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Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, neuroendocrine differentiated (NED) metastatic PCa, which lacks the PSMA antigen, is deprived of effective biomarkers for disease treatment. Magmas, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. Magmas is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, Magmas plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation, but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of Magmas in PCa cells expressing NED markers. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of Magmas compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX- resistant metastatic PCa cell lines and re-sensitize them to DTX. Magmas knockdown in DTX-sensitive and DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of Magmas with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using clonogenic assays, cell migration/invasion assays, and tumorsphere formation assays are currently underway to further establish Magmas as a potential therapeutic target to attenuate the aggressive properties of chemoresistant PCa cells. Lastly, we will investigate the diagnostic potential of Magmas as a biomarker for NED and BT#9 as an imaging/therapeutic-specific agent for NED. Citation Format: Alfonso M. Durán, Christian H. Yoo, Jennifer D. Tran, Krystal Santiago, Adil S. Mohammed, Carlos A. Casiano, Frankis A. Almaguel. Targeting the mitochondrial protein MAGMAS increases sensitivity to docetaxel in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1754.

The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. Interestingly, CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF-1). High serum levels of IGF-1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF-1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. This study is designated to specifically determine and characterize the molecular interaction between CYR61 and IGF-1 in metastatic PCa and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3 and downregulated in the docetaxel-resistant cell line PC3-DR. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF-1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. We also initiated studies to determine the co-localization of CYR61 and IGF-1 using immunofluorescence microscopy (IF). Based on the initial IF observations, we will proceed to examine if their interaction is primarily intra- or extra-cellular. For intracellular interactions, we will co-immunoprecipitate CYR61 with IGF-1 and for extracellular interactions, we will use the AVidity-based EXtracellular Interaction Screen (AVEXIS) system. Our goal is to establish the contribution of CYR61-IGF1 protein-protein interaction to PCa cell proliferation. Specifically, the correlation between PCa aggressiveness and circulating levels of CYR61 and IGF-1 is poorly studied. Their combined role may explain a proportion of aggressive PCa. Citation Format: Greisha L. Ortiz-Hernandez, Mya Walker, Leanne Woods-Burnham, Rick A. Kittles, Carlos A. Casiano, Susan L. Neuhausen. CYR61, a member of the CCN protein family, regulates IGF-1 in metastatic prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3949.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of leukemia in children. B-ALL characterized by cytokine receptor-like factor 2 (CRLF2) overexpression (CRLF2 B-ALL) has a survival rate of Citation Format: Hossam Alkashgari, Cornelia Stoian, Caleb Ruiz-Jimenez, Jacqueline Coats, Carlos A. Casiano, Sinisa Dovat, Kimberly J. Payne. Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia [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 1528.

Prostate cancer (PCa) is the second leading cause of cancer deaths in the U.S., disproportionally affecting African American (AA) men. Glucocorticoids (GCs) are administered to PCa patients and have been implicated in therapy resistance. This may be critical to AA men with PCa since they have elevated endogenous GCs levels compared to Caucasian American (CA) men. GCs bind to the glucocorticoid receptor (GR) to exert their actions. The mechanisms of GR-mediated chemoresistance, and its possible contribution to PCa mortality disparities are unknown. We demonstrated that GCs upregulate the chemoresistance-associated protein and transcription co-activator LEDGF/p75 in PCa cells and identified consensus GR binding sites in the promoter region of this protein. Given that both GR and LEDGF/p75 are components of the RNA polymerase II transcription complex, we hypothesized that GR transcriptionally upregulates LEDGF/p75 and then interacts with it to enhance taxane resistance in PCa cells. Pharmacological and genetic inhibition of GR in a panel of docetaxel (DTX)-sensitive and -resistant PCa cells decreased the expression of LEDGF/p75, confirming its status as a candidate GR target gene. However, silencing of LEDGF/p75 had no effects on GR expression. Immunoprecipitation studies revealed that GR and LEDGF/p75 interact in DTX-resistant PCa cells. This interaction was confirmed by confocal microscopy. Immunohistochemical analysis of GR and LEDGF/p75 expression in normal and tumor prostate tissues was performed, and the results are currently being analyzed. Our studies use a mechanistic approach to evaluate the potential contribution of the GR-LEDGF/p75 axis to PCa chemoresistance. Evaluating the co-expression of these proteins in racially diverse PCa tissues may also reveal race-related differential expression, providing insights into the potential contribution of this axis to PCa chemoresistance and mortality disparities. Citation Format: Evelyn S. Sanchez Hernandez, Greisha L. Ortiz Hernandez, Pedro T. Ochoa, Christian R. Gomez, Carlos A. Casiano. Contribution of the GR/LEDGFp75 axis to prostate cancer chemoresistance [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 2348.

Aberrant lipid metabolism in prostate tumors is linked to aggressive disease and poor outcomes. The purpose of this study was to identify mechanisms by which lipid metabolism in prostate cancer cells influences the tumor immune microenvironment, with emphasis on the activation of macrophages. Our hypothesis that fatty acid synthase-driven de novo lipogenesis promotes immune evasion via alternative M2-like macrophage activation was tested using human monocyte (U937)-derived macrophages co-cultured with prostate cancer cells in the presence of fatty acid synthase inhibitors. Monocytes were differentiated to macrophages in vitro using phorbol-12-myristate-13-acetate for 48 hours prior to co-culture with prostate cancer cells. Macrophage polarization following co-culture with prostate cancer cells was evaluated using multi-parameter flow cytometry to detect established M1 (CD80, CD86) and M2 (CD163, CD206) markers. Furthermore, the expression of fatty acid synthase and pan-macrophage marker CD68 in human prostate tumor tissue microarrays was evaluated using multiplex immunohistochemistry. The lipogenic enzyme and macrophage staining correlated with disease progression, reaching peak levels in metastatic tissues. These findings indicate that tumoral fatty acid synthase is an important mediator of tumor-immune crosstalk, particularly in the context of aggressive and metastatic prostate cancer. These data further suggest that fatty acid synthesis represents a targetable complement to immune-enhancing therapies by modulating tumor cell metabolism, underscoring the need for further evaluation of agents targeting lipid metabolism in prostate cancer. Citation Format: Shannalee R. Martinez, Carla Barrientos Risso, Ralphdy Vergne, Nathan R. Wall, Julie Dutil, Carlos A. Casiano, Gilberto Ruiz-Deya, Carlos Joel Diaz Osterman. Tumor lipogenesis influences macrophage polarization in advanced prostate cancer [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 2559.

The efficacy of anti-androgen receptor (AR) therapies and taxane-based chemotherapy for the treatment of advanced prostate cancer (PCa) is hindered by the development of therapy resistance. Our previous studies demonstrated that docetaxel (DTX)-resistant PCa cells activates a transcriptomic program associated with stemness and display cancer stem cell (CSC) properties, consistent with the notion that CSCs within prostate tumors contribute to chemoresistance. Glucocorticoid receptor (GR) signaling is a critical mediator of resistance to AR-targeted therapy, and has been recently implicated in the development of PCa cell tumorsphere formation and chemoresistance. Like GR, β-catenin is overexpressed in metastatic and therapy-resistant tumors, and is considered a key regulator of cancer stemness and androgen-targeted therapy resistance. Given the structural and functional overlap between GR and AR, a known interacting partner of β-catenin, we hypothesized that GR also interacts with β-catenin to support stemness and chemoresistance in PCa cells. We observed that treatment with the glucocorticoid dexamethasone induces enhanced nuclear accumulation of both GR and β-catenin in DTX-resistant PCa cells compared to their DTX-sensitive counterparts. Knockdown studies revealed that GR and β-catenin do no regulate each other at the protein level. However, using whole cell and nuclear co-immunoprecipitation, we demonstrated the interaction between GR and β-catenin in both DTX-resistant and DTX-sensitive PC3, DU145, and 22RV1 cells. Pharmacological inhibition and RNA interference-mediated silencing of GR with concomitant inhibition of β-catenin enhanced DTX cytotoxicity in resistant PCa cells grown in both adherent and spheroid cultures, and decreased CD44+/CD24- cell populations in tumorspheres. These results indicate that both GR and β-catenin interact and influence stemness, and may be promising therapeutic targets to overcome PCa chemoresistance. Citation Format: Shannalee R. Martinez, Catherine C. Elix, Pedro T. Ochoa, Evelyn S. Sanchez-Hernandez, Hossam R. Alkashgari, Greisha L. Ortiz-Hernandez, Lubo Zhang, Carlos A. Casiano. The glucocorticoid receptor and β-catenin interact in prostate cancer cells and their co-inhibition attenuates stemness and docetaxel resistance [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 399.

Prostate Cancer (PCa) progression leads to an advanced stage called metastatic castration PCa (mCRPC), for which currently there is no cure in spite of advances in treatment with new generation anti-androgen drugs and chemotherapy with taxanes such as docetaxel (DTX). Our group demonstrated previously that the stress oncoprotein Lens Epithelium Derived Growth Factor of 75 kD (LEDGF/p75) is upregulated in clinical prostate tumors and contributes to DTX-resistance in mCRPC cells. However, little is known about the molecular mechanisms by which LEDGF/p75 promotes taxane resistance. The C-terminus of LEDGF/p75 contains a domain called the Integrase Binding Domain (IBD), which in T cells is responsible for tethering the HIV-integrase complex to transcriptionally active chromatin. In non-PCa cells, the LEDGF/p75 IBD interacts with transcription complexes, such as Menin-MLL and the c-MYC binding protein JPO2, to promote cell survival. However, the relevance of these and others protein-protein interactions (PPIs) in the context of PCa chemoresistance has never been explored. This study was designed to test the hypothesis that interactions between LEDGF/p75, the c-MYC binding protein JPO2, Menin and MLL in DTX-resistant mCRPC cells contribute to chemoresistance. We demonstrated by immunoblotting a significant co-upregulation of JPO2, c-MYC, Menin, MLL, and LEDGF/p75 in the DTX-resistant PC3-DR and DU145-DR cell lines, compared to the parental, drug sensitive cells. We also observed nuclear co-localization of these proteins by immunofluorescence microscopy and confocal microscopy. Using an immunoprecipitation approach, we confirmed that endogenous JPO2, c-MYC, Menin, and MLL co-immunoprecipitate with LEDGF/p75 in the DTX-resistant PCa cell lines. We are extending these studies to other known interacting partners of LEDGF/p75 to determine if they are also upregulated in DTX-resistant PCa cells and interact with this protein in the chemoresistance context. In addition, we have initiated studies to evaluate the effects of pharmacologic and genetic inhibition of LEDGF/p75 and its interacting partners on sensitization of DR cells to DTX. We predict that targeting LEDGF/p75 PPIs will disrupt transcriptional activity contributing to DTX resistance. Our goals are to establish the contribution of these PPIs to LEDGF/p75-mediated transactivation of DTX resistance-associated genes, and target these interactions to overcome PCa chemoresistance. Citation Format: Greisha L. Ortiz Hernandez, Evelyn S. Sanchez-Hernández, Carlos A. Casiano. Interaction of LEDGF/p75 with oncogenic transcription factors in docetaxel resistant prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4688.

Prostate Cancer (PCa) progression leads to an advanced stage called metastatic castration PCa (mCRPC), for which currently there is no cure in spite of advances in treatment with new generation androgen deprivation drugs and chemotherapy with taxanes such as docetaxel (DTX). Our group demonstrated previously that the stress oncoprotein Lens Epithelium Derived Growth Factor of 75 kD (LEDGF/p75) is upregulated in clinical prostate tumors and contributes to DTX-resistance in mCRPC cells. However, little is known about the molecular mechanisms by which LEDGF/p75 promotes taxane resistance. The C-terminus of LEDGF/p75 contains a domain called the Integrase Binding Domain (IBD), which in T cells is responsible for tethering the HIV-integrase complex to transcriptionally active chromatin. In cancer cells, the LEDGF/p75 IBD interacts with oncogenic transcription complexes, such as Menin-MLL and the cMYC binding protein JPO2, to promote cell survival. However, the relevance of these protein-protein interactions (PPIs) in PCa and chemoresistance has never been explored. This study is designed to characterize the interaction between LEDGF/p75 and JPO2 in the DTX-resistant mCRPC cell lines PC3-DR and DU145-DR, and determine if this interaction contributes to drug resistance. Also, we want to target this interaction with repurposed HIV-based small molecule inhibitors (SMI’s) of LEDGF/p75, which target the IBD, to abrogate this resistance. We demonstrated by immunoblotting a significant 2-fold co-upregulation of JPO2 and LEDGF/p75 in the DTX-resistant PCa cells. We also observed the nuclear co-localization of these proteins by immunofluorescence microscopy. Using an immunoprecipitation approach, we confirmed that endogenous JPO2 and LEDGF/p75 co-immunoprecipitate in the DTX-resistant PCa cells. In addition, we initiated studies to evaluate SMIs originally designed to target the interaction between LEDGF/p75 and HIV-IN, for their efficacy in sensitizing resistant cells to DTX. After screening over 100 candidate inhibitors, we selected a set of SMIs which demonstrated a 20-30% decrease in viability in DTX-resistant cells when used alone, and 40-60% when used in combination with DTX. Studies are in progress to determine if these SMIs bind to LEDGF/p75 and inhibit its interaction with JPO2 and other oncoproteins. We predict that targeting LEDGF/p75 PPIs with HVI-based SMIs will disrupt transcriptional activity contributing to DTX resistance. Our goals are to establish the contribution of PPIs to LEDGF/p75-mediated transactivation of stress oncoproteins, and target these interactions to overcome PCa chemoresistance. Citation Format: Greisha L. Ortiz Hernandez, Shannalee Martinez, Evelyn Sanchez-Hernandez, Leslimar Rios-Colon, Christina Cajigas-DuRoss, Tino Sanchez, Nouri Neamati, Carlos A. Casiano. The cMYC-associated transcription factor JPO2 is upregulated in taxane resistant prostate cancer cells and interacts with the stress oncoprotein LEDGF/p75 [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 4395.

Prostate cancer (PCa) is the second leading cause of cancer deaths in U.S. men, disproportionately affecting African American men and certain Hispanic/Latino populations. Patients with advanced PCa are treated with androgen deprivation therapy (ADT) and docetaxel (DTX)-based chemotherapy with effective but transient results due to therapy resistance. While the role of glucocorticoid receptor (GR) in promoting ADT resistance has been well characterized, comparatively little is known about its contribution to PCa stemness and chemoresistance. Our group demonstrated recently that DTX-resistance is associated with upregulation of a stemness transcriptomic program and enhanced cancer stem cell (CSC) properties in PCa cells. We also demonstrated that GR signaling upregulates the DTX-resistance associated proteins Clusterin and LEDGF/p75 in a panel of PCa cells. The proto-oncogene β-catenin promotes CSC survival in many cancers, including PCa, and several studies have demonstrated a crosstalk between AR and β-catenin signaling pathways contributing to ADT resistance. Given the structural similarities between AR and GR and the importance of GR in therapy resistance, this study was designed to investigate the interaction between GR and β-catenin and its contribution to CSC survival and DTX resistance. Using a panel of PCa cell lines including PC3, DU145, 22rv1, and their corresponding DTX-resistant cell lines, we showed by quantitative immunoblotting that GR is upregulated and β-catenin is activated in PC3 and 22rv1 DTX-resistant cells compared to sensitive cells. Co-immunoprecipitation studies revealed interaction between endogenous GR and β-catenin in these cells. We next assessed the effect of the potent GR ligand dexamethasone (Dex) on CSC maintenance, and observed that Dex treatment of PC3 cells increased tumorsphere number and expression of established CSC markers measured by multicolor flow cytometry. To identify potential GR target genes that could mediate the acquisition of CSC properties and DTX resistance, we performed RNA-sequencing analysis in four different Dex-treated and -untreated PCa cell lines. This analysis revealed several candidate GR target genes differentially regulated in a cell-type dependent manner. To determine the effects of the GR/β-catenin interaction on downstream GR target genes and β-catenin target genes, we performed immunoblotting of specific candidate target genes on lysates from cells treated with Dex alone or in combination with COR-10829 (selective GR inhibitor, and MSAB (β-catenin inhibitor). Interestingly, we also observed that the dual blockade of GR and β-catenin markedly reduced tumorsphere formation in DTX-resistant cells, a phenomenon not observed with either GR or β-catenin inhibition alone. In summary, our findings revealed a novel interaction between GR and β-catenin in PCa cells, and suggest that co-targeting the GR/β-catenin signaling axis in advanced PCa patients could be a potential strategy to overcome therapy resistance. Citation Format: Shannalee R. Martinez, Carlos J. Diaz Osterman, Xin S. Chen, Charles Wang, Lubo Zhang, Carlos A. Casiano. Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance [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 LB-260.

There is no cure for metastatic prostate cancer (PCa), the most commonly diagnosed cancer and third leading cause of cancer mortality among U.S. men. Patients with advanced PCa often develop resistance to conventional therapies resulting in metastatic castration-resistant PCa (mCRPC). Docetaxel (DTX) chemotherapy is the standard-of-care for mCRPC, followed by secondary chemotherapy with cabazitaxel (CBZ), yet chemoresistance and death inevitably occurs. While pharmacological agents targeting single survival pathways have shown promise at the bench side, clinical trials have failed. This is mostly due to the presence of redundant pathways utilized by PCa tumor cells to maintain chemoresistance. Therefore, we are interested in further defining molecular pathways associated with DTX-resistance in order to identify novel therapeutic targets to overcome this resistance. We hypothesized that RNA sequencing (RNAseq) analysis comparing the transcriptomes of chemosensitive and chemoresistant mCRPC cells will reveal potential targets for combinatorial therapies. RNAseq analysis of DTX-sensitive and -resistant PC3 and DU145 cells revealed a number of differentially regulated genes that were either unique to each cell line or common to both. We found that dipeptidyl peptidase 4 (DPP4), fatty acid binding protein 5 (FABP5), nestin (NES), and tetraspanin 8 (TSPAN8) were among the top most robustly and significantly upregulated genes in both DTX-resistant PC3 and DU145 cell lines. In-house qPCR studies validated the RNAseq results for these genes, and Western blotting analysis showed increased expression of these proteins in DTX-resistant cells, which correlated with increased mRNA expression. Interestingly, NES, TSPAN8, DPP4 are genes associated with development of the cancer stem-cell (CSC) phenotype. Other genes associated with CSCs were also upregulated in the DTX-resistant cells, suggesting that in vitro selection of mCRPC cells for DTX resistance may enrich for CSCs. This novel finding is consistent with recent studies implicating CSCs in PCa chemoresistance. Further analysis and validation of our RNAseq data will increase our understanding of DTX-resistance mechanisms in PCa cells, including the yet undefined functional role of CSC-related genes. These studies also reveal potential therapeutic targets to circumvent DTX resistance in PCa. Citation Format: Christina K. Cajigas-Du Ross, Leanne Woods-Burnham, Joshua Ramirez, Xin Chen, Charles Wang, Carlos A. Casiano. RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5902.

Prostate cancer (PCa) is more aggressive in African American (AA) men compared to European American (EA) men, resulting in increased incidence and mortality disparities. There is a critical need to identify biological determinants contributing to these disparities. Glucocorticoids—a type of stress hormone—have recently been implicated as biological factors driving PCa progression and response to therapy. The underlying mechanism involves endogenous glucocorticoid binding to its receptor (GR), prompting GR to transcriptionally activate genes that are typically androgen-regulated and that promote tumor aggressiveness and therapy resistance. However, a clinical dilemma exists as glucocorticoids, necessary in the palliative care of PCa patients, are now emerging as accelerators of disease progression. In addition, AA men have chronically elevated levels of glucocorticoids linked to stressful life events, and are also hypersensitive to glucocorticoid exposure. We examined the effects of glucocorticoids on the activation of the stress oncoproteins LEDGF/p75 (Lens Epithelium Derived Growth Factor of 75kd) and CLU (Clusterin), linked by our group and others to prostate tumor aggressivenes and taxane resistance. Our goal is to establish the interplay between glucocorticoids and the expression of these proteins in the context of PCa health disparities. We hypothesized that LEDGF/p75 and CLU may be induced by glucocorticoids through GR in PCa cells. Treatment of a racially diverse panel PCa cell lines (MDA-PCa-2b [AA], 22Rv1 [AA/EA], PC3 [EA], and DU145 [EA]) with 10nM cortisol or dexamethasone for up to 48h significantly upregulated both transcript and protein levels of LEDGF/p75 and CLU. This upregulation was more robust in the AA cell lines. Depletion of GR with siRNAs abrogated dexamethasone-induced upregulation of LEDGF/p75 and CLU. Pharmacological inhibition of GR with mifepristone, in the presence of dexamethasone, also abrogated the induction of these proteins in a cell type-dependent manner. We also observed that dexamethasone exposure increased cell migration, a characteristic of tumor aggressiveness. Furthermore, using an ELISA platform we detected increased serum levels of LEDGF/p75 in AA PCa patients, as well as increased levels of CLU in non-PCa AA men, compared to EA patients and controls. These findings show that glucocorticoids induce the expression of oncoproteins associated with PCa aggressiveness and therapy resistance, and this upregulation may occur more robustly in AA men, priming them to develop highly aggressive PCa tumors. Citation Format: Leanne Woods-Burnham, Christina K. Cajigas-Du Ross, Anamika Basu, Evelyn S. Sanchez, Arthur Love, Carlos A. Casiano. Glucocorticoids trigger GR-mediated expression of the chemoresistance-associated oncoproteins LEDGF/p75 and Clusterin: Implications for prostate cancer health disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5043.

Prostate cancer (PCa) is the most commonly diagnosed male cancer and the second leading cause of cancer deaths in U.S. men. Late-stage PCa is characterized by metastasis followed by castration resistant PCa (mCRPC), for which there is no cure. Docetaxel (DTX) is the first-line chemotherapeutic drug for late-stage PCa, and resistance to this drug is inevitable. Our group has previously reported the elevated expression of the stress oncoprotein lens epithelium-derived growth factor (LEDGF/p75) in PCa cells and tissues, and this upregulation promotes resistance to DTX-induced non-apoptotic lysosomal cell death. Another stress oncoprotein, clusterin (CLU), is also upregulated in DTX-resistant PCa, and protects cells from DTX-induced mitochondrial-triggered apoptosis. In this study we observed that targeting LEDGF/75 with siRNAs partially resensitized DTX-resistant (DR) mCRPC cell lines to DTX treatment in clonogenic assays. Similarly, other groups have shown that knockdown of CLU in mCRPC-DR cells resensitizes them to treatment with taxanes. However, recent clinical trials targeting CLU have not been effective, suggesting that blocking this protein alone is insufficient to overcome DTX resistance. We hypothesized that simultaneous knockdown or inhibition of both LEDGF/p75 and CLU could be more effective in resensitizing chemoresisant PCa cells to taxane therapy than targeting each protein alone. Because of LEDGF/p75’s role as a stress transcription co-activator, we first sought to determine if this protein contributes to CLU expression by performing siRNA-mediated knockdown of LEDGF/p75 on the taxane-sensitive and -resistant mCRPC cell lines PC3 and DU145. Western blotting revealed that CLU protein expression was not downregulated by LEDGF/p75 knockdown, compared to treatment with scrambled siRNA control. Similarly, CLU knockdown in these cell lines did not downregulate LEDGF/p75. These results indicated that CLU expression is independent of LEDGF/p75, consistent with previous observations that these two proteins promote chemoresistance via distinct, possibly redundant, mechanisms. We then performed siRNA-mediated knockdown of LEDGF/p75 and CLU, separately or together, in PC3 DR and DU145 DR cells, in the presence and absence of varying concentrations of DTX. MTT and clonogenic assays were used to determine cell death viability and clonogenicity, respectively. Simultaneous knockdown of LEDGF/p75 and CLU, in combination with DTX treatment, resulted in increased sensitization to DTX than single knockdown of each protein. These findings suggest that co-targeting LEDGF/p75 and CLU could be an effective strategy for ameliorating chemoresistance in mCRPC, and set the foundation for combinatorial therapies targeting multiple stress pathways linked to taxane resistance together with taxane chemotherapy for treatment of late-stage PCa. Citation Format: Christina K. Cajigas-Du Ross, Leslimar Rios-Colon, Leanne Woods-Burnham, Carlos A. Casiano. Co-targeting the stress survival proteins LEDGF/p75 and clusterin to resensitize chemoresistant prostate cancer cells to docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4167. doi:10.1158/1538-7445.AM2017-4167

Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of cancer-related deaths in American men. African American (AA) men are more likely to be diagnosed with aggressive PCa at a younger age and twice as likely to die from the disease as European American (EA) men. In order to reduce PCa mortality and its associated racial disparities, there is a critical need to identify and target pathways responsible for PCa aggressiveness. An unexplored target is the plasminogen system, which is key to cancer cell migration and tissue invasion during metastasis. During inflammation, activation of the plasminogen pathway degrades extracellular fibrin networks; however, in the context of cancer, this activation promotes cancer tissue invasion and metastasis. Mounting evidence shows that the glycolytic enzyme alpha-enolase (ENO1) plays a vital role in both increased energy metabolism and plasminogen activation during cancer progression and metastasis. Using immunoseroproteomic profiling of anti-tumor autoantibody responses in AA and EA men with PCa, we identified several tumor-associated autoantigens with functions in glycolysis and plasminogen signaling, including ENO1, annexin A2, fructose bisphosphate aldolase, glucose-regulated protein 78, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In a cohort of PCa (N=157) and non-PCa sera (N=183), we found a significantly higher frequency of autoantibodies to ENO1 in patients with PCa (p Citation Format: Tino Wilson Sanchez, Guangyu Zhang, Jitian Li, Liping Dai, Saied Mirshahidi, Nathan R. Wall, Clayton Yates, Colwick Wilson, Susanne Montgomery, Jian-Ying Zhang, Carlos A. Casiano. Targeting the tumor-associated autoantigen alpha-enolase in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2017-1130

Prostate cancer (PCa) presents the greatest US cancer health disparity in terms of incidence and mortality, disproportionately affecting African American (AA) men. The biological characteristics of prostate tumors are exaggerated in AA men compared to European American (EA) men at time of diagnosis. The mechanisms underlying increased PCa aggressiveness in AA men are not fully understood; therefore, there is a critical need to identify biological determinants contributing to PCa mortality disparities. Glucocorticoids—a type of stress hormone—have been implicated as driving factors in PCa progression. The underlying mechanism involves endogenous glucocorticoid (cortisol) binding to its glucocorticoid receptor (GR) and activating genes that promote tumor aggressiveness and therapy resistance. A clinical dilemma exists as glucocorticoids, important in the palliative care of PCa patients, are now emerging as accelerators of disease progression and shortened survival. Chronically elevated levels of cortisol have been documented in AA men compared to EA men. This unhealthy cortisol production has been linked to stressful life events, which occur more frequently in the AA population. In addition, AA men are more sensitive to glucocorticoid exposure than EA men. These observations support the hypothesis that chronic stress leading to elevated cortisol in AA men may contribute to PCa health disparities. This study focused on examining the effects of glucocorticoid agonists and antagonists on the expression of stress oncoproteins, particularly LEDGF/p75 (Lens epithelial-derived growth factor of 75 kd) and CLU (clusterin), which are upregulated in PCa tumors and promote features of tumor aggressiveness, including resistance to clinically relevant taxanes used for advanced PCa treatment. We exposed a racially diverse panel of PCa cell lines, MDA-PCa-2b, 22Rv1, PC3, and DU145, to glucocorticoids (cortisol and dexamethasone), and observed by immunoblotting a time and dose-dependent upregulation of GR, LEDGF/p75 and CLU, particularly and more robustly in AA cells. Treatment of cells with mifepristone, a GR antagonist, attenuated this glucocorticoid-induced upregulation. In addition, siRNA- and shRNA-knockdown of LEDGF/p75 in PC3 cells resulted in decreased GR expression, suggesting a functional interplay between these two proteins in PCa cells. Interestingly, we also observed, using ELISA, increased circulation levels of LEDGF/p75 in AA PCa patients compared to EA patients and non-PCa controls, suggesting that this stress oncoprotein might play a more prominent role in AA PCa patients. These findings imply that activation of GR-signaling by glucocorticoids contributes to the activation of stress survival pathways associated with PCa aggressiveness and chemoresistance. These results add to the emerging contribution of GR signaling in PCa, particularly in the context of PCa health disparities. Citation Format: Leanne Woods-Burnham, Arthur Love, Christina K. Cajigas-Du Ross, Laura Stiel, Evelyn S. Sanchez, Kwame Amponsah, Susanne Montgomery, Colwick Wilson, Carlos A. Casiano. Glucocorticoid-mediated upregulation of stress oncoproteins: implications for prostate cancer health disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5285. doi:10.1158/1538-7445.AM2017-5285

Prostate specific antigen (PSA) level test in blood has been widely implemented for the early detection and prediction of prostate cancer (PCa). However, lack of specificity led to overdiagnosis, following by unnecessary biopsies and overtreatment. Therefore, novel serological biomarkers are urgently needed to complement PSA by enhancing its sensitivity and specificity in the diagnosis of PCa. Autoantibodies against intracellular tumor associated antigens (TAAs) are commonly found in various types of human cancers. Their utility in providing insights into tumor biology and as potential cancer biomarkers is well documented. Previous studies have demonstrated that sera from patients with PCa contain autoantibodies that react with TAAs. In this study, we identified and characterized nucleophosmin 1 (NPM1) with 33 kDa molecular weight as an autoantigen by using serological proteomics analysis (SERPA) approach. In the further validation study, it was observed that the level of anti-NPM1 in sera from patients with PCa was significantly higher than that from benign prostatic hyperplasia (BPH) and healthy individuals and receiver operating characteristic (ROC) curve analysis showed high diagnostic value for PCa (area under the curve (AUC):0.805). Interestingly, when considering anti-NPM1 combined with PSA level in sera from PCa patients at the early stage as well as BPH, 100% PCa patients could be identified correctly, while 69.2% patients with BPH who has elevated PSA level were found anti-NPM1 negative. The data suggested that anti-NPM1 can elicit humoral immune response in PCa and might be a potential biomarker for the immunodiagnosis of PCa, and it would be a supplementary of PSA examination to distinguish PCa from BPH. Key words: prostate cancer; nucleophosmin 1 (NPM1); tumor-associated antigens (TAAs); autoantibodies; serological proteomics analysis (SERPA) Citation Format: jitian li, Liping Dai, Ningjing Lei, Mengtao Xing, Carlos A. Casiano, Jianying Zhang. Using serological proteomics analysis to identify anti-nucleophosmin 1 autoantibody in sera from prostate cancer patients as potential diagnositic and prognostic biomarker. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4974.

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related male deaths in the U.S. This cancer is more aggressive in African American (AA) men, who are twice as likely to die from the disease when compared to other racial groups. Despite these racial disparities in PCa mortality, studies on PCa biology and biomarker discovery include mostly patients from European American (EA) backgrounds. There is a critical need to find minimally invasive PCa biomarkers that could aid in diagnosis and prognosis, especially in high risk populations like AA men. Immunoproteomics offers a minimally invasive approach to detect early malignant processes that trigger production of autoantibodies to tumor-associated antigens (TAA). We used serum samples from AA (n = 59) and EA (n = 50) men with PCa to probe one- and two-dimensional Western blots of protein lysates from aggressive PCa cell lines. AA PCa patient sera showed stronger immunoreactivity to proteins in PC3 cell lysates compared to the EA sera, with a 50 kD protein band frequently targeted by autoantibodies in several AA PCa sera. Serological proteomic analysis with mass spectrometry showed that these AA PCa autoantibodies recognized alpha-enolase (ENO1), a protein important in tumor formation, expansion, and glucose metabolism. In addition, several other glycolytic enzymes important for tumor proliferation were also identified as candidate prostate TAA, including GAPDH, bisphosphate aldolase, phosphoglycerate kinase, and lactate dehydrogenase. The ENO1 autoantibody frequency, measured by ELISA in sera from 400 racially diverse men with and without PCa, was four-fold higher in PCa compared to non-PCa patients. Interestingly, although sera from AA men with PCa had stronger WB reactivity to ENO1 present in PC3 lysates compared to EA men, and the ENO1 autoantibodies were initially discovered in eight AA PCa sera but only in two EA sera, the anti-ENO1 autoantibody frequency measured by ELISA, which used yeast-purified recombinant human ENO1 as a substrate, was higher in the EA PCa cohort. In addition, several anti-ENO1 positive sera from AA men with PCa showed increased immunoreactivity to this protein in the PCa cell lines LNCaP, MDA-PC2b, PC3, and DU145, but not against the purified ENO1 used in ELISA. The opposite was true in the EA PCa cohort, where strong reactivity against the purified ENO1 did not correlate with an equally strong immunoreactivity against the cellular ENO1. The EA PCa sera did not recognize the pattern of ENO1 expression observed with the AA sera in the same panel of prostate cell lines. These differences of immunoreactivity between AA and EA sera against PC3-ENO1 vs recombinant human ENO1 suggest that select AA sera may predominantly recognize ENO1 epitopes not displayed in the purified protein. These discrepancies point to racial differences in the immune response to prostate tumors. Citation Format: Tino Wilson Sanchez, Jian-Ying Zhang, Liping Dai, Susanne Montgomery, Colwick Wilson, Guangyu Zhang, Saied Mirshahidi, Nathan Wall, Carlos A. Casiano. Immunoproteomic profiling in African American men with prostate cancer: Evidence for an autoimmune response to glycolytic enzymes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3895.

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men and the leading cause of cancer-related deaths in elderly males. Although early stage PCa is treatable, many patients eventually develop castration-resistant disease (mCRPC), characterized by metastasis and resistance to therapy. Furthermore, PCa also presents a major health disparity problem as evidenced by its disproportionately high incidence and mortality in African American men compared to other racial/ethnic groups. Reducing or eliminating these disparities in PCa mortality will require innovative therapeutic approaches to circumvent chemoresistance and increase patient survival. We have previously demonstrated that lens epithelium-derived growth factor of 75 kD (LEDGF/p75) is a stress survival protein that is overexpressed in PCa cells and clinical tumors, and promotes resistance to Docetaxel (DTX), the gold standard for mCRPC chemotherapy. LEDGF/p75 has also been extensively studied as a target to inhibit HIV-1 integration into host chromatin because of its role as a key cellular cofactor of HIV integrase (IN). A series of novel and potent small molecule inhibitors (SMI) of LEDGF/p75 were previously evaluated for their potential to disrupt its interaction with HIV-IN. We hypothesized that repositioning these SMI to target LEDGF/p75 in the context of PCa could be a promising strategy for overcoming taxane resistance. We observed that PC3 and DU145 mCRPC cells selected for DTX resistance expressed high levels of LEDGF/p75, and were also resistant to the taxanes Cabazitaxel (CTX) and Paclitaxel (PTX). This chemoresistance was selective since LEDGF/p75 did not protect cells against the classical apoptotic inducer TRAIL, which triggers caspase-mediated cleavage and inactivation of LEDGF/p75. RNAi-mediated knockdown of LEDGF/p75 in these cell lines sensitized them to taxane therapy. We then evaluated 130 LEDGF/p75 SMI for their cytotoxicity in DTX-resistant and sensitive PC3 and DU145 cells, in the presence and absence of DTX. These experiments yielded a number of inhibitors that induced cell death directly or re-sensitized resistant cells to taxanes only when combined with these drugs. Our results suggest that LEDGF/p75 is a druggable oncoprotein that could be targeted with repositioned HIV-based SMI to overcome PCa chemoresistance. Citation Format: Leslimar Rios Colon, Catherine Elix, Ivana Alicea, Anamika Basu, Christina Du Ross, Tino Sanchez, Nouri Neamati, Carlos Casiano. Repositioning HIV-based small molecule inhibitors of the stress survival oncoprotein LEDGF/p75 to overcome taxane 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 5411. doi:10.1158/1538-7445.AM2015-5411

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related male deaths in the U.S. PCa is diagnosed more frequently in African American men (AA), with a mortality rate that is two-fold higher than in other ethnic groups. The current diagnostic test for PCa relies on the detection of prostate specific antigen (PSA). Although this minimally invasive test has high sensitivity, its limited specificity leads to false positives and unnecessary biopsies. This test may also fail to recognize PCa in men with low PSA levels, especially in obese and young AA men. Therefore, there is a critical need for identifying additional minimally invasive PCa biomarkers, especially in high-risk populations such as AA men. Immunoseroproteomics is a powerful approach for profiling circulating autoantibodies targeting tumor-associated antigens (TAAs) in cancer patients. These autoantibodies may serve as “reporters” to tumorigenic events and potential diagnostic biomarkers relevant to tumor biology. Based on recent reports of racial differences in the immunobiology of PCa, we hypothesized that AA and CC men with PCa may produce different anti-TAA autoantibody profiles. To evaluate this hypothesis, total proteins from aggressive PCa cells were separated by one or two-dimensional (2D) gel electrophoresis and then probed by immunoblotting with sera from AA (n = 40) and CC (n = 50) PCa patients to identify immunoreactive protein bands or spots. Protein spots recognized by the patient sera were then excised from the 2D gels and analyzed by mass spectrometry to profile the anti-TAA autoantibody repertoire in a given patient. Sera from AA PCa patients showed stronger immunoreactivity against PC3 cell proteins than sera from the CC cohort when evaluated under identical conditions in immunoblots. A common immunoreactive band around 45-50kD was produced by several AA sera (n = 8), and by one CC serum, and subsequent analysis by mass spectrometry showed that it corresponded to alpha-enolase. Confirmation that the AA PCa sera recognized 47kD alpha-enolase was obtained by immunoblots showing that these sera recognized the same protein bands recognized by a monoclonal antibody to this protein. Some of these sera also reacted against annexin A2. Additional validation studies are currently under way. Alpha-enolase and annexin A2 have been recently identified as candidate plasminogen receptors with roles in PCa. Alpha-enolase has been shown to promote cancer aggressiveness properties and its expression in tumors is associated with poor clinical outcomes. Alpha-enolase is a promising candidate biomarker that if used in combination with other TAAs for autoantibody profiling in PCa might help in the early detection and management of PCa, particularly in AA men. Citation Format: Tino Wilson Sanchez, Kwame Agyeman, Saied Mirshahidi, Nathan Wall, Colwick Wilson, Susanne Montgomery, Carlos A. Casiano. Identification of alpha-enolase and annexin as candidate tumor-associated antigens by immunoseroproteomic profiling of autoantibodies in African American and Caucasian men with 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 1831. doi:10.1158/1538-7445.AM2015-1831

Autoantibodies against intracellular tumor associated antigens (TAAs) are commonly found in various types of human cancers. Their utility in providing insights into tumor biology and as potential cancer biomarkers is well documented. In this study, serum autoantibodies to the Ras family small GTP binding protein (RalA) were detected by immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), and immunoblotting in 353 sera from patients with prostate cancer (PCa), benign prostatic hyperplasia (BPH), and normal controls. Expression of RalA in prostate tumor tissues was evaluated by immunohistochemistry. The cumulative positive rate of autoantibodies against RalA in PCa reached 18.4%, higher than that in sera with BPH (14.7%) and normal human sera (NHS) (2.25%, P Key words: anti-RalA autoantibody; tumor-associated antigens (TAAs); cancer immunodiagnosis; prostate cancer Citation Format: jitian li, Liping Dai, Carlos A. Casiano, Jianying Zhang. Evaluation and characterization of anti-RalA autoantibody as a potential biomarker in human 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 1564. doi:10.1158/1538-7445.AM2015-1564

Overexpression of self-antigens in malignant tissues may lead to the elicitation of autoantibodies (AAbs) that could serve as tools for the characterization of novel tumor-associated antigens (TAA). We and others have demonstrated that the stress survival protein LEDGF/p75 (lens epithelium derived growth factor p75, also known as DFS70, dense fine speckled nuclear antigen of 70 kD) is targeted by IgG AAbs in some patients with prostate cancer (PCa), leukemia, non-rheumatic inflammatory conditions, and even in healthy individuals. These AAbs are thought to be sensors of events associated with malignancy or inflammation, or protective antibodies that influence host immunosurveillance against LEDGF/p75-overexpressing tumors. LEDGF/p75 is overexpressed in cancer cells and tumors of the prostate, colon, and thyroid, and promotes chemoresistance. A better understanding of the significance of the autoantibody response to LEDGF/p75 requires establishing its specificity. Our objective is to determine whether this response, particularly in healthy individuals, is a component of the host immunosurveillance of LEDGF/p75-overexpressing tumors. Recent studies by our group showed that LEDGFp75 interacts with MeCP2 (methyl CpG binding protein 2) in PCa cells to influence transcriptional activation of Hsp27. Interestingly, non-human antibodies against MeCP2 produce an identical molecular weight and dense fine speckled (DFS) nuclear distribution as AAbs to LEDGF/p75, as assessed by immunoblotting and indirect immunofluorescence (IIF) microscopy, respectively, in cancer cells. These observations led us to determine if human AAbs producing the DFS-IIF pattern, considered specific for LEDGF/p75, are exclusive to this protein or may also target MeCP2. We screened in immunoblots of PC3 cell lysates over 90 human sera producing the LEDGF/p75-associated DFS-IIF pattern to determine if they possess dual reactivity against the two antigens, or are specific for one or the other. Initial results showed that the sera exhibited different reactivity strengths against a 75 kD protein. A group of 20 strongly reacting sera was selected for subsequent studies. Immunoblots of lysates from PC3 cells with LEDGF/p75 knockdown showed that the selected sera did not recognize the 75 kD band, indicating their specificity for LEDGF/p75. We also confirmed the specificity of these sera by demonstrating that they recognized the signature caspase-mediated cleavage pattern of LEDGF/p75 in PC3 cells treated with the apoptosis inducing drug TRAIL. These findings suggest that human AAbs producing the DFS-IIF pattern are specific for LEDGF/p75 and are not reactive with its interacting partner MeCP2. These results enhance our understanding of the specificity of the autoantibody response to this TAA, and have implications for the future development of LEDGF/p75-based vaccines to induce immunity to LEDGF/p75-overexpressing tumors. Citation Format: Anamika Basu, Greisha L. Ortiz-Hernández, Carlos A. Casiano. Specificity of the human autoantibody response against the stress oncoprotein LEDGF/p75. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3619. doi:10.1158/1538-7445.AM2014-3619

Autoantibodies against intracellular antigens are commonly found in a number of systemic autoimmune diseases. Their utility in providing insights into molecular and cellular biology and in the differential diagnosis of autoimmune diseases is well documented. Autoimmune phenomena manifested as antibodies to cellular components have been described in many types of cancer. Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the second leading cause of male cancer deaths in the United States. PCa also presents the greatest racial disparity in this country, with an incidence and mortality in African-American men that is 3 times higher than in White and Latino men. Reducing these disparities would require a multi-faceted approach that includes developing more effective screening method for the early detection of PCa. The objective of this study is to analyze the frequency and specificity of autoantibodies in PCa patients, and further to identify and characterize the targeted tumor-associated antigens. In this study, 34 sera from PCa patients, and 30 sera from normal human individuals were examined for autoantibodies using indirect immunofluorescence (IIF) assay and western blotting. Autoantibodies were detected in 18 of 34 (52.9%) PCa sera by IIF, which was significantly higher than that in normal human sera. Of the interest is that of 34 PCa sera, 9 (26.5%) sera were identified by western blotting analysis containing antibodies against an unknown 70kDa cellular protein and 23.5% PCa patients showed antibody to 45 kDa protein. No reactivity with 70KDa and 45kDa proteins was detected in 30 normal human sera. In the further study, these cellular proteins targeted by serum antibodies in PCa were identified by using two-dimensional gel electrophoresis (2DE) and liquid chromatography-tandem-mass spectrometry (LC-MS/MS). This preliminary data supports that autoimmune responses to certain cellular proteins maybe a by-product in the transformation to PCa, and further studies of novel targeted proteins in PCa may provide insights into how these proteins might be involved in malignancy. Citation Format: Jitian Li, Liping Dai, Carlos A. Casiano, Jianying Zhang. Identification and characterization of tumor-associated antigens as biomarkers in human prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 878. doi:10.1158/1538-7445.AM2014-878

Ikzf1 (Ikaros) encodes a zinc finger protein that binds DNA and regulates gene expression via chromatin remodeling. The loss of Ikaros activity due to genetic or functional inactivation results in leukemia with a poor prognosis. The goal of our project is to determine the mechanism by which Ikaros regulates chromatin remodeling in human leukemia. Previous studies showed that Ikaros function in leukemia is controlled through its direct phosphorylation by Casein Kinase II (CK2). Treatment of leukemia cells with CK2 inhibitors results in enhanced Ikaros activity, which leads to cessation of cell growth. We have studied the mechanism by which inhibition of CK2 regulates Ikaros-induced epigenetic changes in leukemia. The human Nalm6 pre-B cell leukemia was treated with CK2 inhibitor, and the epigenetic signature of the histone modifications H3K9ac and H3K9me3 were determined using chromatin immunoprecipitation coupled with next generation sequencing (ChIP-SEQ). H3K9ac histone modification is associated with positive regulation of gene expression, while H3K9me3 is associated with the formation of heterochromatin and repression. The enrichment of particular histone modifications was confirmed by quantitative chromatin immunoprecipitation (qChIP). The results demonstrated that the inhibition of CK2 activity in leukemia results in a marked alteration in the epigenetic signature of both H3K9ac and H3K9me3 compared to untreated cells. This is associated with altered Ikaros binding to its target genes following CK2 inhibition. Current bioinformatics analysis is directed toward establishing a link between epigenetic modifications and Ikaros binding in leukemia. These results suggest that CK2 and Ikaros regulate gene transcription via epigenetic modifications and chromatin remodeling in leukemia. Citation Format: Jonathon L. Payne, Carlos M. Casiano, Kimberly J. Payne, Justin Sloane, Elanora Dovat, Chunhua Song, Sinisa Dovat. Regulation of chromatin remodeling in leukemia by Ikzf1 and Casein Kinase II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 409. doi:10.1158/1538-7445.AM2014-409

Control of cell cycle progression is achieved by the coordinated function of a large set of genes that are highly conserved in eukaryotic organisms. Malignant cells have impaired regulation of cell cycle progression which results in uncontrolled cellular proliferation. Thus, understanding the regulation of cell cycle progression in malignant cells is essential to advance our knowledge of the process of malignant transformation and for designing novel treatments. Ikaros is a zinc finger protein that acts as a tumor suppressor in leukemia. The loss of Ikaros activity due to deletion or mutation has been associated with the development of high-risk B-cell acute lymphoblastic leukemia (B-ALL), as well as with T-cell ALL and acute myelogenous leukemia (AML). Ikaros binds DNA and regulates transcription of its target genes via chromatin remodeling. The mechanism of Ikaros tumor suppressor activity is largely unknown. Here, we present evidence that Ikaros regulates cell cycle progression in leukemia. Using quantitative Chromatin Immunoprecipitation assay (qChIP), we demonstrate that Ikaros binds in vivo to promoter regions of several genes that regulate cell cycle progression in B-ALL cell lines and in primary cells from patients with B-ALL. To study how Ikaros regulates transcription of these genes, luciferase reporter assays were performed. The promoter regions of three Ikaros target genes were cloned into luciferase reporter constructs. Each of these constructs has been co-transfected with Ikaros or an empty vector (as a negative control) into HEK 293T cells. Results showed that Ikaros represses transcription of the three genes that promote cell cycle progression. Overexpression of Ikaros in leukemia cells by retroviral transduction results in reduced transcription of the cell cycle promoting genes, as evidenced by quantitative real-time PCR (qRT-PCR), as well as cell cycle arrest. These data suggest that Ikaros regulates cell cycle progression in leukemia by direct repression of the transcription of the genes that promote cell cycle progression, and identifies one mechanism of Ikaros function as a tumor suppressor in leukemia. Citation Format: Elanora Dovat, Jonathon Payne, Carlos M. Casiano, Justin Sloane, Chandrika Gowda, Kimberly J. Payne, Sinisa Dovat, Chunhua Song. Regulation of cell cycle progression by Ikaros in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2014-3504

Cancer-associated serum autoantibodies are generally considered as “sensors” of molecular events associated with tumorigenesis, and thus constitute promising biomarkers for cancer detection and management, as well as tools for the identification of novel oncoproteins. Immunoseroproteomics is a standard approach for the identification of serum autoantibodies reacting with tumor-associated antigens (TAA) in human cancer populations. This approach entails using one- and two-dimensional gel electrophoresis of proteins from aggressive PCa cell lines, coupled with immunoblotting and mass spectrometry, to characterize the anti-TAA antibody repertoire in a given patient. Immunoseroproteomics has been applied mostly to cancer patients of European and Asian ancestry, and has not been used in African American (AA) patients with PCa. AA men show a disproportionately high incidence and mortality of PCa, compared to other ethnic groups, and display more aggressive tumors developing at a younger age. Recent epidemiological studies suggest that after adjusting for socioeconomic and health care-related factors, these disparities still persist, pointing to biological factors as contributors to these disparities. Growing evidence indicates that genes associated with immune function and autoimmunity are dysregulated in prostate tumors from AA compared to Caucasian (CC) men. This prompted us to initiate an immunoproteomic analysis of the anti-TAA autoantibody response in AA and CC men with PCa. We hypothesize the anti-TAA autoantibody response might be greater in AA patients with PCa, and that it could be used to identify novel blood biomarkers for early screening and management of the disease. In initial studies, we screened aggressive PCa cell lysates with serum samples from AA (n=39) and CC (n=50) men. Early results showed the autoantibody response in the AA cohort had a greater autoantibody frequency and reactivity than the response in CC patients. Immunoproteomic analysis of select AA sera revealed alpha-enolase, fumarate hydratase, and annexin A11 as candidate TAAs in PCa. These proteins have been implicated in previous PCa studies. Additional validation will come from determining the frequency of the autoantibody response and the expression of candidate TAAs in PCa tissue microarrays. We are currently expanding our immunoseroproteomics analysis to sera from a larger cohort (n>200) of AA men and CC men (n>100) with and without PCa, stratified by age and cancer stage. Comparing the anti-TAA repertoire between AA and CC men with and without PCa will aid in the customization of TAA arrays for autoantibody profiling and lead to the identification of promising biomarkers for early PCa detection and monitoring of tumor progression in AA populations. Our approach may also help identify unique immunoserological signatures in AA and CC men to reveal biological determinants of PCa health disparities. Citation Format: Tino Wilson Sanchez, Saied Mirshahidi, Nathan Wall, Susanne Montgomery, Colwick Wilson, Carlos A. Casiano. Immunoseroproteomic profiling of autoantibodies to tumor associated autoantigens in African American and Caucasian men with prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2502. doi:10.1158/1538-7445.AM2014-2502

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the U.S. It presents a significant health burden among African American men, who tend to develop more aggressive, chemoresistant tumors and experience higher mortality rates compared to men from other ethnic groups. Eliminating these mortality disparities will require innovative therapies for the chemoresistant form of the disease. Our group has shown previously that the stress survival protein lens epithelium-derived growth factor p75 (LEDGF/p75) is overexpressed in PCa cells and clinical tumors, and promotes resistance to the taxane drug docetaxel (DTX). This study was designed to evaluate the hypothesis that LEDGF/p75 overexpression promotes taxane chemoresistance in PCa by antagonizing drug-induced caspase-independent cell death, and that targeting this protein with small inhibitory RNAs (siRNAs) or small molecule inhibitors will restore tumor cell sensitivity. We observed that acquisition of DTX-resistance in metastatic PCa cell lines (PC3 and DU145) is associated with increased expression of LEDGF/p75. Exposure of these cell lines to increasing concentrations of the taxanes DTX, paclitaxel (PTX), and cabazitaxel (CTX), and to the non-taxane drug TRAIL for up to 72 hours revealed that DTX-resistant cells also displayed increased resistance to PTX and CTX, compared to sensitive cells, but not to TRAIL. This was assessed by viability and clonogenic assays, and microscopic examination. To determine if this resistance was associated with overexpression of LEDGF/p75, we knocked down the protein with siRNAs. This knockdown attenuated resistance to the taxanes, but had no effects on TRAIL-induced cell death. We then explored if LEDGF/p75-induced taxane resistance was associated with preservation of the structural integrity of this protein. Immunoblotting analysis revealed that LEDGF/p75 was cleaved into distinct fragments by caspases during TRAIL-induced apoptosis, leading to its inactivation. However, LEDGF/p75 remained intact during treatment with taxanes, suggesting that these drugs do not induce caspase-mediated cleavage and inactivation of this protein. To determine if LEDGF/p75 is a druggable target for overcoming taxane chemoresistance in PCa cells, we repositioned a panel of small molecule inhibitors originally designed to inhibit the interaction of the C-terminal region of this protein with HIV-1 integrase. Screening of 130 inhibitors for their cytotoxic properties in DTX-resistant and sensitive prostate cancer cells, we identified a number of inhibitors that induced cell death directly, or when combined with DTX, re-sensitized the resistant cells to the drug. These promising results provide novel insights into mechanisms by which LEDGF/p75 promotes chemoresistance in PCa cells, and have implications for the development of innovative strategies to overcome this resistance. Citation Format: Leslimar Rios Colon, Catherine Elix, Anamika Basu, Tino W. Sanchez, Nouri Neamati, Carlos A. Casiano. Targeting the stress survival oncoprotein LEDGF/p75 to overcome prostate cancer resistance to taxane therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 775. doi:10.1158/1538-7445.AM2014-775

African-American men have the highest rate of prostate cancer (PCa) incidence in the U.S. In addition, their prostate cancer mortality rate is more than twice as high as the rate for white Americans. The causes of higher rates of prostate cancer among African-American males are largely unknown. Many studies are underway to unravel the impact of a wide variety of potential risk factors, including dietary and other lifestyle differences, occupational exposures, hormonal and genetic differences. Several lines of evidence suggest that one of the main events associated with the conversion to an aggressive phenotype is increased resistance to apoptosis (3) mainly due to upregulation of antiapoptotic genes, including Bcl-2, Bcl-XL, Mcl-1, and Survivin. Recently, we reported that Survivin is released in PCa sera and found to be in the exosomes. Therefore, we speculate that Survivin could be a potential target protein released differentially in prostate cancers in African American (AA) and Caucasian (CC) populations in response to stress. In this study, we have collected serum samples from prostate cancer patients of AA and CC, measured Survivin levels by ELISA. Exosome islation was perfomed by Exoquick approach and quantified by acetylcholinesterase activity assays. We have applied an in-vitro approach to compare the differences between cancer cell lines derived from AA (MDA-MB-PCa) and CC (PC3) patients in terms of stress and stress activated Survivin release by exosomes. Under oxidative stress and drug treatments, we have identified Survivin highly expressed in exosome derived from MDA-MB-PCa compared to PC3 cell lines. In addition, we found a relative overexpression of Survivin protein in AA-PCa compared to CC-PCa both in sera and exosomes. Based on our data, Survivin may be a key stress-induced protein in AA-PCa patients. It is possible that attempts to block Survivin release will not only interrupt the cancer proliferation and interfere with anti-apoptotic pathways, thus preventing cancer progression. Citation Format: Salma Khan, David Turay, Jessica Ms Jutzy, Jonathan R. Aspe, Tino W. Sanchez, Saeid Mirshahidi, Carlos A. Casiano, Nathan R. Wall. Stress-induced differential Survivin release in prostate cancer health disparities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1118. doi:10.1158/1538-7445.AM2014-1118

We have shown that Survivin is exported out of the cancer cell via exosomes despite its lack of a secretory signaling peptide. However, the mechanism of Survivin exosomal loading is not yet known. Exosomal protein sorting can be endosomal sorting complex required for transport (ESCRT)-dependent or ESCRT-independent. ESCRT dependent protein sorting requires monoubiquitination of the target protein, which serves as an exosome sorting signal. The ESCRT independent mechanism is through preferential aggregation of the proteins which is based on intrinsic physical properties to lipid-rafts present in exosomes. Our lab has recently shown that exosomal Survivin is associated with both Hsp70 and Hsp90, as well as PRDX1 which have also been shown associated with detergent-resistant lipid rafts. Here, we determine whether Survivin is lipid-raft dependent and whether Hsp70 and Hsp90 and/or PRDX1 are required for exosomal Survivin loading. Exosomes were collected from conditioned media taken from HeLaS cells and isolated by centrifugation. Colocalizations of Survivin with Hsp70, Hsp90 and PRDX1 in the cell and exosomes were detected by immnocytochemistry visualized by confocal microscopy and immunoprecipitation. HeLaS cells were treated with increasing concentrations of methyl-β-cyclodextrin to disrupt lipid rafts as a way to determine protein lipid raft association. Hsp70, Hsp90 and PRDX1 knock-down using siRNAs will be accomplished to verify the requirement of these proteins for Survivin exosome loading and lipid-raft dependents. Here we show that Survivin colocalizes with Hsp70, Hsp90 and PRDX1 in HeLaS cells as well as their exosomes. Increasing concentrations of methyl-β-cyclodextrin disrupts lipid rafts in the HeLaS cells and releases exosomes which also subsequently contain decreasing PRDX1 and Survivin protein amounts. The release of Survivin into the tumor microenvironment is an important finding as this IAP may contribute to the aggressiveness of the cancer as well as the development of chemoresistance. Despite lacking a secretory signaling peptide, Survivin is being released into the extracellular space via exosomes using chaperones. Knowing how Survivin is exosomally packaged may prove useful in selecting patient specific treatment regimens. Citation Format: Malyn May Asuncion Valenzuela, Heather R. Ferguson Bennit, Carlos Joel Diaz Osterman, Salma Khan, Carlos Casiano, Nathan R. Wall. Survivin packaging into exosomes is lipid raft-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4856. doi:10.1158/1538-7445.AM2014-4856

Prostate cancer (PCa) is the second leading cause of cancer related deaths among men in the United States, with African American men presenting more aggressive tumors and increased mortality compared to other ethnic groups. Eliminating these disparities will require an interdiplinary effort that combines improved access to healthcare and health education, combined with innovative approaches to circumvent therapy resistance. The lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator that transactivates stress protective genes to promote resistance to microenvironmental stressors such as oxidative stress and chemotherapy. Previously, our group established that LEDGF/p75 is overexpressed in PCa cells and clinical tumors and that it promotes resistance to docetaxel (DTX) treatment, the standard of chemotheraoy for metastatic PCa. The present study was designed to determine the effects of LEDGF/p75 overexpression and knockdown on cell viability in different PCa cellular models exposed to various chemotherapeutic drugs used in the treatment of advanced PCa. We also evaluated if exposure to DTX and the other drugs increased expression of LEDGF/p75 in PCa cells. For these studies, we used different cell lines with LEDGF/p75 overexpression and siRNA-mediated knockdown, as well as DTX resistant cell lines with elevated levels of this protein. PCa cells were treated with increasing concentrations of the chemotherapeutic drugs DTX, Doxorubicin (DOXO), Paclitaxel (PTX), and TRAIL for up to 96 hours. Loss of cell viability was assessed by viability assays and morphological microscopic examination. Our preliminary results suggested that elevated expression of LEDGF/p75 promotes selective resistance to chemotherapeutic drugs, particularly to DTX, DOXO, and PTX, but not to TRAIL. This selectivity seems to be related to the ability of LEDGF/p75 to protect against stress-induced lysosomal cell death but not against TRAIL-induced classical death receptor mediated apoptosis. Knockdown of LEDGF/p75 sensitized PCa cells to the chemotherapeutic drugs. In addition, increased cellular stress induced by exposure to DTX treatment resulted in increased expression of LEDGF/p75 in a cell-type dependent manner. These results have implications for the development of novel therapeutic strategies for the treatment of PCa based on targeting LEDGF/p75 and its target genes for the sensitization of tumor cells to chemotherapeutic agents. Citation Format: Leslimar Rios-Colon, Anamika Basu, Catherine Elix, Carlos A. Casiano, Marino De León. The stress oncoprotein LEDGF/P75 promotes selective resistance to multiple chemotherapeutic drugs in prostate cencer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 892. doi:10.1158/1538-7445.AM2013-892

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer related death in men in the US. PCa tumor aggressiveness, response to treatment, and mortality are worst in African-American men, compared to other ethnic groups, thus presenting a major health disparity. Our group reported previously that lens epithelium derived growth factor p75 (LEDGF/p75), a stress survival transcription coactivator, has elevated expression in PCa cells and tissues and promotes resistance to cell death induced by oxidative stress and chemotherapy. This is likely due to its ability to transactivate promoter regions of stress and antioxidant genes such as peroxiredoxin 6 (Prdx6) and heat shock protein 27 (HSP27). We hypothesized that high expression of LEDGF/p75 in prostate tumor cells and tissues contributes to the elevated expression of its putative target genes, some of which were identified in previous studies by our group and others. These include genes associated with stress protection and chemotherapy resistance such as HSP27, endoplasmic reticulum protein 57 (ERp57), and cytoglobin (CYGB). To test this hypothesis we performed an expression analysis of LEDGF/p75, HSP27, ERp57 and CYGB proteins in human PCa cell lines using immunoblotting, and in prostate tumor tissues using immunohistochemistry on tumor tissue microarrays (TMAs). PCa cell lines included stable PC3 clones overexpressing LEDGF/p75, as well as docetaxel (DTX) resistant PC3-DR and DU145-DR cells overexpressing this protein, with their corresponding parental DTX sensitive cell lines. The TMAs contained tissue specimens corresponding to prostate tumor, hyperplasia, normal-adjacent, and disease-free normal prostate tissues. Correlations between the expression of LEDGF/p75 and HSP27 or ERp57 or CYGB in cell lines as well as in tumors and normal tissues was determined. Our results indicated that overexpression of LEDGF/p75 in PC3 stable clones and in DTX resistant PCa cells was associated with increased protein expression of HSP27, ERp57 and CYGB. Statistically significant correlation was also observed between the protein levels of LEDGF/p75 and HSPp27 or ERp57 or CYGB in the TMAs. Luciferase-based transcription reporter assays confirmed that the promoter regions of HSP27 and ERp57 are activated by LEDGF/p75 in PCa cells. Chromatin immunoprecipitation assays also demonstrated binding of LEDGF/p75 to HSP27 promoter in PCa cells. Additional promoter activation studies are in progress to demonstrate that LEDGF/p75 transcriptionally regulates ERp57 and CYGB genes. These results enhance our understanding of the contribution of LEDGF/p75 overexpression to the activation of stress protective genes and its implications for chemotherapy resistance in metastatic PCa. Citation Format: Anamika Basu, Christina Cajigas, Benanette Medina, Heather Rojas, Hiya Banerjee, Melanie Mediavilla-Varela, Laisum Leoh, Carlos Casiano. Overexpression of the stress transcription co-activator LEDGF/p75 contributes to the upregulation of the stress protective genes HSP27, ERp57 and CYGB in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2013-3117

Prostate cancer (PCa) is the second leading cause of cancer death among men in the US, with African American men presenting a disproportionately high incidence and mortality. Reducing this mortality disparity requires developing novel strategies for overcoming prostate tumor chemoresistance. Currently, chemotherapy with Docetaxel (DTX), the standard of care for advanced PCa, is limited because most patients treated with the drug ultimately develop chemoresistance and succumb to the disease. Circumventing this resistance is therefore essential for increasing the effectiveness of DTX and other anti-PCa drugs. Our group has established previously that lens ephitelium-derived growth factor p75 (LEDGF/p75), a stress survival oncoprotein, is overexpressed in human prostate tumors, and that its overexpression in PCa cell models promotes resistance to DTX via inhibition of lysosomal cell death. The present study was designed to evaluate if overexpression of LEDGF/p75 in PCa cells also promotes resistance to other chemotheurapeutic drugs, and if its depletion sensitizes chemoresistant PCa cells to drug-induced cell death. First, we treated PC-3 cells (with and without stable LEDGF/p75 overexpression) with increasing concentrations of DTX, paclitaxel, doxorubicin, and TRAIL for up to 48 hrs. We then assessed cell viability by MTT assays and morphological examination. LEDGF/p75 overexpressing PC-3 cells showed higher viability than parental PC-3 cells in response to DTX, paclitaxel, and doxorubicin. However, LEDGF/p75 did not protect against TRAIL-induced cell death, indicating selectivity in the ability of this stress oncoprotein to induce chemoresistance. We also observed by immunoblotting and quantitative PCR that metastatic PCa cell lines selected for DTX resistance, designated PC3-DR and DU-145DR, naturally express high levels of endogenous LEDGF/p75 relative to their sensitive parental cell lines. To assess the contribution of elevated LEDGF/p75 expression to DTX resistance in these two cell lines, we knocked down this protein with a specific small inhibitory RNA (si-p75). Immunoblots showed that si-p75 depleted cells of LEDGF/p75. Cell viability significantly decreased in both PC3-DR and DU-145DR cell lines in response to combined treatment with DTX and si-p75, relative to scrambled siRNA control. Our preliminary results suggest that LEDGF/p75 overexpression promotes chemoresistance in cellular models of advanced PCa, and that its inhibition increases sensitization to DTX. These promising results have implications for the development of novel theraupetic strategies for chemoresistant PCa. Further studies are in progress to determine the spectrum of anti-PCa drugs antagonized by LEDGF/p75, and if depletion of this protein sensitizes PCa cells to a wide variety of antitumor drugs. 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 775. doi:1538-7445.AM2012-775

The lens epithelium-derived growth factor p75 (LEDGF/p75) is emerging as a stress response oncoprotein in human cancer. Our group and others showed previously that this transcription coactivator is overexpressed in major human cancers, including prostate cancer, and protects against lysosomal cell death and chemotherapeutic drugs. Although LEDGF/p75 is considered a novel anti-apoptotic protein due to its ability to attenuate lysosomal cell death, previous studies from our group demonstrated its inactivation by caspases during apoptosis. We hypothesized that LEDGF/p75 promotes resistance to stress-induced caspase-independent cell death, but not to apoptosis, in cancer cells. To test this hypothesis, we established PC3 and RWPE-2 prostate cancer cell lines stably overexpressing LEDGF/p75, and evaluated its ability to protect these cells against non-apoptotic and apoptotic insults. LEDGF/p75 significantly attenuated cytotoxicity induced by tert-butyl hydroperoxide (TBHP), a strong inducer of oxidative stress. Characterization of TBHP-induced cell death showed that it was caspase-independent since it could not be inhibited with the broad caspase inhibitor z-VAD-fmk and did not involve caspase-3 activation. Furthermore, TBHP-treated cells displayed a necrotic morphology characterized by extensive cytoplasmic fragmentation and shrunken, non-fragmented nuclei. Necrosis was also confirmed by the appearance in immunoblots of a 45 kD cleavage fragment of DNA topoisomerase that was previously established by our group as a marker of necrosis. Overexpression of LEDGF/p75 attenuated the generation of reactive oxygen species (ROS) in TBHP-treated cells. Interestingly, LEDGF/p75 also attenuated ROS in cells treated with the classical apoptosis inducers staurosporine and TRAIL, but failed to protect against these apoptotic insults. These results indicated that the ability of LEDGF/p75 to attenuate ROS is not sufficient for its pro-survival function. Analysis of LEDGF/p75 integrity by immunoblotting analysis showed that the protein remained intact in cells treated with TBHP, but was cleaved into its signature 65 kd apoptotic fragment in cells treated with staurosporine and TRAIL. Our group has shown previously that this caspase-generated fragment lacks pro-survival activity. Taken together, these results strongly suggest that overexpression of LEDGF/p75 can protect tumor cells from caspase-independent cell death processes in which this protein remains structurally intact. This study provides the basis for investigating the contribution of LEDGF/p75 to prostate cancer resistance to therapeutic modalities that induce caspase-independent cell death, as well as its potential as a novel therapeutic target. 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 2083. doi:10.1158/1538-7445.AM2011-2083

Chronic inflammation in the prostate microenvironment, with its associated increase in oxidative stress, is critical for prostate carcinogenesis since it leads to DNA mutations and epigenetic changes, as well as deregulation of stress and antioxidant proteins that protect prostate cells against oxidative damage and cell death. The lens epithelium-derived growth factor p75 (LEDGF/p75), an oxidative stress-induced transcription coactivator, is overexpressed in prostate tumors and other tumor types. LEDGF/p75 is emerging as a stress oncoprotein that protects cancer cells against lysosomal cell death and promotes resistance to chemotherapeutic drugs. We hypothesize that the pro-survival function of LEDGF/p75 is associated with its ability to promote the transcriptional activation of protective stress genes. To date, only very few candidate target genes of LEDGF/p75 have been identified in cancer cells. Towards this goal we initiated a stress focused gene profiling analysis of PC3 prostate cancer cells overexpressing or lacking LEDGF/p75, using the Real Time PCR Array System (SABiosciences). We selected an array that contained 80 genes involved in the cellular stress response and antioxidant defense. siRNA oligos targeting the C-terminus of the LEDGF/p75 mRNA sequence were used to induce transient LEDGF/p75 knockdown in PC3 cell. LEDGF/p75 was also stably overexpressed in PC3 cells by transduction with a lentiliviral vector. Both transient siRNA-mediated knockdown and overexpression of LEDGF/p75 were associated with significant changes in the expression of several stress/antioxidant genes, using a cutoff fold change value of 1.5 (P 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 2092. doi:10.1158/1538-7445.AM2011-2092

The lens epithelium derived growth factor p75 (LEDGF/p75) is an emerging oncoprotein identified as a key player in the cellular response to oxidative stress. LEDGF/p75 is induced by oxidative stress and promotes resistance to stress-induced cell death presumably by transcriptionally activating specific stress and survival genes. LEDGF/p75 has a splice variant, p52, which induces cell death when overexpressed. We have shown elevated expression of LEDGF/p75 in advanced stage prostatic adenocarcinomas, most likely as a result of increased oxidative stress in the prostate tumor microenvironment. To understand the mechanisms by which LEDGF/p75 transcriptionally activate stress genes and promotes protection against oxidative stress-induced cell death, it was necessary to identify endogenous cellular interacting partners of this protein. Screening of a transcription factor protein array identified the Methyl CpG binding Protein 2 (MeCP2) as a possible interacting partner of LEDGF/p75. MeCP2 is mutated in patients with Rett syndrome, both activate and repress transcription and has been implicated in prostate tumor growth. This study was designed to validate MeCP2 as an interacting partner of LEDFG/p75 and to explore its influence on LEDGF/p75's function. To examine the interaction of MeCP2 and LEDGF, pull down assays were performed with purified GST-MeCP2 incubated with His-LEDGF/p75 or His-p52, and with GST-MeCP2 or GST-p75 incubated with U2OS cell lysates. The results suggested binding of MeCP2 to both LEDGF/p75 and p52. Co-immunoprecipitation assays using U2OS cell lysates revealed binding of proteins, detected by immunoblotting using specific anti-LEDGF/p75 or MeCP2 antibodies, suggesting interaction of both proteins in the cellular microenvironment. We also examined the intracellular localization of both proteins in U2OS cells by immunofluorescence microscopy. Both MeCP2 and LEDGF/p75 co-localized in the chromatin, showing a similar dense fine speckled staining pattern. To examine if MeCP2 influences LEDGF/p75's transcriptional activity, luciferase reporter assays were performed. Even though LEDGF/p75, p52 and MeCP2 transactivated the Hsp27 promoter (pr) individually, co-expression of LEDGF/p75 with MeCP2 increased Hsp27pr activity, while co-expression of LEDGF/p52 with MeCP2 decreased activity. In addition, LEDGF/p75 overexpression protected PC3 cells from tert-butyl hydrogen peroxide (TBHP) treatment; but siRNA knockdown of MeCP2 in these cells sensitized cells to TBHP treatment compared to control siRNA. In conclusion, our data suggest that LEDGF/p75 and MeCP2 are interacting partners and MeCP2 influences LEDGF/p75's transcription and survival function. Future studies will explore modulating the interaction between LEDGF/p75 and MeCP2 to sensitize prostate cancer cells to therapy-induced cell death. 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 2945.

Prostate Cancer (PCa) is the most frequently diagnosed male cancer and presents the greatest racial disparity of any cancer in the U.S., with high incidence and mortality among African American (AA) men. Eliminating these disparities requires a multi-prong approach that includes, in addition to aggressive social and public health interventions, a better understanding of the biology of prostate tumors from different ethnic groups, and the development of effective treatments for castration-resistant prostate cancer (CRPC). Our interest is to define the contribution of antioxidant and stress proteins induced by environmental factors to tumor aggressiveness and health disparities in PCa. We analyzed the expression of the peroxiredoxin (PRDX) protein family in prostate cell lines and prostate tissue microarrays (TMA), and observed a dramatic upregulation of PRDX3 in androgen responsive metastatic cell lines, particularly the AA-derived cell line MDA-PCa-2b. There was also a significant correlation between high PRDX3 tissue expression and PCa (when compared to normal tissues), advanced tumor stage, and elevated PSA (P 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 2715.

Long-term cumulative exposure to environmental factors (i.e., dietary habits, unhealthy lifestyle, infectious agents, etc) lead to chronic inflammation and an augmented state of cellular oxidative stress, which contribute to the etiology and progression of various human cancers. We reported previously that lens epithelium derived growth factor p75 (LEDGF/p75), a transcription coactivator, has an elevated expression in human prostate tumors and cell lines. We hypothesize that LEDGF/p75 might be highly expressed in tissues with elevated oxidative stress such as tumors, because of its ability to transcriptionally activate stress genes that promote resistance to stress- and chemotherapy-induced cell death. To date, there have not been any comprehensive studies aimed at investigating the expression of LEDGF/p75 in human cancers. We performed an expression analysis of LEDGF/p75 protein in a panel of human cancers using immunohistochemistry on commercially available tissue microarrays (TMAs). Paraffin embedded tissue specimens were deparaffinized, rehydrated and stained with either a rabbit anti-LEDGF/p75 serum or the rabbit pre-immune serum as an antibody control, using a Biogenic i6000 auto stainer (Biogenex Corp.). Images were acquired using an Olympus BX50 microscope equipped with a Spot RT3TM camera. The TMA slides were assessed for LEDGF/p75 immunoreactivity by a pathologist who was blinded for the tumor/normal status of the tissues. A 4-tier grading system (0-negative, 1-weak, 2-moderate and 3-strong staining intensity) was used to evaluate the staining intensity. Associations between LEDGF/p75 expression in tumors and normal tissues were analyzed using Kruskall Wallis's rank sum test. We used TMAs (Imgenex and NDRI) containing specimens corresponding to over 20 major tumor types and normal adjacent tissues. Our data revealed high expression of this protein in tumors of prostate, pancreas, lungs, liver, thyroid, colon, breast and kidney, as compared to corresponding normal adjacent tissues. To further validate our findings, we analyzed LEDGF/p75 expression in the above-mentioned cancers using TMAs (US BIOMAX), each containing multiple tissues specimens of a specific major tumor type, as well as non-cancerous inflammatory disease and normal post-mortem control tissues. Immunohistochemical analysis revealed high expression of LEDGF/p75 in most of the tumor sections as compared to normal control tissues. The normal tissues showed minimal or no staining. The anti-LEDGF/p75 staining was mostly confined to the nucleus, although cytoplasmic staining was also evident in many cells. These results further validate the high expression of LEDGF/p75 in major human cancers and constitute the first step towards defining the biological significance of LEDGF/p75 expression in human cancers. 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 4667.