Department of Chemistry and Biochemistry, Université de Moncton , Moncton, New Brunswick , Canada.
Atlantic Cancer Research Institute , Moncton, New Brunswick , Canada.
Am J Physiol Cell Physiol. 2019 May 1;316(5):C605-C620. doi: 10.1152/ajpcell.00483.2018. Epub 2019 Feb 13.
Autophagy is a highly conserved, homeostatic process by which cytosolic components reach lysosomes for degradation. The roles played by different autophagic processes in cancer are complex and remain cancer type and stage dependent. Renal cell carcinoma (RCC) is the most common subtype of kidney cancer and is characterized by the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Our previous study identified a small compound, STF-62247, as an autophagy-modulating molecule causing selective cytotoxicity for VHL-inactivated cells. This present study investigates the effects of STF-62247 specifically on the macroautophagic flux to better characterize its mechanism of action in RCC. Our results clearly demonstrate that this compound is a potent blocker of late stages of autophagy. We show that inhibiting autophagy by CRISPR knockouts of autophagy-related genes rendered VHL-deficient cells insensitive to STF-62247, uncovering the importance of the autophagic pathway in STF-selective cell death. By exploiting the autofluorescence of STF-62247, we pinpointed its cellular localization to lysosomes. Finally, in response to prolonged STF treatments, we show that VHL-proficient cells are able to surmount the block in late stages of autophagy by restoring their lysosome numbers. Conversely, an increase in autophagic vesicles accompanied by a time-dependent decrease in lysosomes was observed in VHL-deficient cells. This is the first mechanistic study investigating STF-62447's effects on the autophagic flux in RCC. Importantly, our study reclassifies STF-62247 as a blocker of later stages of autophagy and highlights the possibility of blocking this process through lysosome disruption in VHL-mutated RCCs.
自噬是一种高度保守的、维持细胞内环境稳定的过程，通过该过程细胞质成分被运送到溶酶体进行降解。不同自噬过程在癌症中的作用是复杂的，仍然取决于癌症的类型和阶段。肾细胞癌（RCC）是最常见的肾癌亚型，其特征是抑癌基因 von Hippel-Lindau（VHL）失活。我们之前的研究发现一种小分子化合物 STF-62247 是一种自噬调节分子，可导致 VHL 失活细胞的选择性细胞毒性。本研究专门研究了 STF-62247 对巨自噬通量的影响，以更好地描述其在 RCC 中的作用机制。我们的研究结果清楚地表明，该化合物是晚期自噬的有效阻断剂。我们表明，通过 CRISPR 敲除自噬相关基因来抑制自噬，使 VHL 缺陷细胞对 STF-62247 不敏感，揭示了自噬途径在 STF 选择性细胞死亡中的重要性。通过利用 STF-62247 的自发荧光，我们确定了其在溶酶体中的细胞定位。最后，在长时间的 STF 处理后，我们表明 VHL 功能正常的细胞能够通过恢复溶酶体数量来克服晚期自噬的阻断。相反，在 VHL 缺陷细胞中观察到自噬小泡增加，同时溶酶体数量随时间减少。这是第一个研究 STF-62247 在 RCC 中对自噬通量影响的机制研究。重要的是，我们的研究将 STF-62247 重新归类为晚期自噬的阻断剂，并强调通过溶酶体破坏阻断 VHL 突变的 RCC 中该过程的可能性。
