Matsubara Junichi, Li Yong Fuga, Koul Sanjay, Mukohyama Junko, Salazar Luis E Valencia, Isobe Taichi, Qian Dalong, Clarke Michael F, Sahoo Debashis, Altman Russ B, Dalerba Piero
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA (USA).
Department of Therapeutic Oncology, Graduate School of Medicine, Kyoto University, Kyoto (Japan).
bioRxiv. 2025 Jan 24:2025.01.22.633435. doi: 10.1101/2025.01.22.633435.
(CRCs) are seldom eradicated by cytotoxic chemotherapy. Cancer cells with stem-like functional properties, often referred to as "" (CSCs), display preferential resistance to several anti-tumor agents used in cancer chemotherapy, but the molecular mechanisms underpinning their selective survival remain only partially understood.
In this study, we used (TFTG) enrichment analysis to identify transcriptional regulators (activators or repressors) that undergo preferential activation by chemotherapy in CRC cells with a "" phenotype (EPCAM/CD44/CD166; CSC-enriched) as compared to CRC cells with a "" phenotype (EPCAM/CD44/CD166; CSC-depleted). The two cell populations were purified in parallel by (FACS) from a (PDX) line representative of a moderately differentiated human CRC, following chemotherapy with (CPT-11). The transcriptional regulators identified as differentially activated were tested for differential expression in normal vs. cancer tissues, and in cell populations enriched in stem/progenitor cell-types as compared to differentiated lineages (goblet cells, enterocytes) in the mouse colon epithelium. Finally, the top candidate was tested for mechanistic contribution to drug-resistance by selective down-regulation using (shRNAs).
Our analysis identified E2F4 and TFDP1, two core components of the DREAM transcriptional repression complex, as transcriptional modulators preferentially activated by in EPCAM/CD44/CD166 as compared to EPCAM/CD44/CD166 cancer cells. The expression levels of both genes () were found up-regulated in CRCs as compared to human normal colon tissues, and in a sub-population of mouse colon epithelial cells enriched in stem/progenitor elements (Epcam/Cd44/Cd66a/Kit) as compared to other sub-populations enriched in either goblet cells (Epcam/Cd44/Cd66a/Kit) or enterocytes (Epcam/Cd44/Cd66a). Most importantly, E2F4 down-regulation using shRNAs dramatically enhanced the sensitivity of human CRCs to treatment with , across three independent PDX models.
Our data identified E2F4 and the DREAM repressor complex as critical regulators of human CRC resistance to , and as candidate targets for the development of chemo-sensitizing agents.
细胞毒性化疗很少能根除结直肠癌（CRC）。具有干细胞样功能特性的癌细胞，通常被称为“癌症干细胞”（CSCs），对癌症化疗中使用的几种抗肿瘤药物表现出优先抗性，但其选择性存活的分子机制仍仅部分为人所知。
在本研究中，我们使用转录因子靶基因（TFTG）富集分析来鉴定与具有“非干细胞样”表型（EPCAM/CD44/CD166；CSC耗尽）的CRC细胞相比，在具有“干细胞样”表型（EPCAM/CD44/CD166；CSC富集）的CRC细胞中被化疗优先激活的转录调节因子（激活剂或抑制剂）。在用伊立替康（CPT-11）进行化疗后，通过荧光激活细胞分选（FACS）从代表中度分化人CRC的患者来源异种移植（PDX）系中平行纯化这两种细胞群体。对被鉴定为差异激活的转录调节因子在正常组织与癌组织中，以及与小鼠结肠上皮中分化谱系（杯状细胞、肠细胞）相比富含干/祖细胞类型的细胞群体中的差异表达进行测试。最后，通过使用短发夹RNA（shRNAs）进行选择性下调，测试顶级候选因子对耐药性的机制贡献。
我们的分析确定E2F4和TFDP1，即DREAM转录抑制复合物的两个核心成分，是与EPCAM/CD44/CD166非干细胞样癌细胞相比，在EPCAM/CD44/CD166干细胞样癌细胞中被伊立替康优先激活的转录调节因子。与人类正常结肠组织相比，以及与富含杯状细胞（Epcam/Cd44/Cd66a/Kit）或肠细胞（Epcam/Cd44/Cd66a）的其他亚群相比，在富含干/祖细胞成分（Epcam/Cd44/Cd66a/Kit）的小鼠结肠上皮细胞亚群中，发现这两个基因（E2F4和TFDP1）的表达水平上调。最重要的是，在三个独立的PDX模型中，使用shRNAs下调E2F4显著增强了人CRC对伊立替康治疗的敏感性。
我们的数据确定E2F4和DREAM抑制复合物是人类CRC对伊立替康耐药性的关键调节因子，并且是化疗增敏剂开发的候选靶点。
