Investigating the role of vacuolar-ATPase (V-ATPase) in cancer

Abstract

Overexpression of V-ATPase subunits is linked to cancer cell migration, invasion and metastasis in a number of cancer types including prostate cancer (PCa). Due to expression of androgen receptor (AR) variants, drug resistance is a significant problem in PCa. Various strands of data suggest that the V-ATPase may influence AR function and therefore represents a novel target for which to treat PCa. The central hypothesis addressed in this project is that V-ATPase dysregulation is directly linked to cancer via alterations in activity, and that V-ATPase inhibition influences AR function in prostate cancer.
Somatic missense mutations, glutamine (Q), valine (V) and lysine (K) at glutamate (E) 61 in the human V-ATPase V1E2 isoform were first identified in the COSMIC database (v81). A V-ATPase ΔE null mutant yeast model was complemented with wild-type or mutant forms of human V1E1 or E2 subunits to determine whether the mutations altered V-ATPase activity and function. The results indicated that V-ATPase catalytic activity was significantly increased in the V-ATPases with E61 to V and Q substitutions in the V1E2 subunit compared to the wild-type subunit. The E61Q mutation also substantially increased V-ATPase proton-coupling rate. These results show that cancer-associated V-ATPase mutations can increase enzyme catalytic activity and function.
Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase models. These results were supported by experiments in hormone sensitive PCa cell lines (LNCaP and DuCaP) and mutant AR cell lines (22Rv1 and LNCaP-F877L/T878A), which showed that V-ATPase inhibition reduced AR expression, and expression of AR target genes, at mRNA and protein levels. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to silence the V1C1 subunit in LNCaP and 22Rv1 cells. Results showed that transfection with ATP6V1C1 siRNA significantly reduced AR protein levels and function in 22Rv1 cells. Conversely, CRISPR-Cas9 mediated knockout of V1C1 showed a small increase in AR in both cell lines, which was accompanied by a compensatory increase in protein levels of the alternate V1C2 isoform. Overall, these results are consistent with the hypothesis that V-ATPase dysregulation is directly linked to cancer via alterations in activity. In particular, this work revealed that somatic mutations identified in tumour samples can increase V-ATPase activity, and that V-ATPase inhibition can reduce AR signalling regardless of mutant AR expression.

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ORCID for Simon Crabb: orcid.org/0000-0003-3521-9064

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