Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells

Abstract

Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death.

USP9X Deubiquitylates DVL2 to Regulate WNT Pathway Specification

“…Furthermore, we found that treating cells with WP1130, a small molecule known to inhibit the deubiquitylase activity of USP9X (Kapuria et al, 2010), inhibited canonical WNT activation by WNT3a with a half-maximal inhibitory concentration (IC 50 ) of 2.9 μM (Figure 3C). This result is consistent with our findings that USP9X is required for canonical WNT activation; however, WP1130 is known to target other deubiquitylases (USP24, USP5, USP14, and UCH37) (de Las Pozas et al, 2018; Kapuria et al, 2010; Kushwaha et al, 2015; Peterson et al, 2015). Thus, we cannot exclude the possibility that inhibition of multiple DUB activities drives loss of WNT activation in the presence of WP1130.…”

High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery

“…In 2015, the Hay group used biochemical tools and the MALDI-TOF DUB assay and discovered that UCH DUBs are capable of cleaving peptide-linked N-terminal mono-ubiquitin from several protein substrates 76 . The current HTS platform has also been extensively used to characterize compound selectivity in vitro 74,75,85,86 . In 2017, Kategaya et al used both NMR and HT-activity-based screening to identify USP7 catalytic domain-binding fragments.…”

Dysregulated Transcriptional Control in Prostate Cancer

“…Attempts to reduce AR levels using a specific antisense nucleotide have been evaluated clinically but with little success [159,179]. In another approach, AR transcript and protein levels could be reduced by targeting deubiquitinases, and first in vitro efficacy data based on this approach have been reported [180].…”