Background:Most newly developed anticancer treatments trigger tumor cell death through apoptosis, for which involvement of caspases activity is essential. However, numerous mutations in apoptotic pathways that lead to cancer and favor resistance to apoptosis are known; most are related to caspase-dependent apoptosis pathways and thus have low efficacy. To overcome these limitations, we constructed a novel chimeric protein, GnRH-AIF, using a gonadotropin-releasing hormone (GnRH) analog as a targeting moiety and the apoptosis-inducing factor (AIF) in its cleaved form as a killing moiety, fused at the cDNA level. AIF has a crucial role in the caspase-independent apoptotic pathway. A wide variety of solid tumors overexpress GnRH-receptors (GnRH-R) that are targeted by the new GnRH-AIF chimeric protein.Methods and Results:In this study, we constructed, expressed, and highly purified GnRH-AIF chimeric proteins. We demonstrated the ability of the chimera to enter and specifically and very efficiently kill solid cancer cell lines overexpressing GnRH-R. Most importantly, upon its entry, GnRH-AIFs translocate to the nucleus where it causes DNA fragmentation leading to a direct caspase-independent apoptotic death. As AIFs lack nuclease activity, our findings also emphasize that cell death induced by GnRH-AIF is dependent on the presence of the ENDOG and PPIA proteins, known to participate in the formation of a DNA–degradosome complex. Finally, we demonstrated the high anti-tumor efficacy of the GnRH-AIF ex vivo, in a human, colon cancer organoid model.Conclusions:Our study shows the potential of using a GnRH-AIF chimeric protein as a novel approach to treat solid cancers that overexpress GnRH-R, via a caspase-independent apoptotic pathway.
背景：大多数新开发的抗癌疗法通过细胞凋亡触发肿瘤细胞死亡，这一过程依赖于caspase活性的参与。然而，已知凋亡通路中存在多种导致癌症并促进凋亡抵抗的突变；这些突变大多与caspase依赖性凋亡通路相关，因此治疗效果有限。为克服这些限制，我们构建了一种新型嵌合蛋白GnRH-AIF，该蛋白以促性腺激素释放激素（GnRH）类似物作为靶向模块，以剪切形式的凋亡诱导因子（AIF）作为杀伤模块，并在cDNA水平进行融合。AIF在caspase非依赖性凋亡通路中起关键作用。多种实体瘤过度表达GnRH受体（GnRH-R），而新型GnRH-AIF嵌合蛋白可靶向这些受体。 方法与结果：本研究成功构建、表达并高纯度纯化了GnRH-AIF嵌合蛋白。我们证实该嵌合蛋白能够进入并特异性高效杀伤过度表达GnRH-R的实体癌细胞系。最重要的是，GnRH-AIF进入细胞后易位至细胞核，引起DNA片段化，直接导致caspase非依赖性凋亡死亡。由于AIF缺乏核酸酶活性，我们的研究还强调GnRH-AIF诱导的细胞死亡依赖于ENDOG和PPIA蛋白的存在，这两种蛋白已知参与DNA降解体复合物的形成。最后，我们在人结肠癌类器官模型中验证了GnRH-AIF在离体条件下具有显著的抗肿瘤效果。 结论：本研究揭示了通过caspase非依赖性凋亡通路，使用GnRH-AIF嵌合蛋白作为治疗过度表达GnRH-R实体瘤新策略的潜力。
肿瘤(癌症)患者之家