Masakazu Yashiro

FGFR2-Amplified Gastric Cancer Cell Lines Require FGFR2 and Erbb3 Signaling for Growth and Survival

We have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific small-molecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification.

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.

Role of the stemness factors sox2, oct3/4, and nanog in gastric carcinoma.

BACKGROUNDnCancer stem cells have been proposed to be responsible for cancer tumorigenicity, and then to persist in tumors as a distinct population and cause relapse and metastasis. Recently, the stemness factors Sox2, Oct3/4, and Nanog were associated with induced pluripotent stem cells, suggesting a correlation between these stemness factors and cancer stem cells. We therefore investigated the role of stemness factors in the tumorigenesis of human gastric cancer.nnnMATERIALS AND METHODSnA total of 290 patients who had undergone resection of a primary gastric cancer at our institute were enrolled. A curative R0 resection was performed for 253 of 290 patients, and the remaining 37 patients were treated with a palliative resection. The expression levels of Sox2, Oct3/4, and Nanog were analyzed by immunohistochemistry.nnnRESULTSnSox2, Oct3/4, and Nanog expression were positive in 159 (55%), 129 (44%), and 28 (10%) of 290 gastricxa0cancers, respectively. There was a statistically significantxa0correlation between Sox2-positive or Oct3/4-negative expression and invasion depth, lymph node metastasis, or lymphatic invasion. In 253 patients with a curative resection, the prognosis of patients with Sox2-positive tumors or Oct3/4-negative tumors was significantly (P < 0.01 or P = 0.04, log-rank) worse than that of patients with Sox2-negative or Oct3/4-positive tumors, respectively. A multivariate analysis revealed the expression of Sox2 or Oct3/4 toxa0be an independent prognostic factor (P = 0.01 or P = 0.04).nnnCONCLUSIONSnSox2-positive expression or Oct3/4-negative expression might be associated with invasion of gastric cancer. Sox2 and Oct3/4 might be independent prognostic factors for patients with gastric cancer.

Foretinib (GSK1363089), a multi-kinase inhibitor of MET and VEGFRs, inhibits growth of gastric cancer cell lines by blocking inter-receptor tyrosine kinase networks

SummaryTo explore the mechanism of action of foretinib (GSK1363089), an oral multi-kinase inhibitor known to target MET, RON, AXL, and vascular endothelial growth factor receptors (VEGFRs), in gastric cancer, we evaluated the effects of the agent on cell growth and cell signaling in the following panel of gastric cancer cell lines: KATO-III, MKN-1, MKN-7, MKN-45, and MKN-74. Of these, only MKN-45 and KATO-III, which harbor MET and fibroblast growth factor receptor 2 (FGFR2) amplification, respectively, were highly sensitive to foretinib. In MKN-45, 1xa0μM of foretinib or PHA665752, another MET kinase inhibitor, inhibited phosphorylation of MET and downstream signaling molecules as expected. In KATO-III, however, PHA665752 inhibited phosphorylation of MET independently of downstream molecules. Further, 1xa0μM of foretinib or PD173074, a selective FGFR kinase inhibitor, inhibited phosphorylation of FGFR2 and downstream molecules, suggesting that foretinib targets FGFR2 in KATO-III. We confirmed this novel activity of foretinib against FGFR2 in OCUM-2M, another FGFR2-amplified gastric cancer cell line. Using a phospho-receptor tyrosine kinase array, we found that foretinib inhibits phosphorylation of epidermal growth factor receptor (EGFR), HER3 and FGFR3 via MET inhibition in MKN-45, and EGFR, HER3 and MET via FGFR2 inhibition in KATO-III. Knockdown of HER3 and FGFR3 in MKN-45 with siRNA resulted in the partial inhibition of cell signaling and cell growth. In conclusion, foretinib appears effective against gastric cancer cells harboring not only MET but also FGFR2 amplification, and exerts its inhibitory effects by blocking inter-RTK signaling networks with MET or FGFR2 at their core.

HER2 overexpression correlates with survival after curative resection of pancreatic cancer.

HER2 overexpression has been linked to clinical outcomes in several solid tumors, such as breast cancer. However, the correlation between HER2 overexpression and survival in pancreatic carcinoma remains unclear. The impact of HER2 overexpression on survival in pancreatic ductal cancer was examined. Immunohistochemical staining of 129 pancreatic cancers without hematogenous metastases or peritoneal dissemination treated by macroscopically curative resection were analyzed in association with survival data. To determine HER2 overexpression in this pancreatic cancer series, the polyclonal antibody included in HercepTest, which is used worldwide for clinical examination of HER2 overexpression in breast cancer, was used. Immunoreactivity was classified according to the scale presented in the HercepTest Scoring Guidelines. Twenty‐two cases (17.1%) had a score of 0, 28 cases (21.7%) had of a score of 1+, 41 cases (31.8%) had a score of 2+, and 38 cases (29.4%) had a score of 3+. Therefore, HER2 overexpression (score 2+ or 3+) was observed in 79 cases (61.2%). Patients with HER2 overexpression tumors had significantly shorter survival times than those with HER2 normal expression (score 0 or 1+) tumors (median survival time, 14.7 vs 20.7 months, respectively; P = 0.0078 on the log‐rank test). On multivariate survival analysis, HER2 overexpression remained an independent prognostic factor (hazard ratio, 1.806; P = 0.0258). A significant percentage of pancreatic cancers were demonstrated to have HER2 overexpression, and overexpression of this tyrosine kinase receptor proved to be an independent factor for a worse prognosis. These results should encourage further investigation of treatments using new molecular targeting agents against HER2 protein to improve the survival of pancreatic cancer patients. (Cancer Sci 2009; 100: 1243–1247)

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)

Monoclonal Antibodies to Fibroblast Growth Factor Receptor 2 Effectively Inhibit Growth of Gastric Tumor Xenografts

Purpose: Overexpression of fibroblast growth factor receptor 2 (FGFR2) may be a causative factor of a number of human tumors, especially gastric tumors of the poorly differentiated type. We investigated whether monoclonal antibodies (mAbs) directed against FGFR2 can inhibit the growth of tumors in xenograft models. Experimental Design: We generated and characterized 3 mAbs that recognize different epitopes on FGFR2: GAL-FR21, GAL-FR22, and GAL-FR23. The ability of the mAbs to recognize the FGFR2IIIb and FGFR2IIIc isoforms of FGFR2 was determined, as was their ability to block binding of FGF ligands to FGFR2. The capability of the mAbs to inhibit FGF-induced FGFR2 phosphorylation and to downmodulate FGFR2 expression was also investigated. Finally, the ability of the anti-FGFR2 mAbs to inhibit tumor growth was determined by establishing xenografts of SNU-16 and OCUM-2M human gastric tumor cell lines in nude mice, treating with each mAb (0.5–5 mg/kg intraperitoneally twice weekly) and monitoring tumor size. Results: Of the 3 mAbs, GAL-FR21 binds only the FGFR2IIIb isoform, whereas GAL-FR22 and GAL-FR23 bind to both the FGFR2IIIb and FGFR2IIIc forms, with binding regions respectively in the D3, D2-D3, and D1 domains of FGFR2. GAL-FR21 and GAL-FR22 blocked the binding of FGF2, FGF7 and FGF10 to FGFR2IIIb. GAL-FR21 inhibited FGF2 and FGF7 induced phosphorylation of FGFR2, and both mAbs downmodulated FGFR2 expression on SNU-16 cells. These mAbs effectively inhibited growth of established SNU-16 and OCUM-2M xenografts in mice. Conclusions: Anti-FGFR2 mAbs GAL-FR21 and GAL-FR22 have potential for the treatment of gastric and other tumors. Clin Cancer Res; 16(23); 5750–58. ©2010 AACR.

Polymeric micelles incorporating (1,2-diaminocyclohexane)platinum (II) suppress the growth of orthotopic scirrhous gastric tumors and their lymph node metastasis

Nano-scaled drug carriers have great potential for the treatment of solid tumors. Nevertheless, hypovascularity and fibrosis in some types of solid tumors have been demonstrated to reduce the penetration and accumulation of nano-scaled drug carriers. Diffuse-type scirrhous gastric cancers present such characteristics as well as frequent metastasis to the lymph nodes; therefore, it remains a great challenge to eradicate scirrhous gastric cancers based on the drug targeting using nanocarriers. Herein, we demonstrated that polymeric micelles with 30-nm diameter incorporating (1,2-diaminocyclohexane)platinum(II) (DACHPt), the parent complex of the anticancer drug oxaliplatin, efficiently penetrated and accumulated in an orthotopic scirrhous gastric cancer model, leading to the inhibition of the tumor growth. Moreover, the elevated localization of systemically injected DACHPt-loaded micelles in metastastic lymph nodes reduced the metastatic tumor growth. These results suggest DACHPt-loaded micelles as a promising nanocarrier for the treatment of scirrhous gastric cancers and their lymphatic metastases.

Epigenetic silencing of miR-210 increases the proliferation of gastric epithelium during chronic Helicobacter pylori infection

Persistent colonization of the gastric mucosa by Helicobacter pylori (Hp) elicits chronic inflammation and aberrant epithelial cell proliferation, which increases the risk of gastric cancer. Here we examine the ability of microRNAs to modulate gastric cell proliferation in response to persistent Hp infection and find that epigenetic silencing of miR-210 plays a key role in gastric disease progression. Importantly, DNA methylation of the miR-210 gene is increased in Hp-positive human gastric biopsies as compared with Hp-negative controls. Moreover, silencing of miR-210 in gastric epithelial cells promotes proliferation. We identify STMN1 and DIMT1 as miR-210 target genes and demonstrate that inhibition of miR-210 expression augments cell proliferation by activating STMN1 and DIMT1 . Together, our results highlight inflammation-induced epigenetic silencing of miR-210 as a mechanism of induction of chronic gastric diseases, including cancer, during Hp infection.

Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-β.

Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer‐initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse‐type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5–8% of the human diffuse‐type gastric carcinoma cells, OCUM‐2MLN and HSC‐39; were more tumourigenic than ALDH1− cells; and were able to self‐renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet‐derived family, member 4) as one of the genes up‐regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony‐forming ability of OCUM‐2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF‐β signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse‐type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF‐β signalling. These findings illustrate that, in diffuse‐type gastric carcinoma, REG4 is up‐regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF‐β down‐regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse‐type gastric carcinoma. Copyright