Quantitative proteomics to study a small molecule targeting the loss of von Hippel–Lindau in renal cell carcinomas

Abstract

Inactivation of the tumor suppressor gene, von Hippel-Lindau (VHL), is known to play an important role in the development of sporadic clear cell renal cell carcinomas (ccRCCs). Even if available targeted therapies for metastatic RCCs (mRCCs) have helped to improve progression-free survival rates, they have no durable clinical response. We have previously shown the feasibility of specifically targeting the loss of VHL with the identification of a small molecule, STF-62247. Understanding its functionality is crucial for developing durable personalized therapeutic agents differing from those available targeting hypoxia inducible factor (HIF-) pathways. By using SILAC proteomics, we identified 755 deregulated proteins in response to STF-62247 that were further analyzed by ingenuity pathway analysis (IPA). Bioinformatics analyses predicted alterations in 37 signaling pathways in VHL-null cells in response to treatment. Validation of some altered pathways shows that STF-62247's selectivity is linked to an important inhibition of mTORC1 activation in VHL-null cells leading to protein synthesis arrest, a mechanism differing from two allosteric inhibitors Rapamycin and Everolimus. Altogether, our study identified signaling cascades driving STF-62247 response and brings further knowledge for this molecule that shows selectivity for the loss of VHL. The use of a global SILAC approach was successful in identifying novel affected signaling pathways that could be exploited for the development of new personalized therapeutic strategies to target VHL-inactivated RCCs.

Loperamide, pimozide, and STF-62247 trigger autophagy-dependent cell death in glioblastoma cells

“…Moreover, our study suggests that loperamide, pimozide, and STF-62247 regulate autophagy through dephosphorylation of mTORC1, a master regulator of autophagy 69 . This is consistent with recent findings showing that STF-62247 inhibits mTORC1 70 . Loperamide and pimozide may lead to a decrease in intracellular Ca 2+ levels through inhibition of voltage-gated Ca 2+ channels.…”

STF-62247 blocks late stages of autophagy by disrupting lysosomal physiology

“…Western Blot were performed as previously described. (23,34) Briefly, cells were lysed in M-PER buffer and quantified using Pierce BCA protein assay Kit (Thermo Scientific, #23225).…”

“…Indeed, we highlighted the ability of VHL-proficient cells to restore mTOR activity following prolonged exposition to STF-62247 compared to VHL-mutated RCC4 cells. (34) To characterize earlier effects of STF on this signaling, immunoblot analyses were performed to measure phosphorylation states of mTOR and of its downstream effector RPS6KB1/p70S6 kinase in response to a 4 hr STF treatment ( Fig. 3D).…”

“…34) Briefly, five thousand cells were seeded per well in 96-well plates. Cells were treated with serial dilutions of STF-62247 after which plates were incubated at 37°C under 5% CO 2 for 48 hr or 96 hr.…”

Narciclasine induces autophagy‐dependent apoptosis in triple‐negative breast cancer cells by regulating the AMPK‐ULK1 axis

“…In fact, autophagy is an essential mechanism through which chemotherapeutics kill cancer cells . For example, STF‐62 247 induces autophagy and apoptosis by inhibiting the proliferation of clear‐cell renal carcinoma cells and perturbing protein trafficking . Chemotherapeutics that can promote apoptosis and autophagy may be powerful anticancer drugs …”