Ubiquitination by TOPORS Regulates the Prostate Tumor Suppressor NKX3.1

Abstract

The NKX3.1 gene located at 8p21.2 encodes a homeodomaincontaining transcription factor that acts as a haploinsufficient tumor suppressor in prostate cancer. Diminished protein expression of NKX3.1 has been observed in prostate cancer precursors and carcinomas. TOPORS is a ubiquitously expressed E3 ubiquitin ligase that can ubiquitinate tumor suppressor p53. Here we report interaction between NKX3.1 and TOPORS. NKX3.1 can be ubiquitinated by TOPORS in vitro and in vivo, and overexpression of TOPORS leads to NKX3.1 proteasomal degradation in prostate cancer cells. Conversely, small interfering RNA-mediated knockdown of TOPORS leads to an increased steady-state level and prolonged half-life of NKX3.1. These data establish TOPORS as a negative regulator of NKX3.1 and implicate TOPORS in prostate cancer progression.Prostate cancer is the second leading cause of cancer deaths and the most frequently diagnosed malignancy in American men (1). Prostate carcinomas are considered to arise from cancer precursors including prostatic intraepithelial neoplasia (PIN) 2 and proliferative inflammatory atrophy (2). Molecular alterations, including hereditary and somatic gene mutations, gene deletions, gene amplification, chromosomal rearrangements, as well as epigenetic changes, have been implicated in prostate cancer initiation and progression (3).The NK class homeobox gene NKX3.1 has been studied extensively over the past decade for its roles in prostate development and carcinogenesis (4). The expression of the murine Nkx3.1 gene is androgen-dependent and is restricted largely to prostate epithelial cells in adults (5-7). Deletion of Nkx3.1 by gene targeting leads to prostate ductal morphological defects, as well as prostatic dysplasia and hyperplasia that resembles human PIN (8 -10). Interestingly, heterozygous Nkx3.1 mice also develop hyperplasia and PIN-like lesions (8). The human NKX3.1 gene maps to 8p21.2 within a region where loss of heterozygosity occurs in PIN and is common in prostate carcinomas; however, no mutations have been found in the coding region of the NKX3.1 allele remaining (11,12). In light of these observations, NKX3.1 has been proposed to function as a haploinsufficient tumor suppressor. In support of a dose-dependent growth regulatory function of NKX3.1, reduced but not complete loss of NKX3.1 protein expression is now well documented in most human prostate cancer samples in a manner inversely correlated with Gleason score (13) and also with disease progression (14). Diminished NKX3.1 expression is thought to be an early event in prostate carcinogenesis, and reduced NKX3.1 immunostaining was observed in most proliferative inflammatory atrophy and PIN lesions analyzed (13). The diminished NKX3.1 expression may be partly attributed to selective CpG methylation of the NKX3.1 promoter in some prostate cancer cases (15). Nevertheless, quantitative analyses of mRNA and protein levels of NKX3.1 in prostate carcinoma lesions revealed a lack of concordance between mRNA and protein levels (13). In particular, decre...

Proline-mediated Proteasomal Degradation of the Prostate-specific Tumor Suppressor NKX3.1

“…Other studies have demonstrated that amino acid residues 184 to 196 harbor phosphorylation sites that regulate ubiquitin-mediated NKX3.1 degradation in response to the inflammatory cytokine TNF-␣ (24). In addition, the E3 ubiquitin ligase topoisomerase 1 binding, arginine/serine-rich, E3 ubiquitin ligase has been shown to polyubiquitinate and target NKX3.1 for proteasomal degradation (25). Interactions with TOPORS and other unidentified E3 ligases require specific regions within the NKX3.1 N and C termini (24,25).…”

“…In addition, the E3 ubiquitin ligase topoisomerase 1 binding, arginine/serine-rich, E3 ubiquitin ligase has been shown to polyubiquitinate and target NKX3.1 for proteasomal degradation (25). Interactions with TOPORS and other unidentified E3 ligases require specific regions within the NKX3.1 N and C termini (24,25).…”

NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines

“…Androgen-responsive LNCaP cells (passage number [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] were propagated in RPMI1640 (Invitrogen, UK) medium supplemented with 10% heat-inactivated FBS (Invitrogen, UK). Cells were serum starved in the presence of 2% for 48 h and 0.5% for an additional 24 h in CT-FBS containing RPMI1640.…”

“…However, as part of the Groucho complex, NKX3.1 is required to repress transcription with its loss resulting in prostatic epithelial dysplasia and benign hyperplasia of the rat prostate [10,11]. Ouyang et al, have shown that NKX3.1 suppresses tumor initiation by protecting the cell against oxidative damage via transcriptional regulation of the pro-oxidant enzyme levels [12,13]. TOPORS, a strong E3 ubiquitin ligase, promotes proteasomal degradation of NKX3.1 through direct interaction, predisposing cells to oncogenic transformation by providing an irreversible growth advantage as the first steps in prostate carcinogenesis [14].…”

“…Thus, the DNA damage response is more efficient in cells in the presence of NKX3.1 expression; this report provides useful insights into our understanding of the DDR as well as therapy resistance of LNCaP cells upon DNA damage. We hypothesize that NKX3.1 basal expression protects cells against oxidative damage and/or maintains the constitutive DNA damage response in prostate cells through an unknown mechanism and thus is required to suppress cancer initiation in prostate epithelia [12,13].…”