Inhibition of C-terminal binding protein attenuates transcription factor 4 signaling to selectively target colon cancer stem cells

Selective targeting of cancer stem cells (CSCs), implicated in tumor relapse, holds great promise in the treatment of colorectal cancer. Overexpression of C-terminal binding protein (CtBP), an NADH dependent transcriptional regulator, is often observed in colon cancer. Of note, TCF-4 signaling is also up-regulated in colonic CSCs. We hypothesized that CtBP, whose dehydrogenase activity is amenable to pharmacological inhibition by 4-methylthio-2-oxobutyric acid (MTOB), positively regulates TCF-4 signaling, leading to CSC growth and self-renewal. CSCs demonstrated significant upregulation of CtBP1 and CtBP2 levels (mRNA and protein) and activity partly due to increased NADH/NAD ratio, as well as increased TCF/LEF transcriptional activity, compared to respective controls. Depletion of CtBP2 inhibited, while its overexpression enhanced, CSC growth (1° spheroids) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of spheroid growth and self-renewal in a dose dependent manner. MTOB displayed significantly greater selectivity for growth inhibition in the spheroids, at least in part through induction of apoptosis, compared to monolayer controls. Moreover, MTOB inhibited basal as well as induced (by GSK-3β inhibitor) TCF/LEF activity while suppressing mRNA and protein levels of several β-catenin target genes (CD44, Snail, C-MYC and LGR5). Lastly, CtBP physically interacted with TCF-4, and this interaction was significantly inhibited in the presence of MTOB. The above findings point to a novel role of CtBPs in the promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a novel approach for treatment of colorectal cancer focused on targeting of CSCs.

靶向参与肿瘤复发的肿瘤干细胞（cancer stem cells, CSCs），为结直肠癌治疗带来巨大应用潜力。C末端结合蛋白（CtBP）作为一类烟酰胺腺嘌呤二核苷酸（NADH）依赖的转录调控因子，在结肠癌中常呈现过表达状态。值得注意的是，结肠CSCs中TCF-4信号通路同样存在上调现象。我们提出如下假说：CtBP的脱氢酶活性可被4-甲硫基-2-氧代丁酸（MTOB）通过药理学手段抑制，其可正向调控TCF-4信号通路，进而促进CSCs的增殖与自我更新。与相应对照组相比，CSCs中CtBP1与CtBP2的mRNA、蛋白水平及活性均显著上调，这在一定程度上源于NADH/NAD比值升高，同时CSCs的TCF/LEF转录活性亦有所提升。敲低CtBP2会抑制CSCs的增殖（1级球状体形成）与自我更新能力（2级/3级球状体形成），而过表达CtBP2则可增强这两项过程。类似地，MTOB可通过剂量依赖性方式，显著抑制球状体的增殖与自我更新。与单层培养的对照组相比，MTOB对球状体的增殖抑制具有更显著的选择性，这一效应至少部分源于其诱导细胞凋亡的作用。此外，MTOB可抑制基础状态以及糖原合成激酶3β（GSK-3β）抑制剂诱导的TCF/LEF转录活性，同时下调多个β-连环蛋白靶基因（CD44、Snail、C-MYC及LGR5）的mRNA与蛋白表达水平。最后，CtBP可与TCF-4发生物理相互作用，且该相互作用在MTOB存在时会被显著抑制。上述研究结果揭示了CtBPs通过直接调控TCF/LEF转录，进而促进CSCs增殖与自我更新的全新作用机制。此外，通过小分子化合物抑制CtBP功能可选择性靶向CSCs，为以CSCs为靶点的结直肠癌治疗提供了全新策略。