cells, respectively. There was no detectable active form of either TGF-beta isoform in the conditioned media of preconfluent cells, whereas a significant amount of active TGF-beta 1 and beta 2 was detected in the conditioned media of quiescent cells. Quantitative RNase protection assays were developed to compare the effects of cell crowding vs quiescence on TGF-beta expression. TGF-beta 1 was primarily induced by quiescence. TGF-beta 2 was induced by both quiescence and cell crowding. Increased TGF-beta 1 mRNA levels appeared to be exclusively due to an increase in stability, while increased TGF-beta 2 mRNA levels were due to increased transcription. This growth state-related induction of TGF-beta's was also observed in two other colon carcinoma cell lines. These studies show that TGF-beta 1 and beta 2 are autocrine-negative factors which can be situationally expressed by cells as a function of their growth state. Autocrine expression of the TGF-beta's in this model system appears not to affect exponentially growing cells, but rather to function by maintaining a quiescent state and/or by blocking progression through the cell cycle.

Topics: Carcinoma; Cell Communication; Cell Count; Cell Division; Colonic Neoplasms; Gene Expression Regulation; Humans; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells has shown some promise in the treatment of certain cancers that are unresponsive to conventional treatment approaches. However, colon adenocarcinomas tend to respond poorly to LAK therapy, possibly as a result of tumor-induced immunosuppression. Recently, in vivo administration of anti-CD3 antibody has been shown to induce mouse T lymphocytes to mediate major-histocompatibility-complex(MHC)-unrestricted tumoricidal activity which is distinct from natural-killer-cell-derived LAK activity. It has therefore been suggested that anti-CD3 therapy may find application in tumor immunotherapy in humans. However, the effectiveness of anti-CD3-activated killer cell induction within the environment found in the vicinity of colon adenocarcinoma cells has not been evaluated. The present report demonstrates that colon cancer cells of human (HT-29) and mouse (MCA-38) origin markedly inhibit the generation of activated killer cells in murine spleen cell cultures. DNA synthesis and interleukin-2 production by spleen cells following stimulation with anti-CD3 antibody are also profoundly depressed in the presence of MCA-38 and HT-29 adenocarcinoma cells. MCA-38- and HT-29-mediated inhibition of activated killer cell development is exerted through the production of a tumor-associated soluble factor that is distinct from transforming growth factor beta or prostaglandins. Local immunosuppression associated with sites of tumor growth may therefore represent a major obstacle to successful anti-CD3 immunotherapy of certain colon adenocarcinomas.

Topics: Adenocarcinoma; Animals; CD3 Complex; Colonic Neoplasms; Female; Humans; Immune Tolerance; Indomethacin; Interleukin-2; Killer Cells, Lymphokine-Activated; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Transforming Growth Factor beta; Tumor Cells, Cultured

We describe the spontaneous progression of a colon adenoma cell line to tumorigenicity and growth factor independence. This system allows direct comparison of biologic stages of malignant progression with alterations of colon cancer suppressor genes and oncogenes. VACO-235, a human colon adenoma cell line, is at early passages nontumorigenic in the nude mouse, unable to grow in soft agar, growth stimulated by serum and EGF, and growth inhibited by TGF-beta. VACO-235 daughter passages 93 and higher have in culture spontaneously progressed to being weakly tumorigenic, but retain all other growth characteristics of VACO-235 early passages. A mouse xenograft from late passage VACO-235 was reestablished in culture as the granddaughter cell line, VACO-411. VACO-411 is highly tumorigenic, clones in soft agar, and is unresponsive to serum, EGF, and TGF-beta. Early passage VACO-235 bears a mutant K-ras allele, bears only mutant APC alleles, expresses no DCC transcripts, and expresses only wild type p53 transcripts. VACO-411 retains the identical genotype, still expressing only wild type p53. Colonic cells after ras mutation, APC mutation, and DCC inactivation remain nontumorigenic and growth factor dependent. Malignant progression involves at least two additional steps, and in VACO-411 can proceed by a novel pathway not requiring p53 inactivation.

Topics: Adenoma; Animals; Base Sequence; Cell Division; Chromosome Aberrations; Colonic Neoplasms; Female; Genes, p53; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutation; Oncogenes; Transforming Growth Factor beta; Tumor Cells, Cultured

Mutation of the p53 gene is thought to be a late event in human colorectal carcinogenesis, involved in the malignant conversion of the adenoma to the carcinoma. One of the questions that we hoped to address was whether, in vivo, a single mutational event in one p53 gene is sufficient to confer a significant growth advantage on a colonic epithelial cell. Such a growth advantage could result either from an increase in growth rate and/or loss of response to inhibitory growth signals naturally present in the colonic crypt. We therefore introduced the pC53-SCX3 143 (Val-Ala) p53 mutation into a non tumorigenic adenoma derived cell line, AA/C1, which contained a truncating APC mutation, activating K-ras mutation but was wild-type for the p53 protein. High levels of mutant p53 protein were detected in the pC53-SCX3 transfected AA/C1 cell lines but was found not to affect either the in vitro (colony forming efficiency, anchorage independence) or in vivo (tumorigenicity in nude mice) growth, when compared to vector control or the parental AA/C1 cell line. In addition, to test whether the cells become less sensitive to inhibitory growth factors, the response of the cell lines to the naturally occurring growth inhibitor TGF beta was also investigated. Even though TGF beta had previously been implicated in the control of growth of intestinal epithelium, expression of the mutant p53 protein did not affect the sensitivity of the parental AA/C1 cell line to TGF beta. Under the experimental conditions tested expression of the 143 (Val-Ala) p53 protein was unable to affect the in vitro or in vivo growth characteristics of the adenoma derived AA/C1 cell line. When compared to other studies, these results suggest that the genetic background of the individual recipient cell may greatly influence the effect of expression of a particular p53 mutation.

Topics: Adenoma; Animals; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Humans; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Phenotype; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Transforming growth factor (TGF)-beta 1 modulates the expression of extracellular matrix (ECM) glycoproteins, fibronectin and laminin and the adhesion of Moser colon cancer cells to these glycoproteins. Since adhesion can be altered through expression of cell-surface receptors, binding affinities of adhesion molecules for receptors, or both, we investigated the effect of TGF-beta 1 on the binding properties of fibronectin and laminin to their cell-surface receptors by saturation binding and Scatchard analyses using radiolabeled fibronectin and laminin. Fibronectin bound to its cell-surface receptor with high affinity (Kd = 1.25 x 10(-9) M), Moser cells had approximately 7.1 x 10(4) fibronectin-binding sites per cell. TGF-beta 1 treatment rapidly up-modulated the number of cell-surface fibronectin-binding sites by 1.9-fold. The binding affinity of fibronectin for the receptor, however, was not altered. Laminin was found to bind to a higher-affinity and a lower-affinity receptor. Moser cells expressed approximately 1.1 x 10(3) higher-affinity laminin-binding sites and approximately 3.1 x 10(4) lower-affinity-binding sites per cell. TGF-beta 1 rapidly increased the expression of the higher-affinity sites 3-fold and the lower-affinity sites 5-fold. The binding affinity of both the higher-affinity and lower-affinity laminin receptors increased 3-fold after 2 and 6 hr of TGF-beta 1 treatment respectively. Concurrent with receptor modulation, TGF-beta 1 induced the secretion of fibronectin and laminin from Moser cells. Northern hybridization analyses showed a concurrent stimulation of the expression of the mRNAs for ligands (fibronectin and laminin) and the mRNAs for the integrin species of the fibronectin and laminin receptors (alpha 5 and alpha 6 subunits). Thus the production of fibronectin and laminin and the expression of their receptors were tightly co-regulated by TGF-beta 1.

Topics: Colonic Neoplasms; Fibronectins; Gene Expression; Humans; In Vitro Techniques; Integrins; Laminin; Receptors, Fibronectin; Receptors, Laminin; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

A non-transformed small-intestinal cell line from the rat (IEC-6) and a human colon cancer cell line (HT 29) were examined for their trophic response to sensory neuropeptides. Substance P, neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and peptide YY (PYY) were tested. Epidermal growth factor (EGF), insulin, and somatostatin-14 were also used. Interaction studies were performed on IEC-6 cells by combining EGF or insulin with somatostatin-14. The sensory neuropeptides had no effect either on IEC-6 cell growth and DNA synthesis or on HT29 cell growth. EGF and insulin stimulated cell growth and DNA synthesis in IEC-6 cells and cell growth in HT 29 cells in a dose-dependent fashion. Somatostatin-14 had no effect either alone or in combination with EGF or insulin on IEC-6 cell growth and DNA synthesis. HT 29 cell growth was inhibited by somatostatin-14 only in the presence of serum with a maximal and significant response at 10(-7) M. Our observations suggest that the sensory neuropeptides do not exert a direct growth-regulatory effect either on IEC-6 cells or on HT 29 cells. Somatostatin, however, inhibits serum-induced HT 29 cell growth but does not interfere directly with the proliferative effect of serum, EGF, or insulin on IEC-6 cells in this model.

Topics: Animals; Calcitonin Gene-Related Peptide; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelium; Gastrointestinal Hormones; Humans; Insulin; Intestinal Mucosa; Intestines; Neurokinin A; Neurons, Afferent; Neuropeptides; Peptide YY; Peptides; Rats; Somatostatin; Substance P; Transforming Growth Factor beta; Vasoactive Intestinal Peptide

Effect of interleukin 2 (IL-2) on the proliferative rate of a human colon adenocarcinoma line (HT-29 D4) and of an untransformed rat intestinal cell clone (IEC-6) was studied in vitro. IL-2 had an antiproliferative effect which was cytotoxic-free: interleukin 2 did not induce any significant cell death, apoptosis or change in cellular protein content. Northern blot characterization of cellular mRNA revealed that interleukin 2 enhances the expression of JUN, FOS and TGF beta which are known to be involved in antiproliferative processes. Kinetic analyses suggest that the aforedescribed biological effect is mediated by the AP1 complex (JUN/FOS) when dealing with rapid inhibition (HT-29 D4 and IEC-6) and by an autocrin TGF beta loop when dealing with slow inhibition (IEC-6).

Topics: Adenocarcinoma; Animals; Blotting, Northern; Colonic Neoplasms; Depression, Chemical; DNA; DNA, Neoplasm; Electrophoresis, Agar Gel; Genes, fos; Genes, jun; Humans; Interleukin-2; Intestinal Mucosa; Rats; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Two types of human fibroblasts have been isolated from a patient with a colon cancer with metastasis, one type was derived from a healthy part of the colon, and the other one isolated from a metastasized lymph node close to the intestine. These fibroblasts have been characterized for their expression of collagens type I, III and IV, vimentin, fibronectin, alpha-smooth muscle actin, laminin and desmin. The effects of conditioned media of human colon cancer cell lines, HT29, SW1116, LS180 and HCT8R, on the metabolism of these fibroblasts were tested. All the conditioned media stimulate both types of fibroblasts, as reflected by their incorporation of radiolabelled methionine and proline. Normal fibroblasts were highly sensitive to the conditioned media as compared to the activated fibroblasts. Additionally, the production of TGF beta 1 by the four colorectal cancer cell lines has been quantified, and significant qualitative (production of latent and/or active form) and quantitative differences were observed. The effects of the conditioned media of the four tumoral cell lines and exogenous TGF beta 1 on the proliferation of the two types of fibroblasts were compared. Our data indicated that the two types of fibroblasts respond differently to TGF beta 1 whereas they are both growth stimulated by the conditioned media, apart from the LS180 conditioned medium. We conclude that if TGF beta 1 acts in the fibroblastic reaction, additional factors are required.

Topics: Cell Division; Colonic Neoplasms; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

Samples of colorectal carcinoma, adenoma and normal colorectal mucosa were examined for the expression of TGF-beta by immunohistochemistry. Immunoreactivity for TGF-beta was present in 52 out of a total of 58 samples of normal mucosa examined. In adenomas and carcinomas TGF-beta expression was observed in eight out of ten and 46 out of 48 samples respectively and was largely restricted to epithelial cells. In normal mucosa differential expression of TGF-beta was present within epithelial cells, those in the upper parts of the crypts showing enhanced immunoreactivity compared to cells in the proliferative compartment. This pattern of differential staining is consistent with TGF-beta having an important role in the control of growth and differentiation in colonic mucosa.

Topics: Adenoma; Cell Differentiation; Colon; Colonic Neoplasms; Epithelial Cells; Epithelium; Humans; Immunohistochemistry; Intestinal Mucosa; Transforming Growth Factor beta

FET cells are well differentiated human adenocarcinoma cells whose growth is partially inhibited (50-60%) by transforming growth factor-beta 1 (TGF-beta 1). In exponentially growing cultures, TGF-beta 1 induces the expression of transforming growth factor-alpha (TGF-alpha) by 3-fold. To determine whether this induction is the result of increased TGF-alpha promoter activity, FET cells were transiently transfected with a plasmid containing 2816 base pairs of the 5'-flanking region of the TGF-alpha gene linked to luciferase. Transfected FET cells treated with growth-inhibitory concentrations of TGF-beta 1 (10 ng/ml) showed up to a 10-fold increase in luciferase activity. The increase in luciferase activity was dose dependent through the normal physiological range of TGF-beta 1 (0.5-20 ng/ml), saturating at 10 ng/ml. This effect was also TGF-alpha promoter specific, inasmuch as the Rous sarcoma virus long terminal repeat used as a control remained relatively insensitive to the effects of TGF-beta 1. By using progressively smaller portions of the TGF-alpha promoter region, the TGF-beta 1-responsive element was mapped between base pairs -77 and -201 of the 5'-flanking region. TGF-beta 1 treatment also affected epidermal growth factor receptor levels. FET cells treated with TGF-beta 1 (10 ng/ml) for 48 h showed a 20% decrease in the number of epidermal growth factor receptors and a 2-fold increase in the number of high affinity epidermal growth factor receptors on their surface. These results indicate that TGF-beta 1 acts as a positive regulator of TGF-alpha transcription, and they suggest a possible mechanism by which these cells circumvent the growth-inhibitory effects of TGF-beta 1.

Topics: Adenocarcinoma; Base Sequence; Colonic Neoplasms; Culture Media, Serum-Free; Enzyme Induction; Humans; Luciferases; Molecular Sequence Data; Promoter Regions, Genetic; Transfection; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Selected G1 events associated with butyrate-induced differentiation were examined in HT-29 colon adenocarcinoma cells. [3H]Thymidine incorporation by HT-29 cells was decreased to 40% of control levels by treatment with 5 mM butyrate for 24 h, and cell numbers decreased to 21% of control levels after 48 h of treatment. Cells released from butyrate arrest entered S phase approximately 24 h after release, and serum-deprived HT-29 cells escaped growth inhibition if butyrate was added 8 h or more after serum restimulation. Northern analysis of RNA isolated from rapidly growing HT-29 cells showed a marked induction of alkaline phosphatase mRNA expression within 12 h of treatment with 5 mM butyrate. The appearance of alkaline phosphatase mRNA was temporally associated with a 5-fold increase in expression of transforming growth factor beta 1 (TGF-beta 1) mRNA. Expression of the nuclear protooncogene c-myc began to decrease 30 min after treatment with butyrate and was decreased 4.5-fold 4 h after treatment; however, expression of other immediate-early genes (nup/475 and zif/268) was not significantly affected. Histochemical staining of HT-29 monolayers showed that no cells were positive for alkaline phosphatase protein prior to treatment, and 90% were positive 48 h after treatment. TGF-beta 1 and TGF-beta 2 had no effect on HT-29 cell growth. TGF-beta 1 did not induce alkaline phosphatase mRNA or histochemical positivity. These data indicate that butyrate arrests HT-29 cell growth early in G1.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Alkaline Phosphatase; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Colonic Neoplasms; DNA-Binding Proteins; Early Growth Response Protein 1; Epithelium; Fructose-Bisphosphate Aldolase; Humans; Immediate-Early Proteins; Interphase; Intestinal Mucosa; Neoplasm Proteins; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; RNA, Neoplasm; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Tristetraprolin; Tumor Cells, Cultured

The purpose of this study was to determine whether cultured colonic adenoma and carcinoma cells undergo apoptosis (programmed cell death) in vitro and whether specific growth and dietary factors, thought to be involved in the control of growth and differentiation of human colonic cells, could induce cell death through apoptosis. In cell lines originating from 6 colorectal adenomas and 7 carcinomas, spontaneous apoptosis was observed. Sodium butyrate, a naturally occurring fatty acid, is present in the human large bowel in millimolar amounts as a result of bacterial fermentation of dietary fibre. Sodium butyrate, at physiological concentrations, induced apoptosis in 2 adenoma cell lines, RG/C2 and AA/Cl, and in the carcinoma cell line PC/JW/FI. In contrast, transforming growth factor beta 1, which is thought to have an important role in the control of growth in colonic epithelium, did not induce apoptosis. Neither RG/C2 nor PC/JW/FI contain wild-type p53, therefore this tumour-suppressor gene is not required to mediate signals for the induction of apoptosis in colonic tumour cells. Our studies report the induction of apoptosis in colonic tumour cells by the naturally occurring fatty acid sodium butyrate. Since sodium butyrate is produced by bacterial fermentation of dietary fibre, the observation that this fatty acid can induce apoptosis could, in part, explain why a high-fibre diet appears to be protective against colon cancer. Escape from the induction of programmed cell death may be an important event in colorectal carcinogenesis.

Topics: Adenoma; Apoptosis; Butyrates; Butyric Acid; Carcinoma; Colonic Neoplasms; Dietary Fiber; DNA, Neoplasm; Genes, p53; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

This report examines the effects of inhibitors of cell proliferation on transforming growth factor alpha (TGF-alpha) expression in low-density cultures of poorly (PD) and well-differentiated (WD) human colon carcinoma cells, continuously maintained in serum-free medium. In contrast to results in certain untransformed cells, growth inhibitors such as transforming growth factor beta 1 (TGF-beta 1) and N,N-dimethylformamide up-regulated TGF-alpha mRNA and protein expression in these human colon carcinoma cells. Treatment of low-density WD cells with TGF-beta 1 (10 ng/ml) resulted in a 1.5-fold increase in TGF-alpha mRNA levels within 4 h of treatment. TGF-alpha mRNA levels increased to 2.7-fold above control values by 48 h after TGF-beta 1 addition. Additionally, over a TGF-beta 1 concentration range of 1-30 ng/ml, TGF-alpha protein levels were increased by 2-10-fold, despite the fact that the growth of the WD cells remained inhibited. Although TGF-beta 1 control of TGF-alpha expression was altered in these WD colon carcinoma cells, relative to that in untransformed cells previously examined, the cells retained the ability to up-regulate TGF-alpha expression in an epidermal growth factor-dependent manner. In similarity to the results with TGF-beta 1 in WD colon carcinoma cells, the differentiation agent N,N-dimethylformamide (0.7%) resulted in an increase of TGF-alpha mRNA of approximately 3.8-fold in PD colon carcinoma cells, as well as a 4.4-fold increase in TGF-alpha protein after 4 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Differentiation; Cell Division; Colonic Neoplasms; Culture Media, Serum-Free; Dimethylformamide; Epidermal Growth Factor; Humans; Phenotype; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The aim of this study was to evaluate the correlations between tumor size and cachexia parameters including cytokine levels in serum. In transplantable colon 26 adenocarcinoma-bearing mice, parameters having negative correlations with tumor size were host weight changes, epididymal adipose tissue weight, glucose and interleukin 3 (IL-3) concentration in serum. Parameters having a positive correlation with tumor size were the number of circulating white blood cells and immunosuppressive acidic protein (IAP), interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) concentration in serum.

Topics: Adenocarcinoma; Animals; Blood Glucose; Cachexia; Colonic Neoplasms; Cytokines; Interleukin-3; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Neoplasm Proteins; Transforming Growth Factor beta

The MOSER human colon carcinoma cell line is significantly growth inhibited by exogenous transforming growth factor-beta (TGF-beta). The secretion of TGF-beta by these cells was examined to determine if endogenous TGF-beta might also regulate MOSER cell growth. MOSER cells secreted 11 ng TGF-beta/10(6) cells, 24% of which was in the active form. Blocking antibodies specific for TGF-beta 2 stimulated growth 1.4-fold, while TGF-beta 1 specific antibodies were without effect. Treatment of MOSER cells with the differentiation agent, N,N-dimethylformamide (DMF), inhibited cell growth and resulted in an 8-fold increase in secreted TGF-beta (20% active). Only antibodies specific for TGF-beta 2 were able to reverse the growth inhibitory effect of DMF on these cells. Therefore, TGF-beta 2 acted as a negative autocrine inhibitory factor for MOSER cells and the growth inhibitory effects of DMF were mediated by the increased secretion of active TGF-beta 2.

Topics: Antibodies; Cell Division; Colonic Neoplasms; Dimethylformamide; Growth Inhibitors; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous work has shown that repression of negative autocrine transforming growth factor (TGF) beta 1 did not alter the growth rate of a human colon carcinoma cell line, but the time required for the cells to enter exponential growth from lag phase was reduced (S. P. Wu, D. Theodorescu, R. Kerbel, J. K. V. Willson, K. M. Mulder, L. E. Humphrey, and M. G. Brattain, J. Cell Biol., 116: 187-196, 1992). These results have led to the hypothesis that the tumor suppressive activity of autocrine TGF-beta 1 was directed at quiescent nondividing cells rather than actively dividing cells. In order to test this hypothesis, a weakly tumorigenic, well-differentiated human colon carcinoma cell line designated CBS, which expressed autocrine TGF-beta 1 and -beta 2 activity in quiescent cells, but not in exponential growth phase cells, was identified. This cell line was stably transfected with a full-length TGF-beta 1 antisense complementary DNA. Constitutive expression of TGF-beta 1 antisense mRNA in CBS cells resulted in repression of autocrine TGF-beta 1 and -beta 2 protein activity in quiescent cells of approximately 10-fold. TGF-beta 2 repression could have been due to interaction with TGF-beta 1 antisense mRNA, since these two isoforms have a high degree of homology, or it could have been indirectly due to TGF-beta 1 repression, since this isoform has been shown to affect transcriptional and posttranscriptional control of TGF-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies, Monoclonal; Cell Cycle; Cell Division; Colonic Neoplasms; Humans; RNA, Antisense; RNA, Messenger; Suppression, Genetic; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.

Topics: Adenocarcinoma; Blotting, Western; Colonic Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Ligands; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

We describe an immunohistochemical technique that makes use of two monoclonal antibodies (MAb), one to detect the transforming growth factor B (TGFB) and another that reacts with iodo- and bromodeoxyuridine. The purpose of this technique is to determine the relationship between TGFB expression and the S-phase cells in human tumors. Since both can be distinctly identified in situ from tissue embedded in plastic, in assessment of the geographic orientation of S-phase cells in relation to such factors as TGFB, contiguity to blood vessels, nerve fibers, and macrophages can also be achieved.

Topics: Antibodies, Monoclonal; Biopsy; Bone Marrow; Breast Neoplasms; Bromodeoxyuridine; Cell Cycle; Colonic Neoplasms; Female; Glioblastoma; Histological Techniques; Humans; Idoxuridine; Indicators and Reagents; Kinetics; Neoplasms; S Phase; Transforming Growth Factor beta

The role of transforming growth factor beta 1 (TGF-beta 1) in enterocytic differentiation was examined by treating two undifferentiated HT29 colon carcinoma sublines, U4 and U9, with hexamethylene bisacetamide to up-regulate their level of TGF-beta 1 mRNA expression. Although both lines after treatment secreted approximately equal levels of biologically active TGF-beta 1, only U4H cells were found to undergo enterocytic differentiation when cultured postconfluence on collagen I-coated transwells, forming polarized monolayer cells with an apical brush border, whereas U9H cells remained multilayered and undifferentiated. Enterocytic U4H cells exhibited four times as much cell surface expression of the collagen I-binding protein alpha 2-integrin, twice as much of the accessory collagen-binding protein carcinoembryonic antigen, and almost twice as much binding to collagen I films as undifferentiated U9H cells. TGF-beta 1 treatment doubled U4 cell collagen I binding, increased expression of alpha 2-integrin 4-fold, but increased carcinoembryonic antigen expression only marginally. U4H cells displayed cell cycle regulation by arresting reversibly at a restriction point in G1 when placed in the postconfluent culture conditions which initiated enterocytic differentiation. In contrast, undifferentiated U9H cells exhibited no restriction point but arrested throughout G1. TGF-beta 1 blocked synchronized U4H cells in G1, whereas it stimulated the growth of U9H cells. Thus, TGF-beta 1 has two roles in enterocytic differentiation: to increase levels of collagen I adhesion proteins and to block enterocytic cells in G1 so that they can differentiate.

Topics: Acetamides; Carcinoembryonic Antigen; Carcinoma; Cell Adhesion; Cell Differentiation; Colon; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Integrins; Interphase; Neoplasm Proteins; Receptors, Cell Surface; Receptors, Collagen; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Growth Substances; Neoplasm Metastasis; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta 1 (TGF-beta 1) regulates the expression of the carcinoembryonic antigen (CEA) gene family in the human colon carcinoma cell line Moser. The mechanisms through which it acts, however, are unknown. In this communication, several lines of evidence are presented to show that the induction of CEA expression and secretion (collectively called CEA responses) by TGF-beta 1 is associated with protein kinase C (PKC) pathway of signal transduction. Treatment of intact cells with the PKC-specific inhibitor calphostin C down-modulated cellular PKC phosphotransferase activity and blocked the induction of the CEA responses by TGF-beta 1. Depletion of PKC by treatment of intact cells with phorbol ester also blocked the action of TGF-beta 1. The induction of the CEA responses by TGF-beta 1 was also blocked by the protein kinase inhibitor 1-(isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which also inhibited cellular PKC activity. However, TGF-beta 1 did induce the CEA responses in intact cells treated with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), the calmodulin-dependent phosphodiesterase inhibitor calmidazolium, the diacylglycerol kinase inhibitor R59 022, and the G-protein inhibitors cholera toxin and pertussis toxin. Treatment of intact cells with TGF-beta 1 induced a rapid and transient increase in PKC phosphotransferase activity. TGF-beta 1, however, was unable to induce PKC enzymatic activity in cells pretreated with calphostin C. Therefore, it is concluded that TGF-beta 1 regulates the CEA responses through a signal transducing pathway associated with PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Carcinoembryonic Antigen; Colonic Neoplasms; Humans; Isoquinolines; Naphthalenes; Piperazines; Polycyclic Compounds; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured

We analyzed several factors which could influence the immunogenicity of colon tumor cells, using a series of clones derived from a single chemically induced rat adenocarcinoma cell line. These clones display variable tumorigenic potential in syngeneic immunocompetent animals, and it has been established that in this model the tumorigenicity of the cells depends on their ability to escape immune surveillance. The results show an absence of relationship between tumorigenicity and expression of MHC-class-I antigens, cell adhesion to rat fibroblasts or fibroblast extracellular matrix. The secretion of latent and active TGF beta I appeared to be quite variable from one clone to the other, but was unrelated to tumorigenicity. Unexpectedly, some regressive clones produced elevated levels of this cytokine, suggesting that in this model, spontaneous secretion of TGF beta I is not sufficient to impair the immune system of the host. In contrast, the more tumorigenic clones were more resistant than less tumorigenic ones to cytotoxicity mediated by NK or LAK cells. They also showed arrest of cell proliferation after reaching confluence, something not observed in the less tumorigenic clones. Finally, the strongest relationship with tumorigenicity was found for expression of blood-group carbohydrate antigens. Increased expression of blood-group-H antigen and, conversely, decreased expression of beta-galactoside precursors of this antigen correlated with increased tumorigenicity.

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Antigens, Surface; Cell Adhesion; Cell Division; Clone Cells; Colonic Neoplasms; Flow Cytometry; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Rats; Regression Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured

The effects of sodium butyrate (NaB) on the response of the RCA human colon carcinoma cell line to transforming growth factor-beta 1 (TGF-beta 1) were examined. NaB induced differentiation, as judged by an increase in cellular alkaline phosphatase, in the RCA cells and this differentiation was accompanied by a decreased growth rate. TGF-beta 1 did not significantly alter the growth or state of differentiation of the RCA cells. The growth rate of cells treated simultaneously with NaB and TGF-beta 1 was similar to that of control untreated cells while the alkaline phosphatase levels remained comparable to cells treated with NaB. Addition of TGF-beta 1 to cells grown in the presence of NaB resulted in a stimulation of growth. Cells pretreated with TGF-beta 1 remained sensitive to the growth inhibitory and differentiation inducing effects of NaB. These results suggest that NaB may alter the expression of proteins responsible for a stimulatory signal response to TGF-beta 1 in RCA cells.

Topics: Alkaline Phosphatase; Butyrates; Butyric Acid; Carcinoma; Cell Differentiation; Cell Division; Colonic Neoplasms; Drug Screening Assays, Antitumor; Enzyme Induction; Epidermal Growth Factor; Humans; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The regulatory effect of transforming growth factor beta 1 (TGF-beta 1) on the adhesion of human colon-carcinoma cells to the extracellular matrix (ECM) was investigated. ECMs used in this study included tissue-culture wells coated with fibronectin, laminin, collagen and BSA, as well as plastic wells. Three phenotypically different human colon-carcinoma cell lines (Moser, HCT116, and KM12SM) were used. The Moser cell line is moderately differentiated and, in terms of the diversity of responses elicited by TGF-beta 1, is the human colon-carcinoma cell line most responsive to TGF-beta 1 as reported to date. By comparison, the undifferentiated HCT116 and the highly metastatic KM12SM cells are unresponsive to this growth factor. We showed that TGF-beta 1 regulated the adhesion responses of all 3 cell lines. However, the response profiles as well as the endogenous adhesive properties of each cell line were quite different from those of the others. Endogenous Arg-Gly-Asp(RGD)-related receptors were present on the HCT116 but not on the other cells. The observed regulatory effect of TGF-beta 1 was contingent on the cell line, the type of ECM, and the growth-factor treatment protocol used. When cells were treated with TGF-beta 1 for 16 hr prior to exposure to ECM in a 4-hr adhesion assay, a significant increase in adhesion to fibronectin and collagen was observed for the Moser cells. For the identical experimental protocol, the KM12SM cells responded by increasing adhesion to fibronectin, while the HCT116 cells responded by decreasing adhesion to collagen. Kinetic analyses of TGF-beta 1 treatment showed that increased adhesion response to laminin was induced in the Moser cells after 2 hr of growth-factor treatment. This response declined rapidly upon further exposure of the cells to TGF-beta 1. Simultaneous exposure of cells to both TGF-beta 1 and ECM negated the adhesion responses described above. The up-modulation of adhesion to fibronectin, laminin and collagen by TGF-beta 1 was mediated through RGD-related integrin receptors. RGD-containing peptides effectively blocked the enhanced adhesion responses induced by TGF-beta 1.

Topics: Amino Acid Sequence; Cell Adhesion; Cell Line; Colonic Neoplasms; Extracellular Matrix; Humans; Kinetics; Oligopeptides; Receptors, Immunologic; Receptors, Peptide; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The response of the retinoblastoma (RB) gene and its product (pRB) to transforming growth factor beta 1 (TGF-beta 1) was studied in three types of colon carcinoma cells derived from the same parental line. TGF-beta 1 was a growth inhibitor for two enterocytic-differentiated lines, a growth stimulator for two undifferentiated lines, and had no effect on two goblet cell-differentiated lines. TGF-beta 1 treatment for 3 days decreased RB gene expression and pRB level two- to threefold in each responsive line. When treated with TGF-beta 1 beginning in early G1, enterocytic cells were arrested in G1 and pRB remained under-phosphorylated and in low abundance. Neither goblet cell line exhibited these responses to TGF-beta 1 because they were shown to lack TGF-beta 1 type I and II receptors. Thus during colonocyte differentiation goblet cells lose responsiveness to TGF-beta 1 by down-regulating TGF-beta 1 receptors, while enterocytic cells retain and exhibit responsiveness to TGF-beta 1 through modulations of pRB. Both of the undifferentiated lines exhibited mixed responses to TGF-beta 1: a decrease in total amount of RB mRNA and pRB protein yet an increase in pRB phosphorylation consistent with increased cell cycling. Therefore, TGF-beta 1 controls RB function by two separable mechanisms, the regulation of pRB phosphorylation and the control of RB mRNA and protein level.

Topics: Carcinoma; Cell Cycle; Cell Differentiation; Colonic Neoplasms; Gene Expression; Genes, Retinoblastoma; Humans; In Vitro Techniques; Phosphorylation; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.

Topics: Breast Neoplasms; Cell Division; Colonic Neoplasms; Drug Synergism; Estradiol; Growth Inhibitors; Humans; In Vitro Techniques; Interleukin-4; Recombinant Proteins; Tamoxifen; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression.

Topics: Animals; Antibodies; Cell Division; Cell Line; Cloning, Molecular; Colonic Neoplasms; Genetic Vectors; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Plasmids; RNA, Antisense; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous

Topics: Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gastrointestinal Neoplasms; Humans; Neoplasm Invasiveness; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Stomach Neoplasms; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) stimulated the synthesis of heparan sulfate proteoglycan in cultured human colon carcinoma cells without affecting its rates of intracellular degradation or secretion. The overall hydrodynamic size, electrophoretic mobility, and degree of sulfation of the TGF-beta-induced proteoglycan was indistinguishable from that of untreated cells. The synthesis of the protein core was significantly stimulated by TGF-beta, although total cellular protein was unaltered. The stimulatory effects of TGF-beta were prevented by the inhibitors of protein synthesis and DNA transcription, cycloheximide, or actinomycin D, respectively. Analysis of protein core mRNA levels using a murine cDNA encoding a basement membrane protein core, revealed a marked elevation of the steady state levels of mRNA for this gene product. In contrast, the mRNA levels for glyceraldehyde-3-phosphate dehydrogenase or beta-actin genes were not significantly affected by TGF-beta. Finally, nuclear run-off experiments showed increases in neither protein core-specific transcription nor in general transcriptional activity. Taken together, these results indicate that TGF-beta is a potent modulator of heparan sulfate proteoglycan expression in human colon carcinoma cells and that its effect is mediated primarily through an increase in mRNA levels encoding the protein core, perhaps a result of enhanced RNA stability. The TGF-beta-induced elevation of heparan sulfate proteoglycan may contribute to the control of stromal cell proliferation and matrix production by human colon carcinoma cells.

Topics: Cell Line; Cell Nucleus; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Cycloheximide; Cysteine; Dactinomycin; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kinetics; Molecular Weight; Poly A; RNA; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor beta

Previously, we reported that exponentially proliferating cultures of well-differentiated human colon carcinoma cells responded to transforming growth factor beta 1 (TGF-beta) with growth inhibition, alterations in morphology, and increased secretion of the differentiation marker, carcinoembryonic antigen. Poorly differentiated cultures were unresponsive. Here we show that TGF-beta was ineffective in repressing nutrient-stimulated mitogenesis in quiescent, poorly differentiated cells. However, in quiescent, well-differentiated cells, TGF-beta repressed the mitogenic responses to both nutrients alone (by 90%) and to nutrients plus the exogenous stimulatory factors epidermal growth factor (E), insulin (I), and transferrin (T) (by 55-65%). Thymidine incorporation experiments indicated that TGF-beta reduced both the onset and peak mitogenic response to growth factors and/or nutrients in the well-differentiated cells. Additionally, TGF-beta repressed the growth factor (E + I + T)-stimulated upregulation of expression of both c-myc and of transforming growth factor alpha (TGF-alpha) mRNAs in quiescent, well-differentiated cells. TGF-beta also elicited a rapid (t1/2 approximately 1h) down-regulation of c-myc expression in the absence of prior growth factor (E + I + T) stimulation. In contrast, TGF-beta had no effect on c-myc or TGF-alpha mRNA expression in the poorly differentiated cells. The results suggest that TGF-beta exerts rapid inhibitory effects on proliferation-associated genes in quiescent and restimulated, well-differentiated cells. Expression of these genes (c-myc and TGF-alpha) may otherwise (in the absence of TGF-beta) play roles in the cellular signaling of mitogenic responses by growth stimulatory factors in well-differentiated colon carcinoma cells.

Topics: Blotting, Northern; Cell Line; Colonic Neoplasms; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Insulin; Mitosis; Proto-Oncogene Proteins c-myc; RNA; Transferrin; Transforming Growth Factor alpha; Transforming Growth Factor beta

Previously, we described a model culture system for comparing responsiveness of poorly differentiated and well-differentiated human colon carcinoma cells to exogenous growth factors. While polypeptide growth stimulators elicited an up-regulation of c-myc, as well as a mitogenic response in the well-differentiated cells, the poorly differentiated cells were insensitive to exogenous growth stimulators. We now show, by thymidine incorporation experiments and autoradiographic analysis, that transforming growth factor beta 1 (TGF-beta) abrogated the mitogenic responses to the growth factors epidermal growth factor + insulin + transferrin (IC50 = 0.8 ng/ml), as well as to nutrients (basal medium; IC50 = 0.2 ng/ml) in the well-differentiated cells. The poorly differentiated cells did not respond to TGF-beta. Moreover, TGF-beta (10 ng/ml) completely abrogated the growth factor-stimulated up-regulation of c-myc in the TGF-beta responsive, well-differentiated colon carcinoma cells. Addition of TGF-beta to the TGF-beta-responsive, well-differentiated cells, at a time after c-myc had been transiently up-regulated in response to growth stimulatory factors, resulted in a loss of responsiveness to TGF-beta. Addition of TGF-beta to these cells at increasing time periods after EIT stimulation also resulted in a loss of the TGF-beta-induced repression of c-myc. The results suggest an important role for c-myc in the mechanism of action of TGF-beta in well-differentiated human colon carcinoma cells.

Topics: Cell Differentiation; Cell Division; Cell Line; Colonic Neoplasms; Culture Media; Exons; Genes, myc; Humans; Proto-Oncogene Proteins c-myc; RNA, Neoplasm; Signal Transduction; Transforming Growth Factor beta

Previous studies from this laboratory (Schroy, P., Rifkin, J., Coffey, R.J., Winawer, S., and Friedman, E. (Cancer Res., 50: 261-265, 1990; Schroy, P.C., Winawer, S., and Friedman, E. Cancer Lett., 48: 53-58, 1989) found that a 7-day treatment of the human colon carcinoma cell line HT29 with the differentiation agent hexamethylene bisacetamide (HMBA) induces both a 4-5-fold increase in transforming growth factor beta 1 (TGF beta 1) mRNA levels and reduced tumorigenicity in vivo. A series of 15 cloned lines with different commitments to differentiation has been isolated from 20-day HMBA-treated HT29 cells, maintained without HMBA, and utilized to study the role of TGF beta 1 in colon carcinoma differentiation. Two such lines, HD6 and HD8, differentiate to 97 and 76% mucus-secreting goblet cells, respectively, in columnar monolayers in postconfluent culture. Both HD6 and HD8 cells exhibit low TGF beta 1 mRNA levels, little different from the undifferentiated HT29 parental line, and exhibit no growth modulation in response to exogenous TGF beta 1. In contrast, two other lines, HD3 and HD4, differentiate to fluid-transporting enterocytic cells with functional brush borders and exhibit autocrine-negative growth response to TGF beta 1. Both lines express TGF beta 1 mRNA at levels 11-12-fold higher than the parental line and respond to exogenous TGF beta 1 by growth inhibition. HD3 cells secrete biologically active TGF beta 1 into conditioned media, which inhibited growth of a TGF beta 1-sensitive mink cell line. This inhibition was blocked by antisera to TGF beta 1, proving the specificity of the inhibition. A range of concentrations of this TGF beta 1 antiserum stimulated HD3 cell growth in a dose-dependent manner, further documenting the autocrine-negative response of the cells to TGF beta 1. Another cell line, HI1, was blocked in enterocytic differentiation. HI1 cells synthesized as much TGF beta 1 mRNA as HD3 and HD4 cells, yet they responded to exogenous TGF beta 1 with less growth inhibition, suggesting some impairment in their response to TGF beta 1. A third class of response to TGF beta 1 was exhibited by the HP1 cell line, which was resistant to HMBA-induced differentiation, remaining undifferentiated with a multilayered growth pattern. HP1 cells synthesized TGF beta 1 mRNA at levels over 20 times the parental level but were stimulated to divide by TGF beta 1, exhibiting autocrine-positive response to this growth factor.(ABSTRACT TRUNCATED AT 400 WORDS

Topics: Acetamides; Cell Differentiation; Clone Cells; Colon; Colonic Neoplasms; Growth Inhibitors; Humans; Mucins; RNA, Messenger; Time Factors; Transforming Growth Factor beta