Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERKMEK Pathway

الملخص EN

Ovarian cancer (OVCA) patients often develop tolerance to standard platinum therapy that accounts for extensive treatment failures.

Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress.

In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress.

To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization.

Autophagy requires a tightly regulated intracellular pH.

Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators.

V-ATPase ‘a2’ isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells.

Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage.

Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible factor for cisplatin sensitization.

Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders.

In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment.

The apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin.

Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells.

These cells showed downregulated ERK/MEK pathway that is known to repress autophagy.

Interestingly, upon cisplatin treatment of sh-V0a2-cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells.

However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy flux.

Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired.

Taken together, this study provides a novel insight into the mechanism by which V-ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer.

We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer.