Diffuse-Type Gastric Carcinoma: Progression, Angiogenesis, and Transforming Growth Factor β Signaling

Abstract

BackgroundDiffuse-type gastric carcinoma is a cancer with poor prognosis that has high levels of transforming growth factor β (TGF-β) expression and thick stromal fibrosis. However, the association of TGF-β signaling with diffuse-type gastric carcinoma has not been investigated in detail.MethodsWe used a lentiviral infection system to express a dominant-negative TGF-β type II receptor (dnTβRII) or green fluorescent protein (GFP) as a control in the diffuse-type gastric carcinoma cell lines, OCUM-2MLN and OCUM-12. These infected cells and the corresponding parental control cells were subcutaneously or orthotopically injected into nude mice. Angiogenesis was inhibited by infecting cells with a lentivirus carrying the gene for angiogenic inhibitor thrombospondin-1 or by injecting mice intraperitoneally with the small-molecule angiogenic inhibitor sorafenib or with anti-vascular endothelial growth factor (VEGF) neutralizing antibody (six or eight mice per group). Expression of phospho-Smad2 and thrombospondin-1 was investigated immunologically in human gastric carcinoma tissues from 102 patients. All statistical tests were two-sided.ResultsExpression of dnTβRII into OCUM-2MLN cells did not affect their proliferation in vitro, but it accelerated the growth of subcutaneously or orthotopically transplanted tumors in vivo (eg, for mean volume of subcutaneous tumors on day 10 relative to that on day 0: dnTβRII tumors = 3.49 and GFP tumors = 2.46, difference = 1.02, 95% confidence interval [CI] = 0.21 to 1.84; P = .003). The tumors expressing dnTβRII had higher levels of angiogenesis than those expressing GFP because of decreased thrombospondin-1 production. Similar results were obtained with OCUM-12 cells. Expression of thrombospondin-1 in the dnTβRII tumor or treatment with sorafenib or anti-VEGF antibody reduced tumor growth, whereas knockdown of thrombospondin-1 expression resulted in more accelerated growth of OCUM-2MLN tumors than of GFP tumors (eg, mean tumor volumes on day 14 relative to those on day 0: thrombospondin-1–knockdown tumors = 4.91 and GFP tumors = 3.79, difference = 1.12, 95% CI = 0.80 to 1.44; P < .001). Positive association between phosphorylated Smad2 and thrombospondin-1 immunostaining was observed in human gastric carcinoma tissues.ConclusionsDisruption of TGF-β signaling in diffuse-type gastric carcinoma models appeared to accelerate tumor growth, apparently through increased tumor angiogenesis that was induced by decreased expression of thrombospondin-1.

Transforming growth factor-β decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells

“…Tumor growth of diffuse-type gastric carcinoma OCUM-2MLN cells is reported to be enhanced by inhibition of the TGF-b signaling pathway in cancer cells (Kiyono et al, 2009;Komuro et al, 2009). In accordance with these reports, TGF-b-induced phosphorylation of Smad2 was completely inhibited in OCUM-2MLN cells, which stably expressed dominant negative form of TbRII (2MLN-dnTbRII), but not in control cells, which expressed green fluorescent protein (2MLN-GFP) (Figure 1a).…”

“…We previously reported that deregulation of the TGFb signaling pathway in the diffuse-type gastric carcinoma OCUM-2MLN cells, which were derived from lymph node metastasis of orthotopically implanted primary carcinoma cells, promotes the growth of primary tumors in mouse xenograft models through acceleration of angiogenesis (Kiyono et al, 2009;Komuro et al, 2009). In addition to this mechanism, we here present evidence that TGF-b has a novel role in regulation of the maintenance of CICs in diffuse-type gastric carcinoma cells.…”

“…Immunoblotting Immunoblotting was performed as described (Ehata et al, 2007b;Komuro et al, 2009). Anti-phospho-phosphorylated RB antibody was obtained from BD Pharmingen (San Jose, CA, USA).…”

Overexpression of miR-370 and downregulation of its novel target TGFβ-RII contribute to the progression of gastric carcinoma

“…A previous study has identified that upregulation of the miR-106b/miR-93/miR-25 cluster impairs G 1 /S arrest and apoptosis in GC cells and that this is associated with TGFb signaling (Petrocca et al, 2008). Disruption in TGFb signaling facilitates the growth of diffuse-type GC (Komuro et al, 2009). Furthermore, TGFb suppresses the progression of diffuse-type GC by inducing the differentiation of cancer-initiating cell sub-population (Ehata et al, 2011).…”

“…SB431542 blocks Smad signaling by inactivating TGFb1-RI receptor kinase (Inman et al, 2002). Impairment of TGFb signaling in GC models appeared to accelerate tumor growth (Komuro et al, 2009) and resistance to the TGFb suppressor seems to be an important event in gastric carcinogenesis (Ju et al, 2003). A recent study has shown that E2F2 upregulates the expression of the miR-106b-25 polycistronic cluster, which targets TGFb signaling and enhances gastric carcinogenesis (Petrocca et al, 2008).…”