Sensitive Prostate Cancer Cell Lines Research Articles

Abstract 3294: The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer

Abstract We have previously described our GSPT1 molecular glue degrader MRT-2359, which was optimized to achieve preferential antiproliferative activity in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) expressing high N-MYC or L-MYC mRNA. A clinical trial with this new drug candidate is ongoing (NCT05546268). Here, we present pre-clinical profiling of MRT-2359 across hundreds of cancer cell lines representing multiple cancer types, aiming at identifying other cancers where preferential sensitivity is associated with high expression of a MYC family member. These studies showed that prostate cancer cell lines clearly segregate into groups that are either sensitive or insensitive. Sensitive prostate cancer cell lines could be further divided into two subgroups: (1) androgen receptor positive cell lines displaying higher c-MYC mRNA expression levels than the other prostate cancer cell lines, (2) neuroendocrine prostate cancer cell lines with or without high N-MYC. The latter is in line with our previous observation that the neuroendocrine phenotype is generally associated with heightened sensitivity to MRT-2359 independently of MYC status. The insensitive cell lines were androgen receptor and neuroendocrine negative and displayed lower c-MYC mRNA levels. GSPT1 degradation in androgen receptor-positive cell lines led to rapid and deep loss of c-MYC protein as well as reduction of androgen receptor, including the splice variant AR-V7 that is associated with resistance to agents targeting androgen signaling. In androgen receptor-negative cell lines, the levels of c-MYC protein decreased only modestly upon GSPT1 degradation. Finally, in immunocompromised mice, xenografts of the AR-V7-positive cell line 22RV1 and the neuroendocrine prostate cell line NCI-H660, both of which are sensitive to MRT-2359 in vitro, were treated with several MRT-2359 dose regimens including continuous as well as intermittent (5 days on/9 days off) dosing. Most regimens led to marked tumor regression. Tumors of both models fully regressed after a 4-week course of 10 mg/kg MRT-2359 once daily, and no tumor regrowth could be detected after cessation of treatment. No significant in vivo response was observed for xenografts of the insensitive cell line PC-3. These data support the clinical investigation of MRT-2359 in prostate cancer. Citation Format: Ralph Tiedt, Martin Schillo, Arnaud Osmont, Débora Bonenfant, Rajiv Narayan, Owen Wallace, Markus Warmuth. The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3294.

Abstract 5540: Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer

Abstract Androgen receptor (AR)-targeted therapies are routinely used to treat prostate cancer, including the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these therapies are initially effective, a significant proportion of patients exhibit resistance to treatment, therefore developing castration-resistant prostate cancer (CRPC). While immune checkpoint blockade therapies have led to remarkable clinical responses in patients with several tumor types, CRPC remains resistant to immunotherapy. The characterization of tumor resistance mechanisms has led to the identification of various tumor variants that could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity. Our laboratory has previously shown that phenotypic plasticity is a driver of resistance to immunotherapy. Here we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways these mechanisms can be mitigated. Models of resistance to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR) were derived from the androgen sensitive LNCAP prostate cancer cell line. Resistant and parental LNCAP cells were comparatively evaluated for phenotypic features via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq analysis. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR and LNCAP parental cells were also grown in vivo in NSG MHCI/II-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets. Our results demonstrated that acquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including increased levels of vimentin and fibronectin, and the loss of epithelial E-cadherin. The susceptibility of LNCAP-Enza-R and LNCAP-AbiR cells to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC) were significantly reduced (up to 90%), compared with parental cells. RNAseq analysis identified several upregulated gene and gene pathways, including estrogen receptor 1 (ESR1), which was particularly overexpressed in LNCAP-EnzaR cells. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets. Our data indicates that acquisition of resistance to AR inhibition is associated with significant reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity features. Future studies plan to investigate approaches that revert tumor plasticity, including blockade of increased markers such as ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC. Citation Format: Madeline Dahut, Kristen Fousek, Lucas Horn, Haiyan Qin, Jeffrey Schlom, Claudia Palena. Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5540.

The Dual Effect of the HDAC Inhibitor Romidepsin on Androgen Receptor Signaling and DNA Damage Repair in Prostate Cancer.

Aberrant epigenetic modifications play an important role in prostate cancer development and progression. Histone deacetylase inhibitors (HDACi) are a novel class of small-molecular compounds that have been shown to have activity in several tumor types. Some HDACi's induce a stress response that affects DNA damage repair (radiosensitization) and androgen receptor (AR) signaling (increased sensitivity to androgen blockage). We hypothesized that the HDACi, romidepsin, would increase prostate cancer cell killing through dual effects on AR signaling and radiation DNA damage response.The prostate cancer cell lines LNCaP (androgen sensitive) and 22Rv1 (androgen insensitive) were treated with romidepsin (10 nM) plus or minus 2 Gy radiation. The added effect of enzalutamide (10 mM) was also tested. Cell viability was performed by Real-time Cell assay (RTCA), whole-transcriptome analysis by RNA sequencing, western blot analyses, immunofluorescent quantification of γ-H2AX double strand break foci, and colonogenic survival. Western blot analysis was used to measure the expression of (i) AR signaling pathway proteins that included wild-type AR and its AR-V7 splice variant and (ii) DNA repair genes that included Rad51, DNA-PK and γ-H2AX.Romidepsin reduced cell viability by RTCA of both cell lines in a dose dependent manner. The addition of enzalutamide further reduced cell viability and clonogenic survival. Gene ontology analysis from RNA-Seq data in both LNCaP and 22Rv1 cells revealed that romidepsin suppressed pathways related to prostate cancer aggressiveness including the AR, MYC, DNA damage response, and cell cycle progression pathways. By western blot analysis, romidepsin suppressed AR (LNCaP) and AR-V7 (22Rv1) expression and reduced DNA repair genes including RAD51, DNA-PK and phospho-ATM levels. After 2 Gy radiation, we found that treatment with 10nM romidepsin for 24 hours resulted in an increase in γ-H2AX foci in 35.2% in LNCaP and 27% in 22Rv1 cells respectively. By colonogenic assay, 10 nM romidepsin for 24 hours prior to 2 Gy to 8 Gy radiation resulted in a significant reduction in the surviving fractions. The addition of enzalutamide resulted in the greatest reduction in colonogenic survival.Our study demonstrated for the first time that HDACi romidepsin exerted dual effects on AR signaling and DNA damage response genes in androgen sensitive and insensitive prostate cancer cell lines, and that there is an added suppressive effect with the addition of enzalutamide to HDAC inhibition plus radiation. The use of HDACi's have promise clinically for the treatment of high-risk prostate cancer, for which androgen deprivation plus radiotherapy are the standard treatments.

In vitro and in silico studies of holothurin A on androgen receptor in prostate cancer

The androgen receptor (AR) plays a crucial role in the growth of prostate cancer, and has long been considered the cancer’s primary strategic therapeutic target. However, despite the early susceptibility, patients receiving hormonal therapy targeting AR are likely to develops resistance to the treatment and progresses to the castration-resistant stage as a consequence of the mutation at the ligand binding pocket of AR. Interestingly, the surface pocket of the AR called binding function 3 (BF3) has been reported as a great benefit for treating a recurrent tumor. Herein, we investigate the potential of using a marine triterpenoid saponin, holothurin A, on targeting AR expression of prostate cancer using in vitro and in silico studies. Holothurin A reduced the PSA expression, leading to the growth inhibition of androgen sensitive prostate cancer cell line through a downregulation of AR activity. The molecular docking study demonstrated that holothurin A could bind strongly in the BF3 pocket by energetically favorable hydrogen acceptor and hydrophobic with a calculated binding affinity of −13.90 kcal/mol. Molecular dynamics simulations provided the additional evidence that holothurin A can form a stable complex with the BF3 pocket through the hydrophobic interactions with VAL676, ILE680, and ALA721. As a consequence, holothurin A modulates the activation function-2 (AF2) site of the AR through repositioning of the residues in the AF2 pocket. Targeting alternatives sites on the surface of AR via holothurin A will provide a potential candidate for future prostate cancer treatment. Communicated by Ramaswamy H. Sarma

Abstract LB102: Effect of mistletoe extract on growth and proliferation of prostate cancer cells

Abstract Complementary and alternative medicines are explored for their role in improving the quality of life of patients with cancer. Many anti-cancer drugs have been derived from natural sources. Mistletoe extract, composed of lectins, viscotoxins, flavonoids, and other biologically active substances, has two mechanisms of action: tumor cytotoxicity and immunomodulation. The cytotoxic effects of mistletoe extract are a result of protein synthesis interference, cell cycle inhibition, and apoptosis induction. Further, mistletoe extract has been shown to have anti-angiogenic properties. In Europe, Helixor M, mistletoe extract from Viscum album mali, is used as an anthroposophic medicine in the treatment of malignant and non-malignant tumors, malignant disorders with accompanying impairment of the hematopoietic organs, stimulation of bone marrow activity, reducing the risk of tumor recurrence, and diagnosed pre-cancerous disorders. However, only limited evaluation of mistletoe extract has been conducted in the United States. We studied the effects of Helixor M in prostate cancer. To evaluate its effect on cell proliferation and survival, we treated androgen receptor (AR) positive (LNCaP, LAPC4 and VCaP) and AR negative (PC3 and DU-145) cell lines with Helixor M. Our data indicates that LNCaP and LAPC4 cells were most sensitive to Helixor M while PC3, DU-145, and VCaP cells were relatively resistant to treatment. While AR protein levels and NF-kappaB subunits remained unaffected by treatment, there was an induction of apoptosis along with a concomitant decrease in LC3B, an autophagy marker, in all the sensitive prostate cancer cell lines. In search of pathways that may synergize with growth inhibition by Helixor M, we utilized CRISPR knockouts of TBK1, a kinase involved in autophagy induction, in LNCaP cells and tested whether TBK1 knockouts are synthetically lethal with Helixor M. Helixor M induced increased apoptosis in TBK1 knockouts as compared to parental LNCaP cells, indicating that TBK1 loss might be synthetically lethal with Helixor M. Citation Format: Kavya Boyapati, Rajendra Kumar, Lillian Wilson, Yuhan Yang, Deven Topiwala, Keerti Soundappan, Eleni Panagopoulos, Ivelisse Page, Jennifer Durham, Michael Carducci, Channing Paller, Sushant Kachhap. Effect of mistletoe extract on growth and proliferation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB102.

Abstract 6376: Identification and characterization of tumor types that are driven into apoptosis/regression upon treatment with p300/CBP interactome modulator INTH-454

Abstract Background: Blockage of the CH1/TAZ1 domain of p300/CBP has a profound effect on cell cycle regulation in cancer cells. A small molecule modulator of CH1/TAZ1, INTH-454, has been shown to trigger cell cycle arrest followed by either cell death or senescence causing sustained anti-tumoral effects in xenograft models of several hard-to-treat tumor types like castration-resistant prostate and gastric cancer. Methods: The anti-proliferative effects of INTH-454 were assessed in a panel of five hundred cancer cell lines. A comprehensive analysis of gene expression and mutational data led to the selection of representative cell lines for subsequent in-depth studies of the corresponding molecular events by cell cycle analysis. INTH-454's impact on genes and proteins involved in cell cycle progression and apoptosis e.g. cyclins and caspases were investigated by qPCR, western blotting and reporter assays. The in vitro results were confirmed in cell line and patient derived xenografts models of gastric and prostate cancer. Results: INTH-454 inhibited cell growth at low nanomolar concentrations in one third of the five hundred cell lines tested. In most of these cell lines, the compound caused cytotoxic effects with >90% inhibition of proliferation at submicromolar concentrations. We used information on tissue origin, gene expression and mutations of the panel to select several cell lines for further characterization. Longitudinal analysis of very sensitive prostate, breast, lung and gastric cancer cell lines showed that the cell cycle was heavily dysregulated. INTH-454 induced cell cycle arrest, generated either strong apoptotic responses or senescence, and caused reduction of protein levels of important cellular factors e.g. c-Myc. Accordingly, INTH-454 treatment caused profound growth inhibition of the tumors in the respective xenograft models under various dosing regimens. Apoptotic/cytotoxic effects in vitro translated into tumor regression while senescence in vitro correlated with tumor stagnation in vivo. Conclusions: INTH-454 exhibits strong anti-proliferative effects in a very broad panel of cancer cell lines. Thorough analysis of particularly responsive genotypes in vitro led to the identification of predictive biomarkers for selection of cell lines that undergo apoptosis upon treatment. These in vitro findings were successfully translated to in vivo xenograft models and will facilitate the identification of patients/cancers that are most susceptible to treatment with INTH-454. Citation Format: Valentino Cattori, Beatrice Pilger, Ulrich Kessler. Identification and characterization of tumor types that are driven into apoptosis/regression upon treatment with p300/CBP interactome modulator INTH-454 [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 6376.

4,4′-Diisothiocyanatostilbene-2,2′-disulfonate modulates voltage-gated K+ current and influences cell cycle arrest in androgen sensitive and insensitive human prostate cancer cell lines

The stilbene derivative, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), an anion channel blocker is used in the present study to evaluate its modulatory effect on voltage-gated K+ current (IK) in human prostate cancer cell lines (LNCaP and PC-3). Voltage-gated K+ (KV) channels in the plasma membrane are critically involved in the proliferation of tumor cells. Therefore, KV channels are considered as a novel potential target for cancer treatment. The results of the present study show that the external perfusion of DIDS activates IK in a concentration-dependent manner, although the known K+ channel blocker TEA failed to block the DIDS activated IK in PC-3 cells. Whereas, in LNCaP cells, the higher concentration of DIDS blocked IK, though this effect was not completely recovered after washout. The difference in function of DIDS might be due to the expression of different Kv channel isoforms in LNCaP and PC-3 cells. Further, the anticancer studies show that treatment of DIDS significantly induced G2/M phase cell cycle arrest and induced moderate and low level of cell death in LNCaP and PC-3 cells respectively. This finding reveals that DIDS modulates IK and exerts cell cycle arrest and cell death in LNCaP and PC-3 cells.

Piperine blocks voltage gated K+ current and inhibits proliferation in androgen sensitive and insensitive human prostate cancer cell lines

Piperine is an attractive therapeutic alkaloid from black pepper that exhibits a broad spectrum of pharmacological properties over various pathological disorders including cancer. Voltage gated K+ (KV) channels play an important role in regulating cancer cell proliferation and are considered as potential target for cancer treatment. However, the implication of piperine in KV associated anticancer activities on human prostate cancer cells LNCaP and PC-3 remains unrevealed. The electrophysiological and pharmacological data identifies that both androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cells typically expressed voltage gated K+ current (IK). This current was significantly blocked by piperine in a concentration-dependent manner with an IC50 value 39.91 μM in LNCaP and 49.45 μM in PC-3 cells. Analysis of voltage-dependence of activation kinetics showed that piperine induces a positive shift in the relative activation curve in both the cells. Piperine also depolarized the resting membrane potential by an average of 10.2 mV and 8.3 mV in LNCaP and PC-3 cells, respectively. The anticancer studies showed that, treatment with piperine concentration dependently induced G1 phase cell cycle arrest and apoptosis in LNCaP and PC-3 cells. These results unravel that the IK inhibition might be responsible for the anticancer effect of piperine on androgen sensitive and insensitive human prostate cancer cells.

Abstract 5787: Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models

Abstract Introduction: Therapeutic targeting the PI3K/AKT signaling pathway is an attractive strategy to combat solid tumors that exhibit hyper-AKT activity. Ipatasertib, a potent pan-AKT inhibitor, has exhibited significant inhibitory effects on tumor growth in preclinical studies, including models of Androgen Receptor (AR)-independent prostate cancer (PCa) that lack PTEN, a negative regulator of the PI3K/AKT pathway. It is currently being investigated in several ongoing clinical trials, including evaluating its efficacy in combination with abiraterone in castrate resistant PCa (CRPC) (NCT01485861). There is cross-talk between androgen signaling and PI3K/AKT pathway activation, however the optimal setting for AKT inhibition in PCa is not clear. Here we seek to determine the benefit of ipatasertib for androgen sensitive (AS) PCa alone or in combination with androgen deprivation (AD), as well as the benefit of ipatasertib alone or in combination with the antiandrogen enzalutamide in CRPC. Methods: In vitro studies utilized human androgen sensitive PCa cell lines LAPC, LAPC4shPTEN, and LNCAP, as well as two LNCaP-derived, castrate resistant cell lines. Cell viability was determined using CellTitre Glo assay and apoptosis by flow cytometry. Inhibitory effects of AKT signaling pathway was determined by western blot assays against AKT phosphorylation sites and downstream targets as well as detection for hormone receptor expression. In vivo studies utilized LuCaP 136 and LuCaP 136-derived castrate resistant (LuCaP 136CR) patient derived xenograft (PDX) models. Cells were injected into athymic mice and grouped into 4 arms. Intact mice bearing AS tumors were treated with vehicle, ipatasertib (100mg/Kg), AD, or ipatasertib + AD. Castrated mice bearing castrate-resistant tumors were treated with vehicle, ipatasertib, enzalutamide (10mg/Kg), or ipatasertib + enzalutamide. Tumor growth was determined twice/week via blinded caliper measurements. Results: Ipatasertib effectively inhibited AKT signaling, as indicated by significant decrease in phosphorylation of the downstream targets P70S6K and 4E-BP1. Our in vitro data indicate significant anti-proliferation effects of ipatasertib across all PCa models, both androgen sensitive and castrate resistant. Interestingly, this was irrespective of PTEN status. In addition, we observed a decrease in glucocorticoid receptor (GR) expression in enzalutamide resistant lines, although AR expression was not significantly altered. Preclinical animal studies showed significant response to tumor growth inhibition with ipatasertib monotherapy, which was enhanced with combination treatment. Conclusion: Ipatasertib is beneficial in models of androgen sensitive and castrate resistant PCa and can be effective in the treatment of PCa alone or in combination with current standard therapies targeting the androgen receptor. Citation Format: Remi M. Adelaiye-Ogala, Yen T. Nguyen, Jose A. Rodriguez-Nieves, Aian Neil Alilin, Supreet Agarwal, David Vanderweele. Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5787.

Ginger Phytochemicals Inhibit Cell Growth and Modulate Drug Resistance Factors in Docetaxel Resistant Prostate Cancer Cell.

Ginger has many bioactive compounds with pharmacological activities. However, few studies are known about these bioactive compounds activity in chemoresistant cells. The aim of the present study was to investigate the anticancer properties of ginger phytochemicals in docetaxel-resistant human prostate cancer cells in vitro. In this study, we isolated 6-gingerol, 10-gingerol, 4-shogaol, 6-shogaol, 10-shogaol, and 6-dehydrogingerdione from ginger. Further, the antiproliferation activity of these compounds was examined in docetaxel-resistant (PC3R) and sensitive (PC3) human prostate cancer cell lines. 6-gingerol, 10-gingerol, 6-shogaol, and 10-shogaol at the concentration of 100 μM significantly inhibited the proliferation in PC3R but 6-gingerol, 6-shogaol, and 10-shogaol displayed similar activity in PC3. The protein expression of multidrug resistance associated protein 1 (MRP1) and glutathione-S-transferase (GSTπ) is higher in PC3R than in PC3. In summary, we isolated the bioactive compounds from ginger. Our results showed that 6-gingerol, 10-gingerol, 6-shogaol, and 10-shogaol inhibit the proliferation of PC3R cells through the downregulation of MRP1 and GSTπ protein expression.

Abstract 2102: ONC201 targets AR and AR-V7 signaling pathways, reduces PSA and synergizes with everolimus in castration resistant prostate cancer

Abstract Androgen receptor (AR) signaling plays a key role in prostate cancer progression; thus Androgen Deprivation Therapy (ADT) is a mainstay therapy for patients with advanced prostate cancer. However, in most cases the tumor becomes androgen-independent and resistant to ADT with patients ultimately progressing to metastatic castration resistant prostate cancer (mCRPC). Constitutively activated AR splice variants (AR-Vs) have emerged as major mediators of resistance to AR-targeted therapy and progression of mCRPC, representing an important therapeutic target for mCRPC. Out of at least 15 AR-Vs described thus far, AR-V7 is the most abundant in prostate cancer and its expression correlates with ADT resistance. ONC201 is the founding member of the imipridone class of small molecules that induces apoptosis in a variety of tumor types tested. It is currently in Phase I/II clinical trials for advanced solid tumors and hematological malignancies, including mCRPC with encouraging activity observed in patients in early clinical testing (Stein et al., ASCO, 2016). We demonstrate here that ONC201 treatment induces apoptosis in both castration-resistant and –sensitive human prostate cancer cell lines. Furthermore, an in-vitro synergistic effect is observed with ONC201 and FDA approved drugs for prostate cancer such as enzalutamide, docetaxel and everolimus. Mechanistically, we found that ONC201 inhibits both AR and AR-V7 signaling pathways. Analysis of AR+ cell lines stimulated with DHT showed a significant decrease in both RNA and protein levels of AR, AR-V7, PSA and other AR-target genes following ONC201 treatment. Ongoing experiments are aimed at better understanding the molecular and cellular mechanisms that enable ONC201 to inhibit the AR signaling pathway. In-vivo studies with AR-negative hormone refractory prostate cancer xenografts demonstrate single agent ONC201 anti-tumor efficacy. Ongoing studies are further evaluating the in-vivo efficacy of ONC201 as a single agent or in combination with enzalutamide and everolimus using mouse models of both castration-resistant and -sensitive prostate cancer. These preclinical results indicate that ONC201 is well suited to address mCRPC, including tumors that harbor AR-V7, as a single agent that may be combined synergistically with enzalutamide or everolimus . With these observations we envision further development a combination of ONC201 plus everolimus in CRPC with or without enzalutamide resistance. Citation Format: Avital Lev, Amriti R. Lulla, David T. Dicker, Wafik S. El-Deiry. ONC201 targets AR and AR-V7 signaling pathways, reduces PSA and synergizes with everolimus in castration resistant 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 2102. doi:10.1158/1538-7445.AM2017-2102

Abstract 1516: Androgen-repressed and androgen-induced genes: challenging the traditional dogma of prostate cancer therapy

Abstract Background: Benign and malignant prostate tissues are dependent upon the activity of androgen receptor (AR). The primary function of full-length AR is as a ligand activated transcription factor to increase or repress gene expression. Many androgen-repressed genes regulate the cell cycle and proliferation. With castration, the main therapeutic approach for advanced prostate cancer (PC), these genes are believed to play a role in the initial clinical response. Current approved therapies for advanced PC and castration-resistant PC (CRPC), target the AR C-terminal ligand-binding domain (LBD), such as antiandrogens. Recent antagonists of the AR N-terminal domain (NTD) have been described with EPI-506, the prodrug of EPI-002, now in Phase 1 clinical trials. EPI-002 binds tau-5 in activation function-1 (AF-1) of the NTD that is essential for AR transcriptional activity. As expected with an AF-1 antagonist, EPI-002 is an excellent inhibitor of androgen-induced gene expression and at blocking the transcriptional activities of truncated AR splice variants lacking LBD, such as AR-V7. EPI-002 blocks expression of genes regulated by truncated AR-V7 such as UBE2C while antiandrogens have no effect. Here we reveal that the major difference in gene expression regulated by full-length AR between EPI-002 and antiandrogens is their abilities to de-repress genes that are turned off by androgen. Methods: The androgen sensitive human prostate cancer cell line LNCaP, which expresses full-length AR, was treated with antiandrogens (bicalutamide [BIC] and enzalutamide [ENZA]), EPI-002 and a control vehicle, with and without androgen. Gene expression was analysed using Affymetrix microarrays. Bioinformatical analysis was completed and a selection of androgen-repressed genes that were de-repressed with antiandrogens and/or EPI-002, were selected for validation using qRT-PCR. Results: EPI-002 de-repressed known androgen-repressed genes including SPLTLC3, ST7, PSAT1, TMEM140 and TNFRSF21. EPI-002 was as effective or better than BIC or ENZA in de-repressing a subset of androgen-repressed genes. Importantly, EPI-002 failed to de-repress expression of many androgen-repressed genes that antiandrogens de-repressed, such as SLITRK3, GPR63 and DAB1. Conclusions: EPI binds AR NTD which blocks the transcriptional activities of full-length AR and truncated AR splice variants. EPI-002 was excellent at inhibiting androgen-induced genes. However, EPI did not broadly de-repress expression of genes turned off by androgen when compared to antiandrogens. Such differences between EPI-002, a tau-5/NTD antagonist, and C-terminal LBD antiandrogens probably reflect the complexities of the mechanisms of repression of gene expression that may involve different domains of AR. Citation Format: Daniel P. Caley, Nasrin R. Mawji, Marianne Sadar. Androgen-repressed and androgen-induced genes: challenging the traditional dogma of prostate cancer therapy [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 1516. doi:10.1158/1538-7445.AM2017-1516

Abstract 197: Anti-cancer activity of extract from the Jamaican round-leaf yellow yam (RLY2) (Dioscorea cayenensis)

Abstract The Jamaican Round-Leaf yellow yams (RLY2), Dioscorea cayenensis, are edible tubers, bulbils or rhizomes that are of considerable economic value and possess several health benefits. These RLY2 are a rich source of anti-oxidants, vitamins and phytochemicals. In addition to its nutritional value, the Jamaican populace regards RLY2 as an herbal medicine useful in treatment of diabetes mellitus, hypertension and certain kind of cancers. Chemical analysis of RLY2 revealed the presence of a myriad of bioactive ingredients that are likely to be lost due to the method of preparation for consumption. Thus, we hypothesized that mild extraction of RLY2 will preserved the bioactive analytes that give RLY2 its anti-cancer property. To test this hypothesis, different ethanolic extracts of RLY2 were prepared, and their bioactivity demonstrated in two models of prostate cancer. The present study demonstrates dosage and time-dependence inhibition in both the androgen sensitive LNCaP and androgen insensitive DU-145 prostate cancer cell lines. We observed that DU-145 was three-times more sensitive to RLY2 ethanol extract than LNCaP. Growth of LNCaP for 3-days with varying concentration of RLY2 ethanol extract resulted in an IC50 of 750 ppm (95% C.I. 726-791ppm), whereas DU-145 resulted in an IC 50 of 250 ppm (95% C.I. 236-281ppm). The maximum growth inhibition by RLY2 extract occurs within twenty-four hours for both LNCaP and DU-145 with no significant changes after 5-days. To determine the mechanism of toxicity by RLY2, we induced both LNCaP and DU-145 with RLY2 for 0-24h and prepared cells for flow cytometer analysis. The effect of RLY2 on cell cycle and degree of apoptosis will be discussed. Our data indicated that the ethanol extract of RLY2 was more sensitive toward androgen-insensitive prostate cancer. Thus, this study suggests that RLY2 contain bioactive chemical that may be used in grade II prostate adenocarcinoma represented by the DU-145 phenotype. Citation Format: Sasha-Gay A. Wright, Wesley G. Gray, Helen Asemota. Anti-cancer activity of extract from the Jamaican round-leaf yellow yam (RLY2) (Dioscorea cayenensis) [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 197. doi:10.1158/1538-7445.AM2017-197

Synthesis and Antitumor Activity of New Group 3 Metallocene Complexes

The quest for alternative drugs with respect to the well-known cis-platin and its derivatives, which are still used in more than 50% of the treatment regimens for patients suffering from cancer, is highly needed. In this context, organometallic compounds, which are defined as metal complexes containing at least one direct covalent metal-carbon bond, have recently been found to be promising anticancer drug candidates. A series of new metallocene complexes with scandium, yttrium, and neodymium have been prepared and characterized. Some of these compounds show a very interesting anti-proliferative activity in triple negative breast cancer cell line (MDA.MB231) and the non-hormone sensitive prostate cancer cell line (DU145). Moreover, the interaction of some of them with biological membranes, evaluated using liposomes as bio-membrane mimetic model systems, seems to be relevant. The biological activity of these compounds, particularly those based on yttrium, already effective at low concentrations on both cancer cell lines, should be taken into account with regard to new therapeutic approaches in anticancer therapy.

Probing the prostate tumour microenvironment I: impact of glucose deprivation on a cell model of prostate cancer progression.

In the developed world, prostate cancer is the most common cancer diagnosis in men. Although prostate cancer initially presents as a non life-threatening disease, 90% of patients will develop castration resistant prostate cancer (CRPC), which preludes distant metastasis and is largely accountable for prostate cancer associated deaths. This is because as yet, there are no viable molecular therapeutic targets for effective treatment of CRPC. It is now widely accepted that cancer cells can alter their metabolic profile during the course of tumourgenesis and metastasis such that they are able to survive in oxygen and nutrient-poor environments. This work was aimed towards gaining greater mechanistic understanding of how such stresses in the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under nutrient deficient (low glucose) conditions. This led to the identification of approximately 4,000 proteins - one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of low glucose conditions was established. Novel, intuitive workflows were subsequently implemented to ensure the verification of selected proteins of interest in a robust, reproducible and high throughput manner. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

Probing the prostate tumour microenvironment II: Impact of hypoxia on a cell model of prostate cancer progression

Approximately one in six men are diagnosed with Prostate Cancer every year in the Western world. Although it can be well managed and non-life threatening in the early stages, over time many patients cease to respond to treatment and develop castrate resistant prostate cancer (CRPC). CRPC represents a clinically challenging and lethal form of prostate cancer. Progression of CRPC is, in part, driven by the ability of cancer cells to alter their metabolic profile during the course of tumourgenesis and metastasis so that they can survive in oxygen and nutrient-poor environments and even withstand treatment. This work was carried out as a continuation of a study aimed towards gaining greater mechanistic understanding of how conditions within the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under hypoxic conditions. This led to the identification of over 4,000 proteins – one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of hypoxic conditions was established. Novel, intuitive workflows were subsequently implemented to enable robust, reproducible and high throughput verification of selected proteins of interest. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

Sex Hormone Binding Globulin Modifies Testosterone Action and Metabolism in Prostate Cancer Cells.

Sex Hormone Binding Globulin (SHBG) is the major serum carrier of sex hormones. However, growing evidence suggests that SHBG is internalised and plays a role in regulating intracellular hormone action. This study was to determine whether SHBG plays a role in testosterone uptake, metabolism, and action in the androgen sensitive LNCaP prostate cancer cell line. Internalisation of SHBG and testosterone, the effects of SHBG on testosterone uptake, metabolism, regulation of androgen responsive genes, and cell growth were assessed. LNCaP cells internalised SHBG by a testosterone independent process. Testosterone was rapidly taken up and effluxed as testosterone-glucuronide; however this effect was reduced by the presence of SHBG. Addition of SHBG, rather than reducing testosterone bioavailability, further increased testosterone-induced expression of prostate specific antigen and enhanced testosterone-induced reduction of androgen receptor mRNA expression. Following 38 hours of testosterone treatment cell morphology changed and growth declined; however, cotreatment with SHBG abrogated these inhibitory effects. These findings clearly demonstrate that internalised SHBG plays an important regulatory and intracellular role in modifying testosterone action and this has important implications for the role of SHBG in health and disease.

Mir-15A Reconstitution in Prostate Cancer Cell Line Suppresses Cancer Progression Through Down Regulation of MYB and Androgen Receptor Upregulation

Summary Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. MicroRNAs are noncoding RNAs that have a role of post-transcriptional regulators. In this study we investigated how the tumour suppressor miR-15a modulates main transcription factors like cMYB and AR in androgen sensitive prostate cancer cell line LNCaP. The miR-15a inhibitor, mimic, and their negative controls were transfected into LNCaP cells. Real-time PCR analysis was performed in order to estimate the transcript levels of cMYB and AR. Flow cytometry analysis was performed to measure the protein levels of cMYB and AR. A Cell migration assay was done for cells transfected with miR-15a inhibitor and mimic. We found that cMYB is down-regulated and AR is up-regulated by miR-15a on the transcriptional and protein levels. By reconstituting miR-15a, we found that its down regulation in prostate cancer contributes to cMYB-induced cancer progression and reduced androgen receptivity. The ability of miR-15a to suppress cancer cell viability and migration is a very important phenomenon for understanding cancer heterogeneity in regard to adapted therapeutic approach development.

RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

BackgroundRadiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.MethodsUsing an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.ResultsOpposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).ConclusionsThese findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-808) contains supplementary material, which is available to authorized users.

Peripheral blood microRNAs expression profile: A fingerprint for metastatic prostate cancer.

e22171 Background: We previously have studied differentially microRNA expression levels related to hormone refractory and sensitive prostate cancer cell lines (VCap and LNCap respectively). Now, we investigated circulating miRNAs differentially expressed between metastatic prostate adenocarcinomas (mPA) and healthy controls (HC) that may serve as novel diagnostic and/or prognosis markers. Methods: Using SYBR-green-based custom microRNA RT-qPCR arrays technology (Exiqon), we compared the expression levels of miRNAs in blood samples from 18 HC and 48 mPA. Results: Among a panel of 92 candidates we found 41 underexpressed (UE) and 8 overexpressed (OE) miRNAs. The level of significance was p<0.05. Conclusions: Among the UE miRNAs are miR-203 and 129 which regulate a cohort of metastatic genes. Among the OE is miR-200a, a member of miR-200 family which directly targeted β-catenin mRNA and miR-135b which induce invasion and distant metastasis. The knowledge of miRNAs involved in mPA progression could provide us a profile that could identify patients for adjuvant therapy or even propose new target drugs to avoid disease progression or treat mPA. Our group is actively involved in the study of selected OE miRNAs for their potential as circulating markers for mPA. [Table: see text]