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

Abstract

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.

Figure 1

Regulation of tissue inhibitor of metallo‐proteinase 2 (TIMP2) expression by transforming growth factor (TGF)‐β signaling. (A) Expression levels of human TIMP2 mRNA in tissue from xenografted subcutaneous tumors expressing dominant negative form of the TGF‐β type II receptor (dnTβRII) or green fluorescent protein (GFP) were analyzed by quantitative real‐time PCR. (B) Levels of expression of TIMP2 mRNA in OCUM‐2MLN cells in vitro were determined by quantitative real‐time PCR. OCUM‐2MLN cells expressing GFP or dnTβRII were treated with TGF‐β1 (1 ng/mL) for 0, 1, 6 or 20 h before harvest and RNA isolation.

Figure 2

Establishment of cells overexpressing tissue inhibitor of metalloproteinase 2 (TIMP2). (A,B) Stable expression of TIMP2 in 2MLN‐dnTβRII cells. OCUM‐2MLN green fluorescent protein (GFP)+GFP, OCUM‐2MLN dnTβRII+GFP and OCUM‐2MLN dnTβRII+TIMP2 cell lines were established using a lentiviral system. Proteins and RNA were harvested from the cultured cell lines, and expression of TIMP2 was confirmed by immunoblotting using antibody against TIMP2 (A) and quantitative real‐time PCR using primers against human TIMP2 (B). (C) In vitro growth assay of OCUM‐2MLN cell lines (GFP+GFP, dnTβRII+GFP and dnTβRII+TIMP2 cell lines) cultured in growth medium containing 1% FBS. The cell count was done in triplicate at day 1, 2 and 3 after cell seeding.

Figure 3

In vivo growth of OCUM‐2MLN tumors expressing the dominant negative form of the TGF‐β type II receptor (dnTβRII) and tissue inhibitor of metalloproteinase 2 (TIMP2). (A) green fluorescent protein (GFP)+GFP, dnTβRII+GFP or dnTβRII+TIMP2 expressing OCUM‐2MLN cells were transplanted subcutaneously into nude mice. Relative tumor volumes from the day of starting the evaluation (designated as day 0, 6 days after implantation) are shown. (B) The same cell lines as in (A) were orthotopically transplanted into the gastric walls of nude mice. Left panel: representative images of orthotopic tumors (marked by blue arrows) at day 21 after transplantation. Right panel: relative tumor volumes. Error bars represent standard errors (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6–8).

Figure 4

Effect of tissue inhibitor of metalloproteinase 2 (TIMP2) overexpression on tumor angiogenesis. The vascular density of tumor vasculature in OCUM‐2MLN subcutaneous tumors (n = 3–4 tumors for each condition). The vascular density was determined by immunostaining with an antibody against platelet/endothelial cell adhesion molecule‐1 (PECAM1). Left panel: representative micrographs of immunostained tumor sections photographed at ×40 magnification, with PECAM1‐positive areas shown in green. Right panel: percentage of the PECAM1‐positive area per microscopic field (at least 18 microscopic fields were analyzed for each condition). dnTβRII, dominant negative form of the TGF‐β type II receptor; GFP, green fluorescent protein. Error bars represent standard errors (*P < 0.05, ***P < 0.001).

Figure 5

Effect of tissue inhibitor of metallo‐proteinase 2 (TIMP2) overexpression on the function of tumor vessels. (A) The hypoxic area in OCUM‐2MLN subcutaneous tumors (n = 3–4 tumors for each condition). The hypoxic area of tumors was determined by immunostaining with an antibody against Hypoxyprobe. Left panel: representative micrographs of immunostained tumor sections photographed at ×20 magnification, with areas positive for Hypoxyprobe shown in red. Platelet/endothelial cell adhesion molecule‐1 (PECAM1), green. Right panel: percentage of Hypoxyprobe‐positive area per microscopic field (18–20 microscopic fields were analyzed for each condition). Error bars represent standard errors (***P < 0.001). (B) Vessel structure in OCUM‐2MLN subcutaneous tumors. Representative micrographs of tumor sections photographed at ×40 magnification with immunostaining of endothelial cells and pericytes using an antibody against PECAM1 (green) and neuron‐glial antigen 2 (NG2) (red), respectively. Yellow arrows indicate PECAM1‐positive vessels lacking co‐staining of NG2. dnTβRII, dominant negative form of the TGF‐β type II receptor; GFP, green fluorescent protein.