Introduction:

Autophagy is a lysosome-dependent degradative process, with reports indicating both tumor-suppressing and -promoting effects in cancer, including renal cancer (ccRCC). Loss of the Von Hippel-Lindau tumor suppressor gene (VHL) affects autophagy cellular function and promotes the expression of hypoxia-inducible protein N-myc downstream-regulated gene 1 (NDRG1); however, its regulatory role in the autophagic process is poorly understood. We investigated the influence of autophagy in concert of VHL expression to determine its cytotoxicity and activity associated with NDRG1 expression in ccRCC.

Methods:

An isogenic matched 786-O cell line with restored wild-type VHL cell model was used for this study. These cells were stably lentiviral transduced with shRNA NDRG1 vector, or mCherry-EGFP tandem fluorescent-tagged LC3B reporter for studying autophagosome synthesis and lysosomal degradation (autophagy flux). Cell viability was evaluated by cytotoxic XTT assay. The efficacy of PI3K/AKT/mTOR pathway and its downstream targets inhibition by mTOR inhibitors everolimus (RAD001) and AZD8055 and/or autophagy inhibitors HCQ, bafilomycin A1, and Lys05 were evaluated by immunoblots, electron microscopy, and immunofluorescence.

Results:

In an isogenic matched restored wild-type VHL 786-O cells, VHL downregulated the induction of NDRG1 protein levels, which were found to be modulated by hypoxia, intracellular iron and calcium levels. We demonstrated that VHL-deficient cells exhibited blockade of autophagy flux and impaired lysosomal function upon mTOR kinase inhibition to induce autophagy. These observed effects correlated with the induction of NDRG1 levels concurrent with increased autophagy flux marker p62 and the formation of cytoprotective autophagic vacuoles. Restored VHL in isogenic cells rescued the observed blockade in autophagy and reduced the induced NDRG1 levels to promote autophagy degradation. Interestingly, knockdown of NDRG1 sensitized mTOR-resistant 786-O deficient VHL cells to doxorubicin-induced apoptosis via PARP cleavage and had little effect on cytotoxicity but suppressed the initiation of autophagy induced by mTOR inhibitors. In contrast, silencing NDRG1 sensitized VHL-proficient cells to mTOR and/or autophagy inhibitors-induced cytotoxicity but not VHL-deficient cells. Similarly, combined treatment of mTOR and autophagy inhibitors augmented cytotoxic effects and promoted cell death in a VHL-dependent manner.

Conclusion:

These results reveal that autophagy has different influences on cellular contexts in which autophagy inhibition to augment mTOR inhibition in the presence of VHL can lead to compensatory mechanisms enhancing tumor cell death.

Funding: Mr. Lube Foundation and Canadian Cancer Society