Study On Mechanism Of Epigenetic Reversion Of OCT2 Inhibition And Sensitization Of Renal Cell Carcinoma To Oxaliplatin

Renal cell carcinoma (RCC) is the most common type of urinary tract cancers and accounts for approximately 2-3% of adult malignancies. The intrinsic resistance of RCC results in inefficient chemotherapy (7-10%) as well as high mortality rate. The human renal proximal tubular cells are rich in drug transporters, which confer resistance of cancer cells to anti-cancer drugs. Human organic cation transporter2(SLC22A2), the most abundant organic cation transporter, is specifically localized to the basolateral or apical side of renal epithelial cell. It participates in apical transport (secretion or reabsorption) of some organic cations. Previous studies have shown loss of OCT2 expression in RCC tumor tissues while a high expression in paired normal renal tissues. Transcriptional repression in OCT2 was regulated by two major epigenetic modifications:DNA methylation and histone modifications, which had close links with each other. We speculated signaling pathway based on our results:MLL1 containing SET domain was responsible for catalyzing the active methylation of Lys4 of H3(H3K4me3) at the OCT2 promoter region. MYC, a transcription factors, recruited MLL1 to OCT2 promoter region following transcriptional activation. Aberrant methylation of DNA promoter CpG islands (CGI) was one possible mechanism inducing transcription and protein expression of OCT2 in RCC cell lines and tumor tissues. DNA methylation, blocking the binding between MYC and OCT2 E-Box binding sites, impaired the recruitment of MLL1 at the OCT2 promoter region by MYC. Eventually, transcriptional activation signal H3K4me3 was significantly decreased at OCT2 gene promoter region, which inhibited transcription initiation and decreased protein expression of OCT2.Based on the research of previous studies, we intended to verify the putative regulatory network of DNA methylation and histone methylation and decipher the mechanism of epigenetic regulation to draw more comprehensive signaling pathways. We analyzed OCT2 expression on an RCC tissue microarray(TMA) by Immunohistochemistry (IHC) to confirm OCT2 repression in RCC. To determine the mechanism of regulating OCT2, we constructed 786-O cell lines that conditionally expressed shRNAs specific to MYC and MLL1. Decitabine (DAC), was used to globally inhibit DNA methylation in this two constructed RCC cell lines. Reporter gene assay in vitro and CHIP in vivo were used to confirme our hypothesis.Histone acetylation, another important member of the epigenetic regulation, included histone histone acetylase (HATs) and histone deacetylase (HDACs), which regulated the dynamic state of histone acetylation/deacetylation. Previous research suggested that dysregulation of histone lysine acetylation is intimately linked with the development of cancer in epigenetic level. The expression of HDAC1 in RCC patients was associated with the histological grade and clinical stage. To explore the possible mechanism of OCT2 regulation via HDACs, we screened HDAC7/9 as research subjects, which mRNA levels were both significantly increased in RCC tumor tissue. Real time-qPCR was used to characterize the mRNA levels of HDAC7/9 in RCC tumor tissues and the paired normal renal tissues. Choosing OCT2 silencing expression RCC cell lines 786-0,769-P cells as models, we found that histone deacetylase inhibitor vorinostat (SAHA) significantly induced expression of OCT2 as well as MYC in 786-0 cells but not in 769-P cells. DAC and SAHA sequential combination enhanced OCT2 mRNA expression by about two times in both RCC cells. This result suggested the existence of two different regulation mechanisms in 786-0 and 769-P cells. We proposed DNA methylation and HDACs were involved in transcriptional regulation of OCT2 in RCC cell lines. MYC might play a role of a bridge mediating the crosstalk. SAHA treated 786-0 cell model with stable knockdown of MYC and the expression of OCT2 remained unchanged after treatment, which preliminarily confirmed the essential participation of MYC in histone deacetylation and the critical role of histone acetylation in the reversal of OCT2 expression in RCC cell lines. Furthermore, the RNA interference (RNAi) experiments showed the specific and effective small interfering RNA (siRNA) targeting HDAC7/9 could silence OCT2 in 786-0 and 769-P cells, revealing HDAC7/9 as the target genes of SAHA. The mechanism suggested that MYC recruited HDAC7/9 to mediate the interaction between DNA methylation and histone acetylation.Results from previous in vitro experiment indicated that the combination of DAC and oxaliplatin showed synergie effect on the cytotoxicity to RCC cell lines. To obtain more reliable conclusions, the enhanced effects of combination therapy were also demonstrated in 786-0 and Caki-1 xenograft models. Our findings demonstrated that epigenetic activation of OCT2 by DAC promoted cellular accumulation of oxaliplatin and consequently sensitized RCC cells to oxaliplatin in xenografts.Collectively, epigenetic drugs DAC and SAHA could reverse expression of OCT2 in RCC.MYC acted as a link between DNA methylation and histone modifications (histone methylation and histone acetylation).1. Histone methylation. DAC, a demethylating reagent, blocked cellular DNA methyltransferases (DNMTs) to improve the enrichment of MYC at OCT2 promoter region. This increased recruitment of MLL1 to establish H3K4me3, which promoted OCT2 transcription.2. Histone acetylation. The mRNA levels of HDAC7/9 were abnormal in RCC Ipatients. Inhibition of HDAC7/9 by siRNA increased OCT2 mRNA levels. We proposed SAHA influenced HDAC7/9 enrichment at OCT2 promoter region via MYC, eventually leading to improved level of OCT2 transcription. This mechanical signal could be applied to 786-0 cells but the precise mechanism remained to be studied in 769-P cells. Significant increase of OCT2 transcription was found after a combination of DAC and SAHA in comparison with using DAC alone. It indicated that the decrease in DNA methylation contributed to the MYC regulation of HDACs and the combination of these drugs led to synergistic effects. Based on the regulation mechanism, we treated RCC cells in nude mice with combination therapy, which reduced tumor proliferation and increased tumor cell apoptosis. RCC patients are in urgent need of new treatment options to increase the efficacy of chemotherapeutic agents. The combination therapy, sensitizing RCC cells to oxaliplatin in vivo, highlighted the potential of a novel clinical RCC therapy to improve survival of patients.