Androgen-dependent Prostate Cancer Cells Research Articles

CA916798 affects growth and metastasis of androgen-dependent prostate cancer cells.

Abnormal activation of androgen receptor (AR) signaling pathway is a critical pathogenic mechanism and therapeutic target for prostate cancer (PCa). The CA916798 is a tumor-associated gene and may be regulated by the androgen-AR pathway. This study aims to investigate the function of CA916798 in the growth and metastasis of androgen-dependent PCa cells. CA916798 expression in PCa cell lines was investigated. LNCap cells were divided into 4 groups: LNCap, LNCap+ Dihydrotestosterone (DHT), LNCap+DHT+siCA916798, and LNCap+DHT+siRA group. CA916798 expressions in LNCap cells treated with siCA917698 or siAR were examined. The viability, apoptosis, migration, and invasion of PCa cells were examined. Dual luciferase and ChIP assays were used to examine the interaction between the AR and CA916798. Endogenous CA916798 mRNA levels in PC3 cells were significantly higher than those in LNCap cells (p < 0.05). However, CA916798 was androgen-sensitive in LNCap cells, but not in PC-3 cells. Dual luciferase and ChIP assays showed that AR could specifically bind to the promoter regions of the CA916798. Knockdown of CA916798 (LNCap+DHT+siCA916798) and AR (LNCap+DHT+siAR) resulted in decreased cell viability, migration, and invasion, while it induced apoptosis and G1 cell cycle arrest in LNCap cells. DHT could initiate the transcription of CA916798, which further mediates the androgen-AR signaling pathway-dependent cell growth and metastasis of the prostate cancer cell line LNCap.

Bicalutamide enhances fodrin-mediated apoptosis through calpain in LNCaP.

Prostate cancer is the most common cancer in men, and before it progresses and metastasizes, the anticancer drug bicalutamide is often administered to patients. Many cases of androgen-dependent prostate cancer develop resistance during treatment with bicalutamide. Therefore, the effect of bicalutamide on androgen-dependent LNCaP prostate cancer cells is of clinical interest. The aim of this study was to demonstrate the effects of the anticancer drug bicalutamide on LNCaP prostate cancer cells by using a proteomics approach. Based on the results, 314 proteins were differentially expressed between the LNCaP and LNCaP treated with bicalutamide. The apoptosis pathway associated with differentially expressed proteins was shown in the Kyoto Encyclopedia of Gene and Genome pathway mapper. The Kyoto Encyclopedia of Gene and Genome pathway mapper results revealed that the fodrin-mediated apoptosis pathway is associated with the actions of bicalutamide and Western blotting was performed to validate these results. Impact statement We studied bicalutamide's anticancer action by using proteomics. The effect of bicalutamide on androgen-exposed LNCaP cells was also studied. KEGG identified >1.8-fold differentially expressed proteins between test group cells. KEGG mapper showed fodrin-mediated apoptosis involvement in bicalutamide's action. The anticancer effects of bicalutamide, which was further confirmed using Western blotting. Therefore, this drug is a potential candidate for understanding bicalutamide's effect on LNCaP and fodrin can be used as a biomarker monitoring status in metastatic carcinoma.

Melatonin Analogue Antiproliferative and Cytotoxic Effects on Human Prostate Cancer Cells.

Melatonin has been indicated as a possible oncostatic agent in different types of cancer, its antiproliferative role being demonstrated in several in vitro and in vivo experimental models of tumors. Specifically, melatonin was proven to inhibit cell growth of both androgen-dependent and independent prostate cancer cells, through various mechanisms. A number of melatonin derivatives have been developed and tested for their role in the prevention and treatment of neoplastic diseases. We recently proved the in vitro and in vivo anticancer activity of UCM 1037, a newly-synthetized melatonin analogue, on melanoma and breast cancer cells. In this study we evaluated UCM 1037 effects on cell proliferation, cell cycle distribution, and cytotoxicity in LNCaP, PC3, DU145, and 22Rv1 prostate cancer cells. We demonstrated significant dose- and time-dependent UCM 1037 antiproliferative effects in androgen-sensitive LNCaP and 22Rv1 cells. Data from flow cytometric studies suggest that UCM 1037 is highly cytotoxic in androgen-sensitive prostate cancer cells, although no substantial increase in the apoptotic cell fraction has been observed. UCM 1037 cytotoxic effects were much less evident in androgen-insensitive PC3 and DU145 cells. Experiments performed to gain insights into the possible mechanism of action of the melatonin derivative revealed that UCM 1037 down-regulates androgen receptor levels and Akt activation in LNCaP and 22Rv1 cells.

Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer progression

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of cancer‐derived exosomes is a prerequisite for their better understanding. We have investigated a new biomarker for prostate cancer progression via quantitative proteomics. Exosomes from androgen‐independent PC‐3 and the androgen‐dependent LNCaP prostate cancer cells were isolated by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ‐Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Proteomic characterization showed that 105 proteins from PC‐3 exosomes were 10‐fold higher than those from LNCap. Validation by Western blotting and qRT‐PCR confirmed a higher abundance of PFKL, NRP1 and MCAM(CD146) in PC‐3 cell exosomes, too, indicating that aggressive prostate cancer exhibit higher levels of adhesion or cohesion proteins. We also show that MCAM (CD146) was strongly associated to PC‐3 mobility in vitro. These data provided that MCAM should be useful as a new candidate biomarker for prostate cancer progression.Support or Funding InformationThis work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) (OGM4391711)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Orphan nuclear receptor TLX contributes to androgen insensitivity in castration-resistant prostate cancer via its repression of androgen receptor transcription

The metastatic castration-resistant prostate cancer (CRPC) is a lethal form of prostate cancer, in which the expression of androgen receptor (AR) is highly heterogeneous. Indeed, lower AR expression and attenuated AR signature activity is shown in CRPC tissues, especially in the subset of neuroendocrine prostate cancer (NEPC) and prostate cancer stem-like cells (PCSCs). However, the significance of AR downregulation in androgen insensitivity and de-differentiation of tumor cells in CRPC is poorly understood and much neglected. Our previous study shows that the orphan nuclear receptor TLX (NR2E1), which is upregulated in prostate cancer, plays an oncogenic role in prostate carcinogenesis by suppressing oncogene-induced senescence. In the present study, we further established that TLX exhibited an increased expression in metastatic CRPC. Further analyses showed that overexpression of TLX could confer resistance to androgen deprivation and anti-androgen in androgen-dependent prostate cancer cells in vitro and in vivo, whereas knockdown of endogenous TLX could potentiate the sensitivity to androgen deprivation and anti-androgen in prostate cancer cells. Our study revealed that the TLX-induced resistance to androgen deprivation and anti-androgen was mediated through its direct suppression of AR gene transcription and signaling in both androgen-stimulated and -unstimulated prostate cancer cells. We also characterized that TLX could bind directly to AR promoter and repress AR transcription by recruitment of histone modifiers, including HDAC1, HDAC3, and LSD1. Together, our present study shows, for the first time, that TLX can contribute to androgen insensitivity in CRPC via repression of AR gene transcription and signaling, and also implicates that targeting the druggable TLX may have a potential therapeutic significance in CRPC management, particularly in NEPC and PCSCs.

The promotion on cell growth of androgen-dependent prostate cancer by antimony via mimicking androgen activity

Antimony is a widely used heavier pnictogens in industry, and its toxicity has been a matter of concern. Although previous studies have suggested that antimony may have the function as either a tumor suppressor or an oncogene in several cancers, the molecular basis underlying antimony-mediated transformation is still unclear. In the current study, we attempt to elucidate the potential role of antimony in the development of prostate cancer. Our results showed that the concentration of antimony was much higher in serum of prostate cancer patients, and was closely associated with poor outcome of patients who underwent radical prostatectomy. Additionally, low dose of antimony could promote proliferation and invasion of androgen-dependent prostate cancer cell line LNCaP cells in vitro and in vivo. The mechanistic studies demonstrated that exposure to antimony triggered the phosphorylation of androgen receptor (AR), which transcriptionally regulates the expression of androgen-related targets, including PSA and NKX3.1. Overall, our results unearthed that antimony could promote tumor growth by mimicking androgen activity in androgen-dependent prostate cancer cells. Therefore, these findings expanded our understanding on the molecular mechanism of antimony in tumorigenesis and tumor progression of prostate cancer, and it appears to be an inspiring strategy to restrain prostate cancer by inhibiting antimony-induced androgen-like effects.

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.

Enhanced radiosensitization of enzalutamide via schedule dependent administration to androgen-sensitive prostate cancer cells.

Prostate cancer (PCa) is a progressive disease and the most diagnosed cancer in men. The current standard of care for high-risk localized PCa is a combination of androgen deprivation therapy (ADT) and radiation (XRT). The majority of these patients however become resistant due to incomplete responses to ADT as a result of selective cells maintaining androgen receptor (AR) activity. Improvement can be made if increasing radiosensitivity is realized. Therefore, the aim of this study is to investigate the efficacy of the next-generation PCa drug Enzalutamide (ENZA), as a radiosensitizer in XRT therapy. Using a number of androgen-dependent (LNCaP, PC3-T877A) and androgen-independent (C4-2, 22RV1, PC3, PC3-AR V7) cell lines, the effect of ENZA as a radiosensitizer was studied alone or in combination with ADT and/or XRT. Cell viability and cell survival were assessed, along with determination of cell cycle arrest, DNA damage response and repair, apoptosis and senescence. Our results indicated that either ENZA alone (in AR positive, androgen-dependent PCa cells) or in combination with ADT (in AR positive, hormone-insensitive PCa cells) potentiates radiation response [Dose enhancement factor (DEF) of 1.75 in LNCAP and 1.35 in C4-2] stronger than ADT + XRT conditions. Additionally, ENZA sensitized androgen dependent PCa cells to XRT in a schedule-dependent manner, where concurrent administration of ENZA and radiation lead to a maximal radiosensitization when compared to either drug administration prior or after XRT. In LNCaP cells, ENZA treatment significantly prolonged the presence of XRT-induced phospho-γH2AX up to 24 h after treatment; suggesting enhanced DNA damage. It also significantly increased XRT-induced apoptosis and senescence. Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ADT. We have also observed that its efficacy is schedule dependent and related to increased levels of DNA damage and a delay of DNA repair processes. Finally, the initial abrogation of DNA-PKcs activity by AR inhibition and its subsequent recovery might represent an important mechanism by which PCa cells acquire resistance to combined anti-androgen and XRT treatment. This work suggests a new use of ENZA in combination with XRT that could be applicable in clinical trial settings for patients with early and intermediate hormone responsive disease.

Extracellular vesicles released by mesenchymal-like prostate carcinoma cells modulate EMT state of recipient epithelial-like carcinoma cells through regulation of AR signaling

Extracellular vesicles released from cancer cells may play an important role in cancer progression by shuttling oncogenic information into recipient cells. However, our knowledge is still fragmentary and there remain numerous questions regarding the mechanisms at play and the functional consequences of these interactions. We have recently established a mesenchymal-like prostate cancer cell line (22Rv1/CR-1; Mes-PCa). In this study, we assessed the effects of the extracellular vesicles released by these cells on recipient androgen-dependent epithelial VCaP prostate cancer cells. Mes-PCa derived vesicles were found to promote mesenchymal features in the recipient epithelial-like prostate cancer cells. This transformation was accompanied by a modulation of androgen receptor signaling and activation of TGFβ signaling pathway. Moreover, recipient cells acquiring mesenchymal traits displayed enhanced migratory and invasive features as well as increased resistance to the androgen receptor antagonist, enzalutamide.Our results suggest a previously unappreciated role for Mes-PCa secreted vesicles in cancer promotion by transferring cell-mediated signals and promoting phenotypic changes in recipient prostate cancer cells.

Peptide B targets soluble guanylyl cyclase α1 and kills prostate cancer cells.

Among androgen-regulated genes, soluble guanylyl cyclase α1 (sGCα1) is significant in promoting the survival and growth of prostate cancer cells and does so independent of nitric oxide (NO) signaling. Peptides were designed targeting sGCα1 to block its pro-cancer functions and one peptide is discussed here. Peptide B-8R killed both androgen-dependent and androgen-independent prostate cancer cells that expressed sGCα1, but not cells that do not express this gene. Peptide B-8R induced apoptosis of prostate cancer cells. Importantly, Peptide B-8R does not affect nor its cytotoxicity depend on NO signaling, despite the fact that it associates with sGCα1, which dimerizes with sGCβ1 to form the sGC enzyme. Just as with a previously studied Peptide A-8R, Peptide B-8R induced elevated levels of reactive oxygen species (ROS) in prostate cancer cells, but using a ROS-sequestering agent showed that ROS was not responsible the cytotoxic activity of Peptide B-8R. Interestingly, Peptide B-8R induced elevated levels of p53 and phosphorylated p38, but neither of these changes is the cause of the peptide’s cytotoxicity. Additional drugs were used to alter levels of iron levels in cells and these studies showed that Peptide B-8R activity does not depend on Ferroptosis. Thus, future work will be directed at defining the mechanism of cytotoxic action of Peptide B-8R against prostate cancer cells.

SLC45A3-ELK4 functions as a long non-coding chimeric RNA

Gene fusions in cancer typically lead to the expression of a fusion protein or disrupt the expression of one of the parental genes. Here we report a new phenomenon whereby a fusion transcript functions as a long non-coding chimeric RNA (lnccRNA). This fusion RNA, SLC45A3-ELK4, generated by cis-splicing between neighboring genes, was found in prostate cancer. The fusion RNA encodes the same protein as ELK4. Intriguingly, we found that the fusion RNA level is less than 1% of wild type ELK4, unlikely to perturb the general pool of ELK4 protein. Nonetheless, when the fusion RNA, but not ELK4 is silenced, cell proliferation is inhibited in both androgen-dependent and castration-resistant prostate cancer cells. This growth arrest can be rescued by exogenous expression of the fusion and a mutant designed to prevent translation of the ELK4 protein. In the same setting, the mutant could also suppress CDKN1A and several other targets of SLC45A3-ELK4. In addition, similar to many long non-coding RNAs, the fusion RNA is enriched in the nuclear fraction. Altogether, these results indicate that SLC45A3-ELK4 regulates cancer cell proliferation by its transcript, not translated protein.

Cytotoxic Effects of Sarcophyton sp. Soft Corals-Is There a Correlation to Their NMR Fingerprints?

Sarcophyton sp. soft corals are rich in cembranoid diterpenes, which represent the main chemical defense of corals against their natural predators in addition to their myriad biological effects in humans. Quantitative NMR (qNMR) was applied for assessing the diterpene variation in 16 soft coral specimens in the context of their genotype, origin, and growing habitat. qNMR revealed high diterpene levels in Sarcophyton sp. compared to Sinularia and Lobophyton, with (ent)sarcophines as major components (17–100 µg/mg) of the coral tissues. Multivariate data analysis was employed to classify samples based on the quantified level of diterpenes, and compared to the untargeted NMR approach. Results revealed that qNMR provided a stronger classification model of Sarcophyton sp. than untargeted NMR fingerprinting. Additionally, cytotoxicity of soft coral crude extracts was assessed against androgen-dependent prostate cancer cell lines (PC3) and androgen-independent colon cancer cell lines (HT-29), with IC50 values ranging from 10–60 µg/mL. No obvious correlation between the extracts’ IC50 values and their diterpene levels was found using either Spearman or Pearson correlations. This suggests that this type of bioactivity may not be easily predicted by NMR metabolomics in soft corals, or is not strongly correlated to measured diterpene levels.

Abstract 98: TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells

Abstract Introduction: Translational control is a critical component in development and progression of cancer and could be explored to develop antineoplastic therapies. Results from our laboratory have shown that TET (a derivative of Tetrandrine) inhibits cell growth and promotes apoptosis of prostate cancer (PCa) cells. However, the underlying molecular mechanisms are not clearly understood. We observed that TET inhibited 4EBP phosphorylation, suggesting that it might inhibit translational initiation. In the present study we evaluated the effects of TET on Translation in general and cell cycle related proteins in particular. Methods: Androgen dependent PCa cells LNCaP and Enz resistant CRPCa cells 22Rv1 were maintained in supplemented RPMI as recommended. Where indicated cells were treated with TET (0 or 20µM) for various time points (2h, 8h, 18h and 24h). At the end of the experimental periods cells were pulsed with cycloheximide for 10 minutes in RPMI media. Cytoplasmic RNA was collected by lysing cells in polysome lysis buffer. Cell lysates were centrifuged and the resulting cytosolic supernatant was separated by density (a 10% to 50% sucrose) gradient and centrifugation. Fractions were collected using a TELEDYNE ISCO Density Gradient Fractionation System with continuous monitoring of untranslated mRNAs. Polysome (P)/Monosome (M) ratio were measured. The total RNA in each fraction was extracted using TRIzol LS (Invitrogen) and qRT PCR was done for p21, p27, Cyclin D1 and c-Myc gene for each fraction. Cell viability was measured by MTT assay at various time points. Results: TET treatment resulted in decreased protein synthesis. TET treatment resulted in decrease of RNA associated with Polysomes. There were a significant shift in P/M ratio from 0.82 (control) to 0.48 (24 h. TET) [TET 2hrs (0.75), 8h (0.69) and 18hrs (0.52)] in LNCaP and P/M ratio form 0.91 (control) to 0.32 (8 h TET) [TET 2h (0.78) and 4h (0.53)] in 22Rv1 cells. The decrease of the P/M ratio suggests that TET inhibits translation initiation in prostate cancer cells independent of the androgen dependence. While there was generalized decrease in translation and specific decrease in mRNA for Cyclin D1, c-Myc associated with polysomes following TET treatment. In quite contrast to these findings, we observed an increase in mRNA for p21 and p27 in polysomal fractions of TET treated cells as compare to control at 24 h in LNCaP cells. These data suggest differential effects of TET treatment on polysome loading of cell cycle regulatory proteins. Moreover, TET inhibited growth and proliferation of LNCaP and 22Rv1 cells in dose as well as in time dependent fashion. Conclusions: TET targets translational machinery and differentially effects translation of cell cycle regulatory genes by effecting their polysome loading and effects cell growth and proliferation of androgen dependent and castrate resistant PCa cells. Citation Format: Praveen K. Jaiswal, Sweaty Koul, Qin Dong, Hari Koul. TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells [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 98. doi:10.1158/1538-7445.AM2017-98

Abstract 2406: LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation

Abstract Background: Androgen deprivation therapy (ADT) or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine specific demethylase 1 (LSD1) is a histone demethylase and a key regulator of gene expression in cancer. Prior work demonstrates that LSD1 may act as a cofactor of the AR in androgen-dependent prostate cancer cells. In this report, we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of the AR and independently of histone demethylation. Methods: We used gain and loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and measured gene expression changes with microarrays. To determine the importance of histone demethylation in regulation of these gene networks, we suppressed LSD1 and measured levels of LSD1 canonical histone substrates (H3K4me2 and H3K9me2) genome-wide with chromatin immunoprecipitation-sequencing. Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Microarray studies demonstrated that LSD1 activates androgen-independent genes that comprise cell cycle and embryonic stem cell maintenance gene sets that are enriched in lethal human tumors. Importantly, our global epigenomic studies after LSD1 suppression demonstrated that LSD1 activates these gene sets independently of demethylation of its canonical histone substrates. Conclusions: Our results demonstrate that LSD1 promotes CRPC cell survival independently of the AR and suggest that LSD1 regulates key pathways in CRPC through demethylation of non-histone substrates or via a scaffold function―mechanisms we are currently investigating. In summary, LSD1 contributes to CRPC cell survival through non-canonical mechanisms and represents an attractive therapeutic target in lethal prostate cancer. Citation Format: Archana Sehrawat, Lina Gao, Junior Tayou, Armand Bankhead, Laura M. Heiser, Carly J. King, Yuliang Wang, Jacob Schwartzman, Joshua Urrutia, Daniel J. Coleman, Sheila Weinmann, Bhaskar V. Kallakury, Deborah L. Berry, Reina Haque, Stephen K. Van Den Eeden, Tomasz M. Beer, George V. Thomas, Shannon McWeeney, Joshi J. Alumkal. LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation [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 2406. doi:10.1158/1538-7445.AM2017-2406

Abstract 2837: Differential expression of Protocadherin-7 (PCDH7) in prostate cancer (PCa) cells: a direct association between PCDH7 and castrate resistant PCa

Abstract Introduction: PCa is the most commonly diagnosed disease in American men and the second leading cause of death among them. Most of PCa deaths are a result of distant metastasis and due to emergence of castrate resistant PCa. Protocadherin-7 (PCDH7), a Cadherin superfamily transmembrane protein that is known to function in cell-cell recognition and adhesion, is reported to be overexpressed in breast cancer and non-small cell lung cancer (NSCLC). PCDH7 has been shown to mediate brain metastasis of breast cancer. Overexpression of PCDH7 was reported to be associated with poor clinical outcome of NSCLC. However, there is no report on involvement of PCDH7 in PCa tumorigenesis and metastasis. In the present study we evaluated expression of PCDH7 in androgen dependent and androgen independent PCa cells and in TCGA and Trento/Cornell/Broad 2016 clinical data sets. Materials and Methods: PCa cells- LNCaP, C4-2b, DU145, PC3, 22Rv1 and RWPE1 were used in this study. Protein expression was checked by Western Blotting. mRNA levels were checked by quantitative real time PCR. Results: Our data reveal high PCDH7 protein expression in PC3 cells and low levels of PCDH7 in LNCaP and RWPE1 cells. qRT-PCR confirmed an increased transcript level of PCDH7 in PC3 and low levels in LNCaP cells. We also observed elevated transcript levels for PCDH7 in castrate resistant and Enzalutamide refractory PCa (22Rv1) cells. These data suggest a direct relationship between castrate resistance and PCDH7 expression. Our results also show an inverse relationship between PCDH7 and E-Cadherin and a direct relationship between PCDH7 and EMT markers, indicating expression of PCDH7 is associated with tumor progression. Interestingly, there was no correlation between PCDH7 with tumor grade or metastasis from TCGA data, but analysis of NEPC (Trento/Cornell/Broad 2016) data set revealed amplification of PCDH7 in 9% of NEPC tumors. Collectively, these data suggest PCDH7 expression may be associated with advanced PCa in general and NEPC phenotype PCa in particular. Conclusion: Our results suggest direct association of PCDH7 expression and castrate resistance in PCa cell lines. Role of PCDH7 in castrate resistant PCa needs further evaluation. Citation Format: Gauri Shishodia, Sweaty Koul, Qin Dong, Hari K. Koul. Differential expression of Protocadherin-7 (PCDH7) in prostate cancer (PCa) cells: a direct association between PCDH7 and castrate resistant PCa [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 2837. doi:10.1158/1538-7445.AM2017-2837

Abstract LB-325: Tumor suppressor effects of miR-17-92a cluster miRNAs on prostate cancer

Abstract MicroRNAs in miR-17-92a cluster are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs, miR-17, miR-18a, miR-19a, miR- 19b, miR-20a. These miRNAs are reported to be overexpressed in colon and lung cancers, however, we noted a loss of expression of all members of this miR cluster in prostate cancer tissues compared to uninvolved tissues and in aggressive prostate cancer cells. In this study, we show that replenishment of miR-17-92a cluster as a whole showed antiproliferative effects, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth. Analysis of the expression of mRNA targets showed reduced expression of cell cycle regulatory proteins cyclin D1 and SSH1; and LIMK1 and FGD4 of RhoGTPase signaling pathway through direct targeting. miR-17-92 cluster expression also inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker E-Cadherin. miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP 104-S prostate cancer cells to anti-androgen drug Casodex, AKT inhibitor MK-2206 2HCl, and docetaxel. The androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. RNA seq analysis of prostate cancer cells expressing miR-17-92a cluster revealed altered expression of a number of oncogenic and tumor suppressor mRNAs, and noncoding RNAs, which may have implications in the altered phenotype of these cells. Our data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Ratna Chakrabarti, Richard Ottman, Md Faqrul Hasan. Tumor suppressor effects of miR-17-92a cluster miRNAs on 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 LB-325. doi:10.1158/1538-7445.AM2017-LB-325

Abstract 1473: miR-1207-3p regulates c-Myc in aggressive prostate cancer

Abstract Prostate cancer (PCa) is the most common non-skin cancer and the second leading cause of cancer-related death for men in the U.S. A major challenge is understanding the molecular mechanisms involved in the progression from the asymptomatic androgen-dependent PCa to the lethal castration resistant prostate cancer (CRPC). The chromosomal region 8q24 is associated with aggressive PCa and variants of this region have been identified to interact with the PVT1 non-coding gene in PCa. In previous work we demonstrated that microRNA-1207-3p (miR-1207-3p) has prognostic value in PCa, and directly binds to the 3’ UTR of Fibronectin type III domain containing 1 (FNDC1) to regulate a novel FNDC1/fibronectin (FN1)/androgen receptor (AR) pathway upregulated in metastatic PCa. miR-1207-3p is encoded at the PVT1 gene locus, which is located downstream of c-Myc on the 8q24 human chromosomal region. Studies have suggested amplification at the 8q24 human chromosomal region, which includes C-Myc, as a possible prognostic factor for CRPC. c-Myc is a well-established proto-oncogene that is commonly found to be amplified in up to 72% of CRPCs significant amplication of c-myc has been a consequence of antiandrogen treatment, and c-Myc is downstream of AR in some PCa. miRNAs have also been implicated in the regulation of c-Myc. Nevertheless, the mechanisms regulating c-Myc remain unclear in PCa. Using a panel of five PCa cell lines we discovered that c-Myc expression is higher in aggressive CRPC cell lines compared to non-aggressive CRPC and androgen-dependent prostate cancer cell lines. Furthermore, our data reveals that c-Myc expression is higher in the aggressive E006AA-hT PCa cell line when compared to the indolent E006AA PCa cell line. This suggests that c-Myc is associated with aggressive PCa. Moreover, overexpression of miR-1207-3p suppresses the expression of c-Myc in E006AA-hT PCa cells but not E006AA PCa cell line in which c-Myc is underexpressed compared to the normal prostate epithelial cell line, RWPE-1. Next, using a synthetic biotinylated miR-1207-3p duplex, we discovered that overexpression of miR-1207-3p more effictively inhibited migration, inhibited proliferation and increased apoptosis in the aggressive E006AA-hT PCa cell line with increased c-Myc expression when compared to the indolent E006AA PCa cell line with decreased c-Myc expression. These data demonstrate that the miR-1207-3p/FNDC1/FN1/AR pathway may regulate c-Myc in aggressive PCa. Citation Format: Dibash K. Das, Olorunseun O. Ogunwobi. miR-1207-3p regulates c-Myc in aggressive 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 1473. doi:10.1158/1538-7445.AM2017-1473

A novel deubiquitinase inhibitor b-AP15 triggers apoptosis in both androgen receptor-dependent and -independent prostate cancers

Prostate cancer (PCa) remains a leading cause of cancer-related death in men. Especially, a subset of patients will eventually progress to the metastatic castrate-resistant prostate cancer (CRPC) which is currently incurable. Deubiquitinases (DUBs) associated with the 19S proteasome regulatory particle are increasingly emerging as significant therapeutic targets in numerous cancers. Recently, a novel small molecule b-AP15 is identified as an inhibitor of the USP14/UCHL5 (DUBs) of the 19S proteasome, resulting in cell growth inhibition and apoptosis in several human cancer cell lines. Here, we studied the therapeutic effect of b-AP15 in PCa, and our results indicate that (i) b-AP15 decreases viability, proliferation and triggers cytotoxicity to both androgen receptor-dependent and -independent PCa cells in vitro and in vivo, associated with caspase activation, inhibition of mitochondria function, increased reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress; (ii) pan-caspase inhibitor z-VAD-FMK and ROS scavenger N-acetyl-L-cysteine (NAC) efficiently block apoptosis but not proteasome inhibition induced by exposure of b-AP15; (iii) treatment with b-AP15 in androgen-dependent prostate cancer (ADPC) cells down-regulates the expression of androgen receptor (AR), which is degraded via the ubiquitin proteasome system. Hence, the potent anti-tumor effect of b-AP15 on both androgen receptor-dependent and -independent PCa cells identifies a new promising therapeutic strategy for prostate cancer.

Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands.

Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

P 234 - Glut 1 overexpression prevents glucose deprivation-induced prostate cancer cell death by increasing pentose phosphate pathway and glutathione

Although glucose metabolism was override in prostate cancer, it is recognized that there are metabolic differences between androgen-sensitive and castration-resistant phenotypes that might be responsible of progression or treatment success. Among the targets of glucose metabolism, glucose uptake is on the top of the cascade. The increase of glucose uptake by GLUTs overexpression in oncogenesis or the androgenic regulation of some GLUT transporters in other tissues is demonstrated. The prostate, at the beginning of carcinogenesis, depends on OXPHOS but then, at aggressive stages, tumors become again glycolytic like other cancers. Therefore, their resistance to glucose deprivation is different at the beginning or at later stages. In this work, we demonstrated by using androgen sensitive and insensitive cells that facilitative GLUT1 transporter overexpression protect prostate cancer cells from apoptosis caused by glucose removal. Glucose removal caused an increment in oxidative stress that stimulated androgen receptor activity and GLUT1 overexpression. This protection is mediated by a derivation towards pentose phosphate metabolic pathway and an increment of glutathione in androgen dependent prostate cancer cells.