Distribution of Transforming Growth Factor-β and Its Receptors in Gastric Carcinoma Tissue

The distribution of the three mammalian isoforms of transforming growth factor (TGF)-β (TGF-β1,-β2, and -β3) as well as their signaling receptors, TGF-β type I and type II receptors (TβR-I and TβR-II, respectively), in gastric carcinoma tissue was examined by immunohistochemistry using specific antibodies. Tissue specimens were obtained from 25 cases of gastric carcinoma, which were classified into two groups according to Lauren's classification, i.e. 15 cases of diffuse carcinoma and 10 cases of intestinal carcinoma. In normal gastric mucosa apart from carcinoma nests, all of TGF-β1, -β2, -β3, TβR-I and TβR-II were clearly demonstrated in fundic glands. In sharp contrast, none of them was detectable in surface mucous cells. In carcinoma cells, strong staining for TGF-β1, -β2 and β3 was obtained only in diffuse-type carcinoma. In particular, carcinoma cells scattered as single cells or small nests had a tendency to show strong staining for TGF-βs. The receptors tended to be distributed concomitantly with the ligands, and diffuse-type carcinoma showed stronger receptor staining than intestinal-type carcinoma. In cancer stroma, TGF-βs and receptors were detected in both diffuse and intestinal types, but the area with positive staining was wider and more dispersed in diffuse-type carcinoma than in intestinal carcinoma. These results suggest that TGF-β may contribute in part to the variety of histogenesis and mode of progression of gastric carcinoma.     

Halofuginone inhibits tumor growth in the polyoma middle T antigen mouse via a thrombospondin-1 independent mechanism

Halofuginone inhibits fibrosis by decreasing type I collagen synthesis and tumor growth through an anti-angiogenic mechanism. In vitro data suggested that halofuginone inhibits angiogenesis through upregulating thrombospondin-1 (TSP-1) expression and by inhibiting cell proliferation. To determine whether thrombospondin-1 (TSP-1) is necessary for inhibition of tumor growth and angiogenesis by halofuginone, we tested the effect of halofuginone on mammary tumor growth in polyoma middle T antigen, TSP-1 null (TSP-1-/-PyT) transgenic mice. After 30 days of treatment, we found a significant decrease in tumor weight in these mice and the extent of tumor growth inhibition was comparable to that found in TSP-1 expressing PyT mice (TSP-1+/+PyT). However, no significant difference in tumor weight was observed after 60 days of halofuginone treatment between control and treated mice in both genotypes. Interestingly, type I collagen level was lower in the halofuginone treated TSP-1+/+PyT tumors at 30 days, but this was not observed in the TSP-1-/-PyT mice. Levels of type I collagen did not correlate with blood vessel number as a decrease in the number of vessels was observed in the halofuginone treated tumors from both the TSP-1+/+PyT and TSP-1-/-PyT mice as compared to control tumors. Because halofuginone has been shown to inhibit type I collagen synthesis by inhibiting the TGF-beta signaling pathway, we measured Smad 2/3 phosphorylation levels and found that halofuginone inhibited Smad 2/3 phosphorylation in cells derived from TSP-1+/+PyT tumors. We also found that it inhibited Smad 2/3 phosphorylation in cells treated with the TGF-beta activating sequence of TSP-1, TSR2+RFK. Our data demonstrate that halofuginone inhibits mammary tumor growth in a transgenic mouse model via a TSP-1 independent pathway, by decreasing tumor angiogenesis and by inhibiting TGF-beta signaling.     

Exogenous introduction of tissue inhibitor of metalloproteinase 2 reduces accelerated growth of TGF‐β‐disrupted diffuse‐type gastric carcinoma

Diffuse‐type gastric carcinoma is characterized by rapid progression and poor prognosis. High expression of transforming growth factor (TGF)‐β and thick stromal fibrosis are observed in this type of gastric carcinoma. We have previously shown that disruption of TGF‐β signaling via introduction of a dominant negative form of the TGF‐β type II receptor (dnTβRII) into diffuse‐type gastric cancer cell lines, including OCUM‐2MLN, caused accelerated tumor growth through induction of tumor angiogenesis in vivo. In the present study, we show that TGF‐β induces upregulation of expression of tissue inhibitor of metalloproteinase 2 (TIMP2) in the OCUM‐2MLN cell line in vitro, and that expression of TIMP2 is repressed by dnTβRII expression in vivo. Transplantation of the OCUM‐2MLN cells to nude mice exhibited accelerated tumor growth in response to dnTβRII expression, which was completely abolished when TIMP2 was coexpressed with dnTβRII. Although the blood vessel density of TIMP2‐expressing tumors was only slightly decreased, the degree of hypoxia in tumor tissues was significantly increased and pericytes covering tumor vasculature were decreased by TIMP2 expression in OCUM‐2MLN cells, suggesting that the function of tumor vasculatures was repressed by TIMP2 and consequently tumor growth was reduced. These findings provide evidence that one of the mechanisms of the increase in angiogenesis in diffuse‐type gastric carcinoma is the downregulation of the anti‐angiogenic protein TIMP2. (Cancer Sci 2010; 101: 2398–2403)     

Reduced Levels of Transforming Growth Factor-beta Type I Receptor in Human Gastric Carcinomas

The expressions of transforming growth factor beta (TGF-β) and its receptor and TGF-β inhibitory element (TIE)-binding protein were examined on human gastric carcinomas by Northern blot hybridization, immunohistochemistry, affinity labeling and gel retardation analysis. TGF-β mRNA was expressed in tumor and normal tissues at various levels. Immunohistochemically, TGF-β expression was confirmed to be present within tumor cells. Out of the 17 human gastric carcinoma tissues, 14 (82%) showed a reduction in the level of type I receptor (65 kDa) for TGF-β when compared to corresponding normal mucosas. Interestingly, in seven of the 14 tumors the level of TIE-binding protein in the tumor tissue was lower than that in normal mucosa. Human gastric carcinoma cell line TMK-1, whose growth was inhibited by TGF-β, had only type I receptor for TGF-β and showed a high level of TIE-binding protein. Conversely, MKN-1, a TGF-β -resistant cell line, exhibited an extremely low level of TGF-β receptor and had no TIE-binding protein. These results overall indicate that although human gastric carcinoma cells produced TGF-β, they showed a reduction in TGF-β type I receptor and a low level of TIE-binding protein, resulting in escape from growth inhibition by TGF-β.     

Expression of thrombospondin-1 and Ski are prognostic factors in advanced gastric cancer

Background  

It has been suggested that thrombospondin-1 (TSP-1) plays a role in angiogenesis in many cancers. In addition, TSP-1 has been shown to suppress tumor growth by activating transforming growth factor-β (TGF-β). Recent studies have shown that Ski protein suppresses TGF-β signaling. The aim of this study was to investigate the role of TSP-1 and Ski expression in advanced gastric cancer.     

TGF-² Signaling and the Role of Inhibitory Smads in Non-small Cell Lung Cancer

The signaling pathway mediated by transforming growth factor-² (TGF-²) participates in various biologic processes, including cell growth, differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling. In the context of cancer, TGF-² signaling can inhibit tumor growth in early-stage tumors. However, in late-stage tumors, the very same pathway promotes tumor invasiveness and metastasis. This paradoxical effect is mediated through similar to mothers against decapentaplegic or Smad protein dependent and independent mechanisms and provides an opportunity for targeted cancer therapy. This review summarizes the molecular process of TGF-² signaling and the changes in inhibitory Smads that contribute to lung cancer progression. We also present current approaches for rational therapies that target the TGF-² signaling pathway in cancer.     

Cancer stem cell-like SP cells have a high adhesion ability to the peritoneum in gastric carcinoma

Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, but the evidence to conclusively prove this hypothesis remains uncertain. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC-rich population. The aim of this study was to clarify the characterization of the SP cells in peritoneal metastasis of gastric carcinoma. Gastric cancer cell lines OCUM-2M, OCUM-2D, and OCUM-2MD3 (a daughter cell line with high potential for peritoneal metastasis) were used. We isolated SP cells from OCUM-2M and OCUM-2D using flow cytometry. Serial sorting was performed three times to enrich SP cells, and they were designated as OCUM-2M/SP and OCUM-2D/SP cells. Flow cytometric analysis showed 0.46%, 0.29%, 5.24%, 6.49%, and 11.3% of the SP cells to be found in OCUM-2M, OCUM-2D, OCUM-2MD3, OCUM-2M/SP, and OCUM-2D/SP cells, respectively. The intraperitoneal inoculation of SP cells and OCUM-2MD3 cells produced peritoneal metastasis, but parent cells did not. The adhesion ability of SP and OCUM-2MD3 cells was significantly high in comparison to that of parent cells. The expression level of adhesion molecules α2 -, α5 -, β3 -, and β5-integrin , and CD44 , was high in SP cells compared to parent cells. The expression of stemness markers, Oct3/4 and Sox2, increased in the SP-cell-injected tumors. These findings suggested that CSC-like SP cells expressing α2 -, α5 -, β3 -, and β5-integrin , and CD44 , may play an important role for peritoneal metastasis in gastric carcinoma. Oct3/4 and Sox2 may be associated with CSC in gastric cancer. ( Cancer Sci 2009)     

Transforming Growth Factor-β and Hepatocyte Growth Factor Produced by Gastric Fibroblasts Stimulate the Invasiveness of Scirrhous Gastric Cancer Cells

Scirrhous gastric carcinoma is characterized by cancer cells that infiltrate rapidly in the stroma with extensive growth of fibroblasts. In the present study, we examined the effect of gastric fibroblasts on the invasiveness of a Scirrhous gastric cancer cell line, OCUM-2D, using an invasion assay. Gastric fibroblast-derived conditioned medium (CM) significantly stimulated the invasiveness of OCUM-2D cells, as did transforming growth factor- β (TGF- β ) and hepatocyte growth factor (HGF). The stimulating activity of gastric fibroblast-derived CM was inhibited significantly by anti-TGF- β neutralizing antibody or anti-HGF neutralizing antibody. TGF- β and HGF were detected in the gastric fibroblast-derived CM, and TGF- β receptor and C-met (HGF receptor) were expressed on OCUM-2D cells. Thus, TGF- β and HGF produced by gastric fibroblasts appear to affect the invasiveness of scirrhous gastric cancer cells. TGF- β was also detected in the conditioned medium derived from OCUM-2D cells, though HGF was not. TGF- β appears to affect the invasiveness of OCUM-2D cells in both paracrine and autocrine fashions.     

Thrombospondin 1 Triggers Osteosarcoma Cell Metastasis and Tumor Angiogenesis

Osteosarcomas, especially those with metastatic or unresectable disease, have limited treatment options. The antitumor effects of pharmacologic inhibitors of angiogenesis in osteosarcomas are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here we demonstrated that thrombospondin 1 (TSP-1) is a potent inhibitor of the growth and metastasis of the osteosarcoma cell line MG-63. Moreover, we demonstrate that upregulation of TSP-1 facilitated expression of vasculostatin in MG-63 cells. In angiogenesis assays, overexpression of TSP-1 inhibited MG-63 cells and induced tube formation of human umbilical vein endothelial cells (HUVECs) in a CD36-dependent fashion. Finally, in xenografted tumors, we observed that TSP-1 overexpression inhibited angiogenesis and tumor growth. These results provided strong evidence for an important role of the TSP-1/CD36/vasculostatin signaling axis in mediating the antiangiogenic activity of osteosarcoma.     

Bone marrow-derived stromal cells are associated with gastric cancer progression

Background:

The aim of this study was to clarify the role of bone marrow-derived stromal cells (BM-SCs) expressing CD271 in the development of gastric cancer.

Methods:

The effect of human BM-SCs on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analysed by flow cytometry. We also generated a gastric tumour model by orthotopic inoculation of OCUM-2MLN cells in mice that had received transplantation of bone marrow from the CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumour stroma was examined by immunohistochemistry.

Results:

Numerous BM-SCs infiltrated the gastric tumour microenvironment; CD271 expression was found in ∼25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, whereas migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumours, and tumour invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse compared with that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression.

Conclusions:

Bone marrow-derived stromal cells might have an important role in gastric cancer progression, and CD271-positive BM-SCs might be a useful prognostic factor for gastric cancer patients.     

Overexpression of thrombospondin-1 reduces growth and vascular index but not perfusion in glioblastoma

Little is known about the effects of antiangiogenic therapy on perfusion of human tumors and the mechanisms by which tumors can adapt to these treatments and recur. Here, we examined the effects of serial passaging of LN-229 human glioma xenografts overexpressing thrombospondin (TSP)-1 on tumor growth, vascularity, and perfusion. Persistence of TSP-1 overexpression was confirmed after three serial s.c. passages of small xenografted tumor blocks of cells stably transfected with TSP-1 cDNA (clones C9 and E7) or vector controls (pooled clones A7-A9) in immunodeficient nu/nu mice. The tumor vascularity was estimated by noninvasive near infrared spectroscopy measuring blood volume at 800 +/- 10 nm and by histological vessel scores in CD31-immunostained cryosections. The tumor perfusion was assessed by noninvasive laser Doppler flowmetry. Overexpression of TSP-1 significantly inhibited tumor growth. In size-matched tumors (approximately 300 mm(3)), the blood volume and the histological vessel scores were lower in the TSP-1-transfected tumors than in controls, and this effect was more pronounced in tumors derived from the clone with the highest TSP-1 expression (clone E9). Despite this clear reduction in tumor vascularity, the tumor perfusion was the same in TSP-1-transfected tumors and controls. This study shows that TSP-1 overexpression slows glioma growth in vivo but does not prevent it from reaching a large size (300 mm(3)). Whereas a clear reduction in blood volume during tumor growth and a reduced vascular index at sacrifice are observed in TSP-1-transfected tumors, this did not affect perfusion when size-matched comparisons were performed. Given the increased time needed to reach equal size, it indicates that a fixed rate of perfusion must be maintained in the tumor to allow for growth. Elucidation of the mechanisms that allow this to happen has important consequences for the understanding of tumor recurrence after antiangiogenic therapy.     

Induction of Growth Factor-receptor and Metalloproteinase Genes by Epidermal Growth Factor and/or Transforming Growth Factor-α in Human Gastric Carcinoma Cell Line MKN-28

We examined the effects of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) on EGF receptor (EGFR) phosphorylation and the expression of mRNAs for oncogenes, growth factors, their receptors and metalloproteinase genes by MKN-28 gastric carcinoma cells which express EGF, TGF-α and EGFR genes. Both EGF and TGF-α stimulated EGFR phosphorylation. EGF and TGF-α induced FOS, MYC and ERBB-2 oncogene expression. Interestingly, EGF increased the expression of mRNAs for TGF-α and EGFR. On the other hand, TGF-α increased TGF-α mRNA but decreased the expression of mRNAs for EGFR and TGF-β. Furthermore, mRNAs for interstitial collagenase, stromelysin and procollagen type I genes were also enhanced after treatment with EGF and TGF-α. These results indicate that EGF and TGF-α successively evoke cascade phenomena which favor tumor progression, invasion and extracellular matrix formation, acting as autocrine growth regulators for gastric carcinomas.     

Potentiation of Metastatic Capacity by Transforming Growth Factor-β1 Gene Transfection

This study was designed to assess whether the excessive secretion of transforming growth factor-β1 (TGF-β1) by Chinese hamster ovary (CHO) cells transfected with TGF-β1 gene may be linked to the development of a metastatic phenotype. We observed large numbers of metastatic colonies in the lungs of nude mice inoculated with the transfected CHO cells. The tumors derived from these transfected cells demonstrated marked angiogenesis. We postulate that the overproduction of TGF-β1 by these tumors may participate in the metastatic progression following establishment of angiogenesis at the primary tumor site.