Castration-resistant Prostate Cancer Samples Research Articles

Identification of Genes Required for Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells In Vitro.

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Patterns of stemness‐associated markers in the development of castration‐resistant prostate cancer

Putative castration-resistant (CR) stem-like cells (CRSC) have been identified based on their ability to initiate and drive prostate cancer (PCa) recurrence following castration in vivo. Yet the relevance of these CRSC in the course of the human disease and particularly for the transition from hormone-naive (HN) to castration-resistance is unclear. In this study, we aimed at deciphering the significance of CRSC markers in PCa progression. We constructed a tissue microarray comprising 112 matched HN and CR tissue specimens derived from 55 PCa patients. Expression of eight stemness-associated markers (ALDH1A1, ALDH1A3, ALDH3A1, BMI1, NANOG, NKX3.1, OCT4, SOX2) was assessed by immunohistochemistry and scored as a percentage of positive tumor cells. For each marker, the resulting scores were statistically analyzed and compared to pathological and clinical data associated with the samples. Unsupervised clustering analysis was performed to stratify patients according to the expression of the eight CRSC markers. Publicly-available transcriptional datasets comprising HN and CR PCa samples were interrogated to assess the expression of the factors in silico. Immunohistochemical assessment of paired samples revealed atypical patterns of expression and intra- and intertumor heterogeneity for a subset of CRSC markers. While the expression of particular CRSC markers was dynamic over time in some patients, none of the markers showed significant changes in expression upon the development of castration resistance (CR vs HN). Using unsupervised clustering approaches, we identified phenotypic subtypes based on the expression of specific stem-associated markers. In particular, we found (a) patterns of mutual exclusivity for ALDH1A1 and ALDH1A3 expression, which was also observed at the transcriptomic level in publicly-available PCa datasets, and (b) a phenotypic cluster associated with more aggressive features. Finally, by comparing HN and CR matched samples, we identified phenotypic cluster switches (ie, change of phenotypic cluster between the HN and CR state), that may be associated with clinical and predictive relevance. Our findings indicate stemness-associated patterns that are associated with the development of castration-resistance. These results pave the way toward a deeper understanding of the relevance of CRSC markers in PCa progression and resistance to androgen-deprivation therapy.

When Less Is More: Specific Capture and Analysis of Tumor Exosomes in Plasma Increases the Sensitivity of Liquid Biopsy for Comprehensive Detection of Multiple Androgen Receptor Phenotypes in Advanced Prostate Cancer Patients.

We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor (AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments (AR-V7 transcript, AR T878A point mutation and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patients. The T878A mutation was detected, overall, in nine out of 15 samples and no approach alone was able to reveal mutations in all harboring samples, showing that the employed methods complement each other. AR amplification was detected in the majority of CRPC samples analysed using either cell-free DNA (cfDNA) or exosome isolation kits (80%). We demonstrated that selective isolation of a subset of circulating exosomes enriched for tumor origin, rather than analysis of total plasma exosomes, or total plasma nucleic acids, increases sensitivity and specificity for the detection of specific alterations.

Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR-variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.

A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer.

Increased expression of the full-length androgen receptor (AR-FL) and AR splice variants (AR-Vs) drives the progression of castration-resistant prostate cancer (CRPC). The levels of AR-FL and AR-V transcripts are often tightly correlated in individual CRPC samples, yet our understanding of how their expression is co-regulated is limited. Here, we report a role of c-Myc in accounting for coordinated AR-FL and AR-V expression. Analysis of gene expression data from 159 metastatic CRPC samples and 2142 primary prostate tumors showed that the level of c-Myc is positively correlated with that of individual AR isoforms. A striking positive correlation also exists between the activity of the c-Myc pathway and the level of individual AR isoforms, between the level of c-Myc and the activity of the AR pathway, and between the activities of the two pathways. Moreover, the c-Myc signature is highly enriched in tumors expressing high levels of AR, as is the AR signature in c-Myc-high-expressing tumors. Using shRNA knockdown, we confirmed c-Myc regulation of expression and activity of AR-FL and AR-Vs in cell models and a patient-derived xenograft model. Mechanistically, c-Myc promotes the transcription of the AR gene and enhances the stability of the AR-FL and AR-V proteins without altering AR RNA splicing. Importantly, inhibiting c-Myc sensitizes enzalutamide-resistant cells to growth inhibition by enzalutamide. Overall, this study highlights a critical role of c-Myc in regulating the coordinated expression of AR-FL and AR-Vs that is commonly observed in CRPC and suggests the utility of targeting c-Myc as an adjuvant to AR-directed therapy.

Generation of Whole-Genome Sequencing Data for Comparing Primary and Castration-Resistant Prostate Cancer

Because castration-resistant prostate cancer (CRPC) does not respond to androgen deprivation therapy and has a very poor prognosis, it is critical to identify a prognostic indicator for predicting high-risk patients who will develop CRPC. Here, we report a dataset of whole genomes from four pairs of primary prostate cancer (PC) and CRPC samples. The analysis of the paired PC and CRPC samples in the whole-genome data showed that the average number of somatic mutations per patients was 7,927 in CRPC tissues compared with primary PC tissues (range, 1,691 to 21,705). Our whole-genome sequencing data of primary PC and CRPC may be useful for understanding the genomic changes and molecular mechanisms that occur during the progression from PC to CRPC.

Abstract A077: Chromatin state alterations in human prostate cancer progression

Abstract Understanding the mechanisms of prostate cancer (PC) development into castration-resistant prostate cancer (CRPC) is a key factor in finding better diagnostic and treatment tools. Although a number of studies have tried to explain the molecular evolution of CRPC, more refined understanding on molecular mechanisms is still needed for improved patient care. While increasing amounts of genomic aberrations are accumulating in prostate cancer genome during the disease progression, we still lack understanding on the functional impact of the majority of these aberrations or their combinations. Identification of the regulatory regions from tissue samples enables in-depth analysis of regulatory elements and upstream regulators that are driving tumor development. Thus, we can gain detailed understanding of how the genomic alterations reorganize the chromatin and enable the emergence of cancer phenotype. We have previously characterized a cohort of 60 clinical prostate tissue samples including benign prostatic hyperplasia (BPH), PC, and CRPC with RNA-seq, MeDIP-seq, DNA-seq, small-RNA-seq, and mass spectrometry. To gain insight into the epigenetic regulation during the disease progression, we decided to use assay for transposase-accessible chromatin using sequencing (ATAC-seq). We first developed a method that allows us to use freshly frozen prostate samples as starting material, and performed ATAC-seq for BPH, PC, and CRPC samples from the above-mentioned cohort. High quality peaks were identified from all the samples, ranging from tens of thousands to over one hundred thousand peaks per sample. Large variation in the chromatin structure was observed across the cohort. Unsupervised clustering based on peak intensities was able to separate the three different sample types into separate clusters with distinct chromatin state profile for each sample cluster. Next we extracted nucleosome signals from the data. This signal is able to illustrate nucleosome occupancy and positioning with high resolution in the areas of open chromatin. We observed organized nucleosome patterns in our BPH samples, but this organization start to fall apart in PC and especially in CRPC samples where nucleosome localization is no longer uniform inside the group. The results imply increased heterogeneity in chromatin structure as a result of disease progression. Our data show that ATAC-seq data from clinical prostate material are sufficient to separate different sample groups to their own clusters, and it is possible to have detailed information about nucleosome binding in tumor tissues. These data allow us to reveal novel changes in chromatin state and integrate those changes to other features such as gene expression. With these data we aim to connect specific regulatory elements and upstream regulators to cancer phenotypes and genomic alterations. This new layer of information will bring us closer to understanding the mechanisms that drive molecular evolution of CRPC with potential implications in clinical practice for patients suffering from this devastating disease. Citation Format: Joonas Tuominen, Tomi Häkkinen, Matti Annala, Kati Kivinummi, Teuvo Tammela, Leena Latonen, Kirsi Granberg, Tapio Visakorpi, Matti Nykter. Chromatin state alterations in human prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A077.

Abstract 944: Glucocorticoid receptor (GR)-mediated activation of cyclic-adenosine monophosphate (cAMP) pathway gene expression following androgen receptor (AR) antagonism of prostate cancer

Abstract Background: Despite the recent discovery of several potent androgen receptor (AR)-targeted therapies, progressive castration-resistant prostate cancer (CRPC) remains the second leading cause of male cancer deaths in the United States. While androgen-deprivation therapy (ADT) and AR antagonism continue to be the standard of care for progressive metastatic CRPC, identifying alternative molecular mechanisms is crucial. Our lab and others have shown that increased glucocorticoid receptor (GR) expression and activity can contribute to CRPC progression; this discovery has led to the development of selective GR modulators (SGRMs) as potential therapies for CRPC. Although GR clearly contributes to cell growth in AR-blocked PC cells, it is unclear whether GR serves solely to recapitulate AR-driven cancer-related mechanisms or if GR also regulates critical gene expression pathways unique to GR (and different from AR) allowing a novel growth advantage for tumor cells. Methods: Primary PC and metastatic CRPC samples were normalized and utilized for gene expression analysis. LAPC4 and CWR-22Rv1 PC cell lines were treated with androgen +/- enzalutamide +/- dexamethasone and +/- SGRMs in order to activate or inhibit GR function in both AR active and AR-blocked conditions. For transcriptome analysis we performed next generation sequencing (NGS) of RNA followed by gene expression pathway analysis. To determine GR and AR chromatin binding sites, we performed GR and AR ChIP followed by DNA NGS. Results: Transcriptome analysis of GR-mediated gene expression in LAPC4 PC cells following AR antagonism revealed that the majority of GR-regulated genes were unique to GR activity rather than overlapping with AR target genes.. Furthermore, GR and AR ChIP-sequencing indicated that while important overlap existed between GR and AR ChIP-seq, the majority of GR binding sites were distinct from AR binding sites. Analysis of the GR-unique transcriptomes of both enzalutamide-treated LAPC4 and CWR-22Rv1 cells predicted that cAMP-mediated signaling was activated by GR. Antagonism of GR with SGRMs blocked GR-unique cAMP pathway gene expression. Gene expression analysis of patient samples indicated that GR signaling is also activated in mCRPC and correlated with increased cAMP pathway gene expression. Conclusions: These data suggest that GR activity in progressive CRPC results in novel GR-specific mechanisms of CRPC progression involving signaling pathways distinct from those driven by AR activation. Citation Format: Tiha M. Long, Eva Y. Tonsing-Carter, Wen-Ching Chan, Donald Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Glucocorticoid receptor (GR)-mediated activation of cyclic-adenosine monophosphate (cAMP) pathway gene expression following androgen receptor (AR) antagonism of 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 944.

The expression of AURKA is androgen regulated in castration-resistant prostate cancer

Although second generation endocrine therapies have significantly improved survival, castration-resistant prostate cancer (CRPC) cells are eventually able to escape available hormonal treatments due to reactivation of androgen receptor (AR) signaling. Identification of novel, non-classical and druggable AR-target genes may provide new approaches to treat CRPC. Our previous analyses suggested that Aurora kinase A (AURKA) is regulated by androgens in prostate cancer cells that express high levels of AR. Here, we provide further evidence that AURKA is significantly overexpressed in AR-positive CRPC samples carrying amplification of AR gene and/or expressing AR in high levels. We also demonstrate androgen-induced AR binding in the intronic region of AURKA. The expression of AURKA is increased upon androgen stimulation in LNCaP-ARhi cells that express high levels of AR. The growth of the cells was also significantly inhibited by an AURKA specific inhibitor, alisertib (MLN8237). Together, these findings suggest that the expression of AURKA is regulated by androgen in prostate cancer cells that highly express AR, emphasizing its potential as a therapeutic target in patients with CRPC.

Transcriptomic features of primary prostate cancer and their prognostic relevance to castration-resistant prostate cancer.

Although various mechanisms of castration-resistant prostate cancer (CRPC) have been discovered, reliable biomarkers for monitoring CRPC progression are lacking. We sought to identify molecules that predict the progression of advanced prostate cancer (AdvPC) into CRPC. The study used primary-site samples (N=45 for next-generation sequencing (NGS); N=243 for real-time polymerase chain reaction) from patients with prostate cancer (PC). Five public databases containing microarray data of AdvPC and CRPC samples were analyzed. The NGS data showed that each progression step in PC associated with distinct gene expression profiles. Androgen receptor (AR) associated with tumorigenesis, advanced progression, and progression into CRPC. Analysis of the paired and unpaired AdvPC and CRPC samples in the NGS cohort showed that 15 genes associated with progression into CRPC. This was validated by cohort-1 and public database analyses. Analysis of the third cohort with AdvPC showed that higher serine peptidase inhibitor, Kazal type 1 (SPINK1) and lower Sp8 transcription factor (SP8) expression associated with progression into CRPC (log-rank test, both P<0.05). Multivariate regression analysis showed that higher SPINK1 (Hazard Ratio (HR)=4.506, 95% confidence intervals (CI)=1.175–17.29, P=0.028) and lower SP8 (HR=0.199, 95% CI=0.063–0.632, P=0.006) expression independently predicted progression into CRPC. Gene network analysis showed that CRPC progression may be mediated through the AR-SPINK1 pathway by a HNF1A-based gene network. Taken together, our results suggest thatSPINK1 and SP8 may be useful for classifying patients with AdvPC who have a higher risk of progressing to CRPC.

C-MYC drives histone demethylase PHF8 during neuroendocrine differentiation and in castration-resistant prostate cancer.

Epigenetic factors play critical roles in prostate cancer (PCa) development. However, how they contribute to neuroendocrine differentiation (NED) and castration-resistant PCa (CRPC) is not fully understood. Using bioinformatics and biochemical approaches to analyze cell-based models of NED and CRPC, we found a cluster of epigenetic factors whose expression is downregulated during NED and upregulated in CRPC (i.e. follow a Down-Up pattern). Two histone demethylases within this cluster, PHF8 and KDM3A, are post-transcriptionally regulated by c-MYC through miR-22, which targets both PHF8 and KDM3A. We also found that the c-MYC/miR-22/PHF8 axis is downstream of androgen receptor (AR) signaling in CRPC cells. The co-expression of PHF8 with AR in clinical CRPC samples, normal mouse prostate, and adenocarcinomas of the prostate during PCa progression in a transgenic (TRAMP) mouse model supports the connection between PHF8 and AR. Knockdown of PHF8 impedes cell cycle progression in CRPC cells and has more profound effects on their growth than on the parental LNCaP cell line. Furthermore, PHF8 knockdown sensitizes LNCaP-Abl cells to the AR antagonist enzalutamide. Our data reveal novel mechanisms that underlie the regulation of PHF8 and KDM3A during NED and in CRPC, and support the candidacy of PHF8 as a therapeutic target in CRPC.

Abstract 426: Identifying a predictive biomarker for chemotherapy (taxane) in patients with metastatic castration resistance prostate cancer

Abstract In 2015, American Cancer Society predicts that 220,800 men will be diagnosed with prostate cancer (PC). Patients diagnosed with metastatic PC are first treated with androgen deprivation therapies (ADT) in order to lower testosterone to castration levels. Unfortunately, it is only a matter of time before becoming metastatic castration resistant prostate cancer (mCRPC). Subsequent treatments include combined androgen deprivation therapies such as (abiraterone, enzalutamide), immunotherapies, investigational drugs, and/or chemotherapy (docetaxel and cabazitaxel). Individualizing therapy for prostate cancer patients is an overarching goal of research. Currently, no valid practical biomarkers exist that can predict resistance to the taxane in PC. Identifying a marker that predicts taxane sensitivity would delineate patients who would benefit from taxane regimens from those who would suffer from added side-effects without any improved response. Previous studies have confirmed TLE3 is associated with a response to taxane therapy in breast and ovarian cancers. Preclinical studies have also shown that TLR4, RAGE, and HMGB1 confers chemo resistance to docetaxel on PC-3 human PC cells. After obtaining IRB approval (protocol IUCRO-0499), 8 metastatic castration resistant prostate cancer samples and 6 control samples were pulled for this study after scrutinous review of the H&amp;E slides from clinical cases. This study then consisted of investigating the proteins TLE3, TLR4, RAGE, and HMBG1. Immunostaining was performed using the automated immunostainer, Dako FLEX System, and the slides were scanned into the whole slide Aperio Scanscope CS digital imaging system. Computer-assisted morphometric analysis of digital images was then performed using the Aperio ImageScope software. Review of the TLE3 and TLR4 expression in metastatic castration resistance prostate cancer samples showed a difference between the expressions of these two proteins. TLR4 positivity was found to be 43.88% higher than the expression of TLE3. This is suggestive of resistance to docetaxel treatments. Review of the RAGE and HMGB1 expression in metastatic castration resistance prostate cancer samples compared to normal prostate epithelium showed a 32.9% increase in RAGE and 36.6% increase in HMGB1 expression. The findings of this study are suggestive that there is a possible correlation between the overexpression of these protein biomarkers and the patient's response to a specific drug treatment. Citation Format: Greg A. Rothchild, George Sandusky, Constance J. Temm, Costantine Albany. Identifying a predictive biomarker for chemotherapy (taxane) in patients with metastatic castration resistance prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 426.

Microseminoprotein-Beta Expression in Different Stages of Prostate Cancer.

Microseminoprotein-beta (MSMB, MSMB) is an abundant secretory protein contributed by the prostate, and is implicated as a prostate cancer (PC) biomarker based on observations of its lower expression in cancerous cells compared with benign prostate epithelium. However, as the current literature on MSMB is inconsistent, we assessed the expression of MSMB at the protein and mRNA levels in a comprehensive set of different clinical stages of PC. Immunohistochemistry using monoclonal and polyclonal antibodies against MSMB was used to study protein expression in tissue specimens representing prostatectomies (n = 261) and in diagnostic needle biopsies from patients treated with androgen deprivation therapy (ADT) (n = 100), and in locally recurrent castration-resistant PC (CRPC) (n = 105) and CRPC metastases (n = 113). The transcript levels of MSMB, nuclear receptor co-activator 4 (NCOA4) and MSMB-NCOA4 fusion were examined by qRT-PCR in prostatectomy samples and by RNA-sequencing in benign prostatic hyperplasia, PC, and CRPC samples. We also measured serum MSMB levels and genotyped the single nucleotide polymorphism rs10993994 using DNA from the blood of 369 PC patients and 903 controls. MSMB expression in PC (29% of prostatectomies and 21% of needle biopsies) was more frequent than in CRPC (9% of locally recurrent CRPCs and 9% of CRPC metastases) (p<0.0001). Detection of MSMB protein was inversely correlated with the Gleason score in prostatectomy specimens (p = 0.024). The read-through MSMB-NCOA4 transcript was detected at very low levels in PC. MSMB levels in serum were similar in cases of PC and controls but were significantly associated with PC risk when adjusted for age at diagnosis and levels of free or total PSA (p<0.001). Serum levels of MSMB in both PC patients and controls were significantly associated with the rs10993994 genotype (p<0.0001). In conclusion, decreased expression of MSMB parallels the clinical progression of PC and adjusted serum MSMB levels are associated with PC risk.

Abstract 4351: Identification of molecular signature associated with the castration-resistant prostate cancer (CRPC) development by next-generation sequencing (NGS)-based transcriptomic profiling

Abstract Backgrounds: Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer produced by disease progression following surgical or pharmaceutical castration. The development process of CRPC becoming castrate resistant is unclear. The objective of this study is to identify a gene set and putative signaling pathway for mediating the development of CRPC. Methods: RNA-seq data was generated using 54 prostate samples [primary prostate cancer (PCa), CRPC, and benign prostatic hyperplasia (BPH)], among which 8 were paired samples of PCa and CRPC obtained from 4 patients. Diverse statistical methods, including a generalized linear model likelihood ratio test, were applied to identify the signature associated with CRPC development. The interconnectivity between genes in the signature was verified by Ingenuity Pathway Analysis TM. We also confirmed a relevance of the signature with experimental assays using prostate cancer cell lines. Results: Unsupervised hierarchical cluster analysis of RNA-seq data revealed a clear gene expression pattern of tumorigenesis in PCa with heterogeneous molecular characteristics of CRPC. Comparing paired cancer samples, 309 genes were differentially expressed between PCa and CRPC samples. We also tested unpaired sample groups (PCa vs. CRPC) and found that 182 genes had significantly differences of expression (a generalized linear model likelihood ratio test, P &amp;lt; 0.001, a 2-fold or greater relative difference). When comparing two gene sets, a total of 15 genes was common in both paired and unpaired CRPC groups. Among them, AR and SPINK1 were significantly increased in the CRPC than in the PCa groups. Gene network analyses and experimental assays of AR and SPINK1 revealed that CRPC development could be mediated by SPINK1-AR pathway. Conclusion: The molecular signature defined by activation of SPINK1-AR may be a genetic determinant of progression from PCa to CRPC. Note: This abstract was not presented at the meeting. Citation Format: Seon-Kyu Kim, Jeong-Hwan Kim, Pildu Jeong, Wun-Jae Kim, Yong Sung Kim, Seok-Joong Yun, Seon-Young Kim. Identification of molecular signature associated with the castration-resistant prostate cancer (CRPC) development by next-generation sequencing (NGS)-based transcriptomic profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4351. doi:10.1158/1538-7445.AM2015-4351

Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells.

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA-/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR+PSA+, AR+PSA-, AR-PSA-, and AR-PSA+ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA-/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA+ versus PSA-/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA-/lo PCa cells possess distinct epigenetic profiles. As the PSA-/lo PCa cell population is heterogeneous, in the second part, we employ two PSA- (Du145 and PC3) and two PSA+ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.

MP66-19 REDUCED AUTOPHAGY LEVELS ARE ASSOCIATED WITH A HIGHER GLEASON SCORE, TUMOR STAGE AND AN INCREASED RATE OF PROSTATE CANCER SPECIFIC DEATH

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2015MP66-19 REDUCED AUTOPHAGY LEVELS ARE ASSOCIATED WITH A HIGHER GLEASON SCORE, TUMOR STAGE AND AN INCREASED RATE OF PROSTATE CANCER SPECIFIC DEATH Ashkan Mortezavi, Souzan Salemi, Niels Rupp, Thomas Hermanns, Tullio Sulser, Peter J. Wild, and Daniel Eberli Ashkan MortezaviAshkan Mortezavi More articles by this author , Souzan SalemiSouzan Salemi More articles by this author , Niels RuppNiels Rupp More articles by this author , Thomas HermannsThomas Hermanns More articles by this author , Tullio SulserTullio Sulser More articles by this author , Peter J. WildPeter J. Wild More articles by this author , and Daniel EberliDaniel Eberli More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2372AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Autophagy is a catabolic cellular process used for degradation of cytoplasmic organelles and preservation of cell viability. Data regarding the role of autophagy in treatment naive prostate cancer (PCa) is sparse. Therefore we aimed to analyze the level of autophagy in benign and malignant prostate tissue and to evaluate the prognostic properties for patients with PCa. METHODS A Tissue microarray containing 479 formalin-fixed, paraffin-embedded prostate tissue cores was stained immunohistochemically for major autophagy proteins ATG5, Beclin1 and LC3b: 41 normal prostate tissues, 382 primary adenocarcinomas (ADC) and 56 prostate cancer metastases or castration resistant prostate cancers. Immunoreactivity was semiquantitatively scored and correlated with pathologic and clinical parameters including survival. RESULTS LC3b expression was significantly upregulated in PCa, especially in metastatic and castration-resistant prostate cancer samples compared to normal prostate tissue (p=0.002). Evaluation of expression in ADCs revealed an inverse association with preoperative serum PSA level (p=0.047), pT-stage (0.033) and Gleason Score (p=0.026). Negative LC3b expression was identified as a novel predictor of prostate cancer specific death after radical prostatectomy (p=0.02). A significant association of ATG-5 and Beclin1 with LC3b expression could be noticed, but no link with clincopathologic parameters. CONCLUSIONS A lower LC3b expression in prostate cancer tissue is significantly associated with a higher rate of prostate cancer specific death. Furthermore, there is a significant association of a lower expression in tumors with extraprostatic extension and higher Gleason Scores. This is the first report evaluating autophagy expression levels and their association with the clinical course in prostate cancer patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e823 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ashkan Mortezavi More articles by this author Souzan Salemi More articles by this author Niels Rupp More articles by this author Thomas Hermanns More articles by this author Tullio Sulser More articles by this author Peter J. Wild More articles by this author Daniel Eberli More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Reactive stroma component COL6A1 is upregulated in castration-resistant prostate cancer and promotes tumor growth.

Castration-resistant prostate cancer (CRPC) remains the most critical challenge in the clinical management of prostate cancer (PCa). Reactive stromal changes in PCa are likely involved in the emergence of CRPC. In the present study, we identified a novel oncogene termed COL6A1 which was upregulated in the reactive stroma of CRPC. We established an androgen-independent LNCaP (LNCaP-AI) cell line in steroid-reduced (SR) medium within 2 months. We examined COL6A1 expression with western blot during the LNCaP-AI induction, and studied the function of COL6A1 in vitro and in vivo. Immunohistochemical staining of COL6A1 was performed in ten pairs of androgen-sensitive PCa and CRPC samples. We demonstrated that COL6A1 expression was markedly increased in LNCaP-AI cells and CRPC tissues compared with LNCaP cells and paired androgen-sensitive PCa specimens. In vitro, COL6A1 knockdown resulted in G1-S cell cycle arrest and descended vitality. Overexpression of COL6A1 was associated with accelerated S phase entry and elevated vitality in prostate cancer cells. COL6A1 also promoted tumorigenesis of LNCaP cells in vivo. Taken together, these data suggest an important role of COL6A1 in the molecular etiology of castration-resistant prostate cancer, and support the potential use of COL6A1 in CRPC therapy.

173 Reduced autophagy levels are associated with a higher Gleason score, tumour stage and an increased rate of prostate cancer specific death

INTRODUCTION AND OBJECTIVES: Autophagy is a catabolic cellular process used for degradation of cytoplasmic organelles and preservation of cell viability. Data regarding the role of autophagy in treatment naive prostate cancer (PCa) is sparse. Therefore we aimed to analyze the level of autophagy in benign and malignant prostate tissue and to evaluate the prognostic properties for patients with PCa. METHODS: A Tissue microarray containing 479 formalin-fixed, paraffin-embedded prostate tissue cores was stained immunohistochemically for major autophagy proteins ATG5, Beclin1 and LC3b: 41 normal prostate tissues, 382 primary adenocarcinomas (ADC) and 56 prostate cancer metastases or castration resistant prostate cancers. Immunoreactivity was semiquantitatively scored and correlated with pathologic and clinical parameters including survival. RESULTS: LC3b expression was significantly upregulated in PCa, especially in metastatic and castration-resistant prostate cancer samples compared to normal prostate tissue (p1⁄40.002). Evaluation of expression in ADCs revealed an inverse association with preoperative serum PSA level (p1⁄40.047), pT-stage (0.033) and Gleason Score (p1⁄40.026). Negative LC3b expression was identified as a novel predictor of prostate cancer specific death after radical prostatectomy (p1⁄40.02). A significant association of ATG-5 and Beclin1 with LC3b expression could be noticed, but no link with clincopathologic parameters. CONCLUSIONS: A lower LC3b expression in prostate cancer tissue is significantly associated with a higher rate of prostate cancer specific death. Furthermore, there is a significant association of a lower expression in tumors with extraprostatic extension and higher Gleason Scores. This is the first report evaluating autophagy expression levels and their association with the clinical course in prostate cancer patients.

Abstract 3310: N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers

Abstract Introduction: Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance. Previously, different groups have suggested an association between chemoresistance and epithelial to mesenchimal transition (EMT) in certain advanced cancers. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with EMT, is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: Tissue microarrays of CRPC and high grade prostate cancer clinical samples were stained for TLR4. N-cadherin positive and negative cell lines were evaluated for sensitivity against docetaxel. N-cadherin and TLR4 were ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, VCaP). Docetaxel resistant cell lines (LNCaP-DocR and VCaP-DocR) were established through prolonged exposure to 10-20uM Docetaxel. For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. N-cadherin expressing cell lines were treated with 2A9 monoclonal antibody (mAb), which was raised against the extracellular domain of N-cadherin, and by N-cadherin siRNA knockdown. For in vivo studies, N-cadherin positive xenografts were treated with 2A9 mAb and docetaxel, and tumor growth monitored over time. Results: Tissue microarray results showed significant TLR4 expression in CRPC samples (p &amp;lt; 0.005). In vitro studies showed TLR4 induction correlated with N-cadherin expression. This upregulation of N-cadherin and TLR4 expression promoted docetaxel resistance in CRPC by the activation of NF-kB signaling. Monoclonal antibodies against N-cadherin and TLR4 potentiated docetaxel induced cytotoxicity. Docetaxel resistant cells exhibited EMT phenotypes with increased invasion and castration resistant growth. Treatment with 2A9 antibody restored sensitivity to docetaxel. Combination therapy with 2A9 monoclonal antibody and docetaxel significantly affected tumor growth in N-cadherin positive CRPC xenograft models. Conclusions: These studies suggest that N-cadherin promotes TLR4-dependent docetaxel resistance in CRPC. Although further investigation will be needed, therapeutic targeting of N-cadherin with monoclonal antibodies in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Citation Format: Tatsuya Shimomura, Evelyn Kono, Chau P. Tran, Joyce Yamashiro, Shu Lin, Sean Hyung-Kwon Lee, Zev A. Wainberg, Robert E. Reiter. N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3310. doi:10.1158/1538-7445.AM2014-3310