Transcriptional Inhibition Of Human Organic Cation Transporter2in Renal Cell Carcinoma:Linking Of Epigenetic Regulation To Chemotherapy Response

Human organic cation transporter2(OCT2/SLC22A2), the most abundant organic cation transporter in the human proximal tubules, plays a key role in the renal secretion and reabsorption of endo-and xeno-biotic cationic compounds. In our study, loss of OCT2expression was found in all of five renal cell carcinoma(RCC) tumor tissues analyzed while showing a high expression in paired normal renal tissues. Transcriptional inactivity of OCT2contributed to the loss expression of OCT2in RCC tumor tissues.Epigenetics plays a significant role in the transcriptional regulation. DNA methylation and histone modification are the most intensely studied epigenetic pathways. DNA methylation and histone modification profiles at promoter region of OCT2gene in RCC tumor tissues and paired normal tissues were mapped in this study. In normal renal tissues, promoter CpG island (CGI) was hypomethylated and transcriptional active histone modifications such as H3K4Me2, H3K4Me3, H3K18Ac and H3K27Ac were enriched around transcription start site (TSS) of OCT2. However, in paired RCC tumor tissues, the promoter CGI showed aberrant hypermethylation and the methylation level of some CpG sites inversely correlated with of OCT2transcripts expression. Moreover, those transcriptional active histone modifications around TSS of OCT2mentioned above were also significantly impaired in RCC tumor tissues.To determine the effect of DNA methylation on the transcription of OCT2, RCC cell lines were used as a model in our study. Treatment with DNA methylation inhibitor5-Aza-2’-deoxycitidine (5-Aza-dC) resulted in hypomethylation at OCT2promoter CGI in3different RCC cell lines. Transcription and protein expression of OCT2were dramatically induced in primary RCC cell lines. The uptake function to organic cations was also restored in primary RCC cells; In addition, luciferase assays showed promoter CpG methylation in vitro directly repressed OCT2promoter transcriptional activities in HEK293cells.To investigate the crosstalk between DNA methylation and histone modifications in the transcriptional regulation of OCT2, differential histone modification patterns were determined after DNA methylation was inhibited in786-0cells with the treatment of5-Aza-dC. H3K4methylation around TSS was found to be more enriched as the DNA methylation level decreased at the promoter CGI of OCT2. Therefore, we hypothesized that DNA methylation may impair H3K4methylation by blocking the recruitment of H3K4methylation "writers" to OCT2promoter. This hypothesis was confirmed by reporter gene assay. Both c-Myc, a bHLHZ transcription factor and MLL, a H3K4tri-methylation "writer" were found to be involved in the transcriptional activation of OCT2promoter. The transcriptional activation of MLL to OCT2was dependent on its methyltransferase activity as well as the two E-Box binding sites at OCT2promoter. Moreover, the transcriptional activation of c-Myc and MLL to OCT2could be blocked by CpG methylation in vitro.Based on these data, promoter CGI hypermethylation seems to be one of the direct mechanisms for the transcriptional inactivity of OCT2in RCC tumor tissues and RCC cells. We proposed the relationship between DNA methylation, histone modification and transcription factors as following:c-Myc helps to recruit MLL to the OCT2promoter to set up H3K4Me3modification around TSS and this in turn facilitates transcription initiation at OCT2promoter. Hypermethylation on OCT2promoter CGI in RCC tumor tissues and RCC cells blocks the recognition and binding of c-Myc to E-Box in the proximal promoter region followed by the decreased recruitment of MLL at the OCT2promoter, which impairs H3K4Me3modification around TSS and results in inhibition of the transcription initiation.OCT2is known to dominate the transport of platinum chemotherapeutic agents especially oxaliplatin to renal epithelial cells in human proximal tubules. We hypothesized that the loss expression of OCT2might contribute to the resistance of RCC cells to oxaliplatin. Reactivation of OCT2expression through DNA methylation inhibition might enhance the cytotoxicity of oxaliplatin to RCC cells. Results from in vitro experiment showed that combination of Decitabine, a DNA methylation inhibitor and Oxaliplatin synergied in the cytotoxic response to RCC cell lines in a OCT2expression dependent level. It highlighted the potential of a novel combinational chemotherapy in clinical RCC therapy.