SPOP Deregulation Improves the Radiation Response of Prostate Cancer Models by Impairing DNA Damage Repair.

Rihan El Bezawy,Valentina Zuco,Alessandro Cicchetti,Monica Tortoreto,Stella Tinelli,Nadia Zaffaroni,Valentina Doldi,Riccardo Valdagni,Stefano Percio,Paolo Gandellini,Claudio Stucchi,Martina Tripari

doi:10.3390/cancers12061462

Abstract

Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is the most commonly mutated gene in prostate cancer (PCa). Recent evidence reports a role of SPOP in DNA damage response (DDR), indicating a possible impact of SPOP deregulation on PCa radiosensitivity. This study aimed to define the role of SPOP deregulation (by gene mutation or knockdown) as a radiosensitizing factor in PCa preclinical models. To express WT or mutant (Y87N, K129E and F133V) SPOP, DU145 and PC-3 cells were transfected with pMCV6 vectors. Sensitivity profiles were assessed using clonogenic assay and immunofluorescent staining of γH2AX and RAD51 foci. SCID xenografts were treated with 5 Gy single dose irradiation using an image-guided small animal irradiator. siRNA and miRNA mimics were used to silence SPOP or express the SPOP negative regulator miR-145, respectively. SPOP deregulation, by either gene mutation or knockdown, consistently enhanced the radiation response of PCa models by impairing DDR, as indicated by transcriptome analysis and functionally confirmed by decreased RAD51 foci. SPOP silencing also resulted in a significant downregulation of RAD51 and CHK1 expression, consistent with the impairment of homologous recombination. Our results indicate that SPOP deregulation plays a radiosensitizing role in PCa by impairing DDR via downregulation of RAD51 and CHK1.

Highlights

Speckle-type POZ protein (SPOP) is an adaptor for the E3 ubiquitin ligase Cullin 3 that selectively binds to and targets substrates for ubiquitination and proteasome degradation [1]
To investigate the potential of prostate cancer (PCa)-specific SPOP mutations as radiosensitizing factors in PCa, we transfected DU145 and PC-3 cell lines with pMCV6 expression vectors encoding for WT or mutant (Y87N, K129E and F133V) SPOP and assessed the effect on cell response to 2–8 Gy γ irradiation via clonogenic assay
The overexpression of WT SPOP did not affect the radiosensitivity profile of either cell line, mutant SPOP ectopic expression enhanced radiosensitivity of both cell lines, independently of the type of mutation, as revealed by the reduction in their clonogenic cell survival compared to WT SPOP, which was statistically significant along the whole dose range (Figure 2A)

Summary

Introduction

Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is an adaptor for the E3 ubiquitin ligase Cullin 3 that selectively binds to and targets substrates for ubiquitination and proteasome degradation [1]. Genome-wide generation sequencing studies have revealed that SPOP is frequently mutated in a number of cancer types such as prostate and endometrial [2,3], suggesting SPOP as a putative tumor suppressor. SPOP binds to its substrates via the N-terminal meprin and traf homology (MATH) domain [1], while it interacts with Cullin 3 through the bric-a-brac/tramtrack/broad complex (BTB) domain at the C-terminus [1]. SPOP mutations observed in human cancers are clustered in the MATH domain [2,3], suggesting that they may promote cancer via altering the stability of its substrates (Figure 1). SPOP mutations define a distinct molecular class of PCa, with a high frequency of characteristic genomic rearrangements, but they are mutually exclusive with rearrangement between

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