Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, W12 0NN, UK.
Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, W12 0NN, UK; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, 40002, Thailand.
Mol Cell Endocrinol. 2020 Sep 15;515:110932. doi: 10.1016/j.mce.2020.110932. Epub 2020 Jun 30.
Pharmaceutical inhibitors of the endoplasmic reticulum (ER)-stress modulator PERK (eIF2AK3) have demonstrated anticancer activities in combination therapies, but their effectiveness as a single agent is limited, suggesting the existence of possible compensatory cellular responses. To explore the potential mechanisms involved, we performed time-course drug treatment experiments on the parental MCF-7 and drug resistant MCF-7Epi and MCF-7Tax breast cancer cells and identified GCN2 (eIF2AK4) as a molecule that can potentially cooperate with PERK to regulate FOXO3 via JNK and AKT to modulate drug response. Consistently, GCN2 knockdown severely impaired the clonal survival of parental and resistant MCF-7 cells and sensitised them to epirubicin and paclitaxel treatment. Western blot, RT-qPCR and ChIP analyses also confirmed that GCN2 inactivation causes an induction of JNK and thereby FOXO3 activity, culminating in an increase in PERK activity and expression at the transcription level. Conversely, PERK-inactivation using GSK2606414-induces an induction in GCN2 expression and activity also associated with JNK. In agreement, we also showed that the perk MEFs, expressing elevated levels of P-JNK, JNK, GCN2 and reduced levels of P-AKT and P-FOXO3, have lower clonogenicity and are more sensitive to epirubicin compared to wild-type MEFs. Similarly, gcn2 MEFs expressing augmented levels of P-JNK, JNK, P-PERK, PERK and lower levels of P-AKT and P-FOXO3 also had lower clonogenicity and were more sensitive to epirubicin and PERK-inhibition. In addition, JNK1/2 deletion in MEFs resulted in reduced levels of GCN2, FOXO3, PERK, P-PERK expression as well as FOXO3 activity and enhanced clonal survival and resistance to PERK-inhibition. Together these results demonstrate that GCN2 cooperates with PERK through the JNK-FOXO3 axis in a reciprocal negative feedback loop to mediate cancer chemotherapeutic drug response and clonal survival, advocating the potential of targeting GCN2 as a therapeutic strategy for treating cancer and for overcoming drug resistance.
内质网（ER）应激调节剂 PERK（eIF2AK3）的药物抑制剂与联合疗法相结合已显示出抗癌活性，但作为单一药物的疗效有限，这表明可能存在代偿性细胞反应。为了探索潜在的相关机制，我们对亲本 MCF-7 和耐药 MCF-7Epi 和 MCF-7Tax 乳腺癌细胞进行了时程药物处理实验，并确定 GCN2（eIF2AK4）是一种可以与 PERK 合作通过 JNK 和 AKT 调节 FOXO3 从而调节药物反应的分子。一致地，GCN2 敲低严重损害了亲本和耐药 MCF-7 细胞的克隆存活能力，并使它们对表柔比星和紫杉醇治疗敏感。Western blot、RT-qPCR 和 ChIP 分析也证实，GCN2 失活导致 JNK 的诱导，从而导致 FOXO3 活性增加，最终导致转录水平 PERK 活性和表达增加。相反，使用 GSK2606414 抑制 PERK 会诱导 GCN2 表达和活性的增加，也与 JNK 相关。我们还表明，表达升高的 P-JNK、JNK、GCN2 和降低的 P-AKT 和 P-FOXO3 的 perk MEFs 具有更低的集落形成能力，并且比野生型 MEFs 对表柔比星更敏感。同样，表达增加的 P-JNK、JNK、P-PERK、PERK 和降低的 P-AKT 和 P-FOXO3 的 gcn2 MEFs 也具有更低的集落形成能力，并且对表柔比星和 PERK 抑制更敏感。此外，MEFs 中 JNK1/2 的缺失导致 GCN2、FOXO3、PERK、P-PERK 表达以及 FOXO3 活性降低，并增强克隆存活和对 PERK 抑制的抗性。这些结果表明，GCN2 通过 JNK-FOXO3 轴与 PERK 合作，形成一个相互负反馈回路，从而介导癌症化学治疗药物反应和克隆存活，这表明靶向 GCN2 作为治疗癌症和克服耐药性的潜在治疗策略的潜力。
