Negative Prostate Cancer Research Articles

Abstract 1775: Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer

Abstract Enzalutamide (ENZ) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and approved for the treatment of castration resistant prostate cancer (CRPC). Studies show increased survival in men with metastatic CRPC after chemotherapy when treated with ENZ. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance and resistance to AR inhibitors may lead to increase in N-cadherin. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR positive and negative prostate cancers. Co-targeting AR and N-cadherin with ENZ and mAb respectively show promise in addressing not only castration resistant tumor progression but also ENZ resistance. Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with ENZ. In vivo studies on promising candidates were performed using castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to ENZ, mAb alone, or in combination. At end-point, tumors were harvested and further analyzed. Final candidate mAbs were then selected and re-formatted into fully humanized N-cadherin monoclonal antibodies and further evaluated. A molecular model of the mouse chimeric antibodies were built based on structure of similar antibodies and the CDR loops were grafted to the human framework region and back-translated into nucleotide sequences. Codon-optimized DNA encoding the humanized variable domains connected by a 15 glycine-rich amino acid linker was synthesized by GeneArt and sub-cloned into expression vectors containing light and heavy chain constant regions. Clones were then screened and selected. Candidates were transiently expressed in 293 cells and supernatant were collected and purified for further analysis. These newly generated humanized mAbs engineered from the promising human anti-mouse N-cadherin antibody candidates exhibited similar outcomes to their chimeric counterparts, revealing potential in providing methods for diagnosis and treatment of advanced diseases. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Keyu Li, Joyce Yamashiro, Chun Wang, Anna Wu, Robert E. Reiter. Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer [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 1775.

Abstract 3837: Bumped kinase inhibitor 1553 selectively inhibits androgen receptor positive prostate cancer

Abstract BACKGROUND: Prostate cancer (PCa) is the second most common cancer in men and one of the leading causes of cancer death. Resistance to current PCa therapies, including androgen deprivation, occurs in almost all patients leading to development of castration resistant prostate cancer (CRPC). Resistance is associated with expression of splice variants of the androgen receptor (AR-Vs) that are constitutively active. The intrinsically disordered N-terminus of the AR/AR-Vs makes targeting the AR-Vs difficult. Thus, therapies that indirectly target the AR by inhibiting factors important in regulating AR levels and activity may be the most successful against inhibiting the constitutively active AR variants. Kinases have been shown to be important in regulating the AR, thus kinase inhibitors have the potential to inhibit androgen receptor signaling and function. OBJECTIVE: To demonstrate that our lead kinase inhibitor targets and inhibits AR positive CRPC. RESULTS: Bumped kinase inhibitors designed at the University of Washington are based on a pyrazolopyrimidine backbone, with R1 and R2 groups that confer a “bump” thereby preventing the BKIs from binding to most human kinase ATP binding pockets. While the majority of BKIs do not have activity against CRPC, a subset have activity against AR+ PCa lines, including CRPC lines expressing AR-Vs, but with no activity against AR negative PCa lines or a broad range of other human cell lines. We next examined the effects of our lead BKI compound (1553) on AR expression, phosphorylation, and function. BKI-1553 blocked transactivation activity of AR as judged by the reporter assays using an ARE-luciferase construct. Expression of canonical AR target genes was downregulated in LNCaP cells treated with BKI-1553. Westerns demonstrated a decrease in total AR 4 and 24 hours after treatment with BKI-1553. Levels of phospho-Serine81on the AR, which is critical for AR activity, were decreased even further than total AR by 4 hours after treatment. Thus, AR signaling appears to constitute BKI-sensitive components. We then decided to examine the effect of BKI-1553 on in vivo tumor growth. Toxicity studies demonstrated that it was non-toxic in mice at oral doses that achieved therapeutic levels. In vivo treatment with BKI-1553 significantly decreased growth of a castrate-sensitive patient-derived PCa xenograft and a castration-resistant PCa cell line (LuCaP35, p < 0.01 and LNCaP95, p < 0.005, respectively). SUMMARY: Our lead bumped kinase inhibitor, BKI-1553, selectively inhibits the growth of prostate cancer cells that express full-length AR and/or AR-Vs. BKI-1553 reduced not only cell proliferation, but decreased levels of AR and AR variants, decreased pSer81 levels, and decreased activation of AR-regulated promoters. Since the majority of CRPCs are AR driven, our targeted BKIs could be a novel therapy for cancers that are resistant to currently available therapies. Citation Format: Takuma Uo, Shihua Sun, Kathleen Haugk, Kathryn Soriano Epilepsia, Mamatha Damodarasamy, Matthew Hulverson, Wesley VanVoorhis, Kayode K. Ojo, RamaSubbaRao Vidadala, Dustin Maly, Stephen Plymate, Cynthia C. Sprenger. Bumped kinase inhibitor 1553 selectively inhibits androgen receptor positive prostate cancer [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 3837.

Comparison of LDR Brachytherapy Combined with Conventionally Fractionated EBRT or SBRT in High Risk Node Negative Prostate Cancer Patients: Early Toxicity and Tumor Control Outcomes

One of the most effective radiotherapeutic interventions for the treatment of high-risk prostate is the combination of brachytherapy with supplemental external beam radiotherapy directed to the prostate and regional nodes. While conventionally fractionated external beam radiotherapy has been a standard approach to supplement the brachytherapy boost, we have recently treated such patients with supplemental SBRT using an ultra-hypofractionated regimen. In this study we compare the early toxicity and tumor control outcomes of high risk, node negative patients treated with combination of brachytherapy and SBRT versus brachytherapy and conventionally fractionation RT (CFRT).

Association between pathologic factors and ERG expression in prostate cancer: finding pivotal networking.

To evaluate associations between pathologic factors and erythroblast transformation-specific (ETS)-related gene (ERG) expression in prostate cancer patients. Using next-generation sequencing, we identified target genes and regulatory networks. ERG expression in 60 radical prostatectomies was compared with pathological findings by association rule mining with the Apriori algorithm. Whole-exome and RNA sequencing were performed on three formalin-fixed, paraffin-embedded ERG-positive and negative prostate cancer samples. A network diagram identifying dominant altered genes was constructed using Cytoscape open-source bioinformatics platform and GeneMania plugin. Pathologic conditions positive for perineural invasion, apical margins, and Gleason score 3 + 4 = 7 were significantly more likely to be ERG-positive than other pathologic conditions (p = 0.0008), suggesting an association between ERG positivity, perineural invasion, apical margins, and Gleason score 3 + 4 = 7 (Firth's logistic regression: OR 42.565, 95% CI 1.670-1084.847, p = 0.0232). Results of whole-exome and RNA sequencing identified 97 somatic mutations containing common mutated genes. Regulatory network analysis identified NOTCH1, MEF2C, STAT3, LCK, CACNA2D3, PCSK7, MEF2A, PDZD2, TAB1, and ASGR1 as pivotal genes. NOTCH1 appears to function as a hub, because it had the highest node degree and betweenness. NOTCH1 staining was found 8 of 60 specimens (13%), with a significant association between ERG and NOTCH1 positivity (p = 0.001). Evaluating the association between ERG expression and pathologic factors, and identifying the regulatory network and pivotal hub may help to understand the clinical significance of ERG-positive prostate cancer.

2467 PRMT5 is a master epigenetic regulator to promote repair of radiation-induced DNA damage

OBJECTIVES/SPECIFIC AIMS: We recently reported that PRMT5 epigenetically activates androgen receptor (AR) in prostate cancer cells. Because targeting AR signaling through androgen deprivation therapy is clinically used as a radiosensitization approach to treat high-risk prostate cancer, our finding raised an exciting possibility that targeting PRMT5 may improve RT for prostate cancer patients. Contrary to our expectation, targeting PRMT5 sensitized both AR expressing and AR negative (AR−) prostate cancer cell lines to radiation. The goal of our study was therefore to determine the role of PRMT5 in repair of IR-induced DSBs and to translate these findings to improving radiation therapy for cancer patients in general (not just prostate cancer patients). METHODS/STUDY POPULATION: The majority of experiments were basic science experiments analyzing PRMT5’s role in the DNA damage response in normal and cancer cell lines. For example, to extend our findings and determine if PRMT5’s role in DSB repair is conserved across multiple cell types, we performed similar experiments in AR− prostate cancer cells, luminal breast cancer cells, glioblastoma cells, and human embryonic kidney cells. To determine the clinical significance of our finding, we also analyzed mRNA expression of PRMT5, AR, and both PRMT5 and AR target genes involved in DSB repair across 43 clinical cancer data sets. RESULTS/ANTICIPATED RESULTS: (1) Targeting PRMT5 sensitizes prostate cancer cells to IR in an AR-independent manner, (2) PRMT5 regulates the repair of IR-induced DSBs in an AR-independent manner, (3) RNA-seq analysis reveals that PRMT5 likely regulates genes involved in the DNA damage response, (4) PRMT5 activates expression of several genes in the DDR including those involved in DSB repair, (5) PRMT5 functions as an epigenetic activator of genes involved in DDR, (6) PRMT5 is required for NHEJ, HR, and G2-Arrest upon IR treatment, (7) Upregulation of PRMT5 correlates with formation and repair of IR-induced DSBs, (8) PRMT5’s role in repair of IR-induced DSBs is conserved in several normal and cancer cell types, and (9) PRMT5 expression correlates with expression of DSB repair proteins in clinical cancer samples. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, we provide evidence that PRMT5 is a master epigenetic regulator of IR-induced DSB repair through epigenetic activation of multiple target genes involved both HR and NHEJ as well as G2 arrest. Interestingly, the majority of genes regulated by PRMT5 are well-characterized, “core repair proteins” involved in HR (RAD51, BRCA1, BRCA2, RAD51D, and RAD51AP1), NHEJ (NHEJ1, Ku80, XRCC4, and DNAPKcs), and G2 arrest (Cdk1, CDC25C, CCNB2, and WEE1), which may explain why PRMT5 is essential to repair IR-induced DSBs in several cell lines. Although AR may also regulate DSB repair via both HR and NHEJ, several pieces of evidence in our study suggest that PRMT5 also regulates DSB repair independent of AR. First, PRMT5 targeting sensitizes both AR+ and AR− prostate cancer cells to IR. Second, exogenous expression of AR only partially rescues the impairment of IR-induced DSB repair by PRMT5 knockdown. Third, PRMT5 knockdown increases IR-induced DSB in AR− DU145 cells and several other cancer cell lines and normal cells. Fourth, PRMT5 expression correlates positively with the expression of its target genes in multiple human cancer tissues. During preparation of this project, Braun et al. reported that PRMT5 post-translationally regulates the splicing out of detained-introns (DI)s of genes to modulate gene expression. However, analysis of their data showed that the majority of DEGs we identified either do not contain DIs or DI splicing was not affected by targeting PRMT5. In addition, Clarke et al. reported that PRMT5 participates in the DSB repair choice process and promotes HR through methylation of RUVBL1. It is therefore likely that PRMT5 regulates repair of IR-induced DSB via multiple mechanisms. As PRMT5 is overexpressed in many human cancers and its overexpression correlates with poor prognosis, our findings suggest that increased DSB repair by PRMT5 overexpression in these human cancers may confer survival advantages particularly following DNA damaging treatment. Because targeting DSB repair has been proven to be a valid therapeutic approach for cancer treatment, our findings here also suggest that PRMT5 targeting may be explored as a monotherapy or in combination therapy with RT or chemotherapy for cancer treatment.

Differences in genomic signatures and opportunities for targeted and immunotherapy treatment between castrate-resistant TMPRSS2:ERG fusion-positive and -negative refractory acinar (CRPC) and neuroendocrine prostate cancer (CRNEPC).

5061Background: We hypothesized that sub-categorization of TMPRSS2 fusion status would impact therapy opportunities in patients with clinically advanced CRPC and CRNEPC. Methods: CGP was performed ...

Intratumoral androgen levels are linked to TMPRSS2-ERG fusion in prostate cancer.

Intratumoral androgen biosynthesis is one of the mechanisms involved in the progression of prostate cancer, and an important target for novel prostate cancer therapies. Using gas chromatography-tandem mass spectrometry and genome-wide RNA sequencing, we have analyzed androgen concentrations and androgen-regulated gene expression in cancerous and morphologically benign prostate tissue specimens and serum samples obtained from 48 primary prostate cancer patients. Intratumoral dihydrotestosterone (DHT) concentrations were significantly higher in the cancerous tissues compared to benign prostate (P < 0.001). The tissue/serum ratios of androgens were highly variable between the patients, indicating individual patterns of androgen metabolism and/or uptake of androgens within the prostate tissue. An unsupervised hierarchical clustering analysis of intratissue androgen concentrations indicated that transmembrane protease, serine 2/ETS-related gene (TMPRSS2-ERG)-positive patients have different androgen profiles compared to TMPRSS2-ERG-negative patients. TMPRSS2-ERG gene fusion status was also associated with an enhanced androgen-regulated gene expression, along with altered intratumoral androgen metabolism, demonstrated by reduced testosterone concentrations and increased DHT/testosterone ratios in TMPRSS2-ERG-positive tumors. TMPRSS2-ERG-positive and -negative prostate cancer specimens have distinct intratumoral androgen profiles, possibly due to activation of testosterone-independent DHT biosynthesis via the alternative pathway in TMPRSS2-ERG-positive tumors. Thus, patients with TMPRSS2-ERG-positive prostate cancer may benefit from novel inhibitors targeting the alternative DHT biosynthesis.

Identification of an IL‐1‐induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR− PCa cells

In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL-1 may also contribute to AR-targeted therapy resistance. Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL-1 reprograms AR positive (AR+ ) PCa cells into AR negative (AR- ) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival and tumor progression in the inflammatory tumor microenvironment. LNCaP and PC3 PCa cells were treated with IL-1β or HS-5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT-QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA-PCa-2b, PC3, and DU145 PCa cell lines were treated with the IL-1 family members, IL-1α or IL-1β, or exposed to HS-5 BMSC in the presence or absence of Interleukin-1 Receptor Antagonist (IL-1RA). Treated cells were analyzed by western blot and/or RT-QPCR. Comparative analysis of sequencing data from the AR+ LNCaP PCa cell line versus the AR- PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment. Our data supports that IL-1 reprograms AR+ PCa cells to mimic AR- PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival.

FABP5 RNA expression as potential marker in TMPRSS2:ERG fusion negative prostate cancer

FABP5 RNA expression as potential marker in TMPRSS2:ERG fusion negative prostate cancer

Immunogenomics: A Negative Prostate Cancer Outcome Associated with TcR-γ/δ Recombinations

We developed a scripted algorithm, based on previous, earlier editions of the algorithm, to mine prostate cancer exome files for T-cell receptor (TcR) recombination reads: Reads representing TcR gene recombinations were identified in 497 prostate cancer exome files from the cancer genome atlas (TCGA). As has been reported for melanoma, co-detection of productive TcR-α and TcR-β recombination reads correlated with an RNA expression signature representing T-cell exhaustion, particularly with high RNA levels for PD-1 and PD-L1, in comparison to several different control sets of samples. Co-detection of TcR-α and TcR-β recombination reads also correlated with high level expression of genes representing antigen presenting functions, further supporting the conclusion that co-detection of TcR-α and TcR-β recombination reads represents an immunologically relevant microenvironment. Finally, detection of unproductive TcR-δ recombinations, and unproductive and productive TcR-γ recombinations, strongly correlated with, and may represent a convenient biomarker for a poor clinical outcome. These results underscore the value of the genomics-based assessment of unproductive TcR recombinations and raise questions about the impact of tumor microenvironment lymphocytes in the absence of antigenicity.

High BCAR1 expression is associated with early PSA recurrence in ERG negative prostate cancer

BackgroundBreast cancer anti-estrogen resistance 1 (BCAR1/p130cas) is a hub for diverse oncogenic signaling cascades and promotes tumor development and progression.MethodsTo understand the effect of BCAR1 in prostate cancer, we analyzed its expression on more than 11,000 prostate cancer samples. BCAR1 expression levels were compared with clinical characteristics, PSA recurrence, molecular subtype defined by ERG status and 3p, 5q, 6q and PTEN deletion.ResultsBCAR1 staining was barely detectable in normal prostate glands but seen in 77.6% of 9472 interpretable cancers, including strong expression in 38.5%, moderate in 23.2% and weak in 15.9% of cases. BCAR1 up regulation was associated with positive ERG status (p < 0.0001), high Gleason score (p < 0.0001), advanced pathological tumor stage (p = 0.0082), lower preoperative PSA level (p < 0.0001), increased cell proliferation (p < 0.0001), early PSA recurrence (p = 0.0008), and predicted prognosis independently from clinico-pathological parameters available at the time of the initial biopsy. However, subset analyses revealed that the prognostic impact of BCAR1 expression was limited to ERG-negative cancer. That BCAR1 up regulation was linked to almost all analyzed deletions (p < 0.0001 each for PTEN, 5q, 6q deletion) may suggest a functional link to genomic instability.ConclusionThe results of our study identify BCAR1 as a prognostic biomarker with potential clinical value for risk stratification of ERG-negative prostate cancer.

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

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

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate

Backgroundc-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines.ResultsEleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive.ConclusionThe present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

The Effect of a Small Molecule EZH2 Methyltransferase Inhibitor GSK126 on p53 Negative Prostate Cancer Cell Line

To quantitatively evaluate the effect of GSK126 (an inhibitor of histone methyltransferase) in inhibiting migration of prostate cancer cell line. And to investigate whether GSK126 can regulate the radiosensitivity and reduce the survival rate of p53 negative prostate cancer cell line. Human prostatic carcinoma cells (PC-3) were separated into 5 groups and incubated with GSK126 of different concentrations (0 μM, 5 μM, 10 μM, 20 μM) for 48h. The protein expression levels of EZH2 in the cells were detected by western blotting. Cell migration was examined by both wound scratch assay and transwell assay. And then the PC-3 cells were exposure to a single dose of 4Gy delivered using a 6MV photon beam of Clinac 600 CD. Cell colony assay was used to determine cell survival rates after irradiation. One group of cells without any treatments was used as the control group. All experiments were repeated 3 times, statistical analysis was performed using independent sample T-test and p value of less than 0.05 was considered to be significant. Western blotting study showed that the expression level of EZH2 in PC-3 cells decreased with increasing concentration of GSK126. Transwell assay study showed that, when the concentration of GSK126 was increased to 10μM or higher, the EZH2 expression decreased and number of cells penetrating cell membrane decreased significantly (p<0.02). Wound scratch assay study showed that, along with decreasing EZH2 expression, cell migration speed and the ability to migrate reduced (p<0.05). This is consistent with the results of transwell experiments. Results mentioned above indicated that GSK126 can regulate migration of PC-3 cells by inhibition of EZH2. Cell colony assay study showed that with the increase of GSK126 concentration, the forming rates of colonies decreased significantly after irradiation (P<0.05). It indicated that GSK126 enhanced radiosensitivity of PC-3 cells.Abstract 3402; Table 1Forming rates of colonies after irradiation of 4GyGSK126 concentrationForming rate of coloniesReference (0μM w/o radiation treatment)10 μM0.36±0.045 μM0.33±0.0510 μM0.29±0.0820 μM0.14±0.07 Open table in a new tab Using GSK126 of 10μM or higher can inhibit migration of PC-3 cells. And GSK126 in combination with radiation can effectively reduce the survival rates of PC-3 cells.

FOXA1 expression is a strong independent predictor of early PSA recurrence in ERG negative prostate cancers treated by radical prostatectomy.

FOXA1 (Fork-head box protein A1, HNF-3a) is a transcription factor involved in androgen signaling with relevance for lineage-specific gene expression of the prostate. The expression was analyzed by immunohistochemistry on a tissue microarray containing 11152 prostate cancer specimens. Results were compared with tumor phenotype, biochemical recurrence, androgen receptor expression, ETS-related gene (ERG) status and other recurrent genomic alterations. FOXA1 expression was detectable in 97.6% of 8227 interpretable cancers and considered strong in 28.5%, moderate in 46.2% and weak in 22.9% of cases. High FOXA1 expression was associated with TMPRSS2:ERG rearrangement and ERG expression (P < 0.0001). High FOXA1 expression was linked to high Gleason grade, advanced pathological tumor (pT) stage and early PSA recurrence in ERG negative cancers (P < 0.0001), while these associations were either weak or absent in ERG positive cancers. In ERG negative cancers, the prognostic role of FOXA1 expression was independent of Gleason grade, pathological tumor stage, lymph node stage, surgical margin status and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features such as biopsy Gleason grade, preoperative PSA, cT stage and FOXA1 expression (P < 0.0001). Within ERG negative cancers, FOXA1 expression was also strongly associated with PTEN and 5q21 deletions (P < 0.0001). High expression of FOXA1 is an independent prognostic parameter in ERG negative prostate cancer. Thus, FOXA1 measurement might provide clinically useful information in prostate cancer.

The Advantages of Transurethral Resection of the Prostate in Patients with an Elevated or Rising Prostate Specific Antigen, Mild or Moderate Lower Urinary Tract Symptoms, Bladder Outlet Obstruction and Negative Prostate Cancer Imaging or Prostate Biopsies: A Prospective Analysis in 105 Consecutive Patients

Objective: To investigate elevated or rising prostate specific antigen (PSA) as a marker for bladder outlet obstruction (BOO) in patients with minor lower urinary tract symptoms (LUTS) and without prostate cancer. Materials and Methods: One hundred and five consecutive patients were prospectively analyzed between 2005 and 2013. All patients were referred to the principal investigator by their general practitioner as a result of an elevated and/or rising PSA. Only patients with minor LUTS [International Prostate Symptom Score (I-PSS) 0-19] and without suspicion for prostate cancer were included. All patients had BOO, shown by full urodynamics, and underwent transurethral resection of the prostate. The resected tissue was histologically examined and PSA and I-PSS were evaluated after 3, 6 and 12 months and later on yearly. Results: Mean pre-operative PSA and I-PSS values were 8.8 ng/ml and 11.1, respectively. The mean detrusor pressure at maximum flow was 93.6 cmH₂O. The mean resected volume was 52 g and the mean prostate biopsy rate was 1.8. Eighty-three of 105 patients (79%) had no malignancy and were diagnosed with BOO due to benign prostate hyperplasia (subgroup 1). Their mean PSA decreased from 9.2 to 0.7 ng/ml and 0.9 ng/ml after 6 and 12 months post-operation, respectively. The mean I-PSS declined from 11 to 3 after 6 and 12 months. Sixteen of 105 patients (15%) were treated for prostate cancer (subgroup 2). Radical prostatectomy was performed in 11 patients, brachytherapy in 3 patients and external beam radiotherapy in 2 patients. Six of 105 patients (5.7%) had active surveillance (subgroup 3). Conclusion: BOO can cause an elevated or rising PSA in patients with minor LUTS and negative screening for prostate cancer. Transurethral resection of the prostate is an adequate treatment for these patients.

Abstract 2076: Targeting the FGFR pathway in androgen receptor negative castration resistant prostate cancer

Abstract Metastatic castration-resistant prostate cancer (CRPC) has a poor prognosis and remains a significant therapeutic challenge. The continued importance of androgen receptor (AR) signaling as a growth and survival pathway in most advanced prostate cancer (PC) has prompted the development of therapeutics directed toward further suppressing AR ligands or the AR itself. However, total androgen blockade can give rise to AR-negative PC and currently there are no effective therapies for AR-negative PC. We have developed two novel patient-derived xenograft (PDX) models from a liver metastasis and a rib metastasis of a patient with CRPC: LuCaP 173.1 (Neuroendocrine; NE) and LuCaP 173.2 (Double negative: DN (AR-negative and NE-negative)). Using RNA-Seq we observed that both of these lines express high levels of fibroblast growth factors (FGF’s) and downstream effectors. We hypothesized that FGF signaling promotes survival and proliferation of NEPC and DNPC. To determine if blocking the FGFR survival pathway can attenuate the growth of NEPC/DNPC tumors, to characterize the molecular response and resistance to FGF signaling inhibition, and to support further evaluation in the clinic, we treated both PDX models with the FGFR inhibitor CH5183284. CH5183284 significantly reduced LuCaP 173.2 tumor volume (TV) (p&amp;lt;0.0001). The reduction in TV in CH5183284-treated animals bearing LuCaP 173.1 tumors was not as significant. To address this difference, we analyzed FGFR1 expression at the transcript and protein levels. Analysis of the FGFRs in the LuCaP 173.1 PDX model revealed a loss of FGFR1 expression. FGFR2 and FGFR3 transcripts were expressed at similar levels in both PDXs. The loss of FGFR1 expression may explain the low efficacy of CH5183284 in the LuCaP 173.1 PDX model. We are currently assessing the molecular pathways impacted by FGFR inhibition and mechanisms of resistance to treatment in these studies. We have demonstrated that FGFR inhibition attenuated tumor growth in FGFhigh AR-negative PC. We intend to target the FGFR survival pathway in additional NE and DNPC PDX models to investigate tumor heterogeneity in response to therapy and identify mechanisms of resistance. These studies will provide translational evidence that FGFR inhibitors could represent a new therapy for CRPC patients with AR-negative PC. Citation Format: Colm Morrissey, Eva Corey, Lisha Brown, Ilsa Coleman, Holly Nguyen, Michael Schweizer, Peter Nelson. Targeting the FGFR pathway in androgen receptor negative 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 2076. doi:10.1158/1538-7445.AM2017-2076

Abstract 2036: In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer

Abstract Introduction: Despite the use of androgen deprivation therapy, the majority of prostate cancer patients will progress to castration resistant disease within 2-3 years, driven by aberrant androgen receptor activation. We have previously shown that combination of carmustine and selenite effectively induces apoptosis by completely reducing AR and AR-variants in AR-dependent castration resistant prostate cancer cells both in vitro and in vivo. Although studies have demonstrated that AR signaling is a central mechanism of castration resistant prostate cancer progression, the cell growth mediated by AR-independent signaling is also operative. EGFR signaling has been implicated in the survival, invasion, and metastasis of prostate cancer cells in an AR-independent manner. Therefore, in this study, we tested whether the combination of carmustine and selenite could inhibit EGFR signaling, induce apoptosis, and inhibit growth of AR-independent prostate cancer cells in an in vitro cell culture and in vivo xenograft models. Methods: AR negative PC-3 prostate cancer cell line was used to study the effect of carmustine and selenite alone or in combination on EGFR signaling. Cell viability, proliferation, and apoptosis in the presence or absence of EGF (50ng/ml) were determined. Dose and time dependent effect of EGF on the activation of EGFR was studied. The carmustine and/or selenite effect on EGF stimulated phospho-EGFR, and its downstream signaling pathways, such as AKT and MAPK, were studied in comparison with inhibitors of EGFR (AG1478) or PI3 kinase (LY294002) or MAPK (PD98059). The in vivo effect of carmustine and selenite was examined on PC-3 tumor growth in athymic nude mice. Results: Combination of carmustine and selenite treatment in the presence of EGF markedly suppressed cell viability and proliferation of PC-3 cells. EGF exposure increased phospho-EGFR (Tyr845, Tyr992, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in PC-3 cells in a dose and time dependent manner. The combination treatment exhibited a strong inhibitory effect on EGF stimulated EGFR, Akt, and ERK1/2 phosphorylation. Combination treatment was able to induce apoptosis even in the presence of EGF. Consistent with in vitro results, combination treatment for 3 weeks reduced PC-3 xenograft tumors in nude mice by 88% without any toxicity. Whereas individual agent treatment showed only partial effect both in vitro and in vivo. Conclusions: Our pre-clinical data demonstrate that the combination treatment of carmustine and selenite substantially inhibits EGFR signaling, induces apoptosis, and reduces tumor growth of AR-independent prostate cancer cells in vitro and in vivo. Our novel findings suggest that the combination of carmustine and selenite is an effective therapeutic agent for successful treatment, survival, and improved quality of life for patients with prostate cancer. Citation Format: Vijayalakshmi Thamilselvan, Mani Menon, Sivagnanam Thamilselvan. In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent 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 2036. doi:10.1158/1538-7445.AM2017-2036

Abstract 2989: Expression of immune checkpoints proteins and their prognostic relevance in prostate cancer

Abstract Immune checkpoint proteins including programmed death ligand 1, 2 (PD-L1, PD-L2) and B7-H3 play important roles for immune evasion in many cancer types. PD-L1 is an established therapeutic target in lung cancer and in a growing number of other indications. The clinical success of PD-L1 inhibitors has accelerated the development of drugs also targeting PD-L2 and B7-H3. In prostate cancer, studies on the prevalence and clinical relevance of these proteins have been inconclusive. Here, we performed immunohistochemical analysis of PD-L1, PD-L2 and B7-H3 in our large prostate cancer prognosis tissue microarray (TMA) containing tissue samples from more than 17,000 patients. Normal prostate glands were negative for PD-L1 and B7-H3, but positive for PD-L2. We found that 47% of 12,808 interpretable cancers showed B7-H3 expression, and 92% of 9,463 cancers showed PD-L2 expression, while expression of PD-L1 was virtually absent (0.2% of 17,392 tumors) in epithelial cells. Stroma cells and immune cells were entirely negative for PD-L1 and B7-H3, while some faint staining of lymphocytes was occasionally observed for PD-L2. In cancers, PD-L2 and B7-H3 staining had different prognostic impact, which depended on the ERG status: B7-H3 expression was linked to young patient age, advanced stage, high Gleason grade, lymph node metastasis (p&amp;lt;0.0001 each), presence of PTEN deletion (p&amp;lt;0.0001), and poor prognosis in ERG negative cancers (p&amp;lt;0.0001), while PD-L2 expression was linked to beneficial tumor features and good prognosis exclusively in tumors with ERG fusion (p&amp;lt;0.0001). In a multivariate analysis including pT stage, Gleason grade, nodal stage, resection margin and preoperative PSA level, B7-H3 expression was an independent prognostic factor (p=0.0004). In summary, the results of our study identify a subset of young patients with ERG negative aggressive prostate cancers that might benefit from anti-B7-H3 therapy in the future. PD-L2 expression appears to protect tumors from progression and does not seem to be suitable as a potential target in prostate cancer. PD-L1 expression is extremely rare in this disease, suggesting that only few prostate cancer patients might benefit from PD-L1 inhibitors. Citation Format: Claudia Hube-Magg, Cornelia Schroeder, Ronald Simon, Martina Kluth, Till Krech, Franziska Büscheck, Frank Jacobsen, Doris Höflmayer, Lars Budäus, Hartwig Huland, Markus Graefen, Guido Sauter, Thorsten Schlomm. Expression of immune checkpoints proteins and their prognostic relevance in 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 2989. doi:10.1158/1538-7445.AM2017-2989

Abstract 3202: Pharmacologic modulation of Sigma1 induces autophagic degradation of programmed death-ligand 1 in cancer cells

Abstract Emerging evidence suggests that Sigma1 (also known as sigma1 receptor) is a unique ligand-operated integral membrane chaperone or scaffolding protein that contributes to cellular protein homeostasis. Previously, we found that treatment of various cancer cell lines with some prototypic small molecule modulators of Sigma1 can engage endoplasmic reticulum (ER) associated protein homeostasis pathways including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type 1 integral membrane glycoprotein that is processed and transported through the ER and secretory pathway of tumor cells. PD-L1 expressed at the surface of tumor cells can act as a T-cell inhibitory checkpoint molecule that inactivates tumor infiltrating immune cells that express PD-1, its cognate receptor. Here, we show that Sigma1 physically associates with PD-L1. In triple negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels are suppressed by both RNAi mediated knockdown of Sigma1 and pharmacological modulation of Sigma1. We observe decreased cell surface and intracellular levels of PD-L1 by flow cytometry and biochemical subcellular fractionation respectively, which corresponds with a dose-responsive decrease in functional PD-L1/PD-1 interaction in a co-culture of cancer cells and T-cells. Inhibitors of autophagy block this suppression of PD-L1 protein levels, suggesting PD-L1 is degraded away by autophagy after Sigma1 modulation. Through confocal microscopy, we show that Sigma1 modulation results in colocalization of PD-L1 and GFP-LC3, a marker of autophagosomes. From these conclusions, we hypothesize that autophagic degradation of nascent PD-L1 after Sigma1 modulation plays a key role in preventing the transport of functional PD-L1 to the plasma membrane. Together, these data demonstrate that Sigma1 modulators have the potential to act as novel therapeutic agents in PD-1/PD-L1 blockade strategies. Citation Format: Christina M. Maher, Jeffrey D. Thomas, Charles G. Longen, Derick A. Haas, Halley M. Oyer, Jane Y. Tong, Felix J. Kim. Pharmacologic modulation of Sigma1 induces autophagic degradation of programmed death-ligand 1 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3202. doi:10.1158/1538-7445.AM2017-3202