FOXC1 induces cisplatin resistance through enhancer activation [ChIP-seq]

We previously reported that an aggressive subpopulation of highly tumorigenic, drug-resistant bladder cancer cells can arise from the bulk tumor cells without mutational events and that this phenotypic plasticity is driven at least in part by epigenetic mechanisms. In the current work, we analyzed the chromatin accessibility of enhancers to identify transcription factors that contribute to this transition to a drug-resistant state. Comparing the drug resistant side population (SP) cells and the less drug resistant non-side population (NSP) cells in bladder cancer cells, we identified differential accessible enhancers and differentially expressed genes. Transcription factor motif analysis showed that FOX family motif was enriched in accessible enhancers near SP-overexpressed genes. Among FOX family transcription factors, FOXC1 was the only overexpressed transcription factor, and it was also the most significantly overexpressed gene in SP cells. FOXC1 ChIP-seq confirmed that FOXC1 binding sites are more accessible in SP cells and showed a significantly more FOXC1 binding near the overexpressed genes in SP cells. When the FOXC1 is knocked out, bladder cancer cells exhibit decreased cisplatin resistance and less percentage of SP cells. This change in cisplatin resistance is partially related to the FOXC1-driven expression of ABCB1 gene. In summary, our observations suggest that differential expression and enhancer binding of FOXC1 promotes the previously observed, mutation-independent shift towards cisplatin resistance in bladder cancer. Overall design: mRNA profile of side-population and non-side population of J82 human bladder cancer cells; mRNA profile of FOXC1 knock-out and vector control J82 cells; FOXC1 ChIP-seq of FOXC1

我们此前曾报道，一类具有高度致瘤性与耐药性的侵袭性膀胱癌细胞亚群，可由肿瘤主体细胞（bulk tumor cells）无需发生突变即可产生，且该表型可塑性至少部分由表观遗传机制驱动。在本研究中，我们通过分析增强子（enhancers）的染色质可及性（chromatin accessibility），以筛选参与介导细胞向耐药表型转变的转录因子（transcription factors）。通过对比膀胱癌细胞中的耐药侧群（side population, SP）细胞与低耐药性非侧群（non-side population, NSP）细胞，我们鉴定出了差异可及增强子与差异表达基因。转录因子基序分析显示，FOX家族基序在侧群细胞高表达基因附近的可及增强子中显著富集。在FOX家族转录因子中，FOXC1是唯一出现高表达的转录因子，同时也是侧群细胞中表达上调最显著的基因。FOXC1染色质免疫共沉淀测序（ChIP-seq）结果证实，侧群细胞中FOXC1结合位点的可及性更高，且在侧群细胞高表达基因附近的FOXC1结合信号显著更强。敲除FOXC1后，膀胱癌细胞的顺铂耐药性降低，且侧群细胞占比下降。顺铂耐药性的这一变化，部分与FOXC1介导的ABCB1基因表达调控相关。综上，本研究结果表明，FOXC1的差异表达及其与增强子的结合，可促进此前已观察到的、不依赖突变的膀胱癌细胞向顺铂耐药表型的转变。整体实验设计：J82人膀胱癌细胞侧群与非侧群的mRNA表达谱（mRNA profile）；FOXC1敲除J82细胞与空载对照（vector control）J82细胞的mRNA表达谱；FOXC1的染色质免疫共沉淀测序（ChIP-seq）。