Stages Of Prostate Cancer Progression Research Articles

Emerging Perspectives in Zinc Transporter Research in Prostate Cancer: An Updated Review.

Dysregulation of zinc and zinc transporters families has been associated with the genesis and progression of prostate cancer. The prostate epithelium utilizes two types of zinc transporters, the ZIP (Zrt-, Irt-related Protein) and the ZnTs (Zinc Transporter), to transport zinc from the blood plasma to the gland lumen. ZIP transporters uptake zinc from extracellular space and organelle lumen, while ZnT transporters release zinc outside the cells or to organelle lumen. In prostate cancer, a commonly observed low zinc concentration in prostate tissue has been correlated with downregulations of certain ZIPs (e.g., ZIP1, ZIP2, ZIP3, ZIP14) and upregulations of specific ZnTs (e.g., ZnT1, ZnT9, ZnT10). These alterations may enable cancer cells to adapt to toxic high zinc levels. While zinc supplementation has been suggested as a potential therapy for this type of cancer, studies have yielded inconsistent results because some trials have indicated that zinc supplementation could exacerbate cancer risk. The reason for this discrepancy remains unclear, but given the high molecular and genetic variability present in prostate tumors, it is plausible that some zinc transporters-comprising 14 ZIP and 10 ZnT members-could be dysregulated in others patterns that promote cancer. From this perspective, this review highlights novel dysregulation, such as ZIP-Up/ZnT-Down, observed in prostate cancer cell lines for ZIP4, ZIP8, ZnT2, ZnT4, ZnT5, etc. Additionally, an in silico analysis of an available microarray from mouse models of prostate cancer (Nkx3.1;Pten) predicts similar dysregulation pattern for ZIP4, ZIP8, and ZnT2, which appear in early stages of prostate cancer progression. Furthermore, similar dysregulation patterns are supported by an in silico analysis of RNA-seq data from human cancer tumors available in cBioPortal. We discuss how these dysregulations of zinc transporters could impact zinc supplementation trials, particularly focusing on how the ZIP-Up/ZnT-Down dysregulation through various mechanisms might promote prostate cancer progression.

Differential tempol effects in prostatic cancer: angiogenesis and short- and long-term treatments.

Prostate cancer (PCa) is the second cause of cancer death among men worldwide. Several processes are involved in the development and progression of PCa such as angiogenesis, inflammation and oxidative stress. The present study investigated the effect of short- or long-term Tempol treatment at different stages of prostate adenocarcinoma progression, focusing on angiogenic, proliferative, and stromal remodeling processes in TRAMP mice. The dorsolateral lobe of the prostate of TRAMP mice were evaluated at two different stages of PCa progression; early and late stages. Early stage was again divided into, short- or long-term. 50mg/kg Tempol dose was administered orally. The results demonstrated that Tempol mitigated the prostate histopathological lesion progressions in the TRAMP mice in all treated groups. However, Tempol increased molecules involved in the angiogenic process such as CD31 and VEGFR2 relative frequencies, particularly in long-term treatment. In addition, Tempol upregulated molecule levels involved in angiogenesis and stromal remodeling process VEGF, TGF-β1, VE-cadherin and vimentin, particularly, in T8-16 group. Thus, it was concluded that Tempol treatment delayed prostatic lesion progression in the dorsolateral lobe of the TRAMP mice. However, Tempol also led to pro-angiogenic effects and glandular stromal microenvironment imbalance, especially, in the long-term treatment.

Study of iron metabolism based on T2* mapping sequences in PI-RADS 3 prostate lesions.

Prostate cancer is one of the most common malignant tumors in Chinese men, which is rich in iron metabolic activity and is closely related to all stages of prostate cancer progression. Since the current diagnostic methods are insufficient, we aimed to evaluate the value of quantitative T2 star values from the T2* mapping sequences in multiparametric magnetic resonance imaging (mpMRI) in the diagnosis and grading of PI-RADS 3 prostate cancer (PCa). We prospectively enrolled patients with PCa or benign prostatic hyperplasia (BPH) admitted to our hospital from January 2021 to November 2022. Imaging indicators, including the T2* value and apparent diffusion coefficient (ADC) value, were collected, and enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of proteins involved in iron metabolism in the patients. ROC curves were drawn to explore whether the T2* value could be used for the diagnosis and grading of PCa. We found that three iron metabolism indexes, ferritin, hepcidin, and the ferric ion (Fe), and the T2* value were significantly different between the PCa group and BPH group and between the low International Society of Urology Pathology (ISUP) group (ISUP ≤ 2) and the high ISUP group (ISUP>2). Additionally, there was a significant correlation between the levels of these three indicators and the T2* value. Further ROC analysis showed that the levels of iron metabolism-related indexes and T2* values performed well in diagnosing and grading PCa. The T2* value has good value in detecting and predicting the grade of prostate cancer and can reflect the iron metabolism of the tumor, which could provide a foundation for the diagnosis and grading of PCa in the future.

Abstract 1343: Mesenchymal cell populations associated with different stages of prostate cancer progression in mice and human

Abstract Mesenchymal cells in the prostate cancer (PCa) tumor microenvironment (TME) contribute to the biological and clinical history of PCa. Indeed, mesenchymal cells heavily interact with cancer cells, immune cells, and the other cellular and non-cellular components of the TME to favor or hinder carcinogenesis and tumor progression. Using a comprehensive array of genetically engineered mouse models (GEMMs) of prostate cancer, 8 mesenchymal populations with different transcriptional programs are preferentially enriched in specific GEMMs at different stages of PCa. Here, we determine the transferability of this mesenchymal cluster designation from mice PCa models to human PCa cases. To this end, we compared: a) Tmprss2-ERG (T-ERG) mouse and ERG+ human cases; b) Pb4-Cre+/-;Ptenf/f;LSL-MYCN+/+;Rb1f/f (PRN) mouse and PCa bone metastasis. We generated scRNA-seq data for > 8000 mesenchymal cells from ERG+ (n=6) and ERG- (n=3) PCa patients, and we retrieved data for bone metastasis mesenchymal cells (osteoblasts, osteoclasts, endothelial cells, pericytes; 1,872 total cells) from GSE143791. To transfer the stromal mouse clusters’ labels to human data, human gene symbols were converted to their mouse counterparts, then both datasets were restricted to overlapping genes. For the human PCa cases, label transfer was performed through ‘ingest’ using the scRNA-seq data from the mouse T-ERG model as reference for the human ERG+ cases and data from the remaining GEMMs as reference for the human ERG- cases. For bone metastases cases, mouse stromal data from all GEMMs were used to project the 8 stromal clusters to the mesenchymal cells in the bone metastases microenvironment. Not surprisingly, ERG+ human samples were enriched (> 60% of total stromal cells) in mouse stroma clusters predominantly present in T-ERG mouse model, characterized by the expression of Wnt regulators and AR. Common populations to all murine models, representing myofibroblasts and immunomodulatory fibroblasts (expressing Gpx3, C3, C7, Cfh), were also commonly present in patients, irrespectively to the ERG status. In the PCa bone metastases, mesenchymal clusters enriched in the PRN model were strongly represented in human bone metastases, comprising > 60% of total stromal cells. These cells were characterized by high expression of POSTN and MKI67, as well as bone-specific genes like BGN. Altogether, these findings suggest that our mesenchymal cluster designation developed using GEMMs can be meaningfully applied to human PCa, and that the different transcriptional programs we identified in distinct mesenchymal population are conserved across species. This lays the foundation for the utilization of defined genetically-engineered models in defining the interactions and cross-talks between different mesenchymal populations in relation to cancer and immune cells and other components of the TME in human prostate cancer. Citation Format: Mohamed Omar, Hubert Pakula, Filippo Pederzoli, Giuseppe N. Fanelli, Tania Panellinni, Ryan Carelli, Silvia Rodrigues, Caroline Fidalgo-Ribeiro, Pier V. Nuzzo, Lucie V. Emmenis, Mohammad Mohammad, Madhavi Jere, Caitlin Unkenholz, David Rickman, Christopher Barbieri, Brian Robinson, Luigi Marchionni, Massimo Loda. Mesenchymal cell populations associated with different stages of prostate cancer progression in mice and human [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1343.

Abstract 2546: Targeting MerTK-mediated efferocytosis in the prostate cancer TME

Abstract The prostate cancer tumor microenvironment (TME) is comprised of many different components and cell types that influence tumor progression and patient outcome. Macrophages are highly abundant immune cells in the prostate cancer TME. Macrophage phenotypes can be modeled on a continuous spectrum of M1-like (anti-tumor macrophages) to M2-like (pro-tumor macrophages) and most macrophages in the prostate cancer TME are M2-like. Efferocytosis, the phagocytosis of apoptotic cells, is a pro-tumor function of M2-like macrophages. We have developed a flow cytometry assay to quantify efferocytosis of the prostate cancer cell line LNCaP. With both cell line-based THP-1 macrophages and human monocyte-derived macrophages (HMDMs), we observe that M2 macrophages efferocytose LNCaP cells more than M1 macrophages. Based on literature from other models and contexts, efferocytosis further supports the M2-like phenotype. Efferocytosis also prevents the apoptotic cell from progressing to secondary necrosis, which would attract an M1-like macrophage phenotype. Due these aspects, we hypothesize efferocytosis in the prostate cancer TME is a tumor-promoting function of macrophages. Following efferocytosis of LNCaP cells by M2 HMDMs, we detect a decrease in the M1-like, anti-tumor marker CD80 and an increase in M2-like, pro-tumor markers CD206 and PDL1. Due to this role in modulating macrophage phenotype, we hypothesize targeting efferocytosis will slow prostate cancer growth and promote an anti-tumor immune infiltrate, including M1-like macrophages. MerTK is a receptor tyrosine kinase that mediates efferocytosis by binding phosphatidylserine on apoptotic cells. At both the protein and mRNA level, we detect higher MerTK expression in M2 than M1 THP-1 macrophages and HMDMs. Upon addition of apoptotic LNCaP cells, we observe an increase in phosphorylated MerTK (active form), suggesting the role of MerTK in prostate cancer cell efferocytosis. Currently, we are targeting MerTK to block prostate cancer cell efferocytosis in vitro and in vivo. We have generated a Mertk-/-, Hi-Myc mouse model on the FVB/N background. This prostate cancer GEMM will be used to assess the role of MerTK across different stages of prostate cancer progression. We will be comparing tumor size and immune infiltration between Mertk+/+ and Mertk-/- Hi-Myc mice aged to 2 months, 6 months and 12 months. We predict that the Mertk-/- mice will have smaller tumors and an overall anti-tumor immune infiltrate compared to Mertk+/+ mice in this model due to lack of efferocytosis. Citation Format: Kayla V. Myers, Amber E. de Groot, Anna L. Gonye, Luke V. Loftus, Sarah R. Amend, Kenneth J. Pienta. Targeting MerTK-mediated efferocytosis in the prostate cancer TME [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 2546.

Establishment of the LNCaP Cell Line - The Dawn of an Era for Prostate Cancer Research.

Among the relatively few established human prostate cancer cell lines, LNCaP cells are unique in their ability to model key stages of prostate cancer progression. Analyses of LNCaP cells and their derivatives have been invaluable for elucidating important translational aspects of prostate tumorigenesis, metastasis, and drug response, particularly in the context of androgen receptor signaling. Here, we present major highlights from a wealth of literature that has exploited LNCaP cells and their derivatives to inform on prostate cancer progression and androgen response for improving the treatment of patients with prostate cancer. See related article by Horoszewicz and colleagues, Cancer Res 1983;43:1809-18.

Exploring the Wnt Pathway as a Therapeutic Target for Prostate Cancer.

Aberrant activation of the Wnt pathway is emerging as a frequent event during prostate cancer that can facilitate tumor formation, progression, and therapeutic resistance. Recent discoveries indicate that targeting the Wnt pathway to treat prostate cancer may be efficacious. However, the functional consequence of activating the Wnt pathway during the different stages of prostate cancer progression remains unclear. Preclinical work investigating the efficacy of targeting Wnt signaling for the treatment of prostate cancer, both in primary and metastatic lesions, and improving our molecular understanding of treatment responses is crucial to identifying effective treatment strategies and biomarkers that help guide treatment decisions and improve patient care. In this review, we outline the type of genetic alterations that lead to activated Wnt signaling in prostate cancer, highlight the range of laboratory models used to study the role of Wnt genetic drivers in prostate cancer, and discuss new mechanistic insights into how the Wnt cascade facilitates prostate cancer growth, metastasis, and drug resistance.

Distinct DNA methylation patterns associated with treatment resistance in metastatic castration resistant prostate cancer

Androgens are a major driver of prostate cancer (PCa) and continue to be a critical treatment target for advanced disease, which includes castration therapy and antiandrogens. However, resistance to these therapies leading to metastatic castration-resistant prostate cancer (mCRPC), and the emergence of treatment-induced neuroendocrine disease (tNEPC) remains an ongoing challenge. Instability of the DNA methylome is well established as a major hallmark of PCa development and progression. Therefore, investigating the dynamics of the methylation changes going from the castration sensitive to the tNEPC state would provide insights into novel mechanisms of resistance. Using an established xenograft model of CRPC, genome-wide methylation analysis was performed on cell lines representing various stages of PCa progression. We confirmed extensive methylation changes with the development of CRPC and tNEPC using this model. This included key genes and pathways associated with cellular differentiation and neurodevelopment. Combined analysis of methylation and gene expression changes further highlighted genes that could potentially serve as therapeutic targets. Furthermore, tNEPC-related methylation signals from this model were detectable in circulating cell free DNA (cfDNA) from mCRPC patients undergoing androgen-targeting therapies and were associated with a faster time to clinical progression. These potential biomarkers could help with identifying patients with aggressive disease.

Differential expression of protein disulfide-isomerase A3 isoforms, PDIA3 and PDIA3N, in human prostate cancer cell lines representing different stages of prostate cancer

Prostate cancer (PCa) is a highly heterogeneous and unpredictable progressive disease. Sensitivity of PCa cells to androgens play a central role in tumor aggressiveness but biomarkers with high sensitivity and specificity that follow the progression of the disease has not yet been verified. The vitamin D endocrine system and its receptors, the Vitamin D Receptor (VDR) and the Protein Disulfide-Isomerase A3 (PDIA3), are related to anti-tumoral effects as well as carcinogenesis and have therefore been suggested as potential candidates for the prevention and therapy of several cancer forms, including PCa. In this study, we evaluated the mRNA expression of VDR and PDIA3 involved in vitamin D signaling in cell lines representing different stages of PCa (PNT2, P4E6, LNCaP, DU145 and PC3). This study further aimed to evaluate vitamin D receptors and their isoforms as potential markers for clinical diagnosis of PCa. A novel transcript isoform of PDIA3 (PDIA3N) was identified and found to be expressed in all PCa cell lines analyzed. Androgen-independent cell lines showed a higher mRNA expression ratio between PDIA3N/PDIA3 contrary to androgen-dependent cell lines that showed a lower mRNA expression ratio between PDIA3N/PDIA3. The structure of PDIA3N differed from PDIA3. PDIA3N was found to be a N-truncated isoform of PDIA3 and differences in protein structure suggests an altered protein function i.e. cell location, thioredoxin activity and affinity for 1,25(OH)2D3. Collectively, PDIA3 transcript isoforms, the ratio between PDIA3N/PDIA3 and especially PDIA3N, are proposed as candidate markers for future studies with different stages of PCa progression.

A bioengineered organotypic prostate model for the study of tumor microenvironment-induced immune cell activation.

The prostate tumor microenvironment (TME) is strongly immunosuppressive; it is largely driven by alteration in cell phenotypes (i.e. tumor-associated macrophages and exhausted cytotoxic T cells) that result in pro-tumorigenic conditions and tumor growth. A greater understanding into how these altered immune cell phenotypes are developed and could potentially be reversed would provide important insights into improved treatment efficacy for prostate cancer. Here, we report a microfluidic model of the prostate TME that mimics prostate ducts across various stages of prostate cancer progression, with associated stroma and immune cells. Using this platform, we exposed immune cells to a benign prostate TME or a metastatic prostate TME and investigated their metabolism, gene and cytokine expression. Immune cells exposed to the metastatic TME showed metabolic differences with a higher redox ratio indicating a switch to a more glycolytic metabolic profile. These cells also increased expression of pro-tumor response cytokines that have been shown to increase cell migration and angiogenesis such as Interleukin-1 (IL-1) a and Granulocyte-macrophage colony-stimulating factor (GM-CSF). Lastly, we observed decreased TLR, STAT signaling and TRAIL expression, suggesting that phenotypes derived from exposure to the metastatic TME could have an impaired anti-tumor response. This platform could provide a valuable tool for studying immune cell phenotypes in in vitro tumor microenvironments.

Shaping Chromatin States in Prostate Cancer by Pioneer Transcription Factors.

The androgen receptor (AR) is a critical therapeutic target in prostate cancer that responds to antagonists in primary disease, but inevitably becomes reactivated, signaling onset of the lethal castration-resistant prostate cancer (CRPC) stage. Epigenomic investigation of the chromatin environment and interacting partners required for AR transcriptional activity has uncovered three pioneer factors that open up chromatin and facilitate AR-driven transcriptional programs. FOXA1, HOXB13, and GATA2 are required for normal AR transcription in prostate epithelial development and for oncogenic AR transcription during prostate carcinogenesis. AR signaling is dependent upon these three pioneer factors both before and after the clinical transition from treatable androgen-dependent disease to untreatable CRPC. Agents targeting their respective DNA binding or downstream chromatin-remodeling events have shown promise in preclinical studies of CRPC. AR-independent functions of FOXA1, HOXB13, and GATA2 are emerging as well. While all three pioneer factors exert effects that promote carcinogenesis, some of their functions may inhibit certain stages of prostate cancer progression. In all, these pioneer factors represent some of the most promising potential therapeutic targets to emerge thus far from the study of the prostate cancer epigenome.

Early intervention exercise training does not delay prostate cancer progression in Pten−/− mice

There is convincing evidence that men with advanced prostate cancer experience improved quality of life as a result of exercise therapy, although there is limited preclinical, and no clinical, data to directly support the notion that exercise training improves prostate cancer prognosis or outcome. The aim of this study was to investigate the effect of regular exercise training on the early stages of prostate cancer progression, as well as assessing whether alterations to prostate cancer metabolism are induced by exercise. Mice with prostate-specific deletion of Pten (Pten-/- ) remained sedentary or underwent 6 weeks of endurance exercise training or high-intensity exercise training involving treadmill running. At the conclusion of the training period, the prostate lobes were excised. A portion of fresh tissue was used to assess glucose, glutamine, and fatty acid metabolism by radiometric techniques and a second portion was fixed for histopathology. Despite the implementation of an effective exercise regime, as confirmed by improvements in running capacity, neither prostate mass, cell proliferation or the incidence of high-grade prostate intraepithelial hyperplasia or noninvasive carcinoma in situ were significantly different between groups. Similarly, neither glucose uptake, oxidation and de novo lipogenesis, glutamine oxidation, or fatty acid uptake, oxidation and storage into various lipids were significantly different in prostate tissue obtained from untrained and exercise trained mice. These results show that 6 weeks of moderate or high-intensity exercise training does not alter substrate metabolism in the prostate or slow the progression of Pten-null prostate cancer. These results question whether exercise is a useful therapy to prevent or delay prostate cancer progression.

Epigenetic regulation of the ITGB4 gene in prostate cancer

BackgroundExamination of epigenetic changes at the ITGB4 gene promoter reveals altered methylation at different stages of prostate tumour progression and these changes may, in part, explain the complex patterns of gene expression of this integrin observed. Transcriptional re-programming perturbs expression of cell adhesion molecules and underpins metastatic tumour cell behaviour. Decreasing expression of the cell adhesion molecule ITGB4, which encodes the beta subunit of the integrin, alpha6 beta4 (α6β4), has been correlated with increased tumour aggressiveness and metastasis in multiple tumour types including prostate cancer. Paradoxically, in vitro studies in tumour cell models demonstrate that ITGB4 mediates cell mobility and invasion. Herein we examined whether transcriptional re-programming by methylation influenced ITGB4 gene expression at different stages of prostate cancer progression. Bisulphite sequencing of a large CpG island in the ITGB4 gene promoter identified differentially methylated regions in prostate cancer cell lines representing a localised tumour (22Rv1), lymph node metastasis (LNCaP), and a bone metastasis (PC-3). The highest levels of methylation were observed in the CpG island surrounding the ITGB4 transcription start site in PC-3 cells, and this observation also correlated with higher gene expression of ITGB4 in these cells. Furthermore, PC-3 cells expressed two distinct transcripts, using an alternate transcription start site, which was not detected in other cell lines. In prostate tumour biopsy samples, patterns of methylation across the ITGB4 promoter were similar overall in matched primary and metastatic samples (n = 4 pairs), with a trend toward loss of methylation at specific sites in metastatic lesions.

Tempol Treatment Inhibits Inflammatory Process and Improves Morphological Aspects in Early and Late‐stage of Prostate Cancer Progression in the Ventral Lobe of TRAMP Model.

Tempol (4‐hydroxy‐TEMPO) is a SOD mimetic and member of the nitroxide compounds family. It is used as a co‐adjuvant or protective factor in cancer treatment due to its anti‐oxidant properties. However, in prostate cancer (PCa) Tempol effects are still poorly understood. Its known that PCa progression requires a sustained inflammation, which generates a broad spectrum of reactive nitrogen and oxygen species, leading to uncontrolled cellular proliferation. We aimed to evaluate the Tempol effects in different stages of PCa progression (a) in vivo using a transgenic adenocarcinoma of the mouse prostate model (TRAMP); (b) and in vitro using human PCa cell lines with different genetic background. For the early‐stage analysis, 8‐weeks‐old mice (TPL1 group) were treated orally with 50 mg/kg of Tempol during 4 weeks. For the late‐stage analysis, 16‐weeks‐old mice were treated with 50mg/Kg (TPL2 group) or 100mg/Kg (TPL3 group) of Tempol during the same period. All control groups received water as vehicle. The ventral lobe of the prostate was collected and submitted to the histopathological, immunohistochemical and Western blotting analysis. PC‐3 and LnCaP cells were exposed to different doses of Tempol (0.5, 1.0 and 2.0 mM) and cell viability assay was performed. In vivo, Tempol treatment promoted a great improvement in the prostatic epithelium in all the analyzed cancer stages, reducing significantly premalignant (high‐grade prostatic intraepithelial neoplasia) and malignant lesions (well differentiated prostatic adenocarcinoma). The PCNA immunostaining confirmed the cellular proliferation decrease between the control and treated groups. Tempol also reduced COX‐2 and NFkB, especially in the early‐stage, whereas iNOS increased in this period. In the late‐stage, both doses of Tempol reduced NFkB levels concomitant with IkB‐α increase, suggesting an inhibitory effect of Tempol on NFkB pathway in association with its anti‐oxidant role. In vitro, Tempol decreased cell viability of both cell lines, being the 1.0 mM dose the most efficient and nontoxic concentration. In conclusion, Tempol had a remarkable action in the prostate, delaying cancer progression in the ventral lobe of TRAMP model and inhibiting PC‐3 and LnCaP growth. This SOD mimetic presented great capability of inhibiting the inflammation process, and consequently the cell proliferation. Its action was effective in different stages of PCa progression, particularly in the early stage, indicating that Tempol treatment can be explored as a preventive approach. Finally, for the late PCa stage, Tempol may be considered as an interesting adjuvant therapy.Support or Funding InformationSão Paulo Research Foundation, FAPESP, process 2018/21647‐6 and FAEPEX/UNICAMP, process 2202/19

Epigenetics and prostate cancer: defining the timing of DNA methyltransferase deregulation during prostate cancer progression

DNA methyltransferases (DNMTs) regulate gene expression by methylating cytosine residues within CpG dinucleotides. Aberrant methylation patterns have been shown in a variety of human tumours including prostate cancer. However, the expression of DNMTs in clinical samples across the spectrum of prostate cancer progression has not been studied before. Tissue microarrays were constructed from the prostatectomy specimens of 309 patients across the spectrum of prostate cancer progression: hormone-naïve low-grade prostate cancer (n=49), hormone-naïve high-grade prostate cancer (n=151), hormonally treated high-grade prostate cancer (n=65), and castrate-resistant prostate cancer (CRPC) including neuroendocrine carcinoma (n=44). Adjacent non-neoplastic parenchyma was also available in 100 patients. In 71 patients with high-grade carcinoma and lymph node metastasis, tissue from the metastasis was also available for analysis. Immunohistochemical staining was performed with antibodies against DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. Our results showed that DNMT1 and DNMT3L were upregulated early in prostate cancer progression, whereas DNMT2 was upregulated as a response to androgen ablation. DNMT1, DNMT3A, and DNMT3B were higher in the late stages of prostate cancer progression, i.e., the emergence of castrate resistance and androgen-independent growth. Lastly, DNMT1, DNMT2, and DNMT3L were upregulated in lymph node metastases compared to primary carcinomas. Our results highlight a cascade of epigenetic events in prostate cancer progression.

Incidence of androgen receptor and androgen receptor variant 7 coexpression in prostate cancer.

Prostate cancer (PRCA) is an androgen-driven disease, where androgens act through the androgen receptor (AR) to induce proliferation and survival of tumor cells. Recently, AR splice variant 7 (ARv7) has been implicated in advanced stages of PRCA and clinical recurrence. With the widespread use of AR-targeted therapies, there has been a rising interest in the expression of full-length AR and ARv7 in PRCA progression and how these receptors, both independently and together, contribute to adverse clinicopathologic outcomes. Despite a multitude of studies measuring the expression levels of AR and ARv7 in PRCA progression, the results have been inconsistent and sometimes contradictory due to technical and analytical discrepancies. To circumvent these inconsistencies, we used an automated multiplexed immunostaining platform for full-length AR and ARv7 in human PRCA samples and objectively quantified expression changes with machine learning-based software. With this technology, we can assess receptor prevalence both independently, and coexpressed, within specific tissue and cellular compartments. Full-length AR and ARv7 expression increased in epithelial nuclei of metastatic samples compared to benign. Interestingly, a population of cells with undetectable AR persisted through all stages of PRCA progression. Coexpression analyses showed an increase of the double-positive (AR+ /ARv7+ ) population in metastases compared to benign, and an increase of the double-negative population in PRCA samples compared to benign. Importantly, analysis of clinicopathologic outcomes associated with AR/ARv7 coexpression showed a significant decrease in the double-positive population with higher Gleason score (GS), as well as in samples with recurrence in under 5 years. Conversely, the double-negative population was significantly increased in samples with higher GS and in samples with recurrence in under 5 years. Changes in AR and ARv7 coexpression may have prognostic value in PRCA progression and recurrence. A better understanding of the prevalence and clinicopathologic outcomes associated with changes in these receptors' coexpression may provide a foundation for improved diagnosis and therapy for men with PRCA.

Abstract 3498: Regulation of AR through an FGF5 driven mechanism

Abstract Purpose To investigate the role of increasing FGF5 expression in the progression of prostate cancer and emergence of castration resistant prostate cancer. Methods Utilizing a cell line model representing different stages of prostate cancer progression, we assessed FGF5 expression and function in vitro and in vivo. We analyzed both an outcomes and progression tissue microarray (TMA) containing ~400 prostate cancer patient cores to assess FGF5 expression in a clinical setting. Results There was a significant increase in the both RNA and protein expression of FGF5 in tumorigenic cell lines compared to nontumorigenic. In TMAs that were analyzed, there was a significant increase of FGF5-positive cells across progression of prostate cancer from benign to metastatic, with the highest percentage occurring in cases of cancer. When performing gain of function experiments with recombinant FGF5 (rFGF5) or overexpression plasmids, we found that FGF5 significantly increased the proliferation of AR-positive cell lines. Conversely, in AR-negative cell lines, FGF5 inhibited proliferation. Because FGF5 is associated with stem cells and differentiation events, we next asked whether the population of FGF5 cells that increases through cancer progression could lead to molecular changes in the total population. In addition to inhibiting proliferation of AR-negative nontumorigenic BPH1 cells, rFGF5 resulted in a robust AR expression. In LNCaP, C4-2, and C4-2B rFGF5 resulted in an increase in AR expression. In C4-2 and C4-2B cells an increase in AR protein coincided with an increase in nuclear YAP1, a potent transcriptional regulator. We hypothesized that androgens could be regulating FGF5 expression in the prostate. When androgen dependent LNCaP cells are subjected to an androgen-depleted environment, FGF5 protein and RNA significantly increased within 24hrs. Additionally, when AR is stimulated using dihyrotestosterone, FGF5 protein and RNA levels significantly decreased. These effects are not observed in cell lines that do not express AR. Conclusions These results suggest that FGF5 is increased in prostate cancer progression cell lines as well as patient samples, with highest expression in aggressive cancer cores. Data also suggests that AR plays a role in the regulation of FGF5 and vice versa. These data suggest that FGF5 driven AR expression could be driven by YAP1 nuclear localization. This mechanism will be further interrogated. Citation Format: Dalton McLean. Regulation of AR through an FGF5 driven mechanism [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 3498.

The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP

MicroRNA (miRNA) deregulation in prostate cancer (PCa) contributes to PCa initiation and metastatic progression. To comprehensively define the cancer-associated changes in miRNA targeting and function in commonly studied models of PCa, we performed photoactivatable ribonucleoside-enhanced cross-linking immunoprecipitation of the Argonaute protein in a panel of PCa cell lines modeling different stages of PCa progression. Using this comprehensive catalogue of miRNA targets, we analyzed miRNA targeting on known drivers of PCa and examined tissue-specific and stage-specific pathway targeting by miRNAs. We found that androgen receptor is the most frequently targeted PCa oncogene and that miR-148a targets the largest number of known PCa drivers. Globally, tissue-specific and stage-specific changes in miRNA targeting are driven by homeostatic response to active oncogenic pathways. Our findings indicate that, even in advanced PCa, the miRNA pool adapts to regulate continuing alterations in the cancer genome to balance oncogenic molecular changes. These findings are important because they are the first to globally characterize miRNA changes in PCa and demonstrate how the miRNA target spectrum responds to staged tumorigenesis.

Abstract B31: Characterization of a novel model of prostate cancer metastasis resulting from androgen receptor suppression

Abstract Background: Prostate cancer (PC) is the second leading cause of male cancer-related mortality: 30,000 men die each year from metastatic disease. The Androgen Receptor (AR) may be the earliest known example of a lineage oncogene – a master regulator of prostate cancer cell survival and growth. The AR protein is detectable at all stages of prostate cancer progression and is the primary target of pharmacological agents capable of suppressing AR signaling to extend survival in men with metastatic prostate cancer (mPC). The CYP17 inhibitor Abiraterone, and the 3rd generation AR antagonist, MDV3100 prolong the survival of men with mPC, though cures remain exceeding rare using these agents individually. In order to develop additional therapies to be used in combination with current AR inhibitors, preclinical models that mimic PC progression need to be established. Tissue microarray studies from hormone-naïve, nodal positive, prostate cancer patients revealed a significant down-regulation of AR protein level in matched nodal metastases versus primary tumors. These results suggest that loss of AR or AR activity could play a role in the progression to metastatic disease. Methods: We used a short-hairpin RNA targeting exon 1 of the AR to reduce AR protein in vivo using a doxycycline-inducible system in the non-metastatic AR-positive human prostate cancer cell line 22Rv1. This cell line model, derived from a primary tumor, is castration-resistant and enzalutamide/MDV3100-resistant. We generated xenografts using 22Rv1shARex1 and evaluated tumor growth and propensity for metastatic spread. We profiled transcriptional changes in the context of AR suppression and compared gene expression changes between grafted tumors and sites of metastatic spread. Results: Xenograft studies in NOD-SCID mice revealed that reduction of the AR protein decreased tumor proliferation rates and promoted cancer metastasis. Tumor-bearing mice exposed to continual reduction of the AR developed large liver metastases and had significantly increased disseminated tumor cells in their bone marrow. Furthermore, intratibial and intracardiac injection of 22Rv1shARex1 followed by AR reduction, resulted in an increased formation of metastases. Transcriptional analysis revealed unique signatures between untreated parental 22Rv1 xenografts, tumors with AR reduction, and liver metastases. Genes up-regulated in xenografts following AR reduction, were also up-regulated in patient metastases versus primary tumors including transcripts encoding EGF, HGF, and ZEB2. Discussion: The AR is the primary therapeutic target for mPC. However, a cure for advanced cancer and metastatic disease remains elusive. Reduction of the AR in the 22Rv1 in vivo model mimics disease progression seen in patients. Molecular changes observed during the transformation of localized cancer to metastatic disease in this model may identify new targets to be used in combination with current therapies intent on ablating AR signaling. Citation Format: Clinton K. Matson, Peter S. Nelson. Characterization of a novel model of prostate cancer metastasis resulting from androgen receptor suppression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B31.

Expression of store-operated channel components in prostate cancer: the prognostic paradox

In vitro studies in prostate cancer (PCa) cell lines have suggested a key and complex role of the store-operated channels (SOCs) in major cancer hallmarks, including proliferation, apoptosis, and migration. In the present study, we investigated in vivo the expression of the SOC components transient receptor potential canonical (TRPC) 1, TRPC4, Orai1, and stromal interaction molecule 1 (STIM1), during all stages of PCa progression, and evaluated their prognostic impact in clinically localized cancer (CLC). The expressions of TRPC1, TRPC4, Orai1, STIM1, and the androgen receptor and the proliferation marker Ki-67 were evaluated by immunohistochemistry on tissue microarrays containing samples of normal prostate tissues (n=91), prostatic intraepithelial neoplasia (n=61), CLC surgically treated (n=238), and castration-resistant prostate cancer (CRPC; n=45). All markers significantly increased in CLC compared with normal tissues and (for Orai1 and STIM1) in advanced pT3 tumors compared with pT2. In contrast, their expression decreased in CRPC, particularly for Orai1. In CLC, staining for TRPC1, Orai1 and STIM1 correlated with androgen receptor expression, and TRPC1 status was associated with lower proliferation and longer recurrence-free survival, after adjusting for classical prognostic markers. Although increased SOC expression during PCa progression supports a role in cancer cell migration, the inverse association between TRPC1 and biochemical relapse suggests a protective effect in CLC. Moreover, the dramatic down-regulation of Orai1 in CRPC supports its role in apoptosis at this stage of the disease. These results call for caution when considering SOCs as potential therapeutic targets for PCa.