Erik Johansson

Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.

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

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-β (TGF-β) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-β repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-β, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-β negatively contributes to maintain the CICs within the cancer.

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

Background Diffuse-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. Methods We 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. Results Expression 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. Conclusions Disruption 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.

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)