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TMPRSS2 gene rearrangements occur at DNA breaks formed during androgen receptor-mediated transcription and activate expression of ETS transcription factors at the early stages of more than half of prostate cancers. NKX3.1, a prostate tumor suppressor that accelerates the DNA repair response, binds to androgen receptor at the ERG gene breakpoint and inhibits both the juxtaposition of the TMPRSS2 and ERG gene loci and also their recombination. NKX3.1 acts by accelerating DNA repair after androgen-induced transcriptional activation. NKX3.1 influences the recruitment of proteins that promote homology-directed DNA repair. Loss of NKX3.1 favors recruitment to the ERG gene breakpoint of proteins that promote error-prone nonhomologous end-joining. Analysis of prostate cancer tissues showed that the presence of a TMPRSS2-ERG rearrangement was highly correlated with lower levels of NKX3.1 expression consistent with the role of NKX3.1 as a suppressor of the pathogenic gene rearrangement. [ABSTRACT FROM AUTHOR]

NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1 S186A/- , Nkx3.1 +/- , and Nkx3.1 +/+ mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1 S186A/- mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1 +/- mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss., (©2020 American Association for Cancer Research.)

NKX3.1 is the most commonly deleted gene in prostate cancer and a gatekeeper suppressor. NKX3.1 is a growth suppressor, mediator of apoptosis, inducer of antioxidants, and enhancer of DNA repair. PTEN is a ubiquitous tumor suppressor that is often decreased in prostate cancer during tumor progression. Steady-state turnover of NKX3.1 is mediated by DYRK1B phosphorylation at NKX3.1 serine 185 that leads to polyubiquitination and proteasomal degradation. In this study, we show PTEN is an NKX3.1 phosphatase that protects NKX3.1 from degradation. PTEN specifically opposed phosphorylation at NKX3.1(S185) and prolonged NKX3.1 half-life. PTEN and NKX3.1 interacted primarily in the nucleus as loss of PTEN nuclear localization abrogated its ability to bind to and protect NKX3.1 from degradation. The effect of PTEN on NKX3.1 was mediated via rapid enzyme-substrate interaction. An effect of PTEN on Nkx3.1 gene transcription was seen in vitro , but not in vivo . In gene-targeted mice, Nkx3.1 expression significantly diminished shortly after loss of Pten expression in the prostate. Nkx3.1 loss primarily increased prostate epithelial cell proliferation in vivo . In these mice, Nkx3.1 mRNA was not affected by Pten expression. Thus, the prostate cancer suppressors PTEN and NKX3.1 interact and loss of PTEN is responsible, at least in part, for progressive loss of NKX3.1 that occurs during tumor progression. SIGNIFICANCE: PTEN functions as a phosphatase of NKX3.1, a gatekeeper suppressor of prostate cancer., (©2019 American Association for Cancer Research.)

The prostate-specific tumor suppressor homeodomain protein NKX3.1 is inactivated by a variety of mechanisms in the earliest phases of prostate carcinogenesis and in premalignant regions of the prostate gland. The mechanisms by which NKX3.1 exercises tumor suppression have not been well elucidated. Here, we show that NKX3.1 affects DNA damage response and cell survival after DNA damage. NKX3.1 expression in PC-3 prostate cancer cells enhances colony formation after DNA damage but has minimal effect on apoptosis. NKX3.1 also diminishes and regulates total cellular accumulation of gammaH2AX. Endogenous NKX3.1 in LNCaP cells localizes to sites of DNA damage where it affects the recruitment of phosphorylated ATM and the phosphorylation of H2AX. Knockdown of NKX3.1 in LNCaP cells attenuates the acute responses of both ATM and H2AX phosphorylation to DNA damage and their subnuclear localization to DNA damage sites. NKX3.1 expression enhances activation of ATM as assayed by autophosphorylation at serine 1981 and activation of ATR as assayed by phosphorylation of CHK1. An inherited mutation of NKX3.1 that predisposes to early prostate cancer and attenuates in vitro DNA binding was devoid of the ability to activate ATM and to colocalize with gammaH2AX at foci of DNA damage. These data show a novel mechanism by which a homeoprotein can affect DNA damage repair and act as a tumor suppressor., ((c) 2010 AACR.)

Inflammation of the prostate is a risk factor for the development of prostate cancer. In the aging prostate, regions of inflammatory atrophy are foci for prostate epithelial cell transformation. Expression of the suppressor protein NKX3.1 is reduced in regions of inflammatory atrophy and in preinvasive prostate cancer. Inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1beta accelerate NKX3.1 protein loss by inducing rapid ubiquitination and proteasomal degradation. The effect of TNF-alpha is mediated via the COOH-terminal domain of NKX3.1 where phosphorylation of serine 196 is critical for cytokine-induced degradation. Mutation of serine 196 to alanine abrogates phosphorylation at that site and the effect of TNF-alpha on NKX3.1 ubiquitination and protein loss. This is in contrast to control of steady-state NKX3.1 turnover, which is mediated by serine 185. Mutation of serine 185 to alanine increases NKX3.1 protein stability by inhibiting ubiquitination and doubling the protein half-life. A third COOH-terminal serine at position 195 has a modulating effect on both steady-state protein turnover and on ubiquitination induced by TNF-alpha. Thus, cellular levels of the NKX3.1 tumor suppressor are affected by inflammatory cytokines that target COOH-terminal serine residues to activate ubiquitination and protein degradation. Our data suggest that strategies to inhibit inflammation or to inhibit effector kinases may be useful approaches to prostate cancer prevention.

The prostate-specific homeodomain protein NKX3.1 is a tumor suppressor that is commonly down-regulated in human prostate cancer. Using an NKX3.1 affinity column, we isolated topoisomerase I (Topo I) from a PC-3 prostate cancer cell extract. Topo I is a class 1B DNA-resolving enzyme that is ubiquitously expressed in higher organisms and many prokaryotes. NKX3.1 interacts with Topo I to enhance formation of the Topo I-DNA complex and to increase Topo I cleavage of DNA. The two proteins interacted in affinity pull-down experiments in the presence of either DNase or RNase. The NKX3.1 homeodomain was essential, but not sufficient, for the interaction with Topo I. NKX3.1 binding to Topo I occurred independently of the Topo I NH2-terminal domain. The binding of equimolar amounts of Topo I to NKX3.1 caused displacement of NKX3.1 from its cognate DNA recognition sequence. Topo I activity in prostates of Nkx3.1+/- and Nkx3.1-/- mice was reduced compared with wild-type mice, whereas Topo I activity in livers, where no NKX3.1 is expressed, was independent of Nkx3.1 genotype. Endogenous Topo I and NKX3.1 could be coimmunoprecipitated from LNCaP cells, where NKX3.1 and Topo I were found to colocalize in the nucleus and comigrate within the nucleus in response to either gamma-irradiation or mitomycin C exposure, two DNA-damaging agents. This is the first report that a homeodomain protein can modify the activity of Topo I and may have implications for organ-specific DNA replication, transcription, or DNA repair.