Khurana, Ashwani

AbstractPurpose: The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines.Experimental design: Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA1 knocked down clones.Results: Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all seven papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a three- to fourfold increase in the invasive potential. Exogenous expression of HtrA1 in Ark1 and Ark2 cells resulted in three- to fourfold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark2 cells. Immunohistochemical analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (P = 0.016).Conclusions: Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. Clin Cancer Res; 17(3); 427–36. ©2010 AACR.

Abstract We recently identified HSulf-1 as a down-regulated gene in ovarian carcinomas. Our previous analysis indicated that HSulf-1 inactivation in ovarian cancers is partly mediated by loss of heterozygosity and epigenetic silencing. Here, we show that variant hepatic nuclear factor 1 (vHNF1), encoded by transcription factor 2 gene (TCF2, HNF1β), negatively regulates HSulf-1 expression in ovarian cancer. Immunoblot assay revealed that vHNF1 is highly expressed in HSulf-1–deficient OV207, SKOV3, and TOV-21G cell lines but not in HSulf-1–expressing OSE, OV167, and OV202 cells. By short hairpin RNA–mediated down-regulation of vHNF1 in TOV-21G cells and transient enhanced vHNF1 expression in OV202 cells, we showed that vHNF1 suppresses HSulf-1 expression in ovarian cancer cell lines. Reporter assay and chromatin immunoprecipitation experiments showed that vHNF1 is specifically recruited to HSulf-1 promoter at two different vHNF1-responsive elements in OV207 and TOV-21G cells. Additionally, down-regulation of vHNF1 expression in OV207 and TOV-21G cells increased cisplatin- or paclitaxel-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays and this effect was reversed by down-regulation of HSulf-1. Moreover, nude mice bearing TOV-21G cell xenografts with stably down-regulated vHNF1 were more sensitive to cisplatin- or paclitaxel-induced cytotoxicity compared with xenografts of TOV-21G clonal lines with nontargeted control short hairpin RNA. Finally, immunohistochemical analysis of 501 ovarian tumors including 140 clear-cell tumors on tissue microarrays showed that vHNF1 inversely correlates to HSulf-1 expression. Collectively, these results indicate that vHNF1 acts as a repressor of HSulf-1 expression and might be a molecular target for ovarian cancer therapy. [Cancer Res 2009;69(11):4843–50]