Liu Peng, Khurana Ashwani, Rattan Ramandeep, He Xiaoping, Kalloger Steve, Dowdy Sean, Gilks Blake, Shridhar Viji
Departments of Experimental Pathology, University of British Columbia, Vancouver, British Columbia, Canada.
Cancer Res. 2009 Jun 1;69(11):4843-50. doi: 10.1158/0008-5472.CAN-08-3065.
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, HNF1beta), 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.
我们最近鉴定出HSulf-1是卵巢癌中一个下调的基因。我们之前的分析表明，卵巢癌中HSulf-1的失活部分是由杂合性缺失和表观遗传沉默介导的。在此，我们表明由转录因子2基因（TCF2，HNF1β）编码的变异肝细胞核因子1（vHNF1）在卵巢癌中负向调节HSulf-1的表达。免疫印迹分析显示，vHNF1在缺乏HSulf-1的OV207、SKOV3和TOV-21G细胞系中高表达，但在表达HSulf-1的OSE、OV167和OV202细胞中不表达。通过短发夹RNA介导的TOV-21G细胞中vHNF1的下调以及OV202细胞中vHNF1的瞬时过表达，我们表明vHNF1在卵巢癌细胞系中抑制HSulf-1的表达。报告基因分析和染色质免疫沉淀实验表明，在OV207和TOV-21G细胞中，vHNF1在两个不同的vHNF1反应元件处被特异性招募到HSulf-1启动子。此外，通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐和克隆形成实验确定，OV207和TOV-21G细胞中vHNF1表达的下调增加了顺铂或紫杉醇介导的细胞毒性，并且这种效应通过HSulf-1的下调而逆转。此外，与携带非靶向对照短发夹RNA的TOV-21G克隆系异种移植相比，携带稳定下调vHNF1的TOV-21G细胞异种移植的裸鼠对顺铂或紫杉醇诱导的细胞毒性更敏感。最后，对包括140例透明细胞肿瘤在内的501例卵巢肿瘤组织芯片进行免疫组织化学分析显示，vHNF1与HSulf-1的表达呈负相关。总体而言，这些结果表明vHNF1作为HSulf-1表达的抑制因子，可能是卵巢癌治疗的分子靶点。
