PCa Cell Lines Research Articles

Abstract 3389: A multi-gene prostate cancer liquid biopsy with > 92% accuracy in diagnosis and assessment of disease status

Abstract Background: There are a paucity of blood-based biomarkers with clinical utility for prostate cancer (PCa). We examined a circulating-free mRNA (27-gene) prostate cancer signature to diagnose PCa. Clinical utility of the assay was then assessed in surgical patients. Methods: Gene identification and validation: Publicly available PCa transcriptome sets (n=5) (E-GEOD-46691, E-GEOD-46602, E-GEOD-62116, E-GEOD-62667, E-GEOD-72220: n=1,159 samples); PCA cell lines (n=7) gene expression in 22Rv1 and E006AA-hT (localized); VCaP; PC-3; LNCaP; DU145 and MDA PCa2b (all metastatic) from two normal prostate epithelial lines: PWR-1E and RWPE-1 and validated in PCa tumor tissue (n=50). Transcriptome analyses: tumor compared with normal blood-based transcriptomes using gene co-expression network enrichment, differential expression and functional enrichment analyses to identify candidate markers. Blood gene expression: PCa set #I: n=132, PCA set #II: n=50 (biochemical recurrence [BCR]), BPH: n=44, controls n=55. We then constructed an artificial intelligence PCa model using classification algorithm analyses. Scoring: normalized algorithmically analyzed gene expression (0 to 100), positive score ≥20. Matched tumor/ blood samples: n=50. PSA: BPH (n=44) and PCa (n=132). Clinical score assessment: Score utility was assessed in surgical cohorts: Surgical: (n=47), scores =pre-surgical and post: 1 week - 14 months. Statistics: Non-parametric (Kruskal-Wallis), Pearson-correlation, Fisher's and AUROC analyses (Mean±SEM). Results: Transcriptomic analysis identified a 27 gene blood signature for PCa. Expression levels were significantly elevated (p<0.001, 2.1-35.8-fold) in cell lines and PCA tumors. The matched tissue/blood correlation was r: 0.56 (p=0.0023). In PCa #I, levels were 47±2 (p<0.0001) compared to BPH (19±1) and controls (18±0.5); AUROC: 0.92 (BPH) and 0.94 (controls), with an accuracy of 85-88%, a sensitivity of 86% and specificities 82 & 93% respectively. For PSA the AUROC (PCa vs. BPH) was 0.51 (p=0.88). PSA was positive in 86% of BPH and was >10ng/ml in 30%. PSA was positive in 83% of PCa and >10ng/ml in 40% (Fisher's exact: p=0.28). PSA accuracy (>10ng/ml) was 48%. Levels in PCA #II (BCR) were 44±3. ProstaTest-was positive in 48 (96%). Surgical cohort (n=47): PCa Prostatest accuracy 100% pre-surgery. Resection significantly decreased levels (KW-statistic: 57.4, p<0.0001) from 52±1 to 23.5±2. Conclusion: The diagnostic accuracy of a PCa molecular blood-signature was 92%; significantly better than PSA (48%, p<0.0001). Surgical resection significantly (p<0.0001) decreased levels. Biochemical recurrence was accurately detected (96%). A multi-gene prostate cancer liquid biopsy seems likely to have clinical utility in the diagnosis and monitoring management of PCa. Citation Format: Kambiz Rahbar, Mark Kidd, Ignat Drozdov, Alexandra Kitz, Anna Malczewska, Pawel Rajwa, Christof Bernemann, Lisa Bodei, Irvin Modlin. A multi-gene prostate cancer liquid biopsy with > 92% accuracy in diagnosis and assessment of disease status [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 3389.

Abstract 1581: Prostate tumor associated NK cells (PTANKs) acquire the decidual-like/pro-angiogenic phenotype and polarize macrophages towards the M2-like/TAM subset

Abstract Prostate cancer (PCa) remains the second most common cancer worldwide in men. Angiogenesis has been shown to play a crucial role in PCa progression in several preclinical models. Production of pro-angiogenic factors have been shown to correlate with metastasis, Gleason score and prognosis in PCa and plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been found to be immunosuppressed in solid cancers, including PCa. We demonstrated that NK cells in lung and colorectal cancer, acquire a pro-angiogenic phenotype, similar to NK cell within the decidua (dNK), and functionally support angiogenesis, in a TGFβ and Stat3-dependent manner. Here, we phenotypically and functionally characterized circulating NK cells from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution was investigated by multicolor flow cytometry (FC) for surface antigens on peripheral blood samples PCa patients. Conditioned media (CM) from FACS-sorted PTANKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of PTANKs. Protein arrays were performed to characterize the secretome on FACS-sorted PTANKs We found that PTANKs acquire the pro-angiogenic/ decidual-likeCD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed on cytolytic NK cells, from healthy donors, exposed to CMs of three different PCa cell lines. These results were confirmed also exposing heathy-donor derived NKs to CMs of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFα, IFNγ and GranzymeA. CMs from PTANKs support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Secretome analysis revealed the ability of PTANKs release pro-angiogenic factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7, CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). Finally, CMs from PTANKs can recruit THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Our results place PTANKS as effector cells able in supporting angiogenesis in PCa by directly interaction with endothelial cells and via macrophage polarization. Citation Format: Antonino Bruno, Denisa Baci, Matteo Gallazzi, Annalisa Bosi, Angelo Naselli, Andrea Guarneri, Lorenzo Mortara, Douglas Noonan, Adriana Albini. Prostate tumor associated NK cells (PTANKs) acquire the decidual-like/pro-angiogenic phenotype and polarize macrophages towards the M2-like/TAM subset [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 1581.

Abstract 5365: Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay

Abstract Introduction and objective: The presence of circulating tumor cells (CTC) is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. The aim of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by gene profiling using the NanoString low RNA input nCounter assay to achieve reliable CTC gene profiling. Methods: Blood samples were collected from 4 mPCa patients and 3 healthy donors. In the LP procedure, different blood elements were separated based on density. The mononuclear cell layer (presumed to contain any CTCs) was collected. The Alere™ CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method for isolation of EpCAM+ CTCs, followed by RT-qPCR for PSA, PSMA, and HOXB13 was used to detect CTCs from LP. The Ct value thresholds for positive CTC signal by RT-qPCR were set based on healthy donor LP subjected to AdnaTest as a negative control (PSA 36.3, PSMA 36.82, HOXB13 40). The cDNA of cells captured by EpCAM+ selection (including putative CTCs) and of the EpCAM+ depleted white blood cells (WBC) were assayed by the nCounter PanCancer Progression Panel to determine expression of 770 selected mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer pool was used for the pre-amplification of cDNA and overnight hybridization with the PanCancer Progression panel. To validate the assay, healthy donor LPs were spiked with known numbers of PCa cell line LN95 cells. Results: For mPCa patients, the LP procedure took 136.2±10.6 min, with a screening volume of 10L of peripheral blood. The mean volume of mPCa LP product was 123.64±12.23 ml. The WBC concentration was 69.75±28.2×109/L. All samples were subjected to AdnaTest in triplicate and CTCs were identified in 3 out of 4 patients. The positive Ct values of PSA were 33.86±0.81, 32.14±0.31 and 33.09±0.34; PSMA were 35.55±1.02, 33.55±0.83 and 35.30±0.65; HOXB13 were 36.25±0.34, 37.15±0.22 and 37.09±0.33. To assess profiling of CTCs isolated from LP using the nCounter platform, 100, 500, and 1000 LN95 cells were spiked in healthy LPs and were analyzed with the AdnaTest. Within each spiking group, pre-spiking WBC, captured CTC, post-capture WBC, and LN95 cells in PBS alone were subjected to the Nanostring assay. Eisen Cluster analysis separated pre- and post- WBCs, captured CTCs, LN95 cells into three groups. The gene expression of captured CTCs correlated with those of LN95 cells, and the correlation increased with the spiking cancer cell number (R2=0.45, 0.57, and 0.70, respectively). The gene expression of pre-spiked WBCs were highly correlated with those of post-capture WBCs regardless of spiking cancer cell number (R2=0.83, 0.84 and 0.85). Conclusions: Analyzing CTCs in LP samples by AdnaTest is feasible and offers a promising method for liquid biopsy of mPCa patients. The NanoString low RNA input nCounter assay can provide reliable gene expression profiling of CTC. Citation Format: Liang Dong, Chung-Ying Huang, Changxue Lu, Diane Reyes, Sarah R. Amend, Jun Luo, Kenneth Pienta. Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay [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 5365.

Abstract 4688: Interaction of LEDGF/p75 with oncogenic transcription factors in docetaxel resistant prostate cancer cells

Abstract 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.

Abstract 1404: MicroRNA-mediated induction of neuroendocrine differentiation states in metastatic castration-resistant prostate cancer

Abstract While the widespread use of androgen receptor pathway inhibitors (APIs) such as enzalutamide has led to transformative impact in the management of metastatic castration-resistant prostate cancer (CRPC) patients, resistance is near-universal leading to significantly increased incidences of highly aggressive, therapy-induced neuroendocrine differentiation (NED). It has been realized that therapy-induced NED represents a continuum of treatment-induced changes at phenotypic and molecular level that involves a series of alterations at genetic, epigenetic, post-transcriptional, post-translational levels and changes in microenvironment. Though several recent studies identified key genomic and epigenomic alterations driving therapy-induced NED, the molecular pathogenesis is still not completely understood contributing to late diagnosis of the disease, poor prognosis and lack of effective therapies. The focused objective of our study was to systematically investigate the key microRNA (miRNAs) alterations that drive therapy-induced NED in PCa. We hypothesized that this lineage switch is associated with significant alterations to the miRNAome, that in turn, drives change in cellular gene expression patterns towards NE states. We tested our hypothesis by performing small RNA-NGS in a clinical cohort of patient samples, patient-derived xenograft and cellular models. We identify for the first time that a characteristic set of miRNA alterations promote plasticity of prostate tumors to NE states. Importantly, we identify downregulation of miR-363 as a prominent molecular alteration underlying PCa NED. To examine the functional role of miR-363, we knocked down its expression in CRPC cell lines C42B and LNCaP followed by functional assays. Decreased miR-363 expression resulted in increased clonogenicity potential and invasiveness of PCa cell lines as compared to control suggesting that miR-363 loss contributes to increased aggressiveness. Importantly, we found that miR-363 directly represses Aurora kinase A (AURKA), a prototypical kinase frequently amplified in NED. Correlation analyses in TCGA database of prostate adenocarcinomas (n=494) showed a significant inverse correlation between miR-363 expression and AURKA mRNA levels. In conclusion, our study identifies crucial molecular determinants of NED with potential clinical and transformative potential. Since AURKA inhibition is currently being examined as a therapeutic modality for NEPC, we propose augmentation of miR-363 levels to repress AURKA as a novel treatment option for therapy-induced NEPC. Citation Format: Divya Bhagirath, Michael Liston, Byron Lui, Dat My To, Thao Ly Yang, Laura Z. Tabatabai, Nikhil Patel, Roni Bollag, Adam Olshen, Sharanjot Saini. MicroRNA-mediated induction of neuroendocrine differentiation states in metastatic castration-resistant prostate cancer [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 1404.

The miR-1908/SRM regulatory axis contributes to extracellular vesicle secretion in prostate cancer.

Targeting extracellular vesicle (EV) secretion can have potential clinical implications for cancer therapy, however the precise regulatory mechanisms of EV secretion are not fully understood. Recently, we have shown a novel pathway of EV biogenesis in PCa cell lines, PC3 and PC3M. However, as the characteristics of EVs are divergent even among PCa cell lines, we hypothesized that other pathways or common regulatory pathways of EV biogenesis still exist. Here, we performed quantitative high‐throughput screening to determine the key regulatory genes involved in EV biogenesis in 22Rv1 cells, which secrete a different type of EVs. In total, 1728 miRNAs were screened and miR‐1908 was selected as the potential miRNA regulating EV biogenesis in 22Rv1 cells. Subsequently, we investigated target genes of miR‐1908 using siRNA screening and identified that spermidine synthase (SRM) was the key regulator of EV secretion in 22Rv1 cells. Attenuation of SRM expression significantly inhibited secretion of EVs in 22Rv1 cells, and overexpression of SRM was confirmed in PCa tissues. Furthermore, we found that the number of endosome compartments was increased in cellular cytoplasm after knockdown of the SRM gene. In conclusion, our results showed that miR‐1908‐mediated regulation of SRM can control secretion of EVs in PCa. In addition, these data suggested that the EV secretion pathway was dependent on cellular characteristics.

MiR-138-5p inhibits the malignant progression of prostate cancer by targeting FOXC1

BackgroundThis study aimed to uncover the effect of miR-138-5p on the proliferation and metastasis of PCa cell lines, and further explore the potential regulatory mechanisms via regulating FOXC1.Methods60 pairs cancer tissues and corresponding paracancerous ones from PCa patients were collected to assess the expression level of miR-138-5p by qRT-PCR. Subsequently, over-expression of miR-138-5p were established to explore the proliferation and metastasis of miR-138-5p in PCa cell lines was analyzed by CCK-8, Transwell assay and Wounding healing assay, respectively. Bioinformatics analysis and luciferase reporter gene assay were performed to search for the target genes of miR-138-5p, and FOXC1 was selected. Finally, the biological role of miR-138-5p and FOXC1 in the progression of PCa was clarified by a series of rescue experiments.ResultsThe results of qRT-PCR revealed that miR-138-5p was lowly expressed in PCa tissues and cell lines. Besides, the PCa patients with low-miR-138-5p had a high Gleason score, lymph node metastasis and poor prognosis of PCa, compared with these patients with high-miR-138-5p. Over-expression of miR-138-5p inhibited the proliferative, migratory and invasive capacities of PC-3 and DU-145 cells. Bioinformatics analysis and luciferase reporter gene assay suggested that FOXC1 was predicted to be the target gene of miR-138-5p. Moreover, FOXC1 expression level was negatively correlated to that of miR-138-5p in PCa tissues. Importantly, over-expression of FOXC1 could reverse miR-138-5p mimic induced-inhibition of PCa malignant progression.ConclusionsDownregulated miR-138-5p was closely associated with high Gleason score, more lymph node metastasis and poor prognosis of PCa patients. In addition, miR-138-5p alleviated the malignant progression of PCa by targeting and downregulating FOXC1.

Abstract A109: MicroRNA-4719 and microRNA-6756-5p correlate with aggressive prostate cancer progression in African American men through regulation of Interleukin-24

Abstract Prostate cancer (PCa) incidence and mortality are highest in African American men (AAM) and the molecular mechanisms underlying the racial disparities in PCa are unclear. Interleukin-24 (IL-24), a tumor suppressor whose expression is lost in most human cancers, correlates with cancer progression. Also, microRNAs (miRs) are dysregulated in cancers. microRNA target prediction algorithm tools identified miRs, miR-4719 and miR-6556-5p, as putative regulators of IL-24. This study elucidates the expression profile and role of miR-4719 and miR-6756-5p as regulators of IL-24 in PCa biology of AAM. Our work compares five different PCa cell lines: E006AA (AAM, indolent), E006AA-hT (AAM, aggressive), DU-145 (CM, aggressive), PC-3 (CM, aggressive) to a nontumorigenic prostate epithelial cell line, RWPE1 (CM). qRT-PCR analysis shows that miR-4719 and miR-6756-5p are significantly overexpressed in all PCa cell lines (by >2-fold) compared to RWPE-1. Both miR-4719 (>2 fold) and miR-6756-5p (>50%) are higher in aggressive PCa compared to the indolent PCa, indicating their gain could be an early event in PCa progression. Moreover, miR-4719 and miR-6756-5p are significantly overexpressed in the aggressive PCa of AAM (E006AA-hT) compared to aggressive PCas for CM (PC-3 and DU-145). Interestingly, both miR-4719 and miR-6756-5p expression were higher by (>3-fold) in the aggressive AAM cell line E006AA-hT compared to indolent AAM cell line- E006AA. Thus, miR-4719 and miR-6756-5p may play a role in racial disparity. Using oligonucleotide mimics and inhibitors, we evaluated the functional role of these miRs in the AAM cell lines, E006AA and E006AA-hT. MTT assays revealed that inhibition of miR-4719 and miR-6756-5p significantly decreases proliferation (>40%), while overexpression of both miRs increases proliferation (>20%). Inhibition of miR-4719 and miR-6756-5p further decreased the proliferation of the aggressive E006AA-hT by an extra 20%, compared to indolent E006AA. In contrast, overexpression of both miRs further increased the proliferation of E006AA-hT also by an extra 20%, compared to E006AA. Wound healing assays reveal that inhibition of miR-4719 and miR-6756-5p reduced migration by ~50% compared to the negative control (NC). Strikingly, overexpression of both miRs increased migration by at least 2- and 4-fold, respectively. We observed that IL-24 was downregulated in all PCa cells compared to RWPE-1, suggesting a possible role of these miRs in suppressing IL-24. qRT-PCR analysis show inhibition of miR-4719 and miR-6756-5p significantly increases the expression of IL-24 (by ~2-fold) in PCa cells compared to the NC. In contrast, overexpression of both miRs reduces the expression of IL-24. Our findings indicate that miR-4719 and miR-6756-5p may regulate IL-24 expression and may be biomarkers that can differentiate between indolent and aggressive PCa in AAM. Strategies to inhibit miR-4719 and miR-6756-5p expression to increase IL-24 in PCa could have therapeutic efficacy in AAM. Citation Format: Dibash K. Das, Leah Persaud, Moira Sauane. MicroRNA-4719 and microRNA-6756-5p correlate with aggressive prostate cancer progression in African American men through regulation of Interleukin-24 [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A109.

Abstract C038: MHC class I polypeptide related sequence A (MICA) race-related differential expression in prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men, with higher incidence and death rates in African Americans (AA) relative to Caucasian Americans (CA); nevertheless, these disparities are still poorly understood. MHC class I polypeptide related sequence A (MICA) is a cell surface protein able to promote activation of natural killer cells. Cleavage of MICA and generation of its soluble form (sMICA) has been described as an immunoevasion mechanism presented by different types of aggressive tumors. In agreement with the role of MICA on tumor immunoevasion, recent studies correlated better prognosis for patients expressing higher levels of MICA in different types of cancer. We speculated that MICA expression would be different between AA and CA PCa tumors. To access MICA expression in PCa patients, we stained a TMA containing 30 AA and 27 CA tumor cores. Our results showed that CA PCa tumor cores had higher expression of MICA than the AA (p=0.0019). In order to establish an in vitro system to study the biologic effects of MICA in the context of disparity in PCa, we assessed the expression of surface MICA and release rate of sMICA in different PCa cell lines: MDA-PCa-2b and E006AA-hT, derived from AA, and DU-145 and LNCaP, derived from CA. Flow cytometry analysis showed baseline cell fraction expression positive for surface MICA of 3% in MDA-PCa-2b, 18% in E006AA-hT, 54% in DU-145, and 67% in LNCaP, with significant difference between each cell line (p<0.0001). Baseline release rate of sMICA was nondetectable for MDA-PCa-2b, 5pg/ml/106 cells in E006AA-hT, 120pg/ml/106 cells in DU-145, and 54pg/ml/106 cells for LNCaP cells. Overall, our results show that MICA expression is race related. Further studies are necessary to address the biologic function of the expression and the impact in the immune system. Citation Format: Charles R. Pound, Marcelo J. Sakiyama, Amit Reddy, Ingrid Espinoza, Eldrin Bhanat, Krista Syrigos, Roya Gordji, Deepak Kumar, Jack Lewin, Xinchun Zhou, Jesus Monico, Christian Gomez. MHC class I polypeptide related sequence A (MICA) race-related differential expression in prostate cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C038.

Abstract D117: Differential reactivity and cell migration inhibitory functions of autoantibodies to Enolase 1 from African American and European American men with prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in the U.S., with African American (AA) men showing a higher mortality than European American (EA) men. While biological determinants contributing to PCa health disparities remain to be clearly established, there is growing evidence of differences in immunobiology between AA and EA patients with PCa. We demonstrated previously, using immunoproteomic profiling of anti-tumor autoantibody responses in AA and EA patients with PCa, that the glycolytic enzyme enolase 1 (ENO1) is the target of an autoantibody response in subsets of these patients. AA patient sera showed immunoreactivity to ENO1 in a panel of PCa cell lines, as detected by immunoblotting, with lack of reactivity in docetaxel (DTX)-resistant PC3 and DU145 cells. By contrast, anti-ENO1 sera from EA patients and a monoclonal anti-ENO1 antibody (MoAB-ENO1) showed immunoreactivity across the same cell line panel, including DTX-resistant cells. We also noted differences in anti-ENO1 reactivity in ELISA and immunoblots of PCa cell lysates between AA-PCa and EA-PCa patients, suggesting a race-related generation of autoantibodies to different ENO1 variants. ENO1 is a surface protein in cancer cells with roles in plasminogen activation, cell migration, and metastasis. Thus, we explored if anti-ENO1 autoantibodies from AA and EA PCa patients exerted different anti-cancer effects against DTX-sensitive and DTX-resistant PCa cells. Using cell migration assays, we observed that anti-ENO1 sera from EA patients and MoAB-ENO1 reduced the migration of DTX-resistant PC3 and DU145 cells but not that of DTX-sensitive cells. This inhibitory effect was intrinsic to the autoantibodies since purified IgG fraction produced similar results. Further, these inhibitory effects were reversed by pre-absorption of anti-ENO1 sera and purified IgG with recombinant human ENO1. Intriguingly, anti-ENO1 sera from AA patients lacked the same inhibitory effects on the migration of DTX-resistant cells, consistent with their lack of immunoreactivity to ENO1 in these cells. In addition, we observed that MoAB-ENO1, which behaves similar to the anti-ENO1 EA sera, decreased viability without evident cell death, measured by MTT assays and imaging analysis, in both DTX-sensitive and DTX-resistant cells, with a more robust effect on the latter. Together, these results suggest that anti-ENO1 autoantibodies from EA-PCa patients may exert a protective effect by decreasing the migration and proliferation of chemoresistant PCa cells. By contrast, anti-ENO1 autoantibodies from AA PCa patients may lack these protective effects, which could contribute to increased cancer cell migration and metastasis. Further studies are needed to dissect the molecular basis for this race-related differential anti-ENO1 immunoreactivity in PCa patients, and exploit the immunotherapeutic potential of anti-ENO1 antibodies for the treatment of chemoresistant PCa. Citation Format: Carlos Diaz-Osterman, Tino Sanchez, Carlos A Casiano. Differential reactivity and cell migration inhibitory functions of autoantibodies to Enolase 1 from African American and European American men with prostate cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D117.

Abstract D116: Glucocorticoid receptor interacts with beta-catenin to promote stemness and therapy resistance in prostate cancer cells

Abstract Prostate cancer (PCa) is the most commonly diagnosed male cancer, with men of African ancestry showing a disproportionately high incidence and mortality. There is increasing evidence that glucocorticoid signaling through glucocorticoid receptor (GR) is amplified in African American men due to cumulative life stress, and that GR signaling is a key driver of resistance to androgen-targeted therapy, radiotherapy, and taxane chemotherapy in advanced PCa. Recent studies indicate that GR also promotes in PCa cells the formation of tumorspheres, a property of cancer stem cells (CSC); however, the mechanisms remain unclear. CSCs are intrinsically resistant to therapies due to their low-frequency, expression of transporters and efflux pumps, quiescent cell cycle and metabolic profile. Our previous studies demonstrated that docetaxel (DTX)-resistant PCa cells exhibit increased capacity for CSC-like properties, suggesting that these properties contribute to chemoresistance. Like GR, beta-catenin is overexpressed in metastatic and therapy-resistant tumors, and is considered a key regulator of cancer stemness and androgen-targeted therapy resistance. Thus, we hypothesized that GR may interact with beta-catenin in PCa cells to support stemness and chemoresistance. Using whole-cell and nuclear co-immunoprecipitation, we demonstrated the interaction between GR and beta-catenin in several DTX-sensitive and -resistant PCa cell line pairs. Pharmacological inhibition of GR using the selective inhibitor CORT-108297 with concomitant inhibition of beta-catenin using the small molecule inhibitor MSAB significantly enhanced DTX cytotoxicity in resistant PCa cells grown in both adherent and spheroid cultures. The BET bromodomain inhibitor JQ1, an antagonist of enhancer-mediated GR upregulation, also significantly reduced tumorsphere formation and stemness in DTX-resistant PCa cells. In order to gain insights into transcriptomic profiles activated by GR signaling that may contribute to stemness and therapy resistance, we performed RNA sequencing in a racially diverse panel of PCa cell lines (MDA-PCa-2b, VCaP, 22RV1, and PC3) treated with and without dexamethasone, a potent glucocorticoid. We observed that the African American cell line MDA-PCa-2b yielded the largest number of differentially regulated genes, perhaps due to the ability of dexamethasone to signal through both AR and GR in this particular cell line. RNAseq data is currently being mined and validated in cellular models treated with glucocorticoids or androgens, in the presence or absence of anti-GR or anti-androgen inhibitors, in order to identify GR-regulated genes. Our results offer novel insights into mechanisms by which GR signaling may influence tumorsphere formation and stemness, and suggest that combinatorial targeting of GR and beta-catenin could be a promising therapeutic strategy to overcome PCa therapy resistance and consequently reduce PCa mortality and its racial disparities. Citation Format: Shannalee R Martinez, Evelyn S Sanchez-Hernandez, Xin Chen, Alfonso M Duran, Charles H Wang, Carlos A Casiano. Glucocorticoid receptor interacts with beta-catenin to promote stemness and therapy resistance in prostate cancer cells [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D116.

A blood-based multi-mRNA liquid biopsy with >90% accuracy for diagnosis and assessment of prostate cancers.

5574 Background: There are a paucity of blood-based biomarkers with clinical utility for prostate cancer (PCa). We developed a circulating mRNA (27-gene) prostate cancer signature to diagnose and manage PCa. Methods: Gene identification: Publicly available PCa transcriptome sets ( n= 1,159 samples) were evaluated and compared with normal blood-based transcriptomes using gene co-expression network enrichment, differential expression and functional enrichment analyses to identify candidate markers. Gene expression evaluation: Seven PCA cell lines and two normal prostate epithelial lines were used to assess candidate genes. Marker genes were determined in PCa tumor tissue ( n= 50) and validated in the TCGA-PRAD ( n= 500) dataset. Blood gene expression: Set #I: PCA: n= 132, BPH: n= 44, controls n= 55. Set #II: n= 50 (biochemical recurrence [BCR]). We constructed an artificial intelligence PCa model using classification algorithm analyses. Scoring: normalized algorithmically analyzed gene expression (0 to 100), positive score >20. PSA: BPH ( n= 44) and PCa ( n= 132). Clinical score assessment: Surgical cohort: ( n= 47), samples: pre-surgical and post: 1 week - 14 months. Statistics: Kruskal-Wallis, Pearson-correlation, Fisher’s and AUROC analyses (Mean±SEM). Results: Transcriptomic analysis identified 27 candidates. Cell lines/tissue: Expression levels were significantly elevated ( p< 0.001, 2.1-35.8-fold) in cell lines and PCa surgical samples. All 27 markers were confirmed in TCGA-PRAD samples (average TPM: 58 to 10,366). Blood: In Set#I, levels in PCa were 47±2 ( p< 0.0001) compared to BPH (19±1) and controls (18±0.5); AUROC: 0.92 (BPH) and 0.94 (controls), with an accuracy of 85-88%, a sensitivity of 86% and specificities 82 and 93%. For PSA, the AUROC (PCa vs. BPH) was 0.51 ( p= 0.88). PSA was positive in 86% of BPH and was > 10ng/ml in 30%. PSA was positive in 83% of PCa and > 10ng/ml in 40% (Fisher’s p= 0.28). PSA accuracy ( > 10ng/ml) was 48%. Levels in Set#II (BCR) were 44±3. ProstaTest-was positive in 48 (96%). Surgical cohort ( n= 47): Prostatest accuracy 100% pre-surgery. Resection decreased levels (KW-statistic: 57.4, p< 0.0001) from 52±1 to 23.5±2. Conclusions: A 27-gene blood signature was developed for PCa that exhibited a diagnostic accuracy of 92%; significantly better than PSA (48%, p< 0.0001). Surgical resection significantly ( p< 0.0001) decreased levels. Biochemical recurrence was accurately detected (96%). A multi-gene prostate cancer liquid biopsy is likely to have clinical utility in both diagnosis and monitoring of PCa.

Sin1 promotes proliferation and invasion of prostate cancer cells by modulating mTORC2-AKT and AR signaling cascades

AimsProstate cancer (PCa) is the most common type of cancer and a major cause of death in men worldwide. Aberrant Androgen receptor (AR) and PI3K-AKT signaling are very frequent in PCa patients and, therefore, considered as therapeutic targets in the clinic. Sin1 is an essential component of mTORC2 complex, which determines full AKT activation and PCa development in PTEN−/− mice. Here we examined the role of Sin1 in human PCa cell lines and respective tumor samples. Main methodsWestern blotting and immunohistochemistry (IHC) were performed to analyze the expression of Sin1-mTORC2-AKT related proteins in human PCa cells, as well as prostate tumors and normal tissue counterparts. Cell viability and invasion assays were also pursued in the presence or not of Sin1 in PCa cells. Immunoprecipitation assays were additionally carried out to examine the interaction of Sin1 with AR. Key findingsWe have presently demonstrated that high levels of Sin1 expression in human PCa tissues correlate with cancer progression. Sin1-mediated cell proliferation and invasion of PCa cells occurs by regulating mTORC2-AKT signaling, epithelial-mesenchymal transition and matrix metalloproteinases. Moreover, androgens are able to induce Sin1 expression, which is further translocated to the nucleus of PCa cells. Finally, Sin1 interacts with AR to suppress its transcriptional activity. SignificanceTaken together, these data indicate that both Sin1-mediated mTORC2-AKT signaling and Sin1-AR interaction regulate PCa development. Hence, Sin1 may be considered a novel biomarker of PCa progression.

The Role of Differentially Expressed miRNAs and Potential miRNA-mRNA Regulatory Network in Prostate Cancer Progression and Metastasis

Purpose: Aberrant expression of microRNAs (miRNAs) has been discovered in prostate cancer progression however their function is not well understood, thereby further investigation is required to understand the importance of underlying mechanisms and their involvement in multiple signaling pathways, as well as their potential as therapeutic targets. In this study the role and expression levels of three miRNAs were evaluated: miR-21, miR-221 and miR-200c in different prostate cancer cell lines. In addition, based on the latest studies on miRNAs function, their association with other target genes and molecules were analyzed using bioinformatic tools. Methods: Three PCa cell lines PC3, LNCaP and VCaP and normal prostate epithelial cell line PNT1A were screened for miRNA expression levels using qPCR. miRNA target genes and their association with signaling pathways were analyzed through several Network and pathway analysis online tools. Findings: Upregulation of miR-21 and miR-221 was observed in PC3 and VCaP prostate cancer cells, respectively. According to KEGG analysis, we found that Hippo signaling pathway and cytokine-cytokine receptor interactions were affected by miR-21 while miR-221 would interfere with ECM-receptor interaction, Fatty acid elongation and Huntington disease molecular networks. Exposure of PC3 cells to TGF-β (10 µM) caused upregulation of miR-21 with the evidence with increased invasion potential. Discussion and Conclusion: miRNAs could regulate several genes in multiple signaling pathways. Here, we demonstrated that in a panel of PCa cell lines, both mir-21 and miR-221 expressions were upregulated. miR-21 may be a dignostic and prognosticbiomarker for PCa.

IL-1-conferred gene expression pattern in ER\u03b1+ BCa and AR+ PCa cells is intrinsic to ER\u03b1\u2212 BCa and AR\u2212 PCa cells and promotes cell survival

BackgroundBreast (BCa) and prostate (PCa) cancers are hormone receptor (HR)-driven cancers. Thus, BCa and PCa patients are given therapies that reduce hormone levels or directly block HR activity; but most patients eventually develop treatment resistance. We have previously reported that interleukin-1 (IL-1) inflammatory cytokine downregulates ERα and AR mRNA in HR-positive (HR+) BCa and PCa cell lines, yet the cells can remain viable. Additionally, we identified pro-survival proteins and processes upregulated by IL-1 in HR+ BCa and PCa cells, that are basally high in HR− BCa and PCa cells. Therefore, we hypothesize that IL-1 confers a conserved gene expression pattern in HR+ BCa and PCa cells that mimics conserved basal gene expression patterns in HR− BCa and PCa cells to promote HR-independent survival and tumorigenicity.MethodsWe performed RNA sequencing (RNA-seq) for HR+ BCa and PCa cell lines exposed to IL-1 and for untreated HR− BCa and PCa cell lines. We confirmed expression patterns of select genes by RT-qPCR and used siRNA and/or drug inhibition to silence select genes in the BCa and PCa cell lines. Finally, we performed Ingenuity Pathway Analysis (IPA) and used the gene ontology web-based tool, GOrilla, to identify signaling pathways encoded by our RNA-seq data set.ResultsWe identified 350 genes in common between BCa and PCa cells that are induced or repressed by IL-1 in HR+ cells that are, respectively, basally high or low in HR− cells. Among these genes, we identified Sequestome-1 (SQSTM1/p62) and SRY (Sex-Determining Region Y)-Box 9 (SOX9) to be essential for survival of HR− BCa and PCa cell lines. Analysis of publicly available data indicates that p62 and SOX9 expression are elevated in HR-independent BCa and PCa sublines generated in vitro, suggesting that p62 and SOX9 have a role in acquired hormone receptor independence and treatment resistance. We also assessed HR− cell line viability in response to the p62-targeting drug, verteporfin, and found that verteporfin is cytotoxic for HR− cell lines.ConclusionsOur 350 gene set can be used to identify novel therapeutic targets and/or biomarkers conserved among acquired (e.g. due to inflammation) or intrinsic HR-independent BCa and PCa.

MALAT1 knockdown inhibits prostate cancer progression by regulating miR-140/BIRC6 axis

BackgroundProstate cancer (PCa) is the second most common cancer among men globally. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to be implicated in tumorigenesis and progression of PCa. However, the pathogenesis of MALAT1 in PCa has not been thoroughly elaborated. MethodsRT-qPCR assay was conducted to measure expression of MALAT1, microRNA-140 (miR-140) and Baculoviral IAP repeat containing 6 (BIRC6) mRNA. Protein expression of BIRC6 was detected by western blot assay. Cell proliferative ability was assessed by MTS and Edu retention assays. Cell migratory and invasive abilities were evaluated by wound healing assay and Transwell invasion assay, respectively. Cell apoptotic rate was examined using a flow cytometry. The interaction between miR-140 and MALAT1 or BIRC6 3′UTR was explored by luciferase, RNA immunoprecipitation (RIP) and RNA pull down assays. Xenograft models of PCa were established to further explore the role and molecular mechanism of MALAT in PCa tumorigenesis in vivo. ResultsMALAT1 and BIRC6 were highly expressed in human PCa tumor tissues and cell lines. MALAT1 or BIRC6 knockdown inhibited cell proliferation, migration and invasion and induced cell apoptosis in PCa. MiR-140 could directly bind with MALAT1 or BIRC6 3′UTR. Moreover, MALAT1 knockdown inhibited BIRC mRNA and protein expression through upregulating miR-140 in PCa cells. Additionally, MALAT1 knockdown inhibited PCa xenograft tumor growth by regulating miR-140/BIRC6 axis in vivo. ConclusionMALAT1 knockdown hindered PCa progression by regulating miR-140/BIRC6 axis in vitro and in vivo, hinting the potential value of MALAT1 in the management of PCa.

The stem cell inhibitor salinomycin decreases colony formation potential and tumor-initiating population in docetaxel-sensitive and docetaxel-resistant prostate cancer cells.

BackgroundProstate cancer (PCa) is one of the most frequently diagnosed tumors in men. In general, therapies for localized PCa are curative. However, treatment of advanced PCa is considered palliative since development of therapy resistance occurs rapidly. It has been shown that tumor‐initiating cells are likely involved in therapy resistance. They are not eliminated by conventional therapies and thereby lead to tumor progression and relapse. The aim of this study was to evaluate the effects of the known stem cell inhibitor salinomycin on this critical subpopulation of cells.MethodsExpression of the cell surface markers CD24 and CD44 was assessed by immunofluorescence and fluorescence‐activated cell sorting. Colony formation efficiency and classification of colony types with varying tumor‐initiating potential (holoclones, meroclones, and paraclones) were analyzed in an automated way by the newly developed CATCH‐colonies software in the absence or presence of salinomycin.ResultsAutomated high‐resolution colony formation analysis consistently identified the various colony types in a broad range of PCa cell lines. Serial clonogenic assays confirmed that holoclones show the highest colony formation potential and maintain their tumor‐initiating capacity over multiple rounds. Furthermore, holoclones showed high expression of CD44, while CD24 was not expressed in these clones, thus representing the well‐described tumor‐initiating CD24−/CD44high population. Salinomycin decreased the CD24−/CD44high population in both docetaxel‐sensitive PC3 and docetaxel‐resistant (DR) PC3‐DR. Moreover, treatment of PC3, DU145, PC3‐DR, and DU145‐DR with salinomycin led to a significant reduction in the colony formation potential by targeting the colonies with high tumor‐initiating potential.ConclusionsTaken together, we demonstrated that salinomycin specifically targets the tumor‐initiating cell population in docetaxel‐sensitive and docetaxel‐resistant PCa cells and may represent a potential therapeutic approach for the treatment of advanced PCa.

Abstract B011: Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion

Abstract Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Current available treatments include chemical castration and therapies targeting androgen receptor (AR) signaling pathways. Although initial responses are observed, 30% of patients have primary resistance to both forms of treatment and the majority of patients progress from androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC). Studies on mechanisms of resistance suggest that AR signaling remains a key driver in PCa even after treatment with AR-targeted therapies. Co-treatment with therapies that modulate alternative signaling pathways involved in oncogenesis in PCa including epigenetic modifiers, have been proposed as a modality to overcome resistance. Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of the polycomb repressive complex 2 and is one of the most highly upregulated genes in CRPC compared to localized PCa. Several reports suggest that EZH2 plays an oncogenic role both in ADPC and CRPC.Tazemetostat is a potent, selective, orally bioavailable, investigative first-in-class small molecule inhibitor of EZH2. Phase 2 clinical trials have demonstrated objective clinical responses in patients with B-cell lymphomas and molecularly defined solid tumors, including epithelioid sarcoma and other INI1-negative tumors. Given the implication of EZH2 in PCa, we explored the combination potential of tazemetostat with current second-line therapies, the androgen signaling inhibitors (ASIs) abiraterone and enzalutamide. Tazemetostat displayed both time- and dose-dependent antiproliferative activity in PCa cell lines dependent on AR signaling or those of the neuroendocrine subtype with IC50 values at or below 1 μM in 14-day assays. Synergistic activity was observed in vitro in a subset of cell lines when tazemetostast was combined with ASIs in a 7-day co-treatment model and this activity was further enhanced when cells were pretreated with tazemetostat prior to the cotreatment. To further validate these findings, we conducted in vivo studies in PCa cell line-derived xenograft models. Tazemetostat and enzalutamide showed single agent antitumor activity in a subcutaneous LNCaP xenograft mouse model. Co-administration of enzalutamide and tazemetostat markedly reduced tumor growth rate compared to either of the single agent(s). Similar results were observed with the combination of tazemetostat and abiraterone. Interestingly in the 22Rv1-derived in vivo xenograft model, a cell line that expresses AR-V7, a splice variant implicated in resistance to ASIs in CRPC, modest tumor growth inhibition was observed with tazemetostat and no significant antitumor activity was observed when given in combination with abiraterone or enzalutamide. A significant decrease in intratumoral H3K27me3 levels were observed in all treatment groups containing tazemetostat in both in vivo models, indicating an on-target activity of EZH2 inhibition. Current efforts are underway to investigate the mechanism of action and cellular inhibitory pathways for these combinations. In summary, our preclinical data demonstrates the importance of EZH2 inhibition in CRPC and provides a rationale for combination of tazemetostat with abiraterone and enzalutamide in the clinic. Citation Format: Vinny Motwani, Dorothy Brach, Chloe Pantano, Vania Estanek, Jeffrey A. Keats, Daniel T. Dransfield, Alejandra Raimondi. Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B011. doi:10.1158/1535-7163.TARG-19-B011

Abstract C081: Targeting CDK9 and MCL1 in castration-sensitive and resistant prostate cancer models

Abstract Prostate cancer is the most prevalent cancer in men, comprising 20% of all new cancer cases in United States, as per the Cancer Statistics, 2019. Many therapies for prostate cancer function by lowering androgen levels and include androgen deprivation therapy alone or in combination with surgical or chemical castration. Hormone therapy has been a mainstay treatment for prostate cancer, ultimately leading to progression free disease in over 80% of patients over short time periods. Unfortunately, these effects are not durable, and the majority of patients experience progressive disease. Ultimately, the disease progresses and becomes invasive and lethal in the form of castration-resistant prostate cancer (CRPC). Understanding the mechanism by which prostate cancer cells lose their inherent dependence on the canonical androgen signaling pathway for survival has been critical in developing new therapeutic options for patients with CRPC. CDK9 may constitute one such mechanism. CDK9 phosphorylates RNA polymerase II (RNA Pol II), resulting in gene transcription of anti-apoptotic proteins such as MCL1, that can influence prostate cancer cell survival. Additionally, CDK9 is known to phosphorylate the androgen receptor (AR) and both AR and RNA Pol II cooperate for transcription of key prostate cancer genes, including PSA. Therefore, inhibition of CDK9 has great potential to halt constitutive activity of both AR and the RNA Pol II -driven transcriptional program that drives CRPC. Here, we describe tumor growth inhibition in CRPC models using the CDK9 inhibitor TP-1287, an oral alvocidib prodrug, alone and in combination with docetaxel or venetoclax, a BCL-2 inhibitor that is currently being evaluated in a Phase II clinical trial for metastatic CRPC. The CDK9 inhibitor alvocidib demonstrates low nanomolar IC50 values in CRPC cell lines PC3, 22Rv1 and castration dependent PCa cell lines VCAP and LnCAP ranging from 25-175 nM. Alvocidib treatment inhibits RNA Pol II in 22Rv1 cells (~50%) at 80 and 160 nM, 3 and 24 hrs post treatment and regulates MCL-1 protein expression in PC3 cells (300 nM, 24 hrs). Alvocidib treatment also inhibited AR phosphorylation at Ser 81, reducing PSA gene expression (~50%) and inducing caspase activation in 22Rv1 cells (80-160 nM for 24 hrs). We explored the tumor growth inhibition of TP-1287, in several PCa xenograft models namely PC3, 22Rv1, LnCAP and C4-2 models. In androgen sensitive models, 1.25 mpk BID TP-1287 combined well with 10 mpk docetaxel, demonstrating 90% and 81% TGI in C4-2 and LnCAP models respectively. TP-1287 treatment (1.25 mpk BID) in combination with 100 mpk venetoclax demonstrated 64% tumor growth inhibition in the 22Rv1 CRPC model that was resistant to docetaxel, enzalutamide and venetoclax alone. These data support the potential of TP-1287 to be used in combination with currently available or novel therapies to achieve better efficacy for androgen sensitive and CRPC patients. Citation Format: Tetyana V Forostyan, Evita Weagel, Yuta Matsumura, Ethika Tyagi, Jason M Foulks, Clifford J Whatcott, Adam Siddiqui-Jain, David J Bearss, Steven L Warner. Targeting CDK9 and MCL1 in castration-sensitive and resistant prostate cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C081. doi:10.1158/1535-7163.TARG-19-C081

Long non-coding RNA NORAD induces cell proliferation and migration in prostate cancer

ObjectivesProstate cancer (PCA) is the deadliest urological disease affecting men worldwide. Long noncoding RNA activated by DNA damage (NORAD) levels are increased in many cancer types, and induce cancer cell progression. However, little is known about the biological functions of NORAD in PCA.MethodsIn this work, the roles of NORAD in cell proliferation, migration, and apoptosis were examined by Cell Counting Kit-8, scratch wound, and annexin V-fluorescein isothiocyanate/propidium iodide staining assays, respectively, in PCA cell lines. Knockdown of NORAD was achieved by small interfering (si)RNA in PCA cell lines, and quantitative real-time PCR was used to detect the expression of NORAD.ResultsCell proliferation and migration rates were significantly lower in the siNORAD group than in the wild-type group, while the apoptosis level was significantly higher in the siNORAD group compared with the wild-type group.ConclusionsThese results suggest that NORAD promotes the proliferation and migration of PCA cells and inhibits their apoptosis.