transforming-growth-factor-beta and Peritoneal-Neoplasms

To identify strategies that enhance tumor-specific immunity in patients with ovarian carcinoma, 22 patients received four to six doses of i.p. recombinant IFN-gamma (rIFN-gamma), 200 microg/m2 on days 1, 3, 5, 8, 10, and 12, and i.p. recombinant interleukin 2 (rIL-2), either 6.0 x 10(5) IU/m2 (group A) or 1.0 x 10(5) IU/m2 (group B), on days 9, 10, and 11. Two patients in group A also received T-cell lines expanded from peritoneal tumor-infiltrating lymphocytes (TILs) obtained after i.p. rIFN-gamma/rIL-2 administration. Toxicity was manageable and included five nonhematological grade 3 or 4 events in 22 patients (23%). A patient had normalization of CA-125 values and a progression-free interval of 18 months, after receiving i.p. rIFN-gamma/rIL-2 without TILs. Another patient who received i.p. rIFN-gamma/rIL-2 plus TILs had stabilization of ascites and intra-abdominal tumors and >50% reduction in serum CA-125 values over 6 months. A third patient who received i.p. rIFN-gamma/rIL-2 had stabilization of intra-abdominal tumors and ascites accompanied by CA-125 values of 50 to 100 units over 6 months. T-cell lines for adoptive immunotherapy were developed for only 3 of 20 patients who were treated with rIFN-gamma/rIL-2. Large numbers of CD3- CD56+ adherent cells were expanded in rIL-2 in the remaining patients, precluding the development of T-cell lines. i.p. rIFN-gamma, either alone or followed by rIL-2, increased proportions of human leukocyte antigen (HLA) class I+ and class II+ tumor cells and increased HLA class I staining intensity on peritoneal carcinoma cells. i.p. rIFN-gamma plus rIL-2 also enhanced cytotoxic activity against Daudi and K562 cells and against allogeneic ovarian tumor cells. Increased cytotoxic activity was associated with an increase in the proportion of CD56+ cells. IFN-gamma and IL-2 transcripts were expressed more frequently after rIFN-gamma and rIL-2 treatment. In addition, the proportions of CD45RA+ (naive lymphocytes) were increased, and CD8+ DR+ lymphocytes were increased relative to CD8+ CD69+ cells, which were decreased. IL-10 concentrations in peritoneal fluids were increased after treatment with rIFN-gamma and the higher rIL-2 dosing (group A) but not in those treated with rIFN-gamma and the lower rIL-2 dosing (group B). These results demonstrated that patients with ovarian carcinoma can tolerate treatment with rIFN-gamma and rIL-2 and that rIFN-gamma alone or rIFN-gamma combined with rIL-2 enhances the expression of HLA cla

Topics: Ascitic Fluid; CA-125 Antigen; CD3 Complex; CD4-Positive T-Lymphocytes; CD56 Antigen; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Membrane; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Genes, MHC Class I; Genes, MHC Class II; Humans; Immunohistochemistry; Immunotherapy, Adoptive; Injections, Intraperitoneal; Interferon-gamma; Interleukin-10; Interleukin-2; K562 Cells; Leukocytes, Mononuclear; Lymphocytes, Tumor-Infiltrating; Neopterin; Ovarian Neoplasms; Peritoneal Neoplasms; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Cells, Cultured

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

Cancer-associated fibroblasts (CAFs) play an important role in cancer progression. However, the origin of CAFs remains unclear. This study shows that macrophages in malignant ascites and pleural effusions (cavity fluid-associated macrophages: CAMs) transdifferentiate into fibroblast-like cells. CAMs obtained from gastrointestinal cancer patients were sorted by flow cytometry and cultured in vitro. CD45

Topics: Animals; Ascites; Cancer-Associated Fibroblasts; Cell Adhesion Molecules; Cell Line, Tumor; Cell Proliferation; Fibroblasts; Humans; Macrophages; Mice; Peritoneal Neoplasms; Pleural Effusion; Thy-1 Antigens; Transforming Growth Factor beta; Tumor Microenvironment

Peritoneal metastasis, a hallmark of incurable advanced gastric cancer (GC), presently has no curative therapy and its molecular features have not been examined extensively. Here we present a comprehensive multi-omic analysis of malignant ascitic fluid samples and their corresponding tumor cell lines from 98 patients, including whole-genome sequencing, RNA sequencing, DNA methylation and enhancer landscape. We identify a higher frequency of receptor tyrosine kinase and mitogen-activated protein kinase pathway alterations compared to primary GC; moreover, approximately half of the gene alterations are potentially treatable with targeted therapy. Our analyses also stratify ascites-disseminated GC into two distinct molecular subtypes: one displaying active super enhancers (SEs) at the ELF3, KLF5 and EHF loci, and a second subtype bearing transforming growth factor-β (TGF-β) pathway activation through SMAD3 SE activation and high expression of transcriptional enhancer factor TEF-1 (TEAD1). In the TGF-β subtype, inhibition of the TEAD pathway circumvents therapy resistance, suggesting a potential molecular-guided therapeutic strategy for this subtype of intractable GC.

Topics: Ascites; Cell Line, Tumor; Humans; Peritoneal Neoplasms; Stomach Neoplasms; Transforming Growth Factor beta

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

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

Peritoneal metastasis is a primary metastatic route for gastric cancers, and the mechanisms underlying this process are still unclear. Peritoneal mesothelial cells (PMCs) undergo mesothelial-to-mesenchymal transition (MMT) to provide a favorable environment for metastatic cancer cells. In this study, we investigated how the exosomal miR-21-5p induces MMT and promotes peritoneal metastasis. Gastric cancer (GC)-derived exosomes were identified by transmission electron microscopy and western blot analysis, then the uptake of exosomes was confirmed by PKH-67 staining. The expression of miR-21-5p and SMAD7 were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, and the interactions between miR-21-5p and its target genes SMAD7 were confirmed by Luciferase reporter assays. The MMT of PMCs was determined by invasion assays, adhesion assays, immunofluorescent assay, and western blot. Meanwhile, mouse model of tumor peritoneal dissemination model was performed to investigate the role of exosomal miR-21-5p in peritoneal metastasis in vivo. We found that PMCs could internalize GC-derived exosomal miR-21-5p and led to increased levels of miR-21-5p in PMCs. Through various types of in vitro and in vivo assays, we confirmed that exosomal miR-21-5p was able to induce MMT of PMCs and promote tumor peritoneal metastasis. Moreover, our study revealed that this process was promoted by exosomal miR-21-5p through activating TGF-β/Smad pathway via targeting SMAD7. Altogether, our data suggest that exosomal miR-21-5p induces MMT of PMCs and promote cancer peritoneal dissemination by targeting SMAD7. The exosomal miR-21-5p may be a novel therapeutic target for GC peritoneal metastasis.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Epithelium; Exosomes; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; Peritoneal Neoplasms; Peritoneum; Signal Transduction; Smad7 Protein; Stomach Neoplasms; Transforming Growth Factor beta

Peritoneal dissemination is the primary metastatic route of ovarian cancer (OvCa), and is often accompanied by the accumulation of ascitic fluid. The peritoneal cavity is lined by mesothelial cells (MCs), which can be converted into carcinoma-associated fibroblasts (CAFs) through mesothelial-to-mesenchymal transition (MMT). Here, we demonstrate that MCs isolated from ascitic fluid (AFMCs) of OvCa patients with peritoneal implants also undergo MMT and promote subcutaneous tumour growth in mice. RNA sequencing of AFMCs revealed that MMT-related pathways - including transforming growth factor (TGF)-β signalling - are differentially regulated, and a gene signature was verified in peritoneal implants from OvCa patients. In a mouse model, pre-induction of MMT resulted in increased peritoneal tumour growth, whereas interfering with the TGF-β receptor reduced metastasis. MC-derived CAFs showed activation of Smad-dependent TGF-β signalling, which was disrupted in OvCa cells, despite their elevated TGF-β production. Accordingly, targeting Smad-dependent signalling in the peritoneal pre-metastatic niche in mice reduced tumour colonization, suggesting that Smad-dependent MMT could be crucial in peritoneal carcinomatosis. Together, these results indicate that bidirectional communication between OvCa cells and MC-derived CAFs, via TGF-β-mediated MMT, seems to be crucial to form a suitable metastatic niche. We suggest MMT as a possible target for therapeutic intervention and a potential source of biomarkers for improving OvCa diagnosis and/or prognosis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Topics: Animals; Ascites; Ascitic Fluid; Carcinoma; Cell Line, Tumor; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Humans; Mice; Ovarian Neoplasms; Peritoneal Neoplasms; Receptors, Transforming Growth Factor beta; Sequence Analysis, RNA; Smad3 Protein; Transforming Growth Factor beta

Peritoneal carcinomatosis (PC) is the most frequent metastatic pattern of gastric cancer and its prognosis is extremely poor. PC is characterized by rich fibrosis and the development of obstructive disorders such as ileus, jaundice and hydronephrosis. Epithelial-mesenchymal transition (EMT) is one of the major causes of tissue fibrosis and transforming growth factor β (TGF-β) has a pivotal function in the progression of EMT. Protein-bound polysaccharide K (PSK) is a biological response modifier that can modulate the TGF-β/Smad signaling pathway in vitro. In the present study, we established a fibrotic tumor model using human peritoneal mesothelial cells (HPMCs) and a human gastric cancer cell line to evaluate whether PSK attenuates tumor fibrosis. HPMCs exposed to PSK did not undergo the morphological change from a cobblestone-like pattern to a spindle-shape pattern normally induced by treatment with TGF-β. Immunofluorescence further demonstrated that PSK suppressed TGF-β-induced overexpression of α-SMA in the HPMCs. We further showed that HPMCs contributed to the proliferation of tumor fibrosis by using a mouse xenograft model. Additionally, PSK treatment of these mice significantly reduced the area of observable tumor fibrosis. These results suggest that seeded cancer cells transformed HPMCs into myofibroblast-like cells through their release of TGF-β in the microenvironment, facilitating the development of fibrous tumors in organs covered with HPMCs. Therefore, our study indicates that PSK has potential utility as an anti-fibrotic agent in the treatment of gastric cancer patients with PC.

Topics: Actins; Animals; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Fungal Proteins; Humans; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Peritoneal Neoplasms; Polysaccharides; Signal Transduction; Stomach Neoplasms; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

A recent study reported that long non-coding RNA activated by TGF-β (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-β (TGF-β)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients.. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-β, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-β1 or TGF-β receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype.. The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-β in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression.. LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Aged; Apoptosis; Biomarkers, Tumor; Carcinoma, Signet Ring Cell; Cell Proliferation; Female; Follow-Up Studies; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Liver Neoplasms; Lymphatic Metastasis; Male; MicroRNAs; Neoplasm Invasiveness; Neoplasm Staging; Peritoneal Neoplasms; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Long Noncoding; RNA, Messenger; Stomach Neoplasms; Survival Rate; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Zinc Finger E-box-Binding Homeobox 1

The peritoneal cavity is a common target of metastatic gastrointestinal and ovarian cancer cells, but the mechanisms leading to peritoneal metastasis have not been fully elucidated. In this study, we examined the roles of cells in peritoneal fluids on the development of peritoneal metastasis. We found that a minor subset of human intraperitoneal cells with CD90(+)/CD45(-) phenotype vigorously grew in culture with mesothelial-like appearance. The mesothelial-like cells (MLC) displayed the characteristics of mesenchymal stem cell, such as differentiating into adipocytes, osteocytes, and chondrocytes, and suppressing T cell proliferation. These cells highly expressed type I collagen, vimentin, α-smooth muscle actin and fibroblast activated protein-α by the stimulation with TGF-β, which is characteristic of activated myofibroblasts. Intraperitoneal co-injection of MLCs with the human gastric cancer cell line, MKN45, significantly enhanced the rate of metastatic formation in the peritoneum of nude mice. Histological examination revealed that many MLCs were engrafted in metastatic nodules and were mainly located at the fibrous area. Dasatinib, a potent tyrosine kinase inhibitor, strongly inhibited the proliferation of MLCs but not MKN45 in vitro. Nevertheless, oral administration of Dasatinib significantly inhibited the development of peritoneal metastasis of MKN45, and resulted in reduced fibrillar formation of metastatic nodules. These results suggest floating MLCs in the peritoneal fluids support the development of peritoneal metastasis possibly through the production of the permissive microenvironment, and thus the functional blockade of MLCs is a reasonable strategy to treat recurrent abdominal malignancies.

Topics: Animals; Cell Differentiation; Collagen Type I; Epithelium; Female; Humans; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Myofibroblasts; Peritoneal Neoplasms; Peritoneum; Stomach Neoplasms; Thy-1 Antigens; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

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

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

The TGFβ-mediated alteration of the tumor microenvironment plays a crucial role in tumor progression. Mesothelial cells are the primary components of the tumor microenvironment for ovarian cancer cells; however, the exact role of TGFβ-stimulated mesothelial cells in ovarian cancer progression remains uncertain. In this report, we examined the effects of TGFβ-treated mesothelial cells on ovarian cancer progression. We show that TGFβ-stimulated human primary mesothelial cells (HPMC) are able to promote cancer cell attachment and proliferation and the activation of the promoter activities of MMP-2 and MMP-9, which are metalloproteinases necessary for tumor invasion. Expression of the miR200 family was downregulated in HPMCs by TGFβ stimulation, and restoration of the expression of miR200 family members in HPMCs suppressed cancer cell attachment and proliferation. Downregulation of the miR200 family by TGFβ induced fibronectin 1 production, which promoted cancer cell attachment to HPMCs. Finally, we demonstrated that the delivery of the miR200s to mesothelial cells in mice inhibited ovarian cancer cell implantation and dissemination. Our results suggest that alteration of the tumor microenvironment by the miR200 family could be a novel therapeutic strategy for ovarian cancer treatment.

Topics: Animals; Base Sequence; Binding Sites; Cell Adhesion; Cell Proliferation; Epithelial Cells; Epithelium; Female; Fibronectins; Gene Expression; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Molecular Sequence Data; Neoplasm Transplantation; Ovarian Neoplasms; Peritoneal Neoplasms; Transforming Growth Factor beta

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

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

TP53 (Tumor Protein 53, previously known as p53) is probably the best known of all tumor suppressor genes, and is mutated in nearly all (96%) high-grade serous ovarian cancer (HGS-OvCa), which is the most common histopathological type of epithelial ovarian cancer (EOC). Recently, TP53 is found to involve in regulating cell metabolic pathways besides its classical tumor suppressive functions. In addition, emerging evidence suggests that mutant TP53 is associated with cancer metastasis. Through summarizing and comparing the roles of wild-type TP53 and mutant TP53 in the progression of various types of cancer, we hypothesize that mutant TP53 in HGS-OvCa cells interacts with sterol regulatory element-binding proteins (SREBPs) and guanidinoacetate N-methyltransferase (GAMT), leading to increased gene expression of key enzymes involved in fatty acids (FAs) and cholesterol biosynthesis and the inhibition of fatty acid oxidation (FAO), thus promotes lipid anabolism to accelerate tumor growth and progression. Elevated platelet number in patients' tumor microenvironment results in increased TGF-β production. Then, TGF-β acts in concert with mutant TP53 to promote HGS-OvCa metastasis by assembling a mutant-TP53/p63/Smads protein complex, in which p63's functions as metastasis suppressor are antagonized, and by enhancing the activities of the Slug/Snail and Twist families to drive induce EMT-like transition. Then adipocyte-derived IL-8 facilitates the metastasis of transformative cancer cells to abdominal adipose tissue (e.g., omentum). Once metastasis is established, mutant TP53 together with adipocyte-derived IL-8 upregulates Fatty acid-binding protein 4 (FABP4) expression and then promotes FAs absorption from adipocytes to support rapid tumor growth in adipocyte-rich metastatic environments. In summary, these indicate that mutant TP53 may play determinant roles in the progression of HGS-OvCa.

Topics: Fatty Acid-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Guanidinoacetate N-Methyltransferase; Humans; Interleukin-8; Lipid Metabolism; Mutation; Neoplasm Metastasis; Omentum; Ovarian Neoplasms; Peritoneal Neoplasms; Sterol Regulatory Element Binding Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Peritoneal dissemination including omental metastasis is the most frequent route of metastasis and an important prognostic factor in advanced ovarian cancer. We analyzed the publicly available microarray dataset (GSE2109) using binary regression and found that the transforming growth factor (TGF)-beta signaling pathway was activated in omental metastases as compared to primary sites of disease. Immunohistochemical analysis of TGF-beta receptor type 2 and phosphorylated SMAD2 indicated that both were upregulated in omental metastases as compared to primary disease sites. Treatment of the mouse ovarian cancer cell line HM-1 with recombinant TGF-β1 promoted invasiveness, cell motility and cell attachment while these were suppressed by treatment with A-83-01, an inhibitor of the TGF-β signaling pathway. Microarray analysis of HM-1 cells treated with TGF-β1 and/or A-83-01 revealed that A-83-01 efficiently inhibited transcriptional changes that are induced by TGF-β1. Using gene set enrichment analysis, we found that genes upregulated by TGF-β1 in HM-1 cells were also significantly upregulated in omental metastases compared to primary sites in the human ovarian cancer dataset, GSE2109 (false discovery rate (FDR) q = 0.086). Therapeutic effects of A-83-01 in a mouse model of peritoneal dissemination were examined. Intraperitoneal injection of A-83-01 (150 μg given three times weekly) significantly improved survival (p = 0.015). In summary, these results show that the activated TGF-β signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Profiling; Humans; Immunoenzyme Techniques; Mice; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Omentum; Ovarian Neoplasms; Peritoneal Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Pyrazoles; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad2 Protein; Survival Rate; Thiocarbamates; Thiosemicarbazones; Transforming Growth Factor beta; Up-Regulation

βig-h3 is an extracellular matrix protein and its expression is highly induced by transforming growth factor (TGF-β). It has also been suggested to play an important role in the growth and invasion of colon and pancreatic cancers. In the present study, we demonstrated that βig-h3 is expressed in mesothelial cells, especially in patients with advanced gastric cancer. The positive rate of βig-h3 was significantly higher in cases with a more invasive and advanced serous-type, with visible peritoneal metastasis, and in peritoneal lavage cytological examination (PLC) (+) and peritoneal lavage fluid CEA mRNA(+) subgroups (p<0.05). Our study also showed that the expression of βig-h3 gradually increased with increasing TGF-β1 concentrations in vitro in a time-dependant manner. In addition, βig-h3 also induced human gastric carcinoma cell line (SGC-7901) cell adhesion in a dose-dependent manner and significantly increased cell migration and proliferation. The results suggest that βig-h3 expression in peritoneal mesothelial cells in gastric cancer patients is a marker of the biological behavior of gastric cancer and plays an important role in the process of peritoneal carcinomatosis.

Topics: Carcinoembryonic Antigen; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial Cells; Extracellular Matrix Proteins; Humans; Peritoneal Lavage; Peritoneal Neoplasms; Recombinant Proteins; Stomach Neoplasms; Transforming Growth Factor beta

Ovarian cancer metastasis is characterized by the shedding of malignant cells from the surface of the ovary and their implantation onto the peritoneal surface, which lines the abdominal cavity. As the factors promoting this process are poorly understood, we investigated the ovarian cancer-peritoneal interaction by means of in vitro coculture experiments with ovarian cancer (OVCAR-5 and SKOV-3) and peritoneal (LP-9) cells. One of the proteins differentially expressed in the coculture secretome was identified by MALDI-TOF/TOF mass spectrometry as the extracellular matrix protein transforming growth factor-beta-induced protein (TGFBIp, also known as βig-H3). Immunohistochemistry showed high TGFBIp levels in normal surface ovarian epithelial and peritoneal cells, whereas TGFBIp levels in primary serous ovarian carcinomas and matching metastatic implants was very low. In functional in vitro experiments, treatment with recombinant TGFBIp significantly increased the motility and invasiveness of OVCAR-5 and SKOV-3 cells and significantly increased ovarian cancer cell (OVCAR-5, OVCAR-3 and SKOV-3) adhesion to LP-9 cells. TGFBIp was found to be processed at both the N- and C-terminus in the secretome of the ovarian cancer-peritoneal cell coculture. Plasmin inhibitors blocked TGFBIp processing and significantly reduced OVCAR-5 cell adhesion to peritoneal cells. We conclude that TGFBIp expressed by peritoneal cells increases the metastatic potential of ovarian cancer cells. TGFBIp is therefore a potential novel therapeutic target against ovarian cancer.

Topics: Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Coculture Techniques; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Neoplasm Metastasis; Ovarian Neoplasms; Ovary; Peritoneal Neoplasms; Transforming Growth Factor beta

Gastric cancer cells leaving the primary tumour are exposed to low oxygen levels in the peritoneal cavity; however, peritoneal metastatic phenotypes of hypoxic cancer cells remain unclear. We used 6 gastric cancer cell lines, including 3 diffuse-type gastric cancer (DGC) and 3 non-DGC cell lines. Using adhesion assay, we examined the effect of hypoxic conditions on their ability to adhere to peritoneal components. The expression level of transforming growth factor-beta (TGF-beta) and integrins mRNA of cancer cells was examined using reverse transcriptase-polymerase chain reaction. We further examined the effect of anti-integrin neutralising antibodies and a TGF-beta receptor inhibitor on the adhesion ability of hypoxic cancer cells. The binding ability of DGC cells was higher than that of non-DGC cells; it was significantly increased by hypoxic (1% O2) conditions compared to normoxic (21% O2) conditions. In contrast, no remarkable change in adhesion ability was observed in the non-DGC cells under normoxic and hypoxic conditions. Integrins and TGF-beta expression of hypoxic DGC cells was significantly higher than that of normoxic cells. TGF-beta increased the adhesion ability and alpha2-, alpha3- and alpha5-integrin expression of hypoxic DGC cells, whereas the TGF-beta receptor inhibitor decreased them. Neutralising antibodies against alpha2-, alpha3- and alpha5-integrin inhibited the adhesion ability of DGC cells. These findings suggested that hypoxic conditions promote the adhesion of DGC cells to the peritoneum. The upregulation of alpha2-, alpha3- and alpha5-integrin by TGF-beta under hypoxic conditions may be one of the mechanisms responsible for the high metastatic potential of hypoxic DGC cells to the peritoneum.

Topics: Antibodies, Neutralizing; Benzamides; Cell Adhesion; Cell Hypoxia; Cell Line, Tumor; Dioxoles; Humans; Integrins; Neoplasm Proteins; Peritoneal Neoplasms; Phosphorylation; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad2 Protein; Smad3 Protein; Stomach Neoplasms; Transforming Growth Factor beta

Our previous results suggested that a lack of RUNX3 function contributed to human gastric carcinogenesis, but the role of RUNX3 in progression and metastasis remains unclear. We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray.. Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100%) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-beta (TGF-beta)-induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of approximately 21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified approximately 28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A1 1, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in the TGF-beta signaling pathway.. These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

Topics: Animals; Apoptosis; Base Sequence; Blotting, Northern; Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 3 Subunit; DNA Methylation; DNA-Binding Proteins; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Neoplasms, Experimental; Oligonucleotide Array Sequence Analysis; Peritoneal Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Nucleic Acid; Stomach Neoplasms; Transcription Factors; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous; Up-Regulation

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.

Topics: Animals; Binding, Competitive; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Cell Membrane; Coloring Agents; Culture Media, Conditioned; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Extracellular Matrix; Fibrinolysin; Flow Cytometry; Genome; Humans; Membrane Glycoproteins; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Oligonucleotides, Antisense; Peritoneal Neoplasms; Protein Binding; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins; Serine Endopeptidases; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin Inhibitor, Kunitz Soybean; Urokinase-Type Plasminogen Activator

The modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean trypsin inhibitor, as dietary supplements on experimental and spontaneous pulmonary metastasis of murine Lewis lung carcinoma 3LL cells as well as peritoneal disseminated metastasis model in human ovarian cancer HRA cells were investigated in i.v., s.c. and i.p. injection models in mice. Seven groups of female C57BL/6 or nude mice were fed a basal diet (control group) or the basal diet supplemented with KTI or BBI (5, 15, or 50 g/kg). Here we show that, in an in vivo spontaneous metastasis assay, the diet supplementation with KTI (15 and 50 g/kg), but not with BBI, for 28 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of KTI. In an in vivo experimental metastasis assay, the diet supplementation with KTI or BBI for 21 days after i.v. tumor cell inoculation did not reduce the number of lung tumor colonies. In addition, KTI (15 or 50 g/kg) treatment in a peritoneal disseminated metastasis model of HRA cells resulted in a 40% reduction in total tumor burden when compared with control animals. Immunoblot analysis revealed that KTI specifically reduced expression of uPA protein as well as phosphorylation of MAP kinase and PI3 kinase proteins in the cells stimulated with agonists (G-CSF for 3LL cells or TGF-beta1 for HRA cells). These results suggest that dietary supplementation of KTI more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation during the metastatic process. KTI treatment may also be beneficial for ovarian cancer patients with or at risk for peritoneal disseminated metastasis; it greatly reduces tumor burden in part by inhibiting phosphorylation of MAP kinase and PI3 kinase, leading to suppression of uPA expression.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Dietary Supplements; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Models, Animal; Ovarian Neoplasms; Peritoneal Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin Inhibitor, Bowman-Birk Soybean; Trypsin Inhibitor, Kunitz Soybean; Trypsin Inhibitors; Urokinase-Type Plasminogen Activator

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.

Topics: Cell Adhesion; Cells, Cultured; Culture Media, Conditioned; Epithelium; Female; Fibroblasts; Humans; MAP Kinase Signaling System; Membrane Glycoproteins; Neoplasm Invasiveness; Ovarian Neoplasms; Peritoneal Neoplasms; Peritoneum; Plasminogen Activator Inhibitor 1; Recombinant Proteins; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin Inhibitor, Kunitz Soybean; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

Different patterns of granulomas have been observed in 6- to 8-week-old mice after ip inoculation with 5 x 10(6) yeast cells of Paracoccidioides brasiliensis. Transforming growth factor-beta (TGF-beta) is a cytokine that has been shown to participate in fibrosis and granuloma formation; its activities seem to be modulated by the small proteoglycan decorin. In the present study, TGF-beta and decorin expression in epiploon granulomas was assessed by immunohistochemistry in susceptible (B10.A) and resistant (A/J) mice after 15, 30, 120 and 150 days of P. brasiliensis ip infection. The epiploon was collected, fixed in Methacarn solution and embedded in paraffin, and 5-microm thick sections were used for immunohistochemical analysis employing the streptavidin-biotin-peroxidase technique. The former mouse strain developed fatal disease with many disseminated lesions increasing in size and number during the infection and the latter developed mild disease with the presence of encapsulated granulomas. In the epiploon, TGF-beta was present on macrophages, giant cells, lymphocytes and fibroblasts, and absent on neutrophils. It was also detected in areas of fibrosis and necrosis, as well as disperse in amorphous extracellular matrix, mostly in resistant mice. Decorin was present circumscribing macrophages and giant cells containing fungi, but absent on these cells. In both mouse strains, decorin was found at the periphery of the lesions, and markedly in milky spot granulomas. In resistant mice, positivity was found around fibrotic and necrotic areas of encapsulated and residual lesions containing lysed fungi. Decorin was found associated with thick fibers around encapsulated lesions. In susceptible mice, the size and number of lesions increased with the progression of the disease and were correlated with the weaker expression of decorin. We suggest an association of decorin with the fibrogenic process observed in paracoccidioidal granulomas.

Topics: Animals; Decorin; Extracellular Matrix; Extracellular Matrix Proteins; Female; Granuloma; Immunohistochemistry; Mice; Mice, Inbred A; Omentum; Paracoccidioides; Peritoneal Neoplasms; Proteoglycans; Transforming Growth Factor beta

In vitro morphologic change of mesothelial cells was observed following the addition of serum-free conditioned medium (SF-CM) from peritoneal dissemination cell line OCUM-2MD3. The same morphologic change of mesothelial cells was observed following the addition of 10 ng/ml TGF-beta1, but not following the addition of b-FGF, IGF-I, VEGF or PDGF-AA. In the in vivo study, mesothelial cells of mice treated with SF-CM from OCUM-2MD3 and TGF- beta1 were separated from one another, resulting in exposure of the submesothelial connective tissue. The molecular size of the mesothelial morphology changing activity was estimated by running the SF-CM from OCUM-2MD3 through a gel filtration column TSK-gel G2000SW. The mesothelial morphology changing activity was recognized at positions equivalent of Mr 6, 500-30,000. 25 kDa TGF-beta1 was detected in the active fraction from the TSK-gel G2000SW column and the SF-CM of OCUM-2MD3 by Western blotting using a monoclonal antibody against TGF-beta1. These findings suggest that TGF-beta1 produced by gastric cancer cells changes the morphology of mesothelial cells and may thus be closely associated with peritoneal dissemination.

Topics: Animals; Epithelium; Female; Humans; Mice; Mice, Nude; Molecular Weight; Peritoneal Neoplasms; Stomach Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Peritoneal dissemination frequently occurs after surgery in patients with gastric cancer. The presence of peritoneal metastasis after surgery affects prognosis. Very little is known about the biochemical processes involved in the initial attachment of gastric cancer cells to peritoneal mesothelial cells. We conducted in vitro and in vivo studies to assess the role of adhesion molecules and TGF-beta1 in this process, using 4 cell lines derived from human gastric cancers. NUGC-4 cells, which disseminate early after inoculation into the abdominal cavity of nude mice, predominantly express CD44H and beta1 integrin. We found that NUGC-4 cells adhered to monolayers of mesothelial cells more firmly than to other cell lines. Adhesion of NUGC-4 cells to mesothelial cells was partially inhibited by antibodies against CD44H or the beta1 subunit of integrin and was completely blocked by a combination of these 2 antibodies. Treatment with ligands for CD44H and beta1 integrin also inhibited adhesion. In the NUGC-4 cell culture medium, larger amounts of TGF-beta1 were detected in relation to the increase in cancer cells than in the other cell lines. TGF-beta1 increased the expression of CD44H in NUGC-4 cells and in mesothelial cells and augmented adhesion and implantation of NUGC-4 cells to mesothelial cells accompanied by accumulation of extracellular matrix (ECM) components. Treatment with antibodies against both CD44H and beta1 integrin inhibited the dissemination of NUGC-4 cells in the peritoneal cavity of nude mice and prolonged their survival time. Our findings suggest that CD44H and integrins mediate the initial attachment of gastric cancer cells to mesothelial cells and that TGF-beta1 participates in the promotion of the disease. Increased expression of CD44H and of the amount of ligands for CD44H and integrins induced by TGF-beta1 promotes early development of peritoneal dissemination.

Topics: Animals; Cell Adhesion Molecules; Flow Cytometry; Humans; Hyaluronan Receptors; Integrins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Peritoneal Neoplasms; Stomach Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured