PDF(Pubmed)
Abstract：
Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.
摘要：
胰腺癌或胰腺导管腺癌（PDAC）的特征是具有高度异质性的深度炎性肿瘤微环境（TME），转移倾向，极度缺氧.整合应激反应（ISR）途径的特点是蛋白激酶家族磷酸化真核起始因子2（eIF2）和调节翻译响应不同的胁迫条件，包括缺氧。我们先前证明了eIF2信号传导途径在人PDAC细胞中响应氧化还原因子-1(Ref-1)敲低而受到深刻影响。Ref-1是一种双重功能酶，具有DNA修复和氧化还原信号的活性，对细胞压力有反应，并调节生存途径。Ref-1的氧化还原功能直接调节多种转录因子，包括HIF-1α，STAT3和NF-κB,在PDACTME中具有高度活性。然而,Ref-1氧化还原信号和ISR通路激活之间的串扰机制细节尚不清楚.在Ref-1击倒之后，在常氧条件下观察到ISR的诱导，而缺氧条件足以激活ISR，而与Ref-1水平无关。抑制Ref-1氧化还原活性在多种人类PDAC细胞系中以浓度依赖性方式增加p-eIF2和ATF4转录活性的表达,对eIF2磷酸化的影响是PERK依赖性的。用PERK抑制剂治疗，高浓度的AMG-44导致替代ISR激酶的激活，GCN2和诱导的p-eIF2和ATF4在肿瘤细胞和癌症相关成纤维细胞（CAF）中的水平。Ref-1和PERK抑制剂的联合治疗增强了3D共培养中人类胰腺癌细胞系和CAF的细胞杀伤作用。但仅在高剂量的PERK抑制剂下。当Ref-1抑制剂与GCN2抑制剂联合使用时，这种作用被完全消除。GCN2iB.我们证明了Ref-1氧化还原信号的靶向激活了多个PDAC细胞系中的ISR，并且ISR的这种激活对于抑制共培养球体的生长至关重要。组合效应仅在生理相关的3D共培养中观察到，这表明所使用的模型系统可以极大地影响这些靶向药物的结果。抑制Ref-1信号通过ISR信号通路诱导细胞死亡，Ref-1氧化还原信号传导阻滞与ISR激活的组合可能是PDAC治疗的新治疗策略。
