Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER *

Activation and transcriptional reprogramming of AR in advanced prostate cancer frequently coincides with the loss of two tumor suppressors, INPP4B and PTEN, which are highly expressed in human and mouse prostate epithelium. While regulation of AR signaling by PTEN has been described by multiple groups, it is not known whether the loss of INPP4B affects AR activity. Using prostate cancer cell lines, we showed that INPP4B regulates AR transcriptional activity and the oncogenic signaling pathways Akt and PKC. Analysis of gene expression in prostate cancer patient cohorts showed a positive correlation between INPP4B expression and both AR mRNA levels and AR transcriptional output. Using an Inpp4b mouse model, we demonstrated that INPP4B suppresses Akt and PKC signaling pathways and modulates AR transcriptional activity in normal mouse prostate. Remarkably, PTEN protein levels and phosphorylation of S380 were the same in Inpp4b and WT males, suggesting that the observed changes were due exclusively to the loss of INPP4B. Our data show that INPP4B modulates AR activity in normal prostate and its loss contributes to the AR-dependent transcriptional profile in prostate cancer.

There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADPribose) polymerase (pARp) and poly(ADp-ribose) glycohydrolase (pARG) are key enzymes in BeR that elongate and degrade pAR polymers on target proteins. While pARp inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the pARG locus and induces pARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. thus, AR and pARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients. Late stage prostate cancers are treated with radiation and other cytotoxic therapies. It was observed that androgen ablation sensitizes prostate tumors to radiation and chemotherapy in prostate cancer patients 1-3. Prostate tumors with high androgen receptor (AR) transcriptional output have increased expression of DNA repair genes in general, and base excision repair (BER) associated proteins in particular 4,5. Conversely, AR upregulates pathways in prostate cancer that increase genomic instability, such as the TMPRSS2-ERG gene fusion, which is present in a significant part of advanced prostate cancers 5,6. Importantly, inhibition of poly(ADP-ribose) polymerase 1 (PARP1) significantly increases levels of DNA damage in such tumors 7 and is currently being tested in clinical trials in metastatic castration resistant prostate cancer (CRPC) with somatic or germline mutations in DNA repair genes. The PARP inhibitors rucaparib (NCT02952534, NCT03533946 and NCT03413995) and olaparib (NCT02316197, NCT03012321, NCT03787680, NCT03432897 and others) have been tested in clinical trials and were granted breakthrough designation by the U.S. Food and Drug Administration (FDA) for metastatic CRPC. Poly(ADP-ribose) glycohydrolase (PARG) and PARP1 are key enzymes required for DNA repair and maintenance of genomic stability. Poly ADP-ribose (PAR) is a heterogeneous branched polymer of ADP-ribose that is attached to proteins in response to various stimuli by a dynamic process called PARylation. PARylation regulates DNA damage detection and repair as PAR acts as a loading platform to recruit a variety of DNA repair factors, in a non-covalent fashion, to the DNA lesions. Multiple ...

A high fat diet and obesity have been linked to the development of metabolic dysfunction and the promotion of multiple cancers. The causative cellular signals are multifactorial and not yet completely understood. In this report, we show that Inositol Polyphosphate-4-Phosphatase Type II B (INPP4B) signaling protects mice from diet-induced metabolic dysfunction. INPP4B suppresses AKT and PKC signaling in the liver thereby improving insulin sensitivity. INPP4B loss results in the proteolytic cleavage and activation of a key regulator in de novo lipogenesis and lipid storage, SREBP1. In mice fed with the high fat diet, SREBP1 increases expression and activity of PPARG and other lipogenic pathways, leading to obesity and non-alcoholic fatty liver disease (NAFLD). Inpp4b−/− male mice have reduced energy expenditure and respiratory exchange ratio leading to increased adiposity and insulin resistance. When treated with high fat diet, Inpp4b−/− males develop type II diabetes and inflammation of adipose tissue and prostate. In turn, inflammation drives the development of high-grade prostatic intraepithelial neoplasia (PIN). Thus, INPP4B plays a crucial role in maintenance of overall metabolic health and protects from prostate neoplasms associated with metabolic dysfunction.

High fat diet leads to obesity, chronic inflammation, accelerated prostate cancer progression, and decreased survival. INPP4B, a dual specificity phosphatase, is a tumor suppressor which opposes Akt and PKC signaling pathways activated in response to obesity and prostate cancer progression. Previous research demonstrated that overexpression of PKC in mouse prostate leads to prostate intraepithelial neoplasia (PIN). Akt pathway stimulates prostate cancer progression in mice and is activated in all advanced prostate cancers in men. Using INPP4B knockout mouse model we tested whether INPP4B regulates prostate physiology in mice on high fat diet. The WT and Inpp4b -/- male mice were fed a low-fat diet (LFD) (11% fat) or high-fat diet (HFD) (58% fat) for 12 weeks. The mouse prostate was dissected into the anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP). We observed that INPP4B and androgen receptor (AR) were expressed primarily in DLP and VP. INPP4B expression was decreased in the DLP of HFD fed mice. In the prostate of mice fed a LFD, we observed similar histological architecture in both WT and Inpp4b -/- mice. All the Inpp4b -/- mice fed a HFD developed PIN, whereas WT mice did not. Cells with polymorphic nuclei were abundant in PIN loci. The staining of α-SMA indicated that prostate epithelial cells invaded the stroma of HFD Inpp4b -/- mice. Consistent with the previous findings, HFD activated PKC, and the loss of INPP4B increased the levels of phospho-PKC βII and ζ in HFD mouse DLP. We previously reported that INPP4B loss changes AR transcriptional output in prostate cancer cells and normal mouse prostate. In HFD group, loss of INPP4B did not affect the level of AR in any of the three prostate lobes but led to an altered AR-dependent transcriptional activity. We show that the pro-inflammatory cytokines Il6 , Tnfα and Il1β are expressed at low or undetectable levels in prostates of mice fed LFD. However, all three mediators were strikingly elevated in DLP of HFD Inpp4b -/- mice, where most of the PIN was also observed. Using an IL6 ELISA, we confirmed increases of IL6 protein levels in the prostates of the Inpp4b -/- HFD group. Consistent with elevated levels of inflammatory cytokines in prostates of Inpp4b -/- mice we detected an increase of macrophage specific biomarkers in the DLP of HFD groups, indicating HFD promotes macrophage infiltration in both WT and Inpp4b -/- mice. Importantly, no changes in PTEN were detected at both RNA and protein levels in Inpp4b -/- mice, indicating that increased inflammation and PIN develop...