Department of Medical Biology, Université du Québec à Trois-Rivières, 3351 boul. Des Forges, Trois-Rivières, Québec, G8Z 4M3, Canada.
Cell Commun Signal. 2018 Jul 4;16(1):39. doi: 10.1186/s12964-018-0252-z.
CRM1 enrichment has been shown to be indicative of invasive as well as chemoresistant tumors. On the other hand, TRAIL, a powerful and specific anti-tumoral agent, has yet to be used effectively to treat gynecological tumors in patients. In the present study, we examined if CRM1, a nuclear exporter capable of mediating protein transport, could be a relevant target to restore chemosensitivity in chemoresistant cells. We thus explored the hypothesis that CRM1-driven nuclear exclusion of tumor suppressors could lead to chemoresistance and that CRM1 inhibitors could present a novel therapeutic approach, allowing sensitization to chemotherapeutic agents.
Ovarian cancer cell lines, as well as endometrial cancer cell lines, were treated with leptomycin B (LMB), cisplatin and TRAIL, either singly or in combination, in order to induce apoptosis. Western blot and flow cytometry analysis were used to quantify caspases activation and apoptosis induction. Immunofluorescence was used to determine nuclear localization of p53. Colony formation assays were performed to determine therapeutic effectiveness; p53 siRNA were used to establish p53 role in sensitization. Additional information from GEO database and Prognoscan allowed us to contextualise the obtained results. Finally, qRT-PCR was performed to measure apoptotic regulators expression.
TRAIL and LMB combination therapy lead to cleavage of caspase-3 as well as the appearance of cleaved-PARP, and thus, apoptosis. Further experiments suggested that sensitization was achieved through the synergistic downregulation of multiple inhibitor of apoptosis, as well as the activation of apoptotic pathways. p53 was enriched in the nucleus following LMB treatments, but did not seem to be required for sensitization; additional experiments suggested that p53 opposed the apoptotic effects of LMB and TRAIL. Results obtained from public data repositories suggested that CRM1 was a driver of chemoresistance and poor prognostic; DR5, on the other hand, acted as as a marker of positive prognostic.
Taken together, our results suggest that the use of CRM1 inhibitors, in combination to chemotherapeutic compounds, could be highly effective in the treatment of gynecological malignancies.
CRM1 富集已被证明与侵袭性和耐药性肿瘤有关。另一方面，TRAIL 是一种强大而特异的抗肿瘤药物，但尚未有效地用于治疗患者的妇科肿瘤。在本研究中，我们研究了核输出蛋白 CRM1 是否可以作为恢复耐药细胞化疗敏感性的相关靶点。因此，我们探索了以下假设：CRM1 驱动的肿瘤抑制因子核输出可能导致化疗耐药，而 CRM1 抑制剂可能提供一种新的治疗方法，使化疗药物敏感。
用莱普霉素 B（LMB）、顺铂和 TRAIL 单独或联合处理卵巢癌细胞系和子宫内膜癌细胞系，以诱导细胞凋亡。Western blot 和流式细胞术分析用于定量 caspase 激活和凋亡诱导。免疫荧光用于确定 p53 的核定位。集落形成实验用于确定治疗效果；p53 siRNA 用于确定 p53 在增敏中的作用。来自 GEO 数据库和 Prognoscan 的额外信息使我们能够对获得的结果进行上下文分析。最后，进行 qRT-PCR 以测量凋亡调节剂的表达。
TRAIL 和 LMB 联合治疗导致 caspase-3 的切割以及 cleaved-PARP 的出现，从而导致凋亡。进一步的实验表明，增敏是通过多种凋亡抑制剂的协同下调以及凋亡途径的激活来实现的。LMB 处理后，p53 在核内富集，但似乎不是增敏所必需的；进一步的实验表明，p53 反对 LMB 和 TRAIL 的凋亡作用。来自公共数据存储库的结果表明，CRM1 是化疗耐药和预后不良的驱动因素；另一方面，DR5 是阳性预后的标志物。
综上所述，我们的结果表明，CRM1 抑制剂与化疗化合物联合使用可能对妇科恶性肿瘤的治疗非常有效。
