Castration-resistant Tumor Growth Research Articles

Antitumor effects of pyrrole-imidazole polyamide modified with alkylating agent on prostate cancer cells

Androgens and androgen receptor (AR) have a central role in prostate cancer progression by regulating its downstream signaling. Although androgen depletion therapy (ADT) is the primary treatment for most prostate cancers, they acquires resistance to ADT and become castration resistant prostate cancers (CRPC). AR complex formation with multiple transcription factors is important for enhancer activity and transcriptional regulation, which can contribute to cancer progression and resistance to ADT. We previously demonstrated that OCT1 collaborates with AR in prostate cancer, and that a pyrrole-imidazole (PI) polyamide (PIP) targeting OCT1 inhibits cell and castration-resistant tumor growth (Obinata D et al. Oncogene 2016). PIP can bind to DNA non-covalently without a drug delivery system unlike most DNA targeted therapeutics. In the present study, we developed a PIP modified with a DNA alkylating agent, chlorambucil (ChB) (OCT1-PIP-ChB). Then its effect on the growth of prostate cancer LNCaP, 22Rv1, and PC3 cells, pancreatic cancer BxPC3 cells, and colon cancer HCT116 cells, as well as non-cancerous MCF-10A epithelial cells, were analyzed. It was shown that the IC50s of OCT1-PIP-ChB for 22Rv1 and LNCaP were markedly lower compared to other cells, including non-cancerous MCF-10A cells. Comprehensive gene expression analysis of CRPC model 22Rv1 cells treated with IC50 concentrations of OCT1-PIP-ChB revealed that the gene group involved in DNA double-strand break repair was the most enriched among gene sets repressed by OCT1-PIP-ChB treatment. Importantly, in vivo study using 22Rv1 xenografts, we showed that OCT1-PIP-ChB significantly reduced tumor growth compared to the control group without showing obvious adverse effects. Thus, the PIP combined with ChB can exert a significant inhibitory effect on prostate cancer cell proliferation and castration-resistant tumor growth, suggesting a potential role as a therapeutic agent.

Abstract 5307: Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling

Abstract According to American Cancer Society, prostate cancer is a slow progressing disease that affects nearly 50 percent of males over the age of 60. Every year, 165,000 new cases are diagnosed and 30,000 deaths are reported. Current therapies such as Androgen Depravation Therapy (ADT) and Anti-Androgen treatments such as Enzalutamide increase quality of life and extend survival but do not provide a definitive treatment and are ineffective in highly metastatic prostate cancer. Castrate Resistant Prostate Cancer (CRPC) is an untreatable highly metastatic form of prostate cancer and once diagnosed the survival rate is 1-2 years. Hence, the understanding of molecular mechanisms of CRPC is of critical importance. We previously linked prostate cancer to Abi1 gene. Abl interactor 1 (Abi1) is a scaffolding protein and an integral member of the WAVE Regulatory Complex (WRC). WRC is actin nucleation promoting factor that acts upstream of Arp2/3 to enhance the speed of actin polymerization and is critical for various cellular functions including cell-cell junction formation. Our previous research using a prostate cancer mouse models shows that loss of Abi1 expression leads to down-regulation of cellular adhesion proteins such as E-cadherin. Studies using our CRISPR KO RWPE cell line indicate that loss of Abi1 disrupts cell-cell junction formation via decreased levels of E-cadherin. Analysis of patient data demonstrated loss of Abi1 in a subset of CRPC patients. Castrate resistant tumor growth is androgen independent therefore anti-androgen therapy is ineffective. In search of a link between the androgen signaling and the role of Abi1 in prostate cancer we found that Abi1 gene contains androgen receptor (AR) binding sites. Consistent with the idea that Abi1 is an AR responsive gene, expression levels of Abi1 are increased following AR stimulation with the synthetic androgen, R1881, of the hormone sensitive cell line LNCaP. We also observed increased levels of E-cadherin at the cell-cell junctions. In contrast, cells lacking Abi1 do not form proper cell junctions neither respond to AR stimulation in the same way as wild type cells. We propose that Abi1 is a critical part of androgen signaling by promoting cell-cell junctions and tissue integrity. Supported by NCI R01 CA161018 (LK). Citation Format: Baylee Porter, Disharee Nath, Susan A. Krum, Gennady Bratslavsky, Vladimir A. Kuznetsov, Leszek Kotula. Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling [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 5307.

PDCD4 Is an Androgen-Repressed Tumor Suppressor that Regulates Prostate Cancer Growth and Castration Resistance.

Androgen receptor (AR) transcriptional activity contributes to prostate cancer development and castration resistance. The growth and survival pathways driven by AR remain incompletely defined. Here, we found PDCD4 to be a new target of AR signaling and a potent regulator of prostate cancer cell growth, survival, and castration resistance. The 3' untranslated region of PDCD4 is directly targeted by the androgen-induced miRNA, miR-21. Androgen treatment suppressed PDCD4 expression in a dose responsive and miR-21-dependent manner. Correspondingly, AR inhibition dose-responsively induced PDCD4 expression. Using data from prostate cancer tissue samples in The Cancer Genome Atlas (TCGA), we found a significant and inverse correlation between miR-21 and PDCD4 mRNA and protein levels. Higher Gleason grade tumors exhibited significantly higher levels of miR-21 and significantly lower levels of PDCD4 mRNA and protein. PDCD4 knockdown enhanced androgen-dependent cell proliferation and cell-cycle progression, inhibited apoptosis, and was sufficient to drive androgen-independent growth. On the other hand, PDCD4 overexpression inhibited miR-21-mediated growth and androgen independence. The stable knockdown of PDCD4 in androgen-dependent prostate cancer cells enhanced subcutaneous tumor take rate in vivo, accelerated tumor growth, and was sufficient for castration-resistant tumor growth. IMPLICATIONS: This study provides the first evidence that PDCD4 is an androgen-suppressed protein capable of regulating prostate cancer cell proliferation, apoptosis, and castration resistance. These results uncover miR-21 and PDCD4-regulated pathways as potential new targets for castration-resistant prostate cancer.

Abstract B037: N-Myc driven cell plasticity in castrate-resistant prostate cancer

Abstract An emerging concept of anti-AR therapy resistance is the induction of epithelial plasticity in castration-resistant prostate adenocarcinoma (CRPC) cells that eventually evolve to a neuroendocrine phenotype (e.g., low to no AR signaling and expression of neuroendocrine markers). Neuroendocrine prostate cancer (NEPC) is clinically aggressive and carries a poor prognosis with an average survival of less than one year. We and others have identified and validated new therapeutic targets and drivers of cell transformation from CRPC to NEPC. These include induction of MYCN (encodes N-Myc) or RB1/TP53 loss of function. While N-Myc is a bona fide driver oncogene in several rare tumor types, the N-Myc-driven molecular reprogramming and acquisition of cell plasticity remains poorly understood. We have previously shown that intact N-Myc+/Pten-null genetically engineered mice (GEM) develop poorly differentiated, highly proliferative, invasive prostate cancer that is molecularly similar to human NEPC tumor (1). Similar morphologic features were observed in organoids derived from our GEM model. Flow analysis of these organoids revealed that N-Myc+/Pten-null GEM-derived organoids are comprised of a heterogeneous population of cells with luminal, basal, or features of both lineages compared to more homogeneous population of cells from Pten-null control organoids. Recently, we also found that N-Myc overexpression induced a faster recurrence in the context of castration in vivo, based on the GEM model and 22Rv1 isogenic xenografts. This is consistent with the significant N-Myc induced increased castrate-resistant tumor growth based on data from GEM organoids grown in vivo as allografts. In the GEM, we found that castration led to invasive prostate tumors that metastasize to multiple locations including the liver and that express the EMT marker vimentin and the NEPC marker neural cell adhesion molecule (NCAM1). Based on RNAseq data from 22Rv1 xenografts in castrated mice, we identified stem cell signatures including neural crest stem genes (GSEA q value = 7.0E-19) that were significantly enriched in the N-Myc upregulated genes including SOX2, SOX10, and SOX11, genes implicated in neural development and NEPC associated genes (e.g., NCAM1 and chromogranin A [CHGA]). N-Myc ChIP-Seq data confirmed N-Myc occupancy enriched at promoters of genes implicated in stem cell-associated pathways. RNAseq data from human NEPC organoid following MYCN depletion revealed similar N-Myc-associated gene expression changes. Altogether, these data led to a model in which N-Myc overexpression induces a castrate-resistant, lineage-plastic phenotype that would give rise to NEPC, consistent with the phenotype induced by RB1/TP53/PTEN loss [2-4].

Abstract A52: Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer

Abstract Introduction: Recent advances improved survival in men with metastatic castration-resistant prostate cancer (CRPC) after chemotherapy when treated with androgen ablation therapies, including the well-known androgen receptor (AR) antagonist enzalutamide. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next-generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR-positive and -negative prostate cancers. Here we report the further development of effective N-cadherin specific monoclonal antibodies against CRPC and their value in combination therapies. Method: Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen-dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with enzalutamide. Further, enzalutamide-resistant cell lines were also generated and evaluated. In vivo studies were performed using castration-resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to enzalutamide or mAb alone or in combination. When the tumors reached 100-200 mm3 in volume the mice were castrated and treated with 5-10 mg/kg enzalutamide and/or 5-10 mg/kg mAb candidates. Tumors were monitored for growth, local invasion, and metastasis. At endpoint, tumors were harvested and further analyzed. Promising mAb candidates, as well as new epitope targets, were then selected and reformatted into full humanized N-cadherin monoclonal antibodies and further analyzed. Results: Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. In vitro studies showed that combination treatment of N-cadherin positive and AR-positive cells reduced invasion and cell proliferation than either treatment alone. Certain enzalutamide-resistant cell lines generated showed an increase in N-cadherin expression. Such cells responded to N-cadherin antibodies in androgen-deprived conditions, suggesting a role for N-cadherin in a subset of enzalutamide resistance. In vivo studies using CRPC xenograft models expressing N-cadherin and AR showed inhibition of tumor growth when the animals were castrated and treated with a combination of enzalutamide and N-cadherin mAb than either treatment alone. Androgen-dependent xenograft models in castrated mice also significantly delayed castration-resistant tumor growth when treated in combination than either treatment alone. The newly generated humanized mAbs showed similar outcomes. Conclusion: Previous studies suggested that therapeutic targeting of N-cadherin with monoclonal antibodies have significant effect in inhibiting tumor progression. We have verified that humanized N-cadherin mAb exhibit significant efficacy, fostering our search for increasingly effective immunotherapy in the clinic. Further, the antibodies showed synergy in combination with enzalutamide, suggesting promise in potential combination therapies for CRPC. Other androgen receptor antagonists and other drugs targeting those potentially involved in downstream pathways may prove to have additional clinical benefit. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Joyce Yamashiro, Wang Chun, Della Z. Hu, Anna M. Wu, Robert E. Reiter. Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A52.

Abstract 5173: Role of Gli2 in mediating the progression of prostate cancer to CRPC

Abstract Background: Prostate cancer is a leading cause of cancer-related death in the US men, largely due to the development of castration resistant prostate cancer (CRPC) in the patients treated with androgen deprivation therapy. There is increasing evidence indicating that Hedgehog pathway is involved in the malignancy of prostate cancer, which includes the finding of two prostate tumors with loss-of-function mutations in the SuFu gene, correlation of tumor grade with high expression of Sonic hedgehog, Gli2 and/or hedgehog target genes including Ptch1 and HIP, and increased Gli2 expression in therapy-resistant tumor cells from patients. In addition, treatment of mice with hedgehog inhibitors significantly attenuated the growth of prostate tumor xenografts. Some recent studies also implicated a crosstalk between Hedgehog/Gli signaling and androgen signaling pathways in prostate cancer. However, the role of Gli2 in CRPC progression has not been investigated. Methods: A human Gli2 specific small hairpin RNA (shRNA) expression vector was constructed using Tet-PLKO-puro lentivector, which was delivered into the androgen-dependent human prostate cancer LNCaP cells. RT-PCR, Western Blot and Luciferase Reporter Assay were performed to confirm doxycycline-inducible Gli2 knockdown. The effect of Gli2 knockdown on anchorage-dependent or -independent growth was assessed with MTT and Soft Agar Assays respectively. Cells were cultured in androgen depleted medium or treated with the anti-androgen, Casodex, to mimic androgen deprivation condition. Mouse xenograft models were used to assess the role of Gli2 in the development of CRPC in vivo. Results: Androgen deprivation promoted the expression of Hedgehog signaling components including Gli2 in the LNCaP cells. Gli2 knockdown inhibited anchorage-dependent and -independent growth of LNCaP cells. Casodex complemented the inhibition of Gli2 knockdown on the growth of LNCaP cells. Gli2 knockdown prevented the outgrowth of androgen-independent cells from LNCaP cells cultured in androgen depleted medium. Induced-knockdown of Gli2 by doxycycline inhibited the growth of castration-resistant tumors in vivo in mice. Conclusions: Our study demonstrated the important role of Gli2 in the development of CRPC. Therefore, it may have the potential to serve as a novel therapeutic target for the prevention of CRPC. Citation Format: Lu Xia, Tai Qin, Luzhe Sun. Role of Gli2 in mediating the progression of prostate cancer to CRPC. [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 5173.

Abstract 334: Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer

Abstract (A) Introduction: Because of the central role of androgen receptor (AR) signaling in prostate cancer (PC), AR inhibition is the primary modality of initial PC treatment. While AR-targeted therapies are often initially effective in castration-resistant prostate cancer (CRPC), disease progression is common. AR and glucocorticoid receptor (GR) are structurally similar nuclear receptors, bind the same DNA response elements, and regulate common genes. Previous work from our lab demonstrated that GR expression is upregulated following AR antagonism, GR expression decreased the effectiveness of AR blockade, and inhibiting GR activity reversed these effects. However, the only FDA-approved GR antagonist, mifepristone, is not GR-specific and has significant drug-drug interactions; therefore, we report our initial data with a new generation of selective GR-antagonists (SGRMs) for PC treatment using models of CRPC. (B) Experimental Procedures: The LAPC4, CWR-22Rv1 and VCAP PC cell lines were utilized. GR target gene expression analysis was performed at various time-points by qRT-PCR and protein immunoblots subsequent to treatment with AR agonist (R1881, 1nM), AR antagonist (enzalutamide, 10 μM), GR agonist (dexamethasone, 100nM) and the new SGRMs CORT108297 and CORT118335(1μM). Quantification of cell numbers in vitro in response to these treatments was determined by trypan blue exclusion/live cell counting and the CellTiter Glo 2.0 ATP-luciferase assay. For in vivo studies, PC cells were injected subcutaneously into male nude mice and grown to a predetermined tumor size, mice were then castrated, and fifteen days later treated with mifepristone (12mg/kg), SGRM (16mg/kg, 20mg/kg), or vehicle by daily intra-peritoneal injection to assess time to CRPC. (C) Results: AR antagonism with concurrent GR activation resulted in increased expression of canonical AR/GR-target genes and SGRMs (1μM) effectively inhibited expression of glucocorticoid-mediated genes at the RNA and protein levels. SGRMs did not affect AR-mediated gene expression. When AR was antagonized, activated GR promoted cell survival, which was inhibited by SGRM treatment to varying degrees. Preliminary studies support the hypothesis that these novel SGRMs delay CRPC tumor xenograft progression in vivo. (D) Conclusion: In vitro, new generation SGRMs inhibit GR-mediated, but not AR-selective gene expression and result in decreased tumor cell number. SGRM administration to nude mice bearing PC xenografts can delay castration-resistant tumor growth. Therefore, new SGRMs may be an effective therapy for CRPC treatment worthy of further exploration. Studies are ongoing to determine how SGRMs modulate GR target gene expression globally and whether they decrease expression of tumorigenic pro-survival genes in vivo. Citation Format: Jacob Kach, Phillip Selman, Diana C. West, Donald J. Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer. [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 334.

Targeting Oct1 genomic function inhibits androgen receptor signaling and castration-resistant prostate cancer growth.

Androgen receptor (AR) functions as a ligand-dependent transcription factor to regulate its downstream signaling for prostate cancer progression. AR complex formation by multiple transcription factors is important for enhancer activity and transcriptional regulation. However, the significance of such collaborative transcription factors has not been fully understood. In this study, we show that Oct1, an AR collaborative factor, coordinates genome-wide AR signaling for prostate cancer growth. Using global analysis by chromatin immunoprecipitation sequencing (ChIP-seq), we found that Oct1 is recruited to AR-binding enhancer/promoter regions and facilitates androgen signaling. Moreover, a major target of AR/Oct1 complex, acyl-CoA synthetase 3 (ACSL3), contributes to tumor growth in nude mice, and its high expression is associated with poor prognosis in prostate cancer patients. Next, we examined the therapeutic effects of pyrrole-imidazole polyamides that target the Oct1-binding sequence identified in the center of the ACSL3 AR-binding site. We observed that treatment with Oct1 polyamide severely blocked the Oct1 binding at the ACSL3 enhancer responsible for its transcriptional activity and ACSL3 induction. In addition, Oct1 polyamides suppressed castration-resistant tumor growth and specifically repressed global Oct1 chromatin association and androgen signaling in prostate cancer cells, with few nonspecific effects on basal promoter activity. Thus, targeting Oct1 binding could be a novel therapeutic strategy for AR-activated castration-resistant prostate cancer.

Abstract 3636: Targeting the MLL complex in castration resistant prostate cancer

Abstract Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration resistant prostate cancer (CRPC). Substantial prior work has focused on targeting AR directly; however, the identification and therapeutic targeting of co-activators of AR signaling remains an underexplored area of potential clinical significance. Here we demonstrate that the MLL (mixed-lineage leukemia) complex, a well-known contributor in MLL-fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via its critical subunit, menin. Small molecule inhibition of the menin-MLL interaction blocks AR signaling and inhibits the growth of castration resistant tumors in vivo. Furthermore, we find that menin is up-regulated in castration resistant prostate cancer and high expression correlates with poor overall survival. Taken together, our study identifies the MLL complex as a critical co-activator of AR that can be targeted in advanced prostate cancer. Citation Format: Rohit Malik, Amjad P. Khan, Irfan A. Asangani, Marcin Cieślik, John R. Prensner, Xiaoju Wang, Matthew K. Iyer, Xia Jiang, Dmitry Borkin, June Escara-Wilke, Rachell Stender, Yi-Mi Wu, Xuhong Cao, Felix Y. Feng, Jolanta Grembecka, Tomasz Cierpicki, Arul M. Chinnaiyan. Targeting the MLL complex in castration resistant 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 3636. doi:10.1158/1538-7445.AM2015-3636

MP66-10 CHARACTERIZATION OF SUSCEPTIBILITY LOCI IN HEREDITARY PROSTATE CANCER FAMILIES USING COPY NUMBER VARIATION AND LINKAGE ANALYSIS

INTRODUCTION AND OBJECTIVES: The intratumoral androgen synthesis is one of the mechanisms by which androgen receptor (AR) is aberrantly re-activated in castration-resistant prostate cancer (CRPC) after androgen ablation. However, pathways controlling steroidogenic enzyme expression and de novo androgen synthesis in prostate cancer (PCa) cells are largely unknown. In this study, we explored the potential roles of DAB2IP/AKR1C3 signaling in androgen synthesis and castration-resistant progression of PCa. METHODS: Tumor growth of two PCa cells (androgenindependent C4-2 and androgen-dependent LAPC-4) with or without DAB2IP expression was compared under castrated condition by MTT assay and subcutaneous xenograft model. Expression levels of steroidogenic enzymes were measured by real-time RT-PCR. Regulation of AKR1C3 expression by DAB2IP, subsequent alteration of AR activity and prostate-specific antigen (PSA) re-expression were examined by luciferase reporter assays or western blotting. Intracrine levels of androgens were measured by EIA in PCa cells, xenografts or castrated DAB2IP-/mice prostate tissues. The correlation between DAB2IP and AKR1C3 expression in PCa xenografts, DAB2IP-/mice prostate and human PCa tissues were examined by immunohistochemical (IHC) staining or western blotting. RESULTS: DAB2IP loss could potentiate the castration-resistant tumor growth of PCa cells in vitro and in vivo, and reprogram the expression profiles of steroidogenic enzymes, such as AKR1C3, CYP17A1, 17BHSD2, 17BHSD3, AKR1C1/2 and UGT2B17. Furthermore, DAB2IP coulddramatically inhibit thepromoter activity ofAKR1C3andsuppress its expression. Consistently, the levels of total testosteronewere significantly higher in DAB2IP-deficient PCa cells, xenografts or DAB2IP-/mice prostate tissues, in which more AR phosphorylation or nuclear staining and higher PSA expression were observed. Moreover, knockdown of AKR1C3 using shRNA could abolish all of these phenomena. In addition, DAB2IP expression inversely correlated with AKR1C3 in PCa xenografts, DAB2IP-/mice prostate and human PCa tissues. CONCLUSIONS: Loss of DAB2IP in PCa cells enhances AKR1C3 expression and de novo androgen synthesis, which may contribute to the development of CPRC.

Targeting the MLL complex in castration-resistant prostate cancer.

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration resistant prostate cancer (CRPC). Although prior work focused on targeting AR directly, co-activators of AR signaling—which may represent new therapeutic targets—are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia (MLL) complex, a well-known driver of MLL-fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin MLL subunit. Menin expression is higher in castration resistant prostate cancer compared to hormone naïve prostate cancer and benign prostate and high menin expression correlates with poor overall survival. Treatment with a small molecule inhibitor of the menin-MLL interaction blocks AR signaling and inhibits the growth of castration resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a critical co-activator of AR and a potential therapeutic target in advanced prostate cancer.

Abstract 3310: N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers

Abstract Introduction: Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance. Previously, different groups have suggested an association between chemoresistance and epithelial to mesenchimal transition (EMT) in certain advanced cancers. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with EMT, is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: Tissue microarrays of CRPC and high grade prostate cancer clinical samples were stained for TLR4. N-cadherin positive and negative cell lines were evaluated for sensitivity against docetaxel. N-cadherin and TLR4 were ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, VCaP). Docetaxel resistant cell lines (LNCaP-DocR and VCaP-DocR) were established through prolonged exposure to 10-20uM Docetaxel. For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. N-cadherin expressing cell lines were treated with 2A9 monoclonal antibody (mAb), which was raised against the extracellular domain of N-cadherin, and by N-cadherin siRNA knockdown. For in vivo studies, N-cadherin positive xenografts were treated with 2A9 mAb and docetaxel, and tumor growth monitored over time. Results: Tissue microarray results showed significant TLR4 expression in CRPC samples (p < 0.005). In vitro studies showed TLR4 induction correlated with N-cadherin expression. This upregulation of N-cadherin and TLR4 expression promoted docetaxel resistance in CRPC by the activation of NF-kB signaling. Monoclonal antibodies against N-cadherin and TLR4 potentiated docetaxel induced cytotoxicity. Docetaxel resistant cells exhibited EMT phenotypes with increased invasion and castration resistant growth. Treatment with 2A9 antibody restored sensitivity to docetaxel. Combination therapy with 2A9 monoclonal antibody and docetaxel significantly affected tumor growth in N-cadherin positive CRPC xenograft models. Conclusions: These studies suggest that N-cadherin promotes TLR4-dependent docetaxel resistance in CRPC. Although further investigation will be needed, therapeutic targeting of N-cadherin with monoclonal antibodies in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Citation Format: Tatsuya Shimomura, Evelyn Kono, Chau P. Tran, Joyce Yamashiro, Shu Lin, Sean Hyung-Kwon Lee, Zev A. Wainberg, Robert E. Reiter. N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers. [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 3310. doi:10.1158/1538-7445.AM2014-3310

Synergistic Targeting of PI3K/AKT Pathway and Androgen Receptor Axis Significantly Delays Castration-Resistant Prostate Cancer ProgressionIn Vivo

The progression to castration-resistant prostate cancer (CRPC) correlates with gain-of-function of the androgen receptor (AR) and activation of AKT. However, as single agents, AR or AKT inhibitors result in a reciprocal feedback loop. Therefore, we hypothesized that combination of an AKT inhibitor with an antiandrogen might result in a more profound, long-lasting remission of CRPC. Here, we report that the AKT inhibitor AZD5363 potently inhibits proliferation and induces apoptosis in prostate cancer cell lines expressing the AR and has anticancer activity in vivo in androgen-sensitive and castration-resistant phases of the LNCaP xenograft model. However, we found that the effect of castration-resistant tumor growth inhibition and prostate-specific antigen (PSA) stabilization is transient and resistance occurs with increasing PSA after approximately 30 days of treatment. Mechanistically, we found that single agent AZD5363 induces increase of AR binding to androgen response element, AR transcriptional activity, and AR-dependent genes such as PSA and NKX3.1 expression. These effects were overcome by the combination of AZD5363 with the antiandrogen bicalutamide, resulting in synergistic inhibition of cell proliferation and induction of apoptosis in vitro, and prolongation of tumor growth inhibition and PSA stabilization in CRPC in vivo. This study provides a preclinical proof-of-concept that combination of an AKT inhibitor with antiandrogen results in prolonged disease stabilization in a model of CRPC.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Estrogen receptor β expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naïve prostate cancer and are elevated in castration-resistant disease

Patients with advanced prostate cancer (PC) are usually treated with androgen withdrawal. While this therapy is initially effective, nearly all PCs become refractory to it. As hormone receptors play a crucial role in this process, we constructed a tissue microarray consisting of PC samples from 107 hormone-naïve (HN) and 101 castration-resistant (CR) PC patients and analyzed the androgen receptor (AR) gene copy number and the protein expression profiles of AR, Serin210-phosphorylated AR (pAR(210)), estrogen receptor (ER)β, ERα and the proliferation marker Ki67. The amplification of the AR gene was virtually restricted to CR PC and was significantly associated with increased AR protein expression (P<0.0001) and higher tumor cell proliferation (P=0.001). Strong AR expression was observed in a subgroup of HN PC patients with an adverse prognosis. In contrast, the absence of AR expression in CR PC was significantly associated with a poor overall survival. While pAR(210) was predominantly found in CR PC patients (P<0.0001), pAR(210) positivity was observed in a subgroup of HN PC patients with a poor survival (P<0.05). Epithelial ERα expression was restricted to CR PC cells (9%). ERβ protein expression was found in 38% of both HN and CR PCs, but was elevated in matched CR PC specimens. Similar to pAR(210), the presence of ERβ in HN patients was significantly associated with an adverse prognosis (P<0.005). Our results strongly suggest a major role for pAR(210) and ERβ in HN PC. The expression of these markers might be directly involved in CR tumor growth.

Zyflamend, a Combination of Herbal Extracts, Attenuates Tumor Growth in Murine Xenograft Models of Prostate Cancer

Prostate cancer (PrC) is the second deadliest cancer of males in the United States Hormone deprivation therapy (HDT), a common therapy for advanced forms of the disease, results in tumor regression; unfortunately, tumors inevitably become castrate-resistant. Diet is not an appropriate primary therapy for refractory forms of the disease; however, diet may be effective as an adjuvant to HDT, potentially extending the latency period and delaying relapse and/or inhibiting refractory growth. Zyflamend® is a combination of extracts from multiple herbs, each with reported anticancer properties. Zyflamend can inhibit growth of various PrC cell lines, but no studies have investigated its potential use in vivo using a model of castrate-resistant PrC. In this study, oral doses of Zyflamend at human equivalent doses inhibited androgen-dependent and castrate-resistant tumor growth in a mouse model that mimics advanced stages of the disease, and reduced the expression of a number of biomarkers linked to PrC progression including pAKT, prostate specific antigen, histone deacetylases, and androgen receptor. In summary, this is the first article to report that Zyflamend, when provided at human equivalent doses, can potentiate the effects of hormone deprivation on tumor regression and growth inhibition of androgen-dependent and castrate-resistant PrC tumors in vivo.

Vascular responses to radiotherapy and androgen-deprivation therapy in experimental prostate cancer

BackgroundRadiotherapy (RT) and androgen-deprivation therapy (ADT) are standard treatments for advanced prostate cancer (PC). Tumor vascularization is recognized as an important physiological feature likely to impact on both RT and ADT response, and this study therefore aimed to characterize the vascular responses to RT and ADT in experimental PC.MethodsUsing mice implanted with CWR22 PC xenografts, vascular responses to RT and ADT by castration were visualized in vivo by DCE MRI, before contrast-enhancement curves were analyzed both semi-quantitatively and by pharmacokinetic modeling. Extracted image parameters were correlated to the results from ex vivo quantitative fluorescent immunohistochemical analysis (qIHC) of tumor vascularization (9 F1), perfusion (Hoechst 33342), and hypoxia (pimonidazole), performed on tissue sections made from tumors excised directly after DCE MRI.ResultsCompared to untreated (Ctrl) tumors, an improved and highly functional vascularization was detected in androgen-deprived (AD) tumors, reflected by increases in DCE MRI parameters and by increased number of vessels (VN), vessel density ( VD), and vessel area fraction ( VF) from qIHC. Although total hypoxic fractions ( HF) did not change, estimated acute hypoxia scores ( AHS) – the proportion of hypoxia staining within 50 μm from perfusion staining – were increased in AD tumors compared to in Ctrl tumors. Five to six months after ADT renewed castration-resistant (CR) tumor growth appeared with an even further enhanced tumor vascularization. Compared to the large vascular changes induced by ADT, RT induced minor vascular changes. Correlating DCE MRI and qIHC parameters unveiled the semi-quantitative parameters area under curve ( AUC) from initial time-points to strongly correlate with VD and VF, whereas estimation of vessel size ( VS) by DCE MRI required pharmacokinetic modeling. HF was not correlated to any DCE MRI parameter, however, AHS may be estimated after pharmacokinetic modeling. Interestingly, such modeling also detected tumor necrosis very strongly.ConclusionsDCE MRI reliably allows non-invasive assessment of tumors’ vascular function. The findings of increased tumor vascularization after ADT encourage further studies into whether these changes are beneficial for combined RT, or if treatment with anti-angiogenic therapy may be a strategy to improve the therapeutic efficacy of ADT in advanced PC.

Effect of dietary polyunsaturated fatty acids on castration-resistant Pten-null prostate cancer

A common treatment of advanced prostate cancer involves the deprivation of androgens. Despite the initial response to hormonal therapy, eventually all the patients relapse. In the present study, we sought to determine whether dietary polyunsaturated fatty acid (PUFA) affects the development of castration-resistant prostate cancer. Cell culture, patient tissue microarray, allograft, xenograft, prostate-specific Pten knockout and omega-3 desaturase transgenic mouse models in conjunction with dietary manipulation, gene knockdown and knockout approaches were used to determine the effect of dietary PUFA on castration-resistant Pten-null prostate cancer. We found that deletion of Pten increased androgen receptor (AR) expression and Pten-null prostate cells were castration resistant. Omega-3 PUFA slowed down the growth of castration-resistant tumors as compared with omega-6 PUFA. Omega-3 PUFA decreased AR protein to a similar extent in tumor cell cytosolic and nuclear fractions but had no effect on AR messenger RNA level. Omega-3 PUFA treatment appeared to accelerate AR protein degradation, which could be blocked by proteasome inhibitor MG132. Knockdown of AR significantly slowed down prostate cancer cell proliferation in the absence of androgens. Our data suggest that omega-3 PUFA inhibits castration-resistant prostate cancer in part by accelerating proteasome-dependent degradation of the AR protein. Dietary omega-3 PUFA supplementation in conjunction with androgen ablation may significantly delay the development of castration-resistant prostate cancer in patients compared with androgen ablation alone.

Abstract CN08-01: Targeting the androgen receptor in castration-resistant prostate cancer.

Abstract CN08-01: Targeting the androgen receptor in castration-resistant prostate cancer.

Abstract 5052: N-cadherin monoclonal antibody as a therapeutic agent against chemoresistance in prostate cancer

Abstract Introduction: The development of chemoresistance is a formidable obstacle in realizing effective therapy against cancer. Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance to the treatment. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with Epithelial to mesenchymal transition (EMT), is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: N-cadherin was ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, MDA PCa 2b). For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. Endogenously expressing N-cadherin were either treated with monoclonal antibodies raised against the extracellular domain of N-cadherin or N-cadherin expression was reduced by siRNA knockdown. For in vivo studies, monoclonal antibodies raised against N-cadherin were used in combination with docetaxel to treat N-cadherin positive xenografts, and tumor growth monitored over time. Results: Ectopic expression of N-cadherin in N-cadherin negative cells resulted in &amp;gt;25-fold increase in resistance to cytotoxicity by docetaxel treatment when compared with control cells. N-cadherin knockdown by siRNA of cells endogenously expressing N-cadherin resulted in an increase of cytotoxicity in response to docetaxel when compared with cells transfected with scramble siRNA. Treatment with monoclonal antibody also resulted in increase of cytotoxicity in response to docetaxel. In vitro studies also show that increase in N-cadherin leads to upregulation in NFkB (&amp;gt;2.5-fold ± 0.17 increase in activity) and Toll-like receptor signaling, which are implicated in chemoresistance. Preliminary in vivo studies show some additive effect monoclonal antibodies against N-cadherin in combination with docetaxel on growth of xenograft tumors expressing N-cadherin. Conclusions: These studies suggest that N-cadherin is involved in the development of chemoresistance in prostate cancers. Therapeutic targeting of N-cadherin with monoclonal antibodies, in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Future experiments involve further elucidating the pathways involved and downstream targets, such as those involved in NFkB and Toll-like receptor signaling. 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 5052. doi:10.1158/1538-7445.AM2011-5052