Androgen-sensitive Prostate Cancer Cell Line Research Articles

Abstract 2185: Mechanism of antitumor effects of saffron in human prostate cancer cells

Abstract Prostate cancer is the most common malignancy in men in the USA and the second leading cause of cancer deaths. Several studies have suggested the antitumor properties of saffron in many types of cancers including prostate. The objective of this study was to carry out in vitro studies of saffron-treated prostate cancer cells to ascertain the effects of saffron on key intermediates in prostate carcinogenesis. Our studies demonstrate significant inhibition of cell proliferation of androgen-sensitive prostate cancer cell lines via apoptotic pathways. We also demonstrate statistically significant down-regulation of DNA methyltransferase; COMT, MGMT, EHMT2, and SIRT1 in saffron-treated prostate cancer cells. In addition, saffron-treated prostate cancer cells displayed statistically significant dysregulation of DNA repair intermediates; WRN, p53, RECQ5, MST1R, and WDR70 in a time-dependent manner. Furthermore, Western blot analysis demonstrated that saffron treatment induced changes in the expression of other key genes; DNMT1, DNMT3b, MBD2, CD44, HDAC3, c-Myc, NF-kB, TNFα, AR, N-RAS and PTEN in prostate cancer cells. Collectively, our findings demonstrate important mechanistic targets of saffron mediating anti-tumor properties in prostate cancer. These findings suggest that the use of saffron supplements alongside standard treatment protocols may yield beneficial effects for individuals with prostate cancer. Citation Format: Mohammad Khan, Kaitlyn Hearn, Christian Parry, Mudasir Rashid, Hassan Brim, Hassan Ashktorab, Bernard Kwabi-Addo. Mechanism of antitumor effects of saffron in human prostate cancer cells [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 2185.

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

Mechanism of Antitumor Effects of Saffron in Human Prostate Cancer Cells.

Prostate cancer is the most common cancer and the second leading cause of cancer deaths among men in the USA. Several studies have demonstrated the antitumor properties of saffron in different types of cancers, including prostate cancer. The oral administration of saffron extract has been reported to have antitumor effects on aggressive prostate-cancer-cell-line-derived xenografts in nude male mice. The objective of this study was to carry out in vitro studies of saffron-treated prostate cancer cells to ascertain the effects of saffron on key intermediates in prostate carcinogenesis. Our studies demonstrated the significant inhibition of cell proliferation for androgen-sensitive prostate cancer cell lines via apoptotic pathways. We also demonstrate the statistically significant down-regulation of DNA methyltransferases (COMT, MGMT, EHMT2, and SIRT1 deacetylase) in saffron-treated prostate cancer cells. In addition, saffron-treated prostate cancer cells displayed a statistically significant dysregulation of DNA repair intermediates (WRN, p53, RECQ5, MST1R, and WDR70) in a time-dependent manner. Furthermore, Western blot analysis demonstrated that saffron treatment induced changes in the expression of other key genes (DNMT1, DNMT3b, MBD2, CD44, HDAC3, c-Myc, NF-kB, TNFα, AR, N-RAS, and PTEN) in prostate cancer cells. Collectively, our findings demonstrate the important mechanisms by which saffron mediates anti-tumor properties in prostate cancer. These findings suggest that the use of saffron supplements alongside standard treatment protocols may yield beneficial effects for individuals with prostate cancer.

Design, Synthesis, and Biological Evaluation of Anti-prostate Cancer Agent

In search of more effective chemotherapeutics for the treatment of castration-resistant prostate cancer. The 1,3- thiazolidine-2,4-diones possess a wide diversity of important biochemical effects and interesting pharmacological properties. 1,9-diarylnona-1,3,6,8-tetraen-5-ones bearing two identical terminal heteroaromatic rings have been successfully synthesized through Wittig reaction followed by Horner-Wadsworth-Emmons reaction. The cell proliferation assay was employed to assess their anti-proliferative effects toward both androgen-sensitive and androgen-insensitive human prostate cancer cell lines. curcumin in inhibiting prostate cancer cell proliferation. It can be concluded from our data that 1,9-diarylnona-1,3,6,8-tetraen-5-one can serve as a new potential scaffold for the development of anti-prostate cancer agents.

Comparative Proteomic and Transcriptomic Analysis of the Impact of Androgen Stimulation and Darolutamide Inhibition.

Several inhibitors of androgen receptor (AR) function are approved for prostate cancer treatment, and their impact on gene transcription has been described. However, the ensuing effects at the protein level are far less well understood. We focused on the AR signaling inhibitor darolutamide and confirmed its strong AR binding and antagonistic activity using the high throughput cellular thermal shift assay (CETSA HT). Then, we generated comprehensive, quantitative proteomic data from the androgen-sensitive prostate cancer cell line VCaP and compared them to transcriptomic data. Following treatment with the synthetic androgen R1881 and darolutamide, global mass spectrometry-based proteomics and label-free quantification were performed. We found a generally good agreement between proteomic and transcriptomic data upon androgen stimulation and darolutamide inhibition. Similar effects were found both for the detected expressed genes and their protein products as well as for the corresponding biological programs. However, in a few instances there was a discrepancy in the magnitude of changes induced on gene expression levels compared to the corresponding protein levels, indicating post-transcriptional regulation of protein abundance. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) and Hi-C chromatin immunoprecipitation (HiChIP) revealed the presence of androgen-activated AR-binding regions and long-distance AR-mediated loops at these genes.

Abstract LB087: Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models

Abstract TMEFF2, a transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is primarily restricted in expression to healthy brain and prostate and is enriched in prostate cancer. JNJ-70218902 (JNJ-902) is a bispecific antibody that engages TMEFF2-expressing tumor cells and CD3 on T cells causing exposure dependent pro-inflammatory responses and target tumor cell lysis. Here we report the preclinical characterization of JNJ-902 using in vitro and in vivo prostate cancer models. Expression of TMEFF2 transcript and protein in brain and prostate was confirmed in healthy human tissue, as was its elevated expression in advanced prostate cancer tumors, including in 41/45 (91%) metastatic castration-resistant prostate cancer (mCRPC) samples. JNJ-902 bound to TMEFF2-positive LNCaP-AR cells (androgen-sensitive human prostate cancer cell line) in a concentration-dependent manner (EC50 = 9.6 nM); no binding to TMEFF2-negative DU145 prostate cancer cells was observed. The binding affinity (KD) of JNJ-902 for CD3 on primary human T cells was determined to be ~151 nM. T cell-mediated killing, as measured by caspase-3 activity, was induced upon incubation of JNJ-902 with healthy human donor T cells (n = 5) and LNCaP-AR cells at a 3:1 effector to target ratio (EC50 = 1.4 nM). No cytotoxicity was detected using DU145 cells or with control antibodies. Corresponding T cell activation was confirmed by concentration-dependent increases in cell surface CD8+CD25+ expression and proinflammatory cytokine production (eg, GM-CSF, IFN-γ, IL-10, TNF-α). Antitumor activity of JNJ-902 at increasing concentrations up to 5 mg/kg was observed in T cell humanized NSG mice bearing LNCaP xenografts (tumor growth inhibition [∆%TGI] of 75-122%) and in LuCaP 86.2 patient-derived xenografts (∆%TGI of 72-88%) as compared with control-treated mice. JNJ-902-dependent intratumoral infiltration of CD8+ T cells was detected in both models with increases in frequency of CD8+granzymeB+ effector cells. The data summarized here are the first report on the pharmacological activity of a novel TMEFF2 × CD3 bispecific antibody, supporting further investigation of TMEFF2 in mCRPC given its expression in advanced prostate cancer. Targeting TMEFF2 with the TMEFF2 × CD3 bispecific antibody JNJ-902 elicited robust T cell-mediated responses in both prostate cancer cells and xenograft models. Based on these data, a phase 1 dose escalation trial of JNJ-902 in patients with mCRPC is currently ongoing (NCT04397276). Citation Format: Subhasree Basu, Mike Russell, Katrin Sproesser, Bethany Mattson, Cassandra L. Lowenstein, Sathya Venkataramani, Maura Diamond, Robin E. Ernst, Stephen Jarantown, Jackson Wong, Gerald Chu, Kathryn Packman, Shaozhou Ken Tian, Scott R. Brodeur, Margaret Yu, Brent A. Rupnow. Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models [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 LB087.

Double-layer omics analysis of castration- and X-ray-resistant prostate cancer cells.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P < 0.01). The Lethal dose 50 values of carbon ion beams were 0.9 Gy and 0.7 Gy, respectively (P = 0.09). On DNA mutation analysis, AR mutation was observed specifically in LNCaP-LA. From RNA-seq, 181 genes were identified as differentially expressed genes (DEGs; FDR <0.10, P < 0.00076) between LNCaP and LNCaP-LA. Function analysis suggested that cell death was suppressed in LNCaP-LA, and pathway analysis suggested that the NRF2-pathway involved in intracellular oxidative stress prevention was activated in LNCaP-LA. LNCaP-LA showed X-ray resistance compared to LNCaP and sensitivity to carbon ion beams. The AR mutation and the NRF2-pathway were suggested as causes of resistance.

Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-β-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

ObjectiveDeleted in liver cancer 1 (DLC1) is a GTPase-activating protein that is reported as a suppressor in certain human cancers. However, the detailed biological function of DLC1 is still unclear in human prostate cancer (PCa). In the present study, we aimed to explore the function of DLC1 in PCa cells. MethodsSilencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line (LNCaP) using RNA interference and lentiviral vector transduction. The Cell Counting Kit-8 assay was performed to determine cell proliferation. The cell cycle was examined by performing a propidium iodide staining assay. ResultsOur results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells. Moreover, DLC1 expression was negatively correlated with Rho-associated protein kinase (ROCK) expression in LNCaP cells. Importantly, this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo. ConclusionOur results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

Suppressive Effect and Molecular Mechanism of Houttuynia cordata Thunb. Extract against Prostate Carcinogenesis and Castration-Resistant Prostate Cancer.

Simple SummaryThis study explored the chemopreventive effects of Houttuynia cordata Thunb. (HCT) extracts against prostate carcinogenesis in both androgen-sensitive prostate cancer and castration-resistant prostate cancer (CRPC) using the Transgenic Rat for Adenocarcinoma of Prostate (TRAP) model, CRPC xenograft mice, and prostate cancer cell lines. HCT suppressed cell proliferation and stimulated apoptosis via inactivation of AKT/ERK/MAPK in both androgen-sensitive prostate cancer and CRPC cell lines. HCT also inhibited cell migration and EMT phenotypes through the STAT3/Snail/Twist pathway. One of the active compounds of HCT was identified as rutin. Consistent with in vitro study, the incidence of adenocarcinoma in the TRAP model and CRPC tumor growth in the xenograft model were suppressed by induction of apoptosis and inactivation of AKT/ERK/MAPK by HCT intake. Our data demonstrated that HCT attenuated androgen-sensitive prostate cancer and CRPC by mechanisms that may involve inhibition of cell growth and caspase-dependent apoptosis pathways.Houttuynia cordata Thunb. (HCT) is a well-known Asian medicinal plant with biological activities used in the treatment of many diseases including cancer. This study investigated the effects of HCT extract and its ethyl acetate fraction (EA) on prostate carcinogenesis and castration-resistant prostate cancer (CRPC). HCT and EA induced apoptosis in androgen-sensitive prostate cancer cells (LNCaP) and CRPC cells (PCai1) through activation of caspases, down-regulation of androgen receptor, and inactivation of AKT/ERK/MAPK signaling. Rutin was found to be a major component in HCT (44.00 ± 5.61 mg/g) and EA (81.34 ± 5.21 mg/g) in a previous study. Rutin had similar effects to HCT/EA on LNCaP cells and was considered to be one of the active compounds. Moreover, HCT/EA inhibited cell migration and epithelial-mesenchymal transition phenotypes via STAT3/Snail/Twist pathways in LNCaP cells. The consumption of 1% HCT-mixed diet significantly decreased the incidence of adenocarcinoma in the lateral prostate lobe of the Transgenic rat for adenocarcinoma of prostate model. Similarly, tumor growth of PCai1 xenografts was significantly suppressed by 1% HCT treatment. HCT also induced caspase-dependent apoptosis via AKT inactivation in both in vivo models. Together, the results of in vitro and in vivo studies indicate that HCT has inhibitory effects against prostate carcinogenesis and CRPC. This plant therefore should receive more attention as a source for the future development of non-toxic chemopreventive agents against various cancers.

Heterogeneous induction of an invasive phenotype in prostate cancer cells by coculturing with patient-derived fibroblasts.

Prostate cancer (PCa) cells frequently invade the surrounding stroma, leading to heterogeneous formation of structural atypia. The surrounding stroma contains multiple functionally diverse populations of fibroblasts that trigger numerous changes in PCa cells including motility. Thus, we hypothesized that direct or indirect contact of PCa cells with fibroblasts determines an invasive phenotype in PCa cells. We investigated the effects of 10 different patient-derived fibroblast lines on the three-dimensional (3D) morphogenesis of PCa cells growing on a viscous substrate in vitro. When grown alone, all 10 patient-derived fibroblast lines clumped on the viscous substrate, whereas the human androgen-sensitive PCa cell line LNCaP did not. Cocultures of LNCaP cells with seven of the patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M7, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a thick fibroblast layer that resembled human prostate stromal structures. In contrast, cocultures of LNCaP cells with the remaining three fibroblast lines (NPF-M13, pcPrF-M10, and pcPrF-M26) did not form a thick fibroblast layer. Of the seven fibroblast lines that caused thick layer formation, four patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) induced an invasive phenotype in LNCaP cells with a cord-like infiltrating growth pattern, whereas the other three fibroblast lines (pcPrF-M7, pcPrF-M23, and pcPrF-M24) induced no or a very weak invasive phenotype. Using cell culture inserts, none of the four patient-derived fibroblast lines that induced an invasive phenotype (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) affected CDH1 mRNA expression in LNCaP cells; yet, two patient-derived fibroblast lines (pcPrF-M5 and pcPrF-M28) increased CDH2 mRNA expression in LNCaP cells, whereas the other two fibroblast lines (PrSC and pcPrF-M31) did not. These results suggest that the existence of multiple functionally diverse populations of fibroblasts in PCa tissue may be responsible for the diversity in PCa cell invasion, leading to heterogeneous formation of structural atypia.

Osteoblasts Generate Testosterone From DHEA and Activate Androgen Signaling in Prostate Cancer Cells.

Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront of treatment. However, most prostate cancers transition to a state whereby reducing testicular androgen action becomes ineffective. A common mechanism of this transition is intratumoral production of testosterone (T) using the adrenal androgen precursor dehydroepiandrosterone (DHEA) through enzymatic conversion by 3β- and 17β-hydroxysteroid dehydrogenases (3βHSD and 17βHSD). Given the ability of prostate cancer to form blastic metastases in bone, we hypothesized that osteoblasts might be a source of androgen synthesis. RNA expression analyses of murine osteoblasts and human bone confirmed that at least one 3βHSD and 17βHSD enzyme isoform was expressed, suggesting that osteoblasts are capable of generating androgens from adrenal DHEA. Murine osteoblasts were treated with 100 nM and 1 μM DHEA or vehicle control. Conditioned media from these osteoblasts were assayed for intermediate and active androgens by liquid chromatography-tandem mass spectrometry. As DHEA was consumed, the androgen intermediates androstenediol and androstenedione were generated and subsequently converted to T. Conditioned media of DHEA-treated osteoblasts increased androgen receptor (AR) signaling, prostate-specific antigen (PSA) production, and cell numbers of the androgen-sensitive prostate cancer cell lines C4-2B and LNCaP. DHEA did not induce AR signaling in osteoblasts despite AR expression in this cell type. We describe an unreported function of osteoblasts as a source of T that is especially relevant during androgen-responsive metastatic prostate cancer invasion into bone. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Pheophorbide a-mediated sonodynamic, photodynamic and sonophotodynamic therapies against prostate cancer.

Anticancer efficiencies and mechanisms of Pheophorbide-a-mediated photodynamic, sonodynamic and sonophotodynamic therapies were investigated in vitro using androgen-sensitive (LNCaP) and androgen insensitive (PC3) prostate cancer cell lines. The cells were incubated in RPMI-1640 media at various concentrations of Pheophorbide-a. The media was treated with 0.5 W/cm2 ultrasound and/or 0.5 mJ/cm2 light irradiation. Cell proliferation in both cell lines was inhibited most effectively by sonophotodynamic therapy in comparison to that of both monotherapies. LNCaP cells were more sensitive to the applied treatments and the cell survival in LNCaP cell line was observed to be less than that of PC3 cell line. The results of histochemical analysis showed that there were more apoptotic cells in the treatment groups in comparison to control group. Additionally, the treatments induced apoptosis deduced by the overexpressed levels of caspase-3, caspase-8, PARP, and Bax proteins, while the expression levels of caspase-9 and Bcl-2 proteins were observed to be lower than those of control group. Treatments led to an increase in the oxidative stress markers, ROS and MDA, but a decrease in the activities of antioxidant enzymes, SOD, CAT and GSH. The results of this study revealed that Pheophorbide a-mediated sonophotodynamic therapy more efficiently activates the apoptotic mechanisms in prostate cancer cells and thus may provide a promising approach for treatment.

RESTORE: A single-arm, open-label phase II trial of bipolar androgen therapy (BAT) in men with metastatic castration resistant prostate cancer (mCRPC)—A comparison of post-abiraterone (Abi) versus post-enzalutamide (Enza) patients (Pts).

5576 Background: A paradoxical inhibition of cell growth has been observed in both androgen-sensitive and castration resistant prostate cancer cell lines following the addition of high-dose testosterone.We have conducted several clinical trials investigating a mode of supraphysiologic testosterone therapy termed, BAT, in which testosterone levels are rapidly driven to the supraphysiologic range followed by a return to near-castrate levels over 28-day treatment cycles with favorable results. We previously reported the efficacy of BAT in mCRPC pts that were progressing on enza. In this study, we compared the effect of BAT in mCRPC pts whose last therapy was abi vs. enza. In addition, we examined the benefit of abi or enza rechallenge after progression on BAT. Methods: 59 mCRPC pts (n = 29 post abi; n = 30 post enza) were enrolled and received at lease one dose of BAT monotherapy, 400mg intramuscularly every 28 days. After clinical or radiographic progression on BAT, pts were rechallenged with the AR targeted therapy to which they were most recently resistant. The co-primary endpoints were a 50% decline in PSA from baseline (PSA50) for BAT and for enza/abi rechallenge. Results: 5/29 (17.2%) of post-abi pts compared to 9/30 (30%) in the post enza group achieved a PSA50 response (P = 0.36). Post BAT rechallenge with abi (n = 19) or enza (n = 22) resulted in a PSA50 response rate of 15.8% (n = 3) and 68.2% (n = 15), respectively (P = 0.001). The total duration of benefit (i.e. PFS on BAT + PFS on rechallenge = “PFS2”) was significantly longer in the post enza vs. post-abi patients (Median PFS2: 12.75 vs. 8.125 months; P = 0.04. Lastly, AR-V7 negative (n = 42) pts has a significantly longer median PFS2 compared to AR-V7 positive (n = 10) pts. (10.3 vs. 7.1 months, P = 0.005). Conclusions: Our data suggest that BAT may be more effective at resensitizing mCRPC to direct AR antagonists (i.e. enza) compared to abi. Detection of AR-V7 portended a worse outcome on BAT/rechallenge. Further clinical study is warranted. Clinical trial information: NCT02090114 . [Table: see text]

Microfluidic-Based Mechanical Phenotyping of Androgen-Sensitive and Non-sensitive Prostate Cancer Cells Lines.

Cell mechanical properties have been identified to characterize cells pathologic states. Here, we report our work on high-throughput mechanical phenotyping of androgen-sensitive and non-sensitive human prostate cancer cell lines based on a morphological rheological microfluidic method. The theory for extracting cells’ elastic modulus from their deformation and area, and the used experimental parameters were analyzed. The mechanical properties of three types of prostate cancer cells lines with different sensitivity to androgen including LNCaP, DU145, and PC3 were quantified. The result shows that LNCaP cell was the softest, DU145 was the second softest, and PC3 was the stiffest. Furthermore, atomic force microscopy (AFM) was used to verify the effectiveness of this high-throughput morphological rheological method.

Abstract 1023: Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant prostate cancer

Abstract Androgen receptor (AR) signaling plays an essential role in all stages of prostate cancer. Androgen-deprivation therapy is generally effective for advanced prostate cancer until progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Most CRPC continues to be driven by AR signaling. AR transcriptional activity requires a functional N-terminal domain (NTD). Transactivation of AR is mediated by ligand-independent activation by cross-talk with signal transduction pathways targeting the AR NTD, gain-of-function mutations in AR ligand-binding domain (LBD), or expression of truncated AR splice variants that lack LBD (e.g., AR-V7). The intrinsically disordered AR NTD is essential for its transcriptional activity. It harbors six putative binding sites for Pin1, a proline isomerase that regulates protein conformation at specific phosphorylated-Ser/Thr-Pro motifs. All-trans retinoic acid (ATRA) is a validated and potent Pin1 inhibitor. Since conformational changes within the AR NTD are required for transactivation, perturbation of its structure may be a promising approach to block its activity. The purpose of this study was to assess a therapy that combined ATRA with antagonists of AR NTD, ralaniten (EPI-002) and its analogues. We hypothesized that targeting Pin1 activity with ATRA would disrupt the AR NTD and enhance inhibition by EPI compounds which bind to Tau-5 in the NTD. Using reporter gene assays, proliferation assays, and cell cycle analysis by flow cytometry, we tested ATRA in isolation and in combination with EPI in androgen-sensitive (LNCaP) and androgen-independent (LN95) prostate cancer cell lines. We found that treatment with ATRA decreased the transcriptional activity of AR and androgen-induced expression of PSA. ATRA also attenuated the transcriptional activity of AR-V7 and androgen-independent growth of LN95 cells expressing both full-length AR and AR-V7. Co-immunoprecipitation studies confirmed interactions between Pin1 and specific regions of AR NTD. In combination, ATRA had synergistic interaction with EPI compounds and lowered the effective inhibitory concentrations for blocking AR transcriptional activity. Furthermore, combinations decreased cell cycle progression of LN95 cells through S-phase, leading to accumulation of cells in G1 and induction of senescence. These preclinical findings showed that ATRA enhanced the potency of AR NTD inhibitors and support a novel therapeutic strategy for CRPC. Future studies will determine the in vivo antitumor effect of ATRA in combination with EPI compounds on the growth of CRPC xenografts. Citation Format: Jacky K. Leung, Marianne D. Sadar. Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1023.

UV Light-inactivated HSV-1 Stimulates Natural Killer Cell-induced Killing of Prostate Cancer Cells.

Herein we demonstrate that ultraviolet light-inactivated Herpes Simplex Virus-1 (UV-HSV-1) stimulates peripheral blood mononuclear cells (PBMCs) to lyse both androgen-sensitive and androgen-independent prostate cancer (PrCA) cell lines, but not the benign prostatic hyperplastic epithelial cell line, BPH-1, and is 1000-10,000-fold more potent at stimulating this killing than ultraviolet light-inactivated Vesicular Stomatitis Virus, adenovirus, reovirus or cytomegalovirus. Among PBMCs, natural killer (NK) cells appear to be a major cell type involved in this killing and UV-HSV-1 appears to directly and potently stimulate NK cell expression of CD69, degranulation, cytokine production, and migration to IL-8 in PC3 conditioned medium. We also found that UV-HSV-1 stimulates glycolysis in PBMCs and NK cells, and that 2-deoxyglucose and the protein kinase C inhibitor, Go6976, and the NFκB inhibitor, Bay 11-7082, all abrogate UV-HSV-1 activated killing of PC3 cells by PBMCs and NK cells. Using neutralizing anti-Toll-like receptor 2 (TLR2) we found that UV-HSV-1, like HSV-1, activates NK cells via TLR2. Taken together, these results are consistent with Toll-like receptor 2 ligands on UV-HSV-1 stimulating TLR2 on NK cells to activate protein kinase C, leading to enhanced glycolysis and NFκB activation, both of which play a critical role in this anti-PrCA innate immune response. Importantly, UV-HSV-1 synergizes with IL-15 to increase the cytolytic activity of PBMCs against PC3 cells and there was considerable donor-to-donor variation in killing ability. These results support the preclinical development of UV-HSV-1 as an adjuvant, in combination with IL-15, for cell infusions of healthy, preselected NK cells to treat PrCA.

The dark side of IL-1: role in prostate cancer progression

Abstract IL-1 is a pro-inflammatory cytokine essential for host defense against pathogens, but its role in cancer progression remains understudied. Recently our lab identified the IL-1 pathway as a molecular mechanism responsible for myeloid-driven prostate cancer development. Here, we report that IL-1 signaling in tumor cells drives prostate cancer progression. To identify the contribution of IL-1 signaling in prostate cancer progression, we downregulated expression of IL1R1 (the only known functional receptor for IL-1α/β) using shRNA or Crisr/Cas9 in androgen-sensitive (LNCaP) and androgen-independent (PC-3M) prostate cancer cell lines. We found that IL-1α inhibited expression of androgen receptor in LNCaP while suppression of IL-1 signaling abrogated this effect. The expression of neuron-specific enolase which was shown to be a prognostic marker associated with poor survival outcome was significantly reduced in PC-3M cells lacking IL1R1. Downregulation of IL-1 signaling in cancer cells resulted in significant inhibition of prostate tumor growth while no effect on cell proliferation was detected. We established this phenomenon and found several possible mechanisms underlying it. We found that knocking down of IL1R1 in cancer cells resulted in suppression of mTOR and ACC signaling in tumors. TUNEL staining revealed increased number of apoptotic cells in shIL1R1-PC-3M tumors. Tumors lacking IL1R1 were characterized by impaired vascularization which was accompanied by reduced COX-2, VEGF-A and IL-8 expression. Taken together, our findings indicate that downregulation of IL-1 signaling modifies prostate cancer cell phenotype and suppresses tumor growth.

Nitrogen-containing derivatives of O-tetramethylquercetin: Synthesis and biological profiles in prostate cancer cell models

Forty-eight nitrogen-containing quercetin derivatives were synthesized from readily available rutin or quercetin for the in vitro evaluation of their biological profiles. The WST-1 cell proliferation assay data indicate that thirty-nine out of the forty-eight derivatives possess significantly improved antiproliferative potency as compared with quercetin and fisetin, as well as the parent 3,3′,4′,7-O-tetramethylquercetin toward both androgen-sensitive (LNCaP) and androgen-insensitive (PC-3 and DU145) human prostate cancer cell lines. 5-O-Aminoalkyl-3,3′,4′,7-O-tetramethylquercetins were established as a better scaffold for further development as anti-prostate cancer agents. Among them, 5-O-(N,N-dibutylamino)propyl-3,3′,4′,7-O-tetramethylquercetin (44) was identified as the optimal derivative with IC50 values of 0.55–2.82 µM, being over 35–182 times more potent than quercetin. The flow cytometry-based assays further demonstrate that 44 effectively activates PC-3 cell apoptosis.

O-Aminoalkyl-O-Trimethyl-2,3-Dehydrosilybins: Synthesis and In Vitro Effects Towards Prostate Cancer Cells.

As part of our ongoing silybin project, this study aims to introduce a basic nitrogen-containing group to 7-OH of 3,5,20-O-trimethyl-2,3-dehydrosilybin or 3-OH of 5,7,20-O-trimethyl-2,3-dehydrosilybin via an appropriate linker for in vitro evaluation as potential anti-prostate cancer agents. The synthetic approaches to 7-O-substituted-3,5,20-O-trimethyl-2,3-dehydrosilybins through a five-step procedure and to 3-O-substituted-5,7,20-O-trimethyl-2,3- dehydrosilybins via a four-step transformation have been developed. Thirty-two nitrogen-containing derivatives of silybin have been achieved through these synthetic methods for the evaluation of their antiproliferative activities towards both androgen-sensitive (LNCaP) and androgen-insensitive prostate cancer cell lines (PC-3 and DU145) using the WST-1 cell proliferation assay. These derivatives exhibited greater in vitro antiproliferative potency than silibinin. Among them, 11, 29, 31, 37, and 40 were identified as five optimal derivatives with IC50 values in the range of 1.40–3.06 µM, representing a 17- to 52-fold improvement in potency compared to silibinin. All these five optimal derivatives can arrest the PC-3 cell cycle in the G0/G1 phase and promote PC-3 cell apoptosis. Derivatives 11, 37, and 40 are more effective than 29 and 31 in activating PC-3 cell apoptosis.