G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression. Cancer Res; 70(20); 7830–40. ©2010 AACR.

Classic signaling by the proinflammatory cytokine interleukin 6 (IL-6) involves its binding to target cells that express the membrane-bound IL-6 receptor α. However, an alternate signaling pathway exists in which soluble IL-6 receptor (sIL-6Rα) can bind IL-6 and activate target cells that lack mIL-6Rα, such as endothelial cells. This alternate pathway, also termed trans-signaling, serves as the major IL-6 signaling pathway in various pathologic proinflammatory conditions including cancer. Here we report that sIL-6Rα is elevated in malignant ascites from ovarian cancer patients, where it is associated with poor prognosis. IL-6 trans-signaling on endothelial cells prevented chemotherapy-induced apoptosis, induced endothelial hyperpermeability, and increased transendothelial migration of ovarian cancer cells. Selective blockade of the MAPK pathway with ERK inhibitor PD98059 reduced IL-6/sIL-6Rα–mediated endothelial hyperpermeability. ERK activation by the IL-6/sIL-6Rα complex increased endothelial integrity via Src kinase activation and Y685 phosphorylation of VE-cadherin. Selective targeting of IL-6 trans-signaling in vivo reduced ascites formation and enhanced the taxane sensitivity of intraperitoneal human ovarian tumor xenografts in mice. Collectively, our results show that increased levels of sIL-6Rα found in ovarian cancer ascites drive IL-6 trans-signaling on endothelial cells, thereby contributing to cancer progression. Selective blockade of IL-6 trans-signaling may offer a promising therapeutic strategy to improve the management of patients with advanced ovarian cancer. Cancer Res; 71(2); 424–34. ©2010 AACR.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the progression of human lung adenocarcinoma. However, the mechanism underlying COX-2’s effect on tumor progression remains largely unknown. Lymphangiogenesis, the formation of new lymphatic vessels, has recently received considerable attention and become a new frontier of tumor metastasis research. Here, we study the interaction between COX-2 and the lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, in human lung cancer cells and their implication in patient outcomes. We developed an isopropyl-β-d-thiogalactopyranoside-inducible COX-2 gene expression system in human lung adenocarcinoma CL1.0 cells. We found that VEGF-C gene expression but not VEGF-D was significantly elevated in cells overexpressing COX-2. COX-2-mediated VEGF-C up-regulation was commonly observed in a broad array of non-small cell lung cancer cell lines. The use of pharmacological inhibitors or activators and genetic inhibition by EP receptor-antisense oligonucleotides revealed that prostaglandin EP₁ receptor but not other prostaglandin receptors is involved in COX-2-mediated VEGF-C up-regulation. At the mechanistic level, we found that COX-2 expression or prostaglandin E₂ (PGE₂) treatment could activate the HER-2/Neu tyrosine kinase receptor through the EP₁ receptor-dependent pathway and that this activation was essential for VEGF-C induction. The transactivation of HER-2/Neu by PGE₂ was inhibited by way of blocking the Src kinase signaling using the specific Src family inhibitor, PP1, or transfection with the mutant dominant negative src plasmid. Src kinase was involved in not only the HER-2/Neu transactivation but also the following VEGF-C up-regulation by PGE₂ treatment. In addition, immunohistochemical staining of 59 lung adenocarcinoma specimens showed that COX-2 level was highly correlated with VEGF-C, lymphatic vessels density, and other clinicopathological parameters. Taken together, our results provided evidence that COX-2 up-regulated VEGF-C and promotes lymphangiogenesis in human lung adenocarcinoma via the EP₁/Src/HER-2/Neu signaling pathway.

Ovarian cancer (OCa) peritoneal metastasis is the leading cause of cancer–related deaths in women with limited therapeutic options available for treating it and poor prognosis, as the underlying mechanism is not fully understood. The clinicopathological correlation of G9a expression was assessed in tumor specimens of ovarian cancer patients. Knockdown or overexpression of G9a in ovarian cancer cell lines was analysed with regard to its effect on adhesion, migration, invasion and anoikis-resistance. In vivo biological functions of G9a were tested by i.p. xenograft ovarian cancer models. Microarray and quantitative RT-PCR were used to analyze G9a-regulated downstream target genes. We found that the expression of histone methyltransferase G9a was highly correlated with late stage, high grade, and serous-type OCa. Higher G9a expression predicted a shorter survival in ovarian cancer patients. Furthermore, G9a expression was higher in metastatic lesions compared with their corresponding ovarian primary tumors. Knockdown of G9a expression suppressed prometastatic cellular activities including adhesion, migration, invasion and anoikis-resistance of ovarian cancer cell lines, while G9a over-expression promoted these cellular properties. G9a depletion significantly attenuated the development of ascites and tumor nodules in a peritoneal dissemination model. Importantly, microarray and quantitative RT-PCR analysis revealed that G9a regulates a cohort of tumor suppressor genes including CDH1, DUSP5, SPRY4, and PPP1R15A in ovarian cancer. Expression of these genes was also inversely correlated with G9a expression in OCa specimens. We propose that G9a contributes to multiple steps of ovarian cancer metastasis and represents a novel target to combat this deadly disease.