DataSheet1_Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.docx

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.地西他滨（Decitabine, DAC）作为一种DNA甲基转移酶（DNA methyltransferase, DNMT）抑制剂，目前正被研究与常规抗肿瘤药物联合使用，作为多种实体瘤的治疗方案。尽管表观基因组调控为耐药癌症的治疗提供了颇具前景的方向，但仍需开展更多研究以评估其安全性，以及纠正异常转录谱的能力。由于脱氧胞苷激酶（deoxycytidine kinase, DCK）介导的磷酸化是DAC代谢激活的限速步骤，我们推测其细胞内过表达可增强DAC对细胞甲基化组的作用，进而提升其治疗效能。因此，我们选取两株分子特征存在差异的乳腺癌细胞系JIMT-1与T-47D，采用DCK表达载体进行转染，并以低剂量DAC（约IC20浓度）处理细胞。尽管转染显著提升了DCK的表达水平，且经DAC处理后该表达进一步增强，但转染并未对全基因组DNA甲基化水平或细胞活力产生显著影响。与此同时，我们采用整合分析方法解析DAC诱导、甲基化介导的转录组重编程过程。除全基因组范围的低甲基化伴随基因表达上调外，DAC还在两株细胞系中诱导了大量基因的高甲基化与表达下调。值得注意的是，经DAC处理后，JIMT-1细胞中TET1与TET2（Ten-eleven translocation 1/2）的表达水平降低了一半，而DNMT的表达变化并不显著。包含大量胶原蛋白与溶质载体基因的蛋白质消化吸收通路，在膜转运蛋白相关通路中排名第二；在纳入低甲基化上调基因的分析中，该通路是两株细胞系均富集程度最高的通路。此外，在耐药性中发挥重要作用的钙信号通路，位列JIMT-1细胞的首要富集通路之列。尽管低剂量DAC可使肿瘤抑制基因的表达恢复正常，但同时也上调了部分癌基因，这一发现支持了此前针对其广泛重编程潜能的相关担忧。值得一提的是，本研究证实了主动去甲基化参与了DAC介导的转录组重编程过程。