Falvey Chloe M, O'Donovan Tracey R, El-Mashed Shereen, Nyhan Michelle J, O'Reilly Seamus, McKenna Sharon L
Cork Cancer Research Centre, University College Cork, Cork, Ireland.
Department of Oncology, Cork University Hospital, Cork, Ireland.
Oncotarget. 2017 Apr 4;8(14):23479-23491. doi: 10.18632/oncotarget.15182.
Esophageal cancer remains a poor prognosis cancer due to advanced stage of presentation and drug resistant disease. To understand the molecular mechanisms influencing response to chemotherapy, we examined genes that are differentially expressed between drug sensitive, apoptosis competent esophageal cancer cells (OE21, OE33, FLO-1) and those which are more resistant and do not exhibit apoptosis (KYSE450 and OE19). Members of the ISG15 (ubiquitin-like) protein modification pathway, including UBE2L6 and ISG15, were found to be more highly expressed in the drug sensitive cell lines. In this study, we evaluated the contribution of these proteins to the response of drug sensitive cells. Depletion of UBE2L6 or ISG15 with siRNA did not influence caspase-3 activation or nuclear fragmentation following treatment with 5-fluorouracil (5-FU). We assessed autophagy by analysis of LC3II expression and Cyto-ID staining. Depletion of either ISG15 or UBE2L6 resulted in enhanced endogenous autophagic flux. An increase in autophagic flux was also observed following treatment with cytotoxic drugs (5-FU, rapamycin). In ISG15 depleted cells, this increase in autophagy was associated with improved recovery of drug treated cells. In contrast, UBE2L6 depleted cells, did not show enhanced recovery. UBE2L6 may therefore influence additional targets that limit the pro-survival effect of ISG15 depletion. These data identify UBE2L6 and ISG15 as novel inhibitors of autophagy, with the potential to influence chemosensitivity in esophageal cancer cells.
由于食管癌就诊时多处于晚期且存在耐药性，其预后仍然较差。为了解影响化疗反应的分子机制，我们检测了在药物敏感、具有凋亡能力的食管癌细胞（OE21、OE33、FLO-1）与耐药且不表现凋亡的细胞（KYSE450和OE19）之间差异表达的基因。发现包括UBE2L6和ISG15在内的ISG15（泛素样）蛋白修饰途径的成员在药物敏感细胞系中表达更高。在本研究中，我们评估了这些蛋白对药物敏感细胞反应的作用。用小干扰RNA（siRNA）敲低UBE2L6或ISG15并不影响5-氟尿嘧啶（5-FU）处理后的半胱天冬酶-3激活或核碎裂。我们通过分析LC3II表达和Cyto-ID染色评估自噬。敲低ISG15或UBE2L6均导致内源性自噬通量增强。在用细胞毒性药物（5-FU、雷帕霉素）处理后也观察到自噬通量增加。在敲低ISG15的细胞中，自噬的这种增加与药物处理细胞的恢复改善相关。相比之下，敲低UBE2L6的细胞未显示恢复增强。因此，UBE2L6可能影响限制ISG15敲低的促生存作用的其他靶点。这些数据确定UBE2L6和ISG15为自噬的新型抑制剂，有可能影响食管癌细胞的化疗敏感性。
