Department of Physiology and Medical Physics, Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
Department of Genetics and Bioengineering, Faculty of Engineering, Izmir University of Economics, Balcova, Izmir, Turkey.
Cell Death Dis. 2020 Nov 30;11(11):1020. doi: 10.1038/s41419-020-03232-z.
Colorectal cancer is a molecularly heterogeneous disease. Responses to genotoxic chemotherapy in the adjuvant or palliative setting vary greatly between patients, and colorectal cancer cells often resist chemotherapy by evading apoptosis. Antagonists of an inhibitor of apoptosis proteins (IAPs) can restore defective apoptosis signaling by degrading cIAP1 and cIAP2 proteins and by inhibition of XIAP. Due to the multiple molecular mechanisms-of-action of these targets, responses to IAP antagonist may differ between molecularly distinct colon cancer cells. In this study, responses to the IAP antagonist Birinapant and oxaliplatin/5-fluorouracil (5-FU) were investigated in 14 colon cancer cell lines, representing the consensus molecular subtypes (CMS). Treatment with Birinapant alone did not result in a substantial increase in apoptotic cells in this cell line panel. Annexin-V/PI assays quantified by flow cytometry and high-content screening showed that Birinapant increased responses of CMS1 and partially CMS3 cell lines to oxaliplatin/5-FU, whereas CMS2 cells were not effectively sensitized. FRET-based imaging of caspase-8 and -3 activation validated these differences at the single-cell level, with CMS1 cells displaying sustained activation of caspase-8-like activity during Birinapant and oxaliplatin/5-FU co-treatment, ultimately activating the intrinsic mitochondrial apoptosis pathway. In CMS2 cell lines, Birinapant exhibited synergistic effects in combination with TNFα, suggesting that Birinapant can restore extrinsic apoptosis signaling in the context of inflammatory signals in this subtype. To explore this further, we co-cultured CMS2 and CMS1 colon cancer cells with peripheral blood mononuclear cells. We observed increased cell death during Birinapant single treatment in these co-cultures, which was abrogated by anti-TNFα-neutralizing antibodies. Collectively, our study demonstrates that IAP inhibition is a promising modulator of response to oxaliplatin/5-FU in colorectal cancers of the CMS1 subtype, and may show promise as in the CMS2 subtype, suggesting that molecular subtyping may aid as a patient stratification tool for IAP antagonists in this disease.
结直肠癌是一种分子异质性疾病。在辅助或姑息治疗环境中，对遗传毒性化疗的反应在患者之间差异很大，结直肠癌细胞常常通过逃避细胞凋亡来抵抗化疗。凋亡蛋白抑制剂（IAPs）的拮抗剂可以通过降解 cIAP1 和 cIAP2 蛋白和抑制 XIAP 来恢复有缺陷的细胞凋亡信号。由于这些靶标的多个分子作用机制，IAP 拮抗剂的反应可能因分子上不同的结肠癌细胞而有所不同。在这项研究中，研究了 IAP 拮抗剂 Birinapant 和奥沙利铂/5-氟尿嘧啶（5-FU）在 14 种代表共识分子亚型（CMS）的结肠癌细胞系中的反应。在这个细胞系面板中，单独使用 Birinapant 并没有导致凋亡细胞的大量增加。通过流式细胞术和高内涵筛选定量的 Annexin-V/PI 测定表明，Birinapant 增加了 CMS1 和部分 CMS3 细胞系对奥沙利铂/5-FU 的反应，而 CMS2 细胞则不能有效地增敏。基于 FRET 的 caspase-8 和 -3 激活的成像验证了这些在单细胞水平上的差异，CMS1 细胞在 Birinapant 和奥沙利铂/5-FU 共同处理期间显示持续的 caspase-8 样活性激活，最终激活了内在的线粒体凋亡途径。在 CMS2 细胞系中，Birinapant 与 TNFα 联合表现出协同作用，表明在这种亚型中，Birinapant 可以在炎症信号的背景下恢复外在的细胞凋亡信号。为了进一步探索这一点，我们将 CMS2 和 CMS1 结肠癌细胞与外周血单核细胞共培养。我们观察到在这些共培养物中，Birinapant 单独处理时细胞死亡增加，而抗 TNFα 中和抗体则消除了这种增加。总的来说，我们的研究表明，IAP 抑制是 CMS1 亚型结直肠癌对奥沙利铂/5-FU 反应的一种有前途的调节剂，并且在 CMS2 亚型中可能显示出前景，这表明分子亚分型可能有助于作为该疾病中 IAP 拮抗剂的患者分层工具。
