Transforming growth factor beta 1 secreted from scirrhous gastric cancer cells is associated with excess collagen deposition in the tissue.

To explore the mechanism of increased collagen deposition in scirrhous carcinoma of the stomach, an attempt was made to define the role of transforming growth factor beta 1 (TGF-beta 1), secreted from tumour cells, as a possible humoral factor which functions in a paracrine manner to stimulate the production of collagen in regional fibroblasts. Immunohistochemical staining revealed that tumour cells in scirrhous carcinomas were generally stained more intensively than those in other types of carcinomas. On Northern blot analysis the tumour cells established from scirrhous carcinoma (KATO-III, OCUM-1 and HSC-39) exhibited relatively strong signals compared with those from non-scirrhous carcinoma (MKN-28 and MKN-45). In the culture media of scirrhous carcinoma cells, the active form of TGF-beta 1 was detected, while in those of the non-scirrhous carcinoma cells the latent form was demonstrated by both colony and radioreceptor assays. The culture medium from KATO-III showed strong stimulating activity of collagen synthesis in fibroblasts, and this activity was partially neutralised by an anti-TGF-beta 1 antibody. These results suggest that tumour cells in scirrhous carcinoma produce more active-form TGF-beta 1 than does non-scirrhous carcinoma and thus is partially responsible for the observed enhanced collagen deposition in the region.     

Regulation of transforming growth factor beta1 by nitric oxide

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.     

Transforming growth factor beta 1 expression in human colorectal tumours: an independent prognostic marker in a subgroup of poor prognosis patients.

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

Cleavage of hepatocyte growth factor activator inhibitor-1 by membrane-type MMP-1 activates matriptase

Co-expression of membrane-type 1 (MT1)-MMP with hepatocyte growth factor activator inhibitor-1 (HAI-1) in HEK293T cells resulted in cleavage of HAI-1 to produce three fragments. Recombinant MT1-MMP was shown to cleave HAI-1 protein in vitro. Hepatocyte growth factor activator inhibitor-1 was initially identified as the cognate inhibitor of matriptase, a transmembrane serine protease that processes urokinase-type plasminogen activator (uPA). Co-expression of HAI-1 with matriptase suppressed matriptase protease activity, and co-expression of MT1-MMP with them resulted in recovery of matriptase activity by stimulating shedding of HAI-1 fragments. Matriptase protein was detected in squamous carcinoma-derived HSC-4 cells, however, matriptase protease activity was undetectable. Transfection of siRNA for HAI-1 enhanced serine protease activity, which was suppressed by cotransfection of matriptase siRNA. Collagen-gel culture or treatment with concanavalin A (ConA) of HSC-4 cells enhanced MT1-MMP activity, which induced shedding of HAI-1 fragments and conversely stimulated uPA activation by these cells. Serine protease activity, including uPA activation of cells treated with ConA, was abrogated by downregulation of either matriptase or MT1-MMP through the transfection of each siRNA. These results suggest that MT1-MMP induced by collagen-gel culture or ConA treatment causes cleavage and shedding of HAI-1 protein, which allows activation of matriptase in HSC-4 cells. HSC-4 cells showed a characteristic invasive growth by forming vacuole-like structures in collagen gel, which was suppressed by transfection of siRNA for either MT1-MMP or matriptase, suggesting that activation of matriptase through the cleavage of HAI-1 is one of the MT1-MMP multifunctions essential for invasive growth of HSC-4 cells.     

Effects of transforming growth factor-beta released from gastric carcinoma cells on the contraction of collagen-matrix gels containing fibroblasts.

To investigate the mechanisms underlying contraction of the stomach wall in cases of gastric scirrhous carcinoma, we have developed an in vitro model for gastric cancer, in which both fibroblasts and gastric carcinoma cells are embedded within a collagen matrix. Gastric carcinoma cells of the scirrhous type (KATO-III) but not the nonscirrhous type (MKN-28) markedly enhanced the ability of human intestine, human lip, and mouse kidney fibroblasts to contract collagen gels. KATO-III cells released transforming growth factor-beta (TGF-beta) into culture media in an activated form, whereas the MKN-28 cells produced a latent form. The role of TGF-beta produced by gastric cancer cells from the scirrhous type was clarified by adding TGF-beta (receptor grade) into collagen gels embedded with fibroblasts, contraction being enhanced. Other growth factors tested, including transforming growth factor-alpha and epidermal growth factor, did not enhance the contraction of collagen gels containing embedded human and rodent fibroblasts. These results suggest that the activated form of TGF-beta released from gastric scirrhous carcinoma cells stimulates fibroblasts to contract the collagenous stroma of the stomach wall, which leads to the so-called "linitis plastica" stomach condition.     

Hepatocyte growth factor activation inhibitors (HAI-1 and HAI-2) regulate HGF-induced invasion of human breast cancer cells

Hepatocyte growth factor (HGF) plays a plethora of roles in cancer metastasis and tumour growth. The interaction between tumour cells and their surrounding stromal environment is a crucial factor regulating tumour invasion and metastasis. Stromal fibroblasts are the main source of HGF in the body, and release HGF as an inactive precursor (pro-HGF). HGF activator (HGFA), matriptase, urokinase-type plasminogen activator and hepsin are the main factors responsible for converting pro-HGF into active HGF. HAI-1 and HAI-2 are 2 novel Kunitz-type serine protease inhibitors that regulate HGF activity through inhibition of HGFA, matriptase and hepsin action. Recent studies demonstrate that HAI-1 and HAI-2 may also potently inhibit a number of other pro-metastatic serine proteases and therefore have direct bearing on the spread of tumours. Our study examined the potential of these HAI's to suppress the influence of HGF and regulate cancer metastasis. We generated a retroviral expression system that induced HAI expression in a human fibroblast cell line. Forced expression of either HAI-1 or HAI-2 in these fibroblasts resulted in a dramatic decrease in the production of bioactive hepatocyte growth factor (HGF). This reduction in HGF activity subsequently suppressed HGF's metastatic influence on breast cancer cells. To further assess the anti-cancer properties of HAI-1 and HAI-2 we generated recombinant HAI proteins. These recombinant HAI proteins possessed the ability to potently quench HGF activity. We also demonstrate that these recombinant HAI's suppressed fibroblast-mediated breast cancer invasion. An additional ribozyme transgenes study revealed that elimination of HAI-1 and HAI-2 expression, in an MDA-MB-231 breast cancer cell line, significantly enhanced the migratory, proliferative and invasive nature of these breast cancer cells. Overall, our data demonstrates the important roles of HAI-1 and HAI-2 in cancer metastasis, and reveals that these serine protease inhibitors display strong therapeutic potential.     

Particular types of tumor cells have the capacity to convert transforming growth factor beta from a latent to an active form.

We investigated the capacities of various tumor types to generate an active versus latent form of transforming growth factor beta (TGF-beta) in its culture supernatants (SNs). Tumor cell lines were divided into three types depending on the form and magnitude of TGF-beta detected in their culture SNs: some (2 of 7 lines) generated mostly an active form (Type A); others (4 of 7) generated exclusively a latent form (Type B); and the remaining line (1 of 7) produced only marginal levels of active/latent TGF-beta (Type C). When Type A tumor cells were cultured at lower numbers, cultures failed to generate active TGF-beta. However, the addition of Type B tumor cell culture SNs containing only a latent form of TGF-beta resulted in the generation of the potent activity of active TGF-beta. This capacity was observed for another Type A tumor but not for other types (Type B and Type C). An active form of TGF-beta was detected in culture SNs of Type A tumor cells as early as 3-6 h after the addition of Type B tumor culture SNs. The emergence of an active form of TGF-beta was also observed in cultures of Type A tumor cells, the protein synthesis of which was almost completely inhibited by pretreatment with cycloheximide. Moreover, the Type B tumor SN used for the induction of active TGF-beta activity was found to contain latent TGF-beta with an apparent molecular weight of about 200,000. Type A tumor cells were also capable of generating active TGF-beta by the addition of recombinant TGF-beta of latent form with a small molecular weight (about 60,000), although the generation of active TGF-beta was much weaker after the addition of small latent TGF-beta than after the addition of large latent TGF-beta. Taken collectively, these results indicate that particular types of tumor cells have the capacity to generate an active form of TGF-beta and that such capacity can be attributed to their potential to convert TGF-beta from a latent (mainly large type) to an active form.     

Transforming growth factor beta 1 and sodium butyrate differentially modulate urokinase plasminogen activator and plasminogen activator inhibitor-1 in human breast normal and cancer cells.

The effects of transforming growth factor beta 1 (TGF-beta1) and sodium butyrate on cell proliferation and the urokinase plasminogen activator (uPA) system were examined in normal human breast epithelial cells (HBECs) and in a breast cancer cell line, MDA-MB-231. In HBECs, TGF-beta1 inhibited cell proliferation and uPA activity, while it augmented plasminogen activator inhibitor-1 (PAI-1) antigen level. Sodium butyrate inhibited both cell proliferation and uPA activity but did not affect the level of PAI-1. In MDA-MB-231, TGF-beta1 had no effect on cell proliferation but increased uPA activity and PAI-1 antigen level; sodium butyrate inhibited both cell proliferation and uPA activity but had no effect on PAI-1 level. Moreover, in the presence of plasminogen, cell detachment could be modulated by the level of cell-associated uPA. Our results indicate (1) that the effects of TGF-beta1 on cell growth can be dissociated from its effects on the uPA/PAI system; (2) that, while TGF-beta1 is a potent inhibitor of cell proliferation and uPA activity in normal cells, it may promote invasion and metastasis of tumour cells by increasing uPA activity and PAI-1 levels; and (3) that sodium butyrate offers a potential approach to preventing tumour development by inhibiting both cell proliferation and invasion.     

Plasminogen activator system and its clinical significance in patients with a malignant disease

Urokinase (uPA) plays an essential role in the activation of plasminogen to plasmin, a serine protease participating in the activation of matrixmetaloproteinases, latent elastases, growth factors and cytokines involved in the degradation of extracellular matrix elements. Together with its receptor (uPAR), tissue activator (tPA) and urokinase inhibitors (PAI-1, PAI-2, PAI-3 and protease nexin), it forms the plasminogen activator system (PAS), a component of metastatic cascade importantly contributing to the invasive growth and angiogenesis of malignant tumours. Plasminogen activator inhibitor 1 inhibits uPA-dependent invasiveness of some cancer cell lines. The vitronectin-PAI-1 complex inhibits migration of smooth muscle cells by binding alpha(v)beta3 integrin to vitronectin. PAI-1 or its deficiency interferes with signalling pathways such as PI3K/Akt and JAK/STAT and it is included in the processes of maintaining the integrity of the endothelial cells and thereby regulation of cell death. PAI-1 affects apoptosis by reducing cell adhesion and functioning of intracellular signalling pathways. The individual components of PAS undoubtedly play an important role in angiogenesis and metastasising of malignant tumours. In the near future, results of published studies with various types of cancer could be reflected in diagnostic and therapeutic algorithms and, at the same time, could serve as the goal for targeted therapies.     

Changes in response to, and production of, transforming growth factor type beta during neoplastic progression in cultured rat tracheal epithelial cells

As rat tracheal epithelial cells progress from a normal to a neoplastic phenotype there are systematic changes in their ability to produce and activate latent transforming growth factor type beta (TGF-beta) as well as systematic changes in their response to this growth factor. Using a TGF-beta radioreceptor binding competition assay it was found that normal proliferating rat tracheal cells in early primary culture produced latent TGF-beta. With the emergence of terminally differentiated cell populations active TGF-beta was also detected in the conditioned medium. When normal cells were cultured under conditions allowing for continued proliferation, no active TGF-beta was detected in the conditioned medium. Colonies of proliferating epithelial cells in 4-6 week primary cultures or subculturable tracheal cell lines did not produce detectable levels of active or latent growth factor. With neoplastic progression there was likewise a change in response to active TGF-beta. Normal tracheal cells in primary culture were highly sensitive to growth-factor-induced decreases in thymidine uptake as well as to the induction of terminal differentiation. Proliferating epithelial cells in late (4-6 week) primary cultures and preneoplastic, subculturable cell lines were often as sensitive as normal cells to the growth factor-induced decline in thymidine uptake. None of these altered populations, however, was induced to differentiate (to form cornified, cross-linked envelopes) in the presence of TGF-beta.     

Detection of amplified DNA sequences in gastric cancers by a DNA renaturation method in gel

A DNA renaturation method in gel was used to detect amplified DNA fragments in eight gastric cancer cell lines, KATO-III, OKAJIMA, SCH, MKN1, MKN7, MKN28, MKN45 and MKN74 cells, and tissues of three metastases of gastric cancers to lymph nodes. There were amplified DNA sequences in three gastric cancer cell lines. Judging from the intensities of the bands, HindIII-digested DNA fragments were amplified several hundred times in the KATO-III and MKN7 cell lines. DNA from OKAJIMA cell line contained multiple bands with less intensity. KATO-III cells were found to contain a homogeneously staining region in chromosome 11.     

Studies on the interaction and exchange of inhibitors and proteases on the surface of tumour cells in frozen sections.

We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface protease associated with tumour cells. This protease has been referred to as guanidinobenzoatase (GB) and is now known to be functionally identical to tissue plasminogen activator (t-PA). The use of a fluorescent competitive inhibitor of GB enabled the enzymic status of GB to be determined, i.e. was the enzyme active, latent or removed from our test system. The cell surface GB was then demonstrated to interact with extractable cytoplasmic inhibitors obtained from these sections. We then used a protected form of the GB in the absence of these internal inhibitors; such sections were used to transfer the GB to fibrin fibrils, thus exposing the presumptive receptor on the tumour cell surfaces. Texas red labelled t-PA was then shown to bind to the tumour cells in these pretreated sections from which the GB had previously been removed. We believe that the surface of tumour cells can be used to study the interaction of the naturally occurring inhibitors with GB and also that the cell surface receptors for GB can be used to study the binding of t-PA to cell surfaces.     

The surface of prostate carcinoma DU145 cells mediates the inhibition of urokinase-type plasminogen activator by maspin

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility. Interestingly, in cell-free solutions, maspin does not inhibit several serine proteases including tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). Despite the recent biochemical evidence that maspin specifically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-lysine, the molecular mechanism underlying the tumor-suppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental system, we chose prostate carcinoma DU145 cells because these cells mediate plasminogen activation primarily by uPA, as shown by two different colorimetric enzyme activity assays. Purified recombinant maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reactive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was comparable to the Ki values for plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinoma.     

Semi-quantitation of urokinase plasminogen activator and its receptor in breast carcinomas by immunocytochemistry.

Urokinase plasminogen activator (uPA) is a serine protease involved in cancer invasion and metastasis. uPA acts in vivo by binding to a membrane receptor known as uPAR. In this study, uPA and uPAR levels were semiquantitated by immunocytochemistry in 36 primary breast carcinomas. Using monoclonal antibody HD-UK 1, uPA was detected both in stromal and in malignant cells. However, the predominant location was in the stromal cells. Using double-staining, cells containing uPA were also found to coexpress either cytokeratin (an epithelial cell marker) or more frequently KP1 (a macrophage/monocyte cell marker). With monoclonal antibody HD-uPAR 13.1, uPAR was localized principally to spindle- or macrophage-like stromal cells, especially when these cells surrounded invasive breast cancer. In contrast, uPAR was only rarely detected in cancer cells and was not detected in normal epithelia surrounding tumour or in areas of adenosis. uPA levels in both stromal and epithelial cells were significantly correlated with those for uPAR. We conclude that both uPA and its receptor are mostly present in stromal cells in invasive breast carcinomas. These results suggest that stromal cells collaborate with malignant cells to mediate metastasis.     

Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors.     

Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines

The regulation of Mr 72,000 type IV collagenase and interstitial collagenase expression was studied in vitro. Three tumorigenic human cell lines were used, together with human fetal lung fibroblasts as a nontumorigenic control. Mr 72,000 type IV collagenase was expressed constitutively by all four cell lines, whereas only A2058 melanoma cells exhibited constitutive expression of interstitial collagenase. Treatment of cells with transforming growth factor beta 1 (TGF-beta 1) and 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed an opposite pattern of regulation of these two metalloproteinases. Specifically, TPA increased interstitial collagenase mRNA levels in each cell line and decreased type IV collagenase mRNA levels in control fibroblasts and the tumorigenic cell lines, HT-1080 and A2058. TGF-beta 1 treatment increased type IV collagenase mRNA levels in each cell line and decreased interstitial collagenase mRNA levels in A2058 melanoma cells. Interstitial collagenase mRNA induction was accompanied in all cell lines by elevated interstitial procollagenase in the conditioned medium, as detected by zymography. Changes in Mr 72,000 type IV collagenase expression revealed a more complex pattern of regulation. TPA and TGF-beta 1 treatment of HT-1080 cells resulted in the appearance of two bands of gelatinolytic activity with a molecular weight of approximately 62,000 and 59,000. The Mr 62,000 species was also induced by TGF-beta 1 treatment of A2058 cells. Addition of affinity-purified radiolabeled Mr 72,000 type IV procollagenase to TPA-treated HT-1080 cells demonstrated that both species were products of the Mr 72,000 proenzyme and that exogenous proenzyme could be processed by these cells. Western blot analysis with specific antipeptide antibodies revealed that both the Mr 62,000 and 59,000 species were derived from the Mr 72,000 proenzyme by amino-terminal cleavage. There was no evidence for cellular processing of either interstitial procollagenase or the Mr 92,000 type IV procollagenase. These results demonstrate that the Mr 72,000 type IV collagenase is under the control of different regulatory elements from interstitial collagenase, at the level of both mRNA expression and cellular processing, and that this processing appears to be the result of a phorbol ester and TGF-beta 1-inducible cellular activation mechanism. The ratio of active enzyme species to latent Mr 72,000 proenzyme may provide a better correlation with invasive potential than overall levels of this widely expressed metalloproteinase.     

The aberrant methylation of TSP1 suppresses TGF-beta1 activation in colorectal cancer

Colorectal cancer arises from the progressive accumulation of mutations and epigenetic alterations in colon epithelial cells. Such alterations often deregulate signaling pathways that affect the formation of colon cancer, such as the Wnt, RAS-MAPK and TGF-beta pathways. The tumor promoting effects of mutations in genes, such as APC, have been demonstrated in cancer cell lines and in mouse models of intestinal cancer; however, the biological effects of most epigenetic events identified in colorectal cancer remain unknown. Consequently, we assessed whether the aberrant methylation of TSP1, the gene for thrombospondin 1, a regulator of TGF-beta ligand activation, is an epigenetic mechanism for inhibiting the TGF-beta signaling pathway. We found methylated TSP1 occurs in colon cancer cell lines (33%), colon adenomas (14%) and colon adenocarcinomas (21%). In primary colorectal cancers, loss of TSP1 expression correlated with impaired TGF-beta signaling as indicated by decreased Smad2 phosphorylation and nuclear localization. Furthermore, methylation-induced silencing of TSP1 expression reduced the concentration of secreted active TGF-beta1 and attenuated TGF-beta signaling. Reversal of TSP1 methylation resulted in increased TSP1 mediated activation of the latent LAP:TGF-beta complex and subsequent TGF-beta receptor activation. Our results demonstrate that the aberrant methylation of TSP1 has biological consequences and provide evidence that the aberrant methylation of TSP1 is a novel epigenetic mechanism for suppressing TGF-beta signaling in colorectal cancer.     

Tissue level, activation and cellular localisation of TGF-beta1 and association with survival in gastric cancer patients

Transforming growth factor-beta1 (TGF-beta1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-beta1 activation and localisation of TGF-beta1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-beta1 staining by immunohistochemistry. Active TGF-beta1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-beta1. Active TGF-beta1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-beta1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-beta1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-beta1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-beta1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-beta1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-beta1 activity levels in gastric cancer.