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The discovery that androgens play an important role in the progression of prostate cancer led to the development of androgen deprivation therapy (ADT) as a first line of treatment. However, paradoxical growth inhibition has been observed in a subset of prostate cancer upon administration of supraphysiologic levels of testosterone (SupraT), both experimentally and clinically. Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive prostate cancer cells. This was initiated by the induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activated nucleic acid sensors converge on NFκB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT resulted in increased migration of cytotoxic immune cells to tumor beds in xenograft models and patient tumors. Collectively, these findings indicate that SupraT may inhibit a subset of prostate cancer by activating nucleic acid sensors and downstream immune signaling.Significance:This study demonstrates that supraphysiologic testosterone induces two parallel autophagy-mediated processes, ferritinophagy and nucleophagy, which then activate nucleic acid sensors to drive immune signaling pathways in prostate cancer.

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Background: Prostate cancer continues to show challenges in disease management and ultimately leads to advanced disease with poor survival. These unmet needs, despite recent advances, are leading researchers to identify novel targets to develop next-generation therapeutics. Considerable evidence from the published reports and TCGA database analysis indicates that CRM1, which functions as a nuclear export protein, amplifies prostate cancer, resulting in dysregulated nuclear export machinery. This dysregulation is also seen to correlate with mislocalization of p53, FOXO-1, and 3a, cyclinD1, p16, p21, and p27 in prostate cancer. Besides these mislocalized proteins, key DNA repair pathway members such as BRCA1 and Rad51 exhibit a nuclear export signal (NES) and are putative CRM1 cargoes. Nuclear-cytoplasmic shuttling of these repair proteins may play an essential role in repairing damaged DNA. We hypothesize that inhibition of CRM1 would result in defective DNA repair through disruption of nuclear-cytoplasmic shuttling of important repair protein cargoes. Methods: PCa cells (both AR-positive and negative lines) were treated with Selinexor to estimate the subtoxic doses. Cell lysates and total mRNA were collected to evaluate the relative levels of BRCA-1, RAD51, p53, ATM, ATR transcripts, and proteins. Activation of the DNA damage pathway was measured through a phosphorylated form of these proteins, and functional assessment of homologous recombination(HR) was measured through an HR sensor plasmid construct. Results: Treatment of PCa cells with Selinexor affected expression, localization, and kinetics of DNA repair leading to unrepaired DNA and induced apoptosis. Citation Format: Rajendra Kumar, Janet Mendonca, Kavya Boyapati, Yuhan Yang, Deven Topiwala, Suthicha Kanacharoen, Keerti Soundappan, Lillian Wilson, Eleni Panagopoulos, Michael Carducci, Sushant K. Kachhap. Exploring the role of CRM1 in DNA repair pathway in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1505.

The discovery that androgens play an important role in the progression of prostate cancer (PCa) has led to the development of androgen deprivation therapy as a first line of treatment against PCa. However, paradoxical growth inhibition has been observed, both experimentally and clinically, in a subset of PCa upon administration of supraphysiological levels of testosterone (SupraT). Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive PCa cells. This is initiated by induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activates nucleic acid sensors that converge on NF-kappaB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT results in increased migration of cytotoxic immune cells to tumor beds of animal xenografts and patient tumors. Collectively, our findings indicate that SupraT may inhibit a subset of PCa by activating nucleic acid sensors and downstream immune signaling.

Experimental and clinical evidence suggests that N‐myc downregulated gene 1 (NDRG1) functions as a suppressor of prostate cancer metastasis. Elucidating pathways that drive survival and invasiveness of NDRG1‐deficient prostate cancer cells can help in designing therapeutics to target metastatic prostate cancer cells. However, the molecular mechanisms that lead NDRG1‐deficient prostate cancer cells to increased invasiveness remain largely unknown. In this study, we demonstrate that NDRG1‐deficient prostate tumors have decreased integrin expression and reduced cell adhesion and motility. Our data indicate that loss of NDRG1 differentially affects Rho GTPases. Specifically, there is a downregulation of active RhoA and Rac1 GTPases with a concomitant upregulation of active Cdc42 in NDRG1‐deficient cells. Live cell imaging using a fluorescent sensor that binds to polymerized actin revealed that NDRG1‐deficient cells have restricted actin dynamics, thereby affecting cell migration. These cellular and molecular characteristics are in sharp contrast to what is expected after loss of a metastasis suppressor. We further demonstrate that NDRG1‐deficient cells have increased resistance to anoikis and increased invasiveness which is independent of its elevated Cdc42 activity. Furthermore, NDRG1 regulates expression and glycosylation of EMMPRIN, a master regulator of matrix metalloproteases. NDRG1 deficiency leads to an increase in EMMPRIN expression with a concomitant increase in matrix metalloproteases and thus invadopodial activity. Using a three‐dimensional invasion assay and an in vivo metastasis assay for human prostate xenografts, we demonstrate that NDRG1‐deficient prostate cancer cells exhibit a collective invasion phenotype and are highly invasive. Thus, our findings provide novel insights suggesting that loss of NDRG1 leads to a decrease in actin‐mediated cellular motility but an increase in cellular invasion, resulting in increased tumor dissemination which positively impacts metastatic outcome.

The original version of this Article contained errors in Fig. 7. In panels e and f, the graph titles incorrectly read ‘LNCaP-AdtNs’ and ‘LAPC4-AdtNs’, respectively. These errors have now been corrected in both the PDF and HTML versions of the Article.

Despite recent advances, the efficacy of androgen/androgen receptor (AR)-targeted therapy remains limited for many patients with metastatic prostate cancer. This is in part because prostate cancers adaptively switch to the androgen/AR-independent pathway for survival and growth, thereby conferring therapy resistance. Tumor hypoxia is considered as a major cause of treatment resistance. However, the exact mechanism is largely unclear. Here we report that chronic-androgen deprivation therapy (ADT) in the condition of hypoxia induces adaptive androgen/AR-independence, and therefore confers resistance to androgen/AR-targeted therapy, e.g., enzalutamide. Mechanistically, this is mediated by glucose-6-phosphate isomerase (GPI), which is transcriptionally repressed by AR in hypoxia, but restored and increased by AR inhibition. In turn, GPI maintains glucose metabolism and energy homeostasis in hypoxia by redirecting the glucose flux from androgen/AR-dependent pentose phosphate pathway (PPP) to hypoxia-induced glycolysis pathway, thereby reducing the growth inhibitory effect of enzalutamide. Inhibiting GPI overcomes the therapy resistance in hypoxia in vitro and increases enzalutamide efficacy in vivo., Prostate cancer often develops resistance to androgen receptor (AR) targeting drugs. Here, the authors show that, under conditions of hypoxia, AR inhibition via enzalutamide increases the expression of the glycolytic enzyme phosphoglucose isomerase (GPI) promoting a metabolic rewiring that allows the cells to survive, and consistent GPI inhibition restores sensitivity to enzalutamide.

// Janet Mendonca 1,* , Anup Sharma 1,* , Hae-Soo Kim 1 , Hans Hammers 1 , Alan Meeker 1 , Angelo De Marzo 1 , Michael Carducci 1 , Michael Kauffman 2 , Sharon Shacham 2 , Sushant Kachhap 1 1 Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Medical Institutions, Baltimore, MD 2 Karyopharm Therapeutics, Natick, MA * These authors contributed equally to this work Correspondence: Sushant Kachhap, email: // Keywords : Nucleocytoplasmic transport, CRM1, XPO 1, SINE inhibitors, prostate cancer Received : February 28, 2014 Accepted : July 05, 2014 Published : July 07, 2014 Abstract Mislocalization of proteins is a common feature of cancer cells. Since localization of proteins is tightly linked to its function, cancer cells can inactivate function of a tumor suppressor protein through mislocalization. The nuclear exportin CRM1/XPO 1 is upregulated in many cancers. Targeting XPO 1 can lead to nuclear retention of cargo proteins such as p53, Foxo, and BRCA1 leading to cell cycle arrest and apoptosis. We demonstrate that selective inhibitors of nuclear export (SINE) can functionally inactivate XPO 1 in prostate cancer cells. Unlike the potent, but toxic, XPO 1 inhibitor leptomycin B, SINE inhibitors (KPT-185, KPT-330, and KPT-251) cause a decrease in XPO 1 protein level through the proteasomal pathway. Treatment of prostate cancer cells with SINE inhibitors lead to XPO 1 inhibition, as evaluated by RevGFP export assay, leading to nuclear retention of p53 and Foxo proteins, consequently, triggering apoptosis. Our data reveal that treatment with SINE inhibitors at nanomolar concentrations results in decrease in proliferation and colonogenic capacity of prostate cancer cells by triggering apoptosis without causing any cell cycle arrest. We further demonstrate that SINE inhibitors can be combined with other chemotherapeutics like doxorubicin to achieve enhanced growth inhibition of prostate cancer cells. Since SINE inhibitors offer increased bioavailability, reduced toxicity to normal cells, and are orally available they can serve as effective therapeutics against prostate cancer. In conclusion, our data reveals that nucleocytoplasmic transport in prostate cancer can be effectively targeted by SINE inhibitors.

Castration resistance prostate cancer (CRPC) first manifests as a sustained rise in the androgen-responsive gene, PSA, consistent with reactivation of a functioning androgen receptor (AR) axis. This observation led to the development of “second-line” therapies aimed at further blocking androgen/AR signaling. Unfortunately, resistance to these agents can also develop quickly. Paradoxically, several studies have suggested that the growth of AR-positive human CRPC cell lines may be inhibited by supraphysiologic levels of testosterone (SupT). These studies suggested that the adaptive reliance on AR signaling by CRPC cells becomes a therapeutic liability that can be exploited through the administration of SupT, which we termed as bipolar androgen therapy (BAT). Understanding how BAT works at the molecular and cellular levels might help in rationally combining BAT with other agents to achieve increased efficacy and tumor responses. Our data indicates that SupT induces DNA double strand breaks (DSBs) in prostate cancer (PCa) cells. Unrepaired DSBs induced by SupT are routed for specialized autophagic degradation, termed nucleophagy. We further show that SupT-induced autophagosomal DNA can activate cytoplasmic DNA sensing pathways and downstream innate immune signaling. Based on our findings, we propose that BAT engages the immune system to inhibit tumor growth. Future combination of BAT with existing immunotherapeutics including immune checkpoint blockade may prove beneficial for treatment of CRPC. Citation Format: Janet Mendonca, Olutosin Owoyemi, Marc Rosen, Michael Carducci, Mark Markowski, Emmanuel Antonarakis, Drew Pardoll, Samuel Denmeade, Sushant K. Kachhap. Supraphysiological androgens activate innate immune signaling in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 517.

Background. The antifungal drug itraconazole inhibits angiogenesis and Hedgehog signaling and delays tumor growth in murine prostate cancer xenograft models. We conducted a noncomparative, randomized, phase II study evaluating the antitumor efficacy of two doses of oral itraconazole in men with metastatic prostate cancer. Patients and Methods. We randomly assigned 46 men with chemotherapy-naïve metastatic castration-resistant prostate cancer (CRPC) to receive low-dose (200 mg/day) or high-dose (600 mg/day) itraconazole until disease progression or unacceptable toxicity. The primary endpoint was the prostate-specific antigen (PSA) progression-free survival (PPFS) rate at 24 weeks; a 45% success rate in either arm was prespecified as constituting clinical significance. Secondary endpoints included the progression-free survival (PFS) rate and PSA response rate (Prostate Cancer Working Group criteria). Exploratory outcomes included circulating tumor cell (CTC) enumeration, serum androgen measurements, as well as pharmacokinetic and pharmacodynamic analyses. Results. The high-dose arm enrolled to completion (n = 29), but the low-dose arm closed early (n = 17) because of a prespecified futility rule. The PPFS rates at 24 weeks were 11.8% in the low-dose arm and 48.0% in the high-dose arm. The median PFS times were 11.9 weeks and 35.9 weeks, respectively. PSA response rates were 0% and 14.3%, respectively. In addition, itraconazole had favorable effects on CTC counts, and it suppressed Hedgehog signaling in skin biopsy samples. Itraconazole did not reduce serum testosterone or dehydroepiandrostenedione sulfate levels. Common toxicities included fatigue, nausea, anorexia, rash, and a syndrome of hypokalemia, hypertension, and edema. Conclusion. High-dose itraconazole (600 mg/day) has modest antitumor activity in men with metastatic CRPC that is not mediated by testosterone suppression.

The physiological processes regulating neuromuscular transmission are highly dependent on the structural features of the motor neuron and motor endplate, and detailing the structure of neuromuscular junctions (NMJs) in muscle biopsies is a powerful method for research and diagnostics. The observation of NMJ ultrastructure, however, is complicated by the difficulty in locating NMJs for analysis by electron microscopy. Consequently, a correlative confocal-transmission electron microscopy method was developed. Fixed muscle samples were cryo-protected in sucrose, sectioned on a cryostat, and stained with fluorescent alpha-bungarotoxin for confocal microscopy. Sections containing junctions were mapped and then processed for transmission electron microscopy (TEM). Cryostat sections allowed large expanses of muscle tissue to be rapidly screened and enabled specific junctions to be targeted for TEM. The morphology of the junctions was well preserved with all essential features of the pre- and postsynaptic elements readily identifiable without freeze damage. Unlike NMJ correlative methods using histochemical stains and DAB photo-oxidation, no electron dense precipitate was deposited over the NMJ, enabling an unobstructed view of the pre- and postsynaptic structures.

The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBPβ, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPβ, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPβ knockdown suppressed autophagosome–lysosome fusion. We also found that C/EBPβ knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPβ levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPβ sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPβ. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPβ knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPβ is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.

Given the dearth of gene mutations in prostate cancer, [1] ,[2] it is likely that genomic rearrangements play a significant role in the evolution of prostate cancer. However, in the search for recurrent genomic alterations, "private alterations" have received less attention. Such alterations may provide insights into the evolution, behavior, and clinical outcome of an individual tumor. In a recent report in "Genome Biology" Wyatt et al. [3] defines unique alterations in a cohort of high-risk prostate cancer patient with a lethal phenotype. Utilizing a transcriptome sequencing approach they observe high inter-tumor heterogeneity; however, the genes altered distill into three distinct cancer-relevant pathways. Their analysis reveals the presence of several non-ETS fusions, which may contribute to the phenotype of individual tumors, and have significance for disease progression.

Combinations of anticancer therapies with high efficacy and low toxicities are highly sought after. Therefore, we studied the effect of polo-like kinase 1 (Plk1) inhibitors on prostate cancer cells as a single agent and in combination with histone deacetylase (HDAC) inhibitors valproic acid and vorinostat. IC50s of Plk1 inhibitors BI 2536 and BI 6727 were determined in prostate cancer cells by MTS assays. Morphological and molecular changes were assessed by immunoblotting, immunofluorescence, flow cytometry, real-time RT-PCR, and pulldown assays. Efficacy of combination therapy was assessed by MTS and clonogenic assays. IC50 values in DU145, LNCaP, and PC3 cells were 50, 75, and 175 nM, respectively, for BI 2536 and 2.5, 5, and 600 nM, respectively, for BI 6727. Human prostate fibroblasts and normal prostate epithelial cells were unaffected at these concentrations. While DU145 and LNCaP cells were solely arrested in mitosis on treatment, PC3 cells accumulated in G2 phase and mitosis, suggesting a weak spindle assembly checkpoint. Combining Plk1 inhibitors with HDAC inhibitors had synergistic antitumor effects in vitro. DMSO-treated prostate cancer cells were used as controls to study the effect of Plk1 and HDAC inhibition. Plk1 inhibitors decreased proliferation and clonogenic potential of prostate cancer cells. Hence, Plk1 may serve as an important molecular target for inhibiting prostate cancer. Combining HDAC inhibitors with BI 2536 or BI 6727 may be an effective treatment strategy against prostate cancer.—Wissing, M. D., Mendonca, J., Kortenhorst, M. S. Q., Kaelber, N. S., Gonzalez, M., Kim E., Hammers, H., van Diest, P. J., Carducci, M. A., Kachhap, S. K. Targeting prostate cancer cell lines with polo-like kinase 1 inhibitors as a single agent and in combination with histone deacetylase inhibitors.

Cerebral palsy (CP) is a static encephalopathy occurring when a lesion to the developing brain results in disordered movement and posture. Patients present with sometimes overlapping spastic, athetoid/dyskinetic, and ataxic symptoms. Spastic CP, which is characterized by stiff muscles, weakness, and poor motor control, accounts for ∼80% of cases. The detailed mechanisms leading to disordered movement in spastic CP are not completely understood, but clinical experience and recent studies suggest involvement of peripheral motor synapses. For example, it is recognized that CP patients have altered sensitivities to drugs that target neuromuscular junctions (NMJs), and protein localization studies suggest that NMJ microanatomy is disrupted in CP. Since CP originates during maturation, we hypothesized that NMJ disruption in spastic CP is associated with retention of an immature neuromotor phenotype later in life. Scoliosis patients with spastic CP or idiopathic disease were enrolled in a prospective, partially-blinded study to evaluate NMJ organization and neuromotor maturation. The localization of synaptic acetylcholine esterase (AChE) relative to postsynaptic acetylcholine receptor (AChR), synaptic laminin β2, and presynaptic vesicle protein 2 (SV2) appeared mismatched in the CP samples; whereas, no significant disruption was found between AChR and SV2. These data suggest that pre- and postsynaptic NMJ components in CP children were appropriately distributed even though AChE and laminin β2 within the synaptic basal lamina appeared disrupted. Follow up electron microscopy indicated that NMJs from CP patients appeared generally mature and similar to controls with some differences present, including deeper postsynaptic folds and reduced presynaptic mitochondria. Analysis of maturational markers, including myosin, syntrophin, myogenin, and AChR subunit expression, and telomere lengths, all indicated similar levels of motor maturation in the two groups. Thus, NMJ disruption in CP was found to principally involve components of the synaptic basal lamina and subtle ultra-structural modifications but appeared unrelated to neuromotor maturational status.

The N-myc downstream regulated gene 1 (NDRG1) has been identified as a metastasis-suppressor gene in prostate cancer (PCa). Compounds targeting PCa cells deficient in NDRG1 could potentially decrease invasion/metastasis of PCa. A cell based screening strategy was employed to identify small molecules that selectively target NDRG1 deficient PCa cells. DU-145 PCa cells rendered deficient in NDRG1 expression by a lentiviral shRNA-mediated knockdown strategy were used in the primary screen. Compounds filtered from the primary screen were further validated through proliferation and clonogenic survival assays in parental and NDRG1 knockdown PCa cells. Screening of 3360 compounds revealed irinotecan and cetrimonium bromide (CTAB) as compounds that exhibited synthetic lethality against NDRG1 deficient PCa cells. A three-dimensional (3-D) invasion assay was utilized to test the ability of CTAB to inhibit invasion of DU-145 cells. CTAB was found to remarkably decrease invasion of DU-145 cells in collagen matrix. Our results suggest that CTAB and irinotecan could be further explored for their potential clinical benefit in patients with NDRG1 deficient PCa.

BACKGROUND: The antifungal drug itraconazole inhibits angiogenesis and Hedgehog signaling, and delays tumor growth in murine prostate cancer xenograft models. Unlike ketoconazole, it does not suppress androgen synthesis. METHODS: A phase 2 study of oral itraconazole in men with mCRPC was conducted at 4 sites. Men were randomized to low dose (LD) or high dose (HD) itraconazole (200 or 600 mg/day) until disease progression or unacceptable toxicity. The primary endpoint was PSA progression-free survival (PPFS) at 24 wk (PSA progression = 25% PSA rise above baseline/nadir); a 45% success rate in either arm was prespecified as constituting clinical significance. Secondary endpoints were progression-free survival (PFS) at 24 wk (progression = clinical/radiographic progression or death, but not rising PSA); median PFS; median PPFS; and max PSA decline. Exploratory outcomes included CTC enumeration, analysis of adrenal androgens (serum testosterone and DHEA), VEGF analysis, and GLI1 expression in skin biopsy samples. RESULTS: The HD arm enrolled to completion (N=29), but the LD arm closed early (N=17) due to a prespecified futility analysis. Table 1 shows efficacy results. In addition, 3/5 men (60%) in the HD arm and 2/3 men (67%) in the LD arm with unfavorable (≥5) CTCs at baseline converted to favorable ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-224. doi:1538-7445.AM2012-LB-224

Cyclin-dependent kinase 5 (CDK5) has long been known to be an enzyme necessary for neuronal migration. In recent years, extraneural functions of CDK5 have been discovered, including CDK5 playing an essential role in invasion and migration in pancreatic and prostate cancer, and in tumor growth and Ras signaling through Ral in pancreatic cancer. This makes CDK5 an interesting target against cancer. We blocked CDK5 activity in prostate cancer cell line PC3 by dominant-negative constructs, and used a library consisting of 3360 compounds to screen for small molecules that differentially inhibit proliferation activity in wildtype PC3 (PC3wt) and CDK5 dominant-negative PC3 (PC3cdn) cells, by performing MTS assays. We selected for compounds in which the difference in proliferation activity between the two cell lines was more than 40% and/or compounds that inhibited both cell lines by more than 50%. With this selection, 32 compounds were identified. Topically acting compounds were excluded, the other compounds were sorted for difference in proliferation activity inhibition between the two cell lines, and a Pubmed search was performed on the compounds with the largest difference. This way, tilorone analog R 9536-DA was selected as the most potent compound, as it inhibited proliferation in PC3wt and PC3cdn cells by 72.5% (SD 0.5%) and 81.1% (SD 2.1%) respectively (a difference in proliferation activity of 32%). MTS and clonogenic assays confirmed the differential sensitivity of PC3wt and PC3cdn cells to tilorone dihydrochloride. The IC50 in PC3wt cells was around 16 μM, while in PC3cdn cells it was around 10 μM. This difference in sensitivity to tilorone dihydrochloride in the presence of CDK5 inhibition was confirmed in pancreatic and other prostate cancer cell lines as well. To examine the toxicity of tilorone dihydrochloride in normal cells, the compound was administered to normal human prostate fibroblasts, in which the IC50 was found to be higher than 20μM. Western blots were performed to investigate the mechanism behind this difference in sensitivity between cell lines. These experiments conclude that administration of tilorone dihydrochloride differentially induces PARP and the p38-MAPK signaling pathway, indicating the involvement of this pathway in tilorone induced cell death. Tilorone analogs previously have been shown in mice to inhibit metastasis and increase survival in several tumor types. This study suggests that the combination of tilorone and inhibition of CDK5 may be a promising therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2976. doi:10.1158/1538-7445.AM2011-2976

Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics.