Transcriptional Inhibition Of Human Concentrative Nucleoside Transporter 2 In Colorectal Cancer Cells

Colorectal cancer (CRC) is one of the most aggressive cancers and a leading cause of cancer deaths worldwide. One of the fundamental processes driving the initiation and progression of CRC is the accumulation of epigenetic changes in colorectal cancer cells. Concentrative nucleoside transporter 2 (CNT2/SLC28A2), an influx transporter expressed in the apical side of intestinal, is mainly responsible for the transport of nucleoside drugs. In our study, using data obtained from the cancer transcriptome database Oncomine, we identified the repression of CNT2 as a potential factor contributing to clinical treatment in CRC. By analyzing CNT2 expression in collected patient tissues, we demonstrated CNT2 repression in CRC tissues at both transcription and protein levels. Transcriptional inactivity of CNT2 contributed to the repression of CNT2 in CRC 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 CNT2 gene in CRC tissues and paired normal tissues were mapped in this study. Epigenetic analysis revealed that the repressed CNT2 promoter in CRC was mainly related to histone modification. There was no significant difference in methylation level of promoter region in colorectal cancer tissues and paired normal tissues. However, in colorectal cancer tissues, transcriptional active histone modifications such as H3K9Ac, H3K18Ac and H4Ac were impaired and transcriptional inhibition histone modifications such as H3K9me3 was increased in the promoter region of CNT2.To determine the effect of histone modification on the transcription of CNT2, CRC cells were used as a model in our study. Treatment with histone deacetylation inhibition trichostatin A (TSA) resulted in acetylation at CNT2 promoter region in 2 different CRC cell lines. Transcription expression of CNT2 were dramatically induced in CRC cell lines. The uptake function to ribavirin was also increased in CRC cells.To investigate the function of histone modifications in the transcriptional regulation of CNT2, differential histone modification patterns were determined after histone deacetylation was inhibited in HCT15 and HT29 cells with the treatment of TSA. H3K18Ac and H4Ac were found to be more enriched, while H3K9me3 level was decreased at the promoter region of CNT2. Then we investigated the changes of histone modification enzymes and transcriptional factors in tissues and cells. It was found that transcription factors, such as HDAC7, SUV39H1, PGCl? may play a vital role in the regulation of CNT2 expression. The siRNA transfections were also involved. Based on these data, histone modifications seems to be one of the direct mechanisms for the transcriptional inactivity of CNT2 in CRC tissues and CRC cells.CNT2 is known to transport the nucleoside drugs. We hypothesized that the reactivation of CNT2 expression through histone deacetylation inhibition might enhance the cytotoxicity of cladribine to CRC cells. Results from in vitro experiment showed that combination of TSA/FK228 and cladribine synergied in the cytotoxic response to CRC cell lines. It highlighted the potential of a novel combinational chemotherapy in clinical CRC therapy.