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Purpose: The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation.Experimental Design: Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells.Results: SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-κB ligand expression was up-regulated in cells expressing PSA compared with controls.Conclusions: Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin–receptor activator of NF-κB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.

Supplementary Table 1 from Prostate-Specific Antigen Modulates Genes Involved in Bone Remodeling and Induces Osteoblast Differentiation of Human Osteosarcoma Cell Line SaOS-2

Immunotherapy targeting the PD-1/PD-L1 receptor has achieved great success in melanoma patients. Although many studies have addressed the underlying mechanisms involved in the blockade of PD-1/PD-L1 and the consequent modulation of the immune system, the mechanisms of PD-L1 upregulation and reliable biomarkers to predict the efficacy of anti-PD-1/PD-L1 therapy remain unknown. The present study demonstrates the correlation between IGFBP2 and PD-L1, revealing a novel immune-associated tumor function of IGFBP2 in facilitating nuclear accumulation of EGFR and activation of the EGFR/STAT3/PD-L1 signaling pathway in melanoma cells. Our results also suggest that combined IGFBP2 and PD-L1 expression has the potential to predict the efficacy of anti-PD-1 treatment for malignant melanoma; because the combination of high IGFBP2 and PD-L1 expression characterizes melanoma patients with worse overall survival and is associated with a better immune ecosystem. These characteristics have been confirmed by both in vitro and in vivo data. Consequently, IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway and its function as a PD-L1 regulator might suggest novel therapeutic approach for melanoma.

Lung cancer is the leading cause of cancer-related death worldwide, with a 5-year survival rate of ~18% (1-3). Non-small cell lung cancer (NSCLC) comprises 85% of all lung cancer cases. Great strides have been made with the development of more advanced diagnosis, prognosis, and treatment strategies. Despite improvements, African American patients (AAs) continue to present with more advanced stages and later-stage metastatic tumors of lung cancer at diagnosis. The severe types of lung cancer can reduce the 5-year survival rate to only 4% (4). The disparities may partially be due to the socioeconomic disadvantages of AAs in receiving cancer services (5). Etiologically, cigarette smoking and exposure to secondhand smoking are leading preventable causes of lung cancer and premature death in the United States (US) (5). Notably, menthol cigarettes, which have been particularly marketed to communities with high AA population (7), can cause deeper inhalation, increased absorption of tobacco toxicants, a higher nicotine dependence and reduced cessation success (6-8). Some genetic alterations have been suggested to contribute to the disparities of breast cancer (9), but genetic factors have not been well studied in lung cancer disparities. Utilizing the genomic data from the Precision Oncology Initiative (POI) cohort at the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC), our initial analysis has shown that TP53 has a significantly higher mutation rate in AA patients, which was validated with the TCGA cohort (10). Furthermore, codons including Cysteine 176 (C176) and C242 mutated more frequently in AA than CA patients. Considering the four amino acids in the zinc-binding region (C176, Histidine 179 [H179], C238, and C242), AA patients have a higher mutation rate than CA patients within both our POI (15% v.s. 5%, P=5E-4) and the TCGA (14% vs. 6%, P Citation Format: Liang Liu, Farideh Mehraein-Ghomi, Elizabeth Forbes, Umit Topaloglu, W. Jeffrey Petty, Stefan Grant, Jimmy Ruiz, Kristie L. Foley, Karen Winkfield, Boris Pasche, Wei Zhang. TP53 mutations reprogram fatty acid metabolism and tumor microenvironment in African American patients with non-small cell lung cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C001.

In the United States, endometrial cancer is the fourth most common malignancy among women, with an estimated 61,380 new cases and 10,920 deaths in 2017 (1). The incidence is increasing more rapidly among African American (AfAm) women than other racial/ethnic groups (2), and the 5-year survival rate in AfAms is much lower than other racial/ethnic groups, including Caucasian Americans (CaAm) (62% vs. 84%) (3, 4). Endometrioid-type endometrial carcinoma (EEC) accounts for about 75% of all endometrial carcinomas. Many patients with early-stage and low-grade EEC can be cured by surgery alone, but for women who present with higher-grade advanced-stage EEC, more aggressive therapy is needed. Studies have shown a close gene/environment interaction in endometrial cancer development and progression (5-7); however, why AfAm women have a higher mortality rate and why their incidence of endometrial cancer is increasing more rapidly remain unknown. To address this issue, we analyzed gene mutation profiles in AfAm and CaAm women with EEC, using samples and genomic data from The Cancer Genome Atlas (TCGA) including 397 patients with EEC (284 CaAm, 67 AfAm, and 46 other races) (8). We found that tumor suppressor gene, TP53, was the top differentially mutated gene that occurred more frequently in AfAm patients with EEC. This is consistent with our recent publication that the TP53 tumor suppressor is more frequently mutated in AfAm patients compared to CaAm patients when several cancer types (e.g., lung, colorectal, bladder, prostate and breast cancer) were analyzed together (9). Moreover, survival analysis of TCGA patients with EEC showed that patients with TP53 mutations have generally poorer outcomes than those with wild-type TP53. Furthermore, our pathway analysis using the RNA sequencing (RNA-Seq) data from TCGA showed that fatty acid metabolism pathway is upregulated in AfAm patients with EEC compared to their CaAm counterparts. Further analysis showed that TP53 mutations are also associated with downregulation of AMP-activated protein kinase (AMPK) and activation of mTOR (mammalian target of rapamycin) pathways, which can lead to dysregulation of fatty acid metabolism and promotion of tumorigenesis (10). Furthermore, obesity is a significant risk factor for EC (11, 12), consistent with our TCGA data analysis showing that AfAm patients with EEC were more likely to be obese than CaAm patients (39.1 vs. 34.6, mean BMI indexes) and with our observation in a cohort of 247 patients, including 47 AfAm and 196 CaAm patients with EEC (35.5 vs. 33.6, mean BMI indexes) from the Precision Oncology Initiative (POI) at Wake Forest Baptist Cancer Center (WFBCC). Of note, inhibition of AMPK activity also leads to obesity and type 2 diabetes (13). Thus, differential TP53 mutations emerge as a major genetic factor that contributes to cancer health disparity. Citation Format: Farideh Mehraein-Ghomi, Liang Liu, Umit Topaloglu, Karen Winkfield, Michael Kelly, Boris Pasche, Wei Zhang. Dysregulation of fatty acid metabolism by TP53 mutations underlies more aggressive endometrial cancers in African American women [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C002.

Accumulating evidence shows that androgen receptor (AR) activation and signaling plays a key role in growth and progression in all stages of prostate cancer, even under low androgen levels or in the absence of androgen in the castration-resistant prostate cancer. Sustained activation of AR under androgen-deprived conditions may be due to its interaction with co-activators, such as p52 NF-κB subunit, and/or an increase in its stability by phosphorylation that delays its degradation. Here we identified a specific inhibitor of AR/p52 interaction, AR/p52-02, via a high throughput screen based on the reconstitution of Gaussia Luciferase. We found that AR/p52-02 markedly inhibited growth of both castration-resistant C4-2 (IC50 ∼6 μM) and parental androgen-dependent LNCaP (IC50 ∼4 μM) human prostate cancer cells under low androgen conditions. Growth inhibition was associated with significantly reduced nuclear p52 levels and DNA binding activity, as well as decreased phosphorylation of AR at serine 81, increased AR ubiquitination, and decreased AR transcriptional activity as indicated by decreased prostate-specific antigen (PSA) mRNA levels in both cell lines. AR/p52-02 also caused a reduction in levels of p21(WAF/CIP1), which is a direct AR targeted gene in that its expression correlates with androgen stimulation and mitogenic proliferation in prostate cancer under physiologic levels of androgen, likely by disrupting the AR signaling axis. The reduced level of cyclinD1 reported previously for this compound may be due to the reduction in nuclear presence and activity of p52, which directly regulates cyclinD1 expression, as well as the reduction in p21(WAF/CIP1), since p21(WAF/CIP1) is reported to stabilize nuclear cyclinD1 in prostate cancer. Overall, the data suggest that specifically inhibiting the interaction of AR with p52 and blocking activity of p52 and pARser81 may be an effective means of reducing castration-resistant prostate cancer cell growth.

BACKGROUND Multiple studies show that reactive oxygen species (ROS) play a major role in prostate cancer (PCa) development and progression. Previously, we reported an induction of Spermidine/Spermine N1-Acetyl Transferase (SSAT) by androgen-activated androgen receptor (AR)-JunD protein complex that leads to over-production of ROS in PCa cells. In our current research, we identify small molecules that specifically block AR-JunD in this ROS-generating metabolic pathway.

Glioblastoma is one of the most lethal central nervous system tumors with the poorest prognosis. Fusion genes are common chromosomal aberrations in malignancies that can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our previous study has shown that F3T3, generated by a rearrangement of chromosome, led to the loss of the 3′-UTR of FGFR3, blocking epigenetic regulation of miR-99a and enhancing expression of the fusion gene. To further explore whether there is a post-translational regulation on F3T3 protein, we immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wild-type FGFR3, followed by LC-MS/MS analyses. Quantitative proteomic analysis suggest a stronger interactions of the F3T3 fusion protein with Hsp90 and Cdc37 proteins when compared with wild-type FGFR3. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 and LN229 cells constitutively expressing F3T3 with either Hsp90 inhibitors or siRNA of Cdc37. Targeting Hsp90-Cdc37 complex significantly suppressed the activation of F3T3 and downstream signaling pathways. Intriguingly, both Hsp90 and Cdc37 inhibition caused a degradation of glycosylation form of F3T3 protein. F3T3 was found highly expressed in both the untreated and matched recurrence glioblastoma, indicating a resistance to the concurrent radiotherapy and temozolomide (TMZ) treatment. Our results provide evidence that the F3T3 fusion gene confers drug resistance to TMZ induced DNA damage and using Hsp90 inhibitor chemosensitizes F3T3 glioma cells to TMZ. In conlusion, our findings establish that F3T3 is an strong Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex and suggests a novel strategy for targeting F3T3 fusion gene in glioma. Citation Format: Tao Li, Farideh Mehraein-Ghomi, Sanjeev V. Namjoshi, Qian Qian Song, Elizabeth A. Ballard, Mary E. Forbes, Ping-Chieh Chou, Xuejun Yang, Wei Zhang. Targeting Hsp90-Cdc37 complex in glioma harboring FGFR3-TACC3 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B036. doi:10.1158/1535-7163.TARG-19-B036

Glioblastoma is one of the most lethal malignancies with the poorest prognosis among tumors of the central nervous system. Fusion genes are common chromosomal aberrations in many cancers that may give rise to an in-frame fusion protein with oncogenic function. These fusion genes can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our studies have shown that glioblastoma cells harboring the F3T3 are resistant to the frontline glioblastoma drug Temozolomide (TMZ). Using the Comet assay, we show that TMZ-mediated DNA damage is repaired more rapidly in cells harboring the F3T3 compared to control. Our studies on the mechanism of this drug resistance show that the F3T3 confers an aberrant activation of FGFR and ERK pathways when treated with TMZ. To further explore druggable target(s) to overcome resistance caused by F3T3, we immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wildtype FGFR3, followed by LC-MS/MS analyses. Our quantitative proteomic analysis revealed interactions of the F3T3 fusion protein with Hsp90α and Cdc37 proteins. These interactions were further validated by reciprocal IPs followed by Western blotting. Activation of many kinases, such as FGFRs, depends on their interaction with the Hsp90 molecular chaperone system. Hsp90 recruitment is mediated by the co-chaperone adaptor protein Cdc37, which simultaneously binds to both the kinase and Hsp90. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 cells constitutively expressing F3T3 with the Hsp90 inhibitor Onalespib. We show that Onalespib treatment at 26 nM significantly suppresses the proliferation of F3T3 expressing U251 cells in comparison to micromolar levels of the pan-FGFR inhibitor BGJ398 and PD173074. Our results provide evidence that the F3T3 fusion gene contributes to drug resistance via multiple chemo-resistance pathways. Importantly, our findings establish that F3T3 is an Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex for cell growth and suggests a novel strategy for targeting F3T3 fusion gene in glioma. Citation Format: Tao Li, Farideh Mehraein-Ghomi, Sanjeev V. Namjoshi, Lynette M. Phillips, Elizabeth A. Ballard, Mary E. Forbes, ping-Chieh Chou, Xuejun Yang, Wei Zhang. Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene [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 1736.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 (SaCas9) mRNA in each lentivirus-like bionanoparticle (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9. Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.

Most prostate, colon and breast cancer cells are resistant to growth inhibitory effects of suberoylanilide hydroxamic acid (SAHA). We have examined whether the high oxidative stress in these cells causes a loss of SAHA activity and if so, whether pretreatment with an anti-oxidant can sensitize these cells to SAHA.A DNA-Hoechst dye fluorescence measured cell growth and dichlorfluorescein-diacetate (DCF-DA) dye fluorescence measured reactive oxygen species (ROS). Growth inhibitory and ROS-generating activities of SAHA in androgen-treated or untreated LNCaP cells and PC-3 prostate cancer cells, HT-29 and HCT-115 colon cancer cells, MDA-MB231 breast cancer cells and A549 and NCI-H460 lung cancer cells with or without pretreatment with an anti-oxidant Vitamin E was determined. SAHA activity against LNCaP cells treated with another anti-oxidant N-acetyl cysteine (NAC) was also determined. Liquid chromatography-mass spectrometry (LC-MS) was used to determine intracellular SAHA level.SAHA treatment markedly inhibits LNCaP cell growth, when the cells are at a low ROS level. SAHA is, however, inactive against the same cell line, when the cells are at a high ROS level. A significant decrease in SAHA level was observed in LNCaP cells with high ROS after 24- and 72-h treatment when compared to cells with low ROS. Vitamin E pretreatment that reduces cellular ROS, synergistically sensitizes oxidatively stressed LNCaP, PC-3, HT-29, HCT-115 and MDA-MB231 cells, but not the A-549 and NCI-H460 cells with low ROS to SAHA. NAC treatment also sensitized androgen-treated LNCaP cells to the growth inhibitory effects of SAHA.Response to SAHA could be improved by combining anti-oxidants such as Vitamin E with SAHA for the treatment of oxidatively stressed human malignancies that are otherwise resistant to SAHA.

BACKGROUND. Numerous and compelling evidence shows that high level of reactive oxygen species (ROS) plays a key role in prostate cancer occurrence, recurrence and progression. The molecular mechanism of ROS overproduction in the prostate gland, however, remains mostly unknown. Unique AP-1 transcription factor JunD has been shown to inhibit cell proliferation, promote differentiation and mediate stress responses in a variety of eukaryotic cells. We previously reported that androgen–androgen receptor induced ROS production in androgen-dependent LNCaP human prostate cancer cells is associated with increased JunD level/AP-1 transcriptional activity. METHODS. LNCaP cells constitutively overexpressing a functionally inactive form of JunD (JunDDTA) or stably transfected with JunD siRNA (siJunD) to suppress JunD protein expression were established. Overexpression of JunD in LNCaP cells using transient transfection method was applied to assess the induction of ROS production in LNCaP cells. DCF assay was used to measure the ROS concentrations in the transfected as well as nontransfected control cells. RT-PCR and Western blot analyses were used to confirm silencing or overexpression of JunD in the transfected cells. RESULTS. In the absence of androgen, LNCaP cells transiently transfected with a JunD overexpressing vector have relatively enhanced cellular ROS levels as compared to LNCaP cells transfected with a vector control. LNCaP cells that fail to express functional JunD (JunDDTA or siJunD) do not exhibit any increase in ROS production in response to androgen. CONCLUSION. Based on these data, we conclude that JunD is an essential mediator of the androgen-induced increase in ROS levels in LNCaP cells. Prostate 68: 924–934, 2008. # 2008 Wiley-Liss, Inc.

Purpose: The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation. Experimental Design: Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells. Results: SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-κB ligand expression was up-regulated in cells expressing PSA compared with controls. Conclusions: Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin–receptor activator of NF-κB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.

Stat3, a member of the signal transduction and activation of transcription (STAT) family, is a key signal transduction protein that mediates signaling by cytokines, peptide growth factors, and oncoproteins and is constitutively activated in numerous cancers including prostate. Previous studies demonstrated that constitutively activated Stat3 plays an important role in the development and progression of prostate cancer by promoting cell proliferation and protecting against apoptosis. The present study was designed to investigate the potential use of RNA interference to block Stat3 expression and activation and the effect on the growth of human prostate cancer cells. We identified a small interfering RNA (siRNA) specific for Stat3 and demonstrate that blockade of Stat3 activation by the Stat3 siRNA suppresses the growth of human prostate cancer cells and Stat3-mediated gene expression and induces apoptotic cell death. The Stat3 siRNA does not inhibit the proliferation nor induces apoptosis of Stat3-inactive human prostate cancer cells. In addition, the Stat3 siRNA inhibits the levels of androgen-regulated prostate specific antigen (PSA) expression in prostate cancer cells. These results demonstrate that targeting Stat3 signaling using siRNA technique may serve as a novel therapeutic strategy for treatment of prostate cancer expressing constitutively activated Stat3.

The goal of this research was to verify the specificity of the inhibition of androgen receptor (AR) ? NFkB/p52 interaction by small molecule AR/p52 inhibitors (selected from prior high throughput screen) in cell culture and AR/p52 activity assays, and determine the efficacy of the compounds against castration resistant prostate cancer (CRPCa) cell / xenograft growth. Data from this research should establish the AR-p52 interaction as a viable new target for preventing progression to CRPCa and identify lead compound(s) to be further developed for preclinical toxicity testing and clinical trials for PCa that fall beyond the scope of this proposal. Cell culture studies identified a lead compound with IC50 of 5 μM against both androgen-dependent and ?independent PCa cell growth that involves reduction in CyclinD1, and significant inhibition of AR transcriptional activity as measured by PSA mRNA as well as inhibition of AR and p52 translocation to nucleus. Efficacy of the lead compound in an oral regimen against CRPCa xenograft tumor development and progression was established. Thus the lead compound shows evidence of specificity for AR-p52 interaction and efficacy against CRPCa. These data help establish AR-p52 interaction as a viable new target for PCa therapy and identify a lead compound that can be further developed toward clinical application as a new therapeutic agent to prevent progression of CRPCa.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The relatively high oxidative stress in the prostate is postulated to be one of the major factors for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we have shown that the transcription factor JunD is essential for the androgen-induced reactive oxygen species (ROS) production in androgen-dependent LNCaP human prostate cancer cells. We have also recently demonstrated that androgen induces the first and the regulatory enzyme spermidine/spermine N1-acetyl transferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present co-immunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with activated AR in situ. Our chromatin immnoprecipitation (ChIP) assay data demonstrate that JunD binds directly with a specific SSAT promoter sequence only in androgen-treated LNCaP cells. Using a vector containing a luciferase reporter gene connected to the SSAT promoter and a JunD silenced LNCaP cell line, we show that JunD is an essential co-activator of androgen-induction of SSAT gene expression. As there is no Androgen Response Element (ARE) in the SSAT promoter, we hypothesize that JunD mediates binding of androgen-activated AR to the SSAT promoter for the induction of SSAT that leads to polyamine oxidation and ROS production in prostate cells. Elucidation of this pathway may help the design of inhibitors of oxidative stress generation specifically in prostate cells to prevent prostate cancer occurrence and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4386.

High levels of reactive oxygen species (ROS) present in human prostate epithelia are an important etiologic factor in prostate cancer (CaP) occurrence, recurrence, and progression. Androgen induces ROS production in the prostate by a yet unknown mechanism. Here, to the best of our knowledge, we report for the first time that androgen induces an overexpression of spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in the polyamine oxidation pathway. As prostatic epithelia produce a large excess of polyamines, the androgen-induced polyamine oxidation that produces H2O2 could be a major reason for the high ROS levels in the prostate epithelia. A small molecule polyamine oxidase inhibitor N,N'-butanedienyl butanediamine (MDL 72,527 or CPC-200) effectively blocks androgen-induced ROS production in human CaP cells, as well as significantly delays CaP progression and death in animals developing spontaneous CaP. These data show that polyamine oxidation is not only a major pathway for ROS production in prostate, but inhibiting this pathway also successfully delays CaP progression. [Cancer Res 2009;69(19):7689–95]

Purpose: Anti-androgens are widely used in androgen deprivation therapy (ADT), a standard-of-care for patients with recurrent prostate cancer (PCa). Unfortunately, most patients ultimately develop resistance to ADT and progress to castrate-resistant prostate cancer (CRPC). Recently, two agents that block androgen receptor activation [abiraterone acetate (Zytiga, J&J) and enzalutamide (Xtandi, Medivation/Astellas)] have been approved for CRPC therapy. While some patients respond to these therapies, many fail. We investigated if sequestration of metabolically aberrant mitochondria in the autophagosomes (mitophagy) imparts anti-androgen resistance. Method: We studied the effects of anti-androgen enzalutamide on the autophagy of androgen-dependent LNCaP and -independent C4-2 cells. Autophagy was monitored by cellular fluorescence in cells treated with monodansylcadavarine (MDC). Cellular fluorescence due to Mitosox dye oxidation was used to identify mitochondria producing high superoxide (O2-). Mitophagy of O2- producing mitochondria was monitored using fluorescence resonance energy transfer (FRET) between MDC and Mitosox in a 96-well plate based high throughput (HTS) assay and by visualization of FRET images and quantitation of FRET image intensities using a Nikon A1 fluorescence confocal microscope and associated software. Results: Our data show that in low androgen media, the degree of autophagy is less in C4-2 cells than in LNCaP cells. Enzalutamide treatment induces autophagy in both cell lines, but the increase in autophagy is more pronounced in the androgen-independent C4-2 than in the -dependent LNCaP cells. FRET data from both HTS and fluorescence microscopy show that while enzalutamide increases mitophagy of O2- producing mitochondria in C4-2 cells in a dose dependent manner, it decreases after an initial increase in LNCaP cells. Mitophagy of such mitochondria has also been observed by FRET based fluorescence microscopy in live circulating tumor cells (CTCs) isolated from blood sample of a patient undergoing enzalutamide therapy. Conclusion: Our data demonstrate that PCa cells resistant to enzalutamide show high degree of mitophagy of O2- mitochondria in autophagosomes. If this effect correlates with CTCs from blood collected from CRPC patients undergoing enzalutamide therapy, it can become a clinically useful method of identifying patients who are most likely to benefit from enzalutamide treatment. Citation Format: Hirak S. Basu, Cynthia L. Schrieber, Jamie M. Sperger, Maryanne Naundorf, Ashley M. Weichman, Farideh Mehraein-Ghomi, Dawn R. Church, Joshua M. Lang, George Wilding. Mitophagy imparts enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2015-2899

Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells. ROS play a major role in the development and progression of PCa. We have published that the activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT). SSAT is the first, and a regulatory, enzyme in a major polyamine catabolism pathway that leads to an over-production of ROS, specifically in the polyamine-rich PCa cells. A focus of our current research is to identify compounds that specifically target and block steps in this newly discovered ROS production pathway downstream to AR activation. Such compounds have the potential to become new targeted therapeutic agents offering minimal side effects for preventing progression to CRPCa in patients with early stage progressing PCa. Here we present data on drug-like small molecule inhibitors of the AR-JunD interaction that initiates this ROS-generating pathway in PCa. A novel high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen NCI Diversity Set and Life Chemicals libraries of drug-like small molecules to identify inhibitors of the AR-JunD interaction. The 27 selected hits were categorized as antiandrogens or non-antiandrogens through the use of a published fluorescence polarization assay. Sixteen non-antiandrogens, acting downstream of AR activation, were chosen for further study. In vitro studies entailed the measurement of the ability of the compounds to inhibit androgen-independent and dependent growth as well as androgen-induced ROS in human PCa cells. The top four compounds exhibited significant inhibition of growth and androgen-induced ROS at concentrations of 5 uM or less. Immunocytochemistry (ICC) studies were performed to determine the ability of the selected compounds to specifically inhibit the AR-JunD co-translocation to the nuclei. Based on the cell culture and ICC data, lead compounds were selected for pharmacokinetics studies to determine oral bioavailability. Detailed pharmacokinetic data of selected top compounds showing their bioavailability after intra venous and oral administration will be presented. 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 2844. doi:1538-7445.AM2012-2844

Development of an effective therapy to prevent PCa occurrence, recurrence and/or progression remains an unmet medical need. This is mainly due to a poor understanding of the mechanism of PCa occurrence and progression. There is accumulating evidence that ROS (Reactive Oxygen Species) such as hydrogen peroxide, superoxide, hydroxyl radical, etc. that are produced in high amounts in PCa cells may be a major factor in PCa occurrence and its progression to androgen-independence. One of the ROS (H2O2) plays a key role in triggering growth signals and expression of transcription factors to sustain growth of androgen-dependent PCa cells in the absence of androgen. One such factor is Nuclear Factor κB (NF-κB) that may sustain cell survival by preventing apoptosis and inducing cell proliferation. NF-κB activation kinetically correlates with androgen-induced ROS production in cultured LNCaP human PCa cells. This activation is also seen in resected PCa tissues from patients, who subsequently progressed to CRPCa. Our published data show that androgen induces an up-regulation of spermidine/spermine acetyl transferase (SSAT) mRNA as well as enzyme activity that initiates polyamine oxidation in LNCaP cells after 72 h exposure. Because of the unusually high polyamine levels in the PCa cells, polyamine oxidation causes a large increase in ROS production that is abrogated by silencing SSAT expression in PCa cells stably transfected with shRNA against SSAT (siSSAT). To determine whether SSAT is required for NF-κB activation following prolonged androgen stimulus, we transfected 3xκB-luciferase vector containing NF-κB consensus binding sites-luciferase reporter construct along with a β-gal expression vector (to control for the transfection efficiency) into both wild type LNCaP cells and its siSSAT clones. While 72 h androgen exposure causes an over 30-fold induction of luciferase activity in wild type LNCaP cells, no activation of NF-κB was observed in the siSSAT clones. These data clearly show a strong SSAT induction is required for a robust activation of NF-κB in androgen-treated PCa cells. Multiple published reports show that NF-κB can also induce SSAT in several human cancer cells. Thus, our data suggest an intriguing mechanism of CRPCa progression, where androgen induced SSAT expression and consequent upregulation of NF-κB can set up an autocrine feed forward loop of SSAT-ROS-NFκB-SSAT that can sustain PCa cell proliferation in the absence of androgen that can lead to CRPCa growth. 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 2165. doi:10.1158/1538-7445.AM2011-2165

Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells and play a major role in PCa development and progression. We have published that activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT), the first and regulatory enzyme in a major polyamine catabolism pathway that yields over-production of ROS specifically in the polyamine-rich PCa cells. Our recent data further suggest an intriguing mechanism of PCa progression, where AR-JunD induced SSAT expression and consequent upregulation of the transcription factor NF-κB may set up an autocrine feed forward loop of SSAT-ROS-NFκB-SSAT that can sustain ROS production and PCa cell proliferation in the absence of androgen. A focus of our current research is to identify compounds that specifically target and block steps in this novel pathway downstream to AR activation and thereby have potential to be new targeted therapeutic agents to prevent PCa progression to CRPCa in early stage progressing PCa patients with minimal side effects. In the research presented here we utilized a novel high throughput screen (HTS) assay to find compounds that prevent the initiating AR-JunD interaction step in this ROS generating pathway and tested their efficacy in PCa cells. A high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen the NCI diversity set library of drug like molecules to identify inhibitors of the AR-JunD interaction. Selected hits were further screened to determine their ability to bind to the AR using a published fluorescence polarization assay in order to categorize the molecules as non-antiandrogens or antiandrogens. As we intend to target the pathway downstream of AR activation, we focused on non-antiandrogens, further testing them for efficacy against androgen induced ROS generation and effect on cell growth in PCa cells in vitro by our published DCFH dye oxidation and DNA fluorescence assays. Of the 14 hits from the HTS assay of the NCI diversity set library, nine small molecules were chosen based on their drug-like chemical characteristics for further studies along with two synthesized analogs of one of the selected small molecules. We have categorized six agents as non-antiandrogens and five as having some antiandrogenic activity. Our ROS assay identified a lead non-antiandrogen compound that blocks androgen induced ROS production in PCA cells at less than 1uM concentration. This agent also significantly inhibited androgen-independent growth of PCa cells at 4uM. Data for all compounds will be presented. 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 2583. doi:10.1158/1538-7445.AM2011-2583

The relatively high oxidative stress in the prostate is postulated to be one of the major factors for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we have shown that the transcription factor JunD is essential for the androgen-induced reactive oxygen species (ROS) production in androgen-dependent LNCaP human prostate cancer cells. We have also recently demonstrated that androgen induces the first and the regulatory enzyme spermidine/spermine N1-acetyl transferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present co-immunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with activated AR in situ. Our chromatin immnoprecipitation (ChIP) assay data demonstrate that JunD binds directly with a specific SSAT promoter sequence only in androgen-treated LNCaP cells. Using a vector containing a luciferase reporter gene connected to the SSAT promoter and a JunD silenced LNCaP cell line, we show that JunD is an essential co-activator of androgen-induction of SSAT gene expression. As there is no Androgen Response Element (ARE) in the SSAT promoter, we hypothesize that JunD mediates binding of androgen-activated AR to the SSAT promoter for the induction of SSAT that leads to polyamine oxidation and ROS production in prostate cells. Elucidation of this pathway may help the design of inhibitors of oxidative stress generation specifically in prostate cells to prevent prostate cancer occurrence and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4386.