Role In Prostate Tumorigenesis Research Articles

Low SMARCD3 expression is associated with poor prognosis in patients with prostate cancer.

SWI/SNF complexes represent a family of multi-subunit chromatin remodelers that are affected by alterations in >20% of human tumors. While mutations of SWI/SNF genes are relatively uncommon in prostate cancer (PCa), the literature suggests that deregulation of various subunits plays a role in prostate tumorigenesis. To assess SWI/SNF functions in a clinical context, we studied the mutually exclusive, paralogue accessory subunits SMARCD1, SMARCD2, and SMARCD3 that are included in every known complex and are sought to confer specificity. Performing immunohistochemistry (IHC), the protein levels of the SMARCD family members were measured using a tissue microarray (TMA) comprising malignant samples and matching healthy tissue of non-metastatic PCa patients (n = 168). Moreover, IHC was performed in castration-resistant tumors (n = 9) and lymph node metastases (n = 22). To assess their potential role as molecular biomarkers, SMARCD1 and SMARCD3 protein levels were correlated with clinical parameters such as T stage, Gleason score, biochemical recurrence, and progression-free survival. SMARCD1 protein levels in non-metastatic primary tumors, lymph node metastases, and castration-resistant samples were significantly higher than in benign tissues. Likewise, SMARCD3 protein expression was elevated in tumor tissue and especially lymph node metastases compared to benign samples. While SMARCD1 levels in primary tumors did not exhibit significant associations with any of the tested clinical parameters, SMARCD3 exhibited an inverse correlation with pre-operative PSA levels. Moreover, low SMARCD3 expression was associated with progression to metastasis. In congruence with previous literature, our results implicate that both SMARCD1 and SMARCD3 may exhibit relevant functions in the context of prostate tumorigenesis. Moreover, our approach suggests a potential role of SMARCD3 as a novel prognostic marker in clinically non-metastatic PCa.

SND1 binds to ERG and promotes tumor growth in genetic mouse models of prostate cancer

SND1 and MTDH are known to promote cancer and therapy resistance, but their mechanisms and interactions with other oncogenes remain unclear. Here, we show that oncoprotein ERG interacts with SND1/MTDH complex through SND1’s Tudor domain. ERG, an ETS-domain transcription factor, is overexpressed in many prostate cancers. Knocking down SND1 in human prostate epithelial cells, especially those overexpressing ERG, negatively impacts cell proliferation. Transcriptional analysis shows substantial overlap in genes regulated by ERG and SND1. Mechanistically, we show that ERG promotes nuclear localization of SND1/MTDH. Forced nuclear localization of SND1 prominently increases its growth promoting function irrespective of ERG expression. In mice, prostate-specific Snd1 deletion reduces cancer growth and tumor burden in a prostate cancer model (PB-Cre/Ptenflox/flox/ERG mice), Moreover, we find a significant overlap between prostate transcriptional signatures of ERG and SND1. These findings highlight SND1’s crucial role in prostate tumorigenesis, suggesting SND1 as a potential therapeutic target in prostate cancer.

THU488 Genetic Alterations In CREBRF Influence Prostate Cancer Survival And Impact Prostate Tissue Homeostasis In Mice

Abstract Disclosure: L.E. Pascal: None. K.A. Frahm: None. K. Skalitzky: None. D.B. DeFranco: None. L. Rigatti: None. R. Lu: None. T. Liu: None. Background: Risk factors for prostate cancer include age, environmental factors, race and ethnicity. Genetic variants in cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) gene are frequently observed in Pacific Islanders, a population with elevated prostate cancer incidence. CREBRF has been shown to play a role in other cancers, however its function in prostate homeostasis and tumorigenesis has not been previously explored. Methods: Alterations in CREBRF were identified in prostate cancer patients via in silico analysis of several publicly available datasets through cBioPortal. Male Crebrf knockout mice were generated and examined for prostate defects at 4 months of age. Immunohistochemical staining of murine prostate sections was used to determine the impact of Crebrf knockout on proliferation, apoptosis, inflammation and blood vessel density in the prostate. Serum adipokine levels were measured using a Luminex Multiplex Assay. Results: CREBRF alterations were identified in up to 4.05% of prostate tumors and the mutations identified were categorized as likely damaging. Median survival of patients with CREBRF alterations was 41 months, compared to 131 months for patients without CREBRF alterations. In the murine model, the prostates of Crebrf knockout mice had reduced epithelial proliferation and increased TUNEL+ apoptotic cells. Circulating adipokines PAI-1 and MCP-1 were also altered in Crebrf knockout mice compared to age-matched controls. Conclusions: Prostate cancer patients with CREBRF alterations were associated with decreased overall survival suggesting that CREBRF may play a role in prostate tumorigenesis and progression. The murine knockout model demonstrated that CREBRF could modulate proliferation and apoptosis and macrophage density in the prostate. Serum levels of adipokines PAI-1 and MCP-1 were also altered and could contribute to the phenotypic changes observed in the prostates of Crebrf knockout mice. Future studies focused on populations susceptible to CREBRF mutations and mechanistic studies will be required to fully elucidate the potential role of CREBRF in prostate tumorigenesis. Presentation: Thursday, June 15, 2023

AR activates YAP/TAZ differentially in prostate cancer.

The Hippo signalling pathway is a master regulator of cell growth, proliferation, and cancer. The transcriptional coregulators of the Hippo pathway, YAP and TAZ, are central in various cancers. However, how YAP and TAZ get activated in most types of cancers is not well understood. Here, we show that androgens activate YAP/TAZ via the androgen receptor (AR) in prostate cancer (PCa), and that this activation is differential. AR regulates YAP translation while inducing transcription of the TAZ encoding gene, WWTR1 Furthermore, we show that AR-mediated YAP/TAZ activation is regulated by the RhoA GTPases transcriptional mediator, serum response factor (SRF). Importantly, in prostate cancer patients, SRF expression positively correlates with TAZ and the YAP/TAZ target genes CYR61 and CTGF We demonstrate that YAP/TAZ are not essential for sustaining AR activity, however, targeting YAP/TAZ or SRF sensitize PCa cells to AR inhibition in anchorage-independent growth conditions. Our findings dissect the cellular roles of YAP, TAZ, and SRF in prostate cancer cells. Our data emphasize the interplay between these transcriptional regulators and their roles in prostate tumorigenesis and highlight how these insights might be exploited therapeutically.

Abstract A056: AR/FOXA1-mediated active gene repression as a mechanism of prostate carcinogenesis

Abstract The identity of the prostate gland is defined by transcription factors (TF) like the androgen receptor (AR), FOXA1, and NKX3.1 that bind regulatory elements early in development to activate the lineage gene program. For instance, KLK3, is a canonical target gene activated by AR. While the mechanistic interactions between pioneer factors, chromatin remodelers, and epigenetic proteins required for gene activation is well understood, repressive activity of lineage-defining TFs remain largely uncharacterized. Here, we document and mechanistically describe the active, repressive transcriptional functions of AR and FOXA1 in the context of prostate tumorigenesis.We identified a shared repertoire of repressed gene targets of AR and FOXA1 by using transcriptomic profiles (RNA-seq) of PCa cells treated with dihydrotestosterone (DHT) or TF targeting siRNA. Chromatin binding of AR and/or FOXA1 (ChIPseq) at cis-regulatory elements (dCREs) within the gene’s topologically-associated domain was used as primary evidence for direct regulation. Here, the KLK3 and DEPTOR gene loci emerged as candidate-activated or repressed AR/FOXA1 targets in PCa cells. DEPTOR, a tumor suppressor gene, negatively regulates the mTORC1/2 complexes by inhibiting their kinase activity. In contrast to the passive model of repression, where sequestration of co-activators ensues in transcriptional down-regulation of genes, we found ligand-bound AR and FOXA1 bind CREs within DEPTORs’ TAD establishing looping interactions (H3K3me3 HiChIP-seq) with the DEPTOR gene promoter. Functional inactivation of AR or FOXA1 led to a 2-4 fold upregulation of DEPTOR in VCaP, LNCaP, and 22RV1 cells. Instead, DHT-stimulation of AR led to DEPTOR downregulation. To assess the rapidness of this effect, we used a SWI/SNF ATPase PROTAC, AU15330, which triggers instant compaction of CREs and dislodges AR/FOXA1 from the chromatin. Treatment of PCa cells with AU15330 for 4 hours led to an immediate and significant upregulation of DEPTOR, substantiating the direct transcriptional repression by the AR/FOXA1 complex. Consistently, AU-15330 treatment also abolished FOXA1 and AR binding and 3D loop formation into the DEPTOR promoter. Using an orthogonal genetic approach, CRISPR-KRAB-targeted heterochromatinization of the AR/FOXA1 repressive CREs led to increased production of the nascent DEPTOR transcript. Finally, monoclones devoid of the CRE had elevated DEPTOR levels. Altogether, our findings position DEPTOR as an actively repressed target of the AR/FOXA1 transcriptional complex, suggesting the “silencer” duties of driver TF oncogenes to play a critical role in prostate tumorigenesis. Furthermore, the repertoire of activated and repressed gene targets identified in this study serve as exciting loci to study differences in the co-factor machinery and/or other cis or trans-attributes of the encompassing TADs that regulate downstream transcriptional outcomes. We posit the active mode of gene repression plays a driver role in prostate carcinogenesis. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James George, Mustapha Jaber, Lanbo Xiao, Fengyun Su, Rui Wang, Xuhong Cao, Arul Chinnaiyan. AR/FOXA1-mediated active gene repression as a mechanism of prostate carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A056.

Abstract A028: Dissecting the role of cellular senescence in prostate cancer initiation and immune suppression

Abstract Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in American men. Although many disease cases remain latent for decades, others progress to malignant prostate cancer with high rates of mortality. The molecular underpinnings driving malignancy are still poorly understood, and there is an urgent need to identify mechanisms and informative biomarkers of disease progression. Using the Pb-Cre+; Ptenfl/fl (CP) mouse model, we and others found that Pten genomic loss, a common occurrence in human prostate cancer, leads to induction of cellular senescence, senescence-associated inflammation, and immune suppression during tumor onset. Similarly, senescence markers have also been identified in benign and early prostate cancer lesions in humans. This suggests that senescence may play a yet uncharacterized functional role in prostate tumorigenesis. Cellular senescence is a damage-induced pathway leading to durable growth arrest and has long been considered a potent tumor suppressive mechanism that limits the ability of pre-malignant cells to proliferate and develop into malignant tumors. However, recent findings suggest that the accumulation of senescent cells can paradoxically lead to chronic inflammation that fosters an environment for eventual tumor development and progression through the induction of growth and inflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). Thus, it remains unclear whether senescence or specific senescent cell populations block or rather promote prostate tumor development. Leveraging the CP prostate cancer mouse model and genetic tools to track senescent cells, we observed that markers of senescence (SA-β-gal, p16, Bcl2) and the inflammatory SASP (NF-𝜅B, IL-1b, CXCL1, CSF-1) were not only present in early prostate intraepithelial neoplasia (PIN) lesions but remained elevated following adenocarcinoma development and progression, and could be found in both epithelial cells as well as other immune and stromal populations. Expression of the canonical senescence marker p16 also correlated with disease progression in human prostate cancer samples. Moreover, senescent cell accumulation was associated with a reduction in cytotoxic lymphocytes and an influx of suppressive myeloid populations that we and others have previously shown contribute to prostate cancer progression. Senolytic approaches to eliminate these senescent cell populations reduced progression to adenocarcinoma and remodeled the immune system to reverse myeloid-mediated immune suppression and activate cytotoxic lymphocyte immunity. Together, our preliminary results suggest that senescence might contribute to prostate cancer initiation and progression, and that removal of senescent cells could delay tumor onset and reverse prostate cancer immune suppression. Citation Format: Lin Zhou, Jarin Snyder, Katherine C. Murphy, Kelly D. DeMarco, Sachliv Chana, Karl Simin, Zhong Jiang, Marcus Ruscetti. Dissecting the role of cellular senescence in prostate cancer initiation and immune suppression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A028.

Abstract 316: Engineering large chromosomal deletions to advance precision oncology for prostate cancer

Abstract Human prostate cancer is notable for its high number of somatic copy number alterations (CNAs), which include both focal and broad arm-level amplifications and deletions. Up to 90% of prostate tumors harbor aberrant CNAs, and genomic deletions affect about 40% of these tumors. Many chromosomal deletions are highly recurrent and occur in more than 10% of prostate tumors, including 8p (40~50%), 6q14-16, 10q23, 13q14, and 16q22-24 (20~30% each). Most of these deletions are large, often including hundreds of genes. Notably, several large deletions, including 6q14-16 and 16q22-24, are significantly associated with poor patient outcomes, suggesting that these large chromosomal deletions play a functional role in prostate tumorigenesis, and highlighting the critical need for elucidation of the mechanisms underlying large-deletion driven tumorigenesis. However, due to the limited cloning capacity of targeting vectors and the rarity of on-target homologous recombination events in traditional gene-targeting technology, modelling large chromosomal deletions has proven highly challenging, and the role of such lesions in tumorigenesis has therefore been significantly understudied. Here, we developed an innovative approach to engineering the chromosome over large genetic distances through CRISPR/Cas9 technology in both mouse embryonic stem (ES) cells and human prostate cancer cells. Upon completion of the desired genome edits, the resulting mouse ES clones make possible the creation of conditional knockout mice that accurately mimic prostate cancer-associated large deletions, while the engineered human cancer cell clones serve as an indispensable in vitro model to validate the biological relevance of large chromosomal deletions in prostate tumorigenesis. Further characterization of the human prostate cell line bearing 10q23 deletion revealed that 10q23 loss conferred RWPE1 cells growth advantage. The development and characterization of these in vivo and in vitro models will contribute greatly to the implementation of precision oncology research, open new opportunities for modelling human diseases associated with large chromosomal deletions, and lead to novel insights into the biology of prostate cancer-associated large deletions, which will facilitate the identification of deletion-specific vulnerabilities that can be exploited for prostate cancer treatment. Citation Format: Jinjin Wu, Chen Wu, Joel A. Lawitts, Cheryl B. Bock, Jiaoti Huang, Ming Chen. Engineering large chromosomal deletions to advance precision oncology for prostate cancer [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 316.

WD Repeat Domain 77 Protein Regulates Translation of E2F1 and E2F3 mRNA.

WD repeat domain 77 protein (WDR77) is required for cellular proliferation of lung and prostate epithelial cells during earlier stages of development and is reactivated during prostate and lung tumorigenesis. WDR77 plays an essential role in prostate tumorigenesis and cell growth mediated by growth regulatory factors. Here, we identified E2F1 and E2F3 mRNAs as translational targets of WDR77. We demonstrated that WDR77 regulated the translation of E2F1 and E2F3 mRNAs through the 5' untranslated regions (UTRs) of E2F1 and E2F3 (E2F1/3) mRNAs. WDR77 physically interacted with programmed cell death 4 (PDCD4) that suppresses translation of mRNAs containing structured 5' UTRs by interacting with eukaryotic translation initiation factor 4A (eIF4A) and inhibiting its helicase activity. Further, we demonstrated that the interaction between WDR77 and PDCD4 prevented the binding of PDCD4 to eIF4A and relieved PDCD4's inhibitory effect on eIF4A1. Overall, our work reveals for the first time that WDR77 is directly involved in translational regulation of E2F1/3 mRNAs through their structured 5' UTRs, PDCD4, and eIF4A1 and provides novel insight into the cell growth controlled by WDR77.

MiR-203 inhibits cell proliferation and ERK pathway in prostate cancer by targeting IRS-1

IntroductionProstate cancer (PCa) is one of the most common types of cancer in men. In the course of the development and progression of this disease, abnormal expression of miR-203 is usually accompanied. However, its role in prostate tumorigenesis and the underlying mechanism are poorly understood.MethodsDual luciferase reporter gene analysis was used to detect miR-203 binding site in insulin receptor substrates 1 (IRS-1). Cell proliferation was assessed by MTT assay in PCa cells with either IRS-1 knockdown or miR-203 overexpression. IRS-1 and other proteins expression in PCa cells was assessed by Western Blot.Resultswe found that the insulin receptor substrates 1 (IRS-1) is a novel target of miR-203 in PCa and miR-203 can specifically bind to the 3′UTR region of the IRS-1 thus suppresses its expression. Moreover, we demonstrate that miR-203 functions as a tumor suppressor by directly targeting IRS-1 to inhibit cell proliferation and migration which results in PCa cell cycle arrest. Importantly, miR-203 overexpression blocks ERK signalling pathway by down-regulating IRS-1 expression.ConclusionsOur results show a novel link between miR-203 and IRS-1, and reveal the importance of strict control of IRS − 1 by miR-203 in the progression of PCa, suggesting miR-203 may act as a promising target for the diagnosis and treatment of advanced PCa.

Long non-coding RNAs in prostate tumorigenesis and therapy (Review).

Prostate cancer (PCa) is one of the most frequently diagnosed malignancy. Although there have been many advances in PCa diagnosis and therapy, the concrete mechanism remains unknown. Long non-coding RNAs (lncRNAs) are novel biomarkers associated with PCa, and their dysregulated expression is closely associated with risk stratification, diagnosis and carcinogenesis. Accumulating evidence has suggested that lncRNAs play important roles in prostate tumorigenesis through relevant pathways, such as androgen receptor interaction and PI3K/Akt. The present review systematically summarized the potential clinical utility of lncRNAs and provided a novel guide for their function in PCa.

Characterization of unique PMEPA1 gene splice variants (isoforms d and e) from RNA Seq profiling provides novel insights into prognostic evaluation of prostate cancer.

Prostate cancer is a disease with heterogeneity of multiple gene transcriptomes and biological signaling pathways involved in tumor development. The prostate transmembrane protein, androgen induced 1 (PMEPA1), a multifunctional protein played critical roles in prostate tumorigenesis. The pleiotropic nature of PMEPA1 in modulating androgen and TGF-β signaling as well as splice variants mechanisms for functional regulations of cancer-associated genes prompted us to investigate the biological roles of PMEPA1 isoforms in prostate cancer. In addition to 4 reported PMEPA1 isoforms (a, b, c and d), one novel isoform PMEPA1-e was identified with RNA Seq analysis of hormone responsive VCaP, LNCaP cells and human prostate cancer samples from The Cancer Genome Atlas (TCGA) dataset. We analyzed the structures, expressions, biological functions and clinical relevance of PMEPA1-e isoform and less characterized isoforms c and d in the context of prostate cancer and AR/TGF-β signaling. The expression of PMEPA1-e was induced by androgen and AR. In contrast, PMEPA1-d was responsive to TGF-β and inhibited TGF-β signaling. Both PMEPA1-d and PMPEA1-e promoted the growth of androgen independent prostate cancer cells. Although PMEPA1-c was responsive to TGF-β, it was found to have no impacts on cell growth and androgen/TGF-β signaling. The TCGA data analysis from 499 patients showed higher expression ratios of PMEAP1-b versus -d or -e strongly associated with enhanced Gleason score. Taken together, our findings first time defined the prostate tumorigenesis mediated by PMEPA1-d and -e isoforms, providing novel insights into the new strategies for prognostic evaluation and therapeutics of prostate tumor.

Divergent Modulation of Proteostasis in Prostate Cancer.

Proteostasis regulates key cellular processes such as cell proliferation, differentiation, transcription, and apoptosis. The mechanisms by which proteostasis is regulated are crucial and the deterioration of cellular proteostasis has been significantly associated with tumorigenesis since it specifically targets key oncoproteins and tumor suppressors. Prostate cancer (PCa) is the second most common cause of cancer death in men worldwide. Androgens mediate one of the most central signaling pathways in all stages of PCa via the androgen receptor (AR). In addition to their regulation by hormones, PCa cells are also known to be highly secretory and are particularly prone to ER stress as proper ER function is essential. Alterations in various complex signaling pathways and cellular processes including cell cycle control, transcription, DNA repair, apoptosis, cell adhesion, epithelial-mesenchymal transition (EMT), and angiogenesis are critical factors influencing PCa development through key molecular changes mainly by posttranslational modifications in PCa-related proteins, including AR, NKX3.1, PTEN, p53, cyclin D1, and p27. Several ubiquitin ligases like MDM2, Siah2, RNF6, CHIP, and substrate-binding adaptor SPOP; deubiquitinases such as USP7, USP10, USP26, and USP12 are just some of the modifiers involved in the regulation of these key proteins via ubiquitin-proteasome system (UPS). Some ubiquitin-like modifiers, especially SUMOs, have been also closely associated with PCa. On the other hand, the proteotoxicity resulting from misfolded proteins and failure of ER adaptive capacity induce unfolded protein response (UPR) that is an indispensable signaling mechanism for PCa development. Lastly, ER-associated degradation (ERAD) also plays a crucial role in prostate tumorigenesis. In this section, the relationship between prostate cancer and proteostasis will be discussed in terms of UPS, UPR, SUMOylation, ERAD, and autophagy.

HCRP-1 regulates cell migration, invasion and angiogenesis via Src/ FAK signaling in human prostate cancer.

Prostate cancer (PCa) is the third leading malignancy engendering mortality among men globally. The present study aimed at determining the expression of hepatocellular carcinoma-related protein-1 (HCRP-1) in PCa, to explore its potential role in prostate tumorigenesis in vitro and in vivo. We evaluated HCRP-1 protein with immunohistochemistry (IHC) technology and found HCRP-1 expression was significantly low in PCa tissues (PCTs); In addition, the decreased HCRP-1 was significantly associated with TNM (tumor node metastasis) stage, advanced histology grade and gleason score. Transwell, tube formation, Western blot and co-immunoprecipitation (Co-IP) assays were utilized to determine the role of down-regulating HCRP-1 in PCa cell migration, invasion and angiogenesis. Meanwhile, we found HCRP-1 depletion induced Src and focal adhesion kinase (FAK) phosphorylation, which could be reversed by Src inhibitor PP2 or FAK inhibitor. Furthermore, down-regulated HCRP-1 evidently induced lung metastasis of PCa cells in xenograft mode. Taken together, our study indicates HCRP-1 plays an important role in PCa metastasis. HCRP-1 may serve as a potential therapeutic target for PCa.

Analysis of PMEPA1 Isoforms (a and b) as Selective Inhibitors of Androgen and TGF-β Signaling Reveals Distinct Biological and Prognostic Features in Prostate Cancer.

Dysfunctions of androgen/TGF-β signaling play important roles in prostate tumorigenesis. Prostate Transmembrane Protein Androgen Induced 1 (PMEPA1) inhibits androgen and TGF-β signaling via a negative feedback loop. The loss of PMEPA1 confers resistance to androgen signaling inhibitors and promotes bone metastasis. Conflicting reports on the expression and biological functions of PMEPA1 in prostate and other cancers propelled us to investigate isoform specific functions in prostate cancer (PCa). One hundred and twenty laser capture micro-dissection matched normal prostate and prostate tumor tissues were analyzed for correlations between quantitative expression of PMEPA1 isoforms and clinical outcomes with Q-RT-PCR, and further validated with a The Cancer Genome Atlas (TCGA) RNA-Seq dataset of 499 PCa. Cell proliferation was assessed with cell counting, plating efficiency and soft agar assay in androgen responsive LNCaP and TGF-β responsive PC3 cells. TGF-β signaling was measured by SMAD dual-luciferase reporter assay. Higher PMEPA1-a mRNA levels indicated biochemical recurrence (p = 0.0183) and lower PMEPA1-b expression associated with metastasis (p = 0.0173). Further, lower PMEPA1-b and a higher ratio of PMEPA1-a vs. -b were correlated to higher Gleason scores and lower progression free survival rate (p < 0.01). TGF-β-responsive PMEPA1-a promoted PCa cell growth, and androgen-responsive PMEPA1-b inhibited cancer cell proliferation. PMEPA1 isoforms -a and -b were shown to be promising candidate biomarkers indicating PCa aggressiveness including earlier biochemical relapse and lower disease specific life expectancy via interrupting androgen/TGF-β signaling.

PMEPA1 gene isoforms to indicate disease progression in solid tumors.

e16580 Background: Dysfuncitons of androgen and TGF-β signaling play important roles in prostate tumorigenesis. PMEPA1 gene has been defined as an androgen and TGF-β responsive gene which inhibits androgen and TGF-β signaling via negative feed-back loops. Our previous data has established that PMEPA1 distinct isoforms ( PMEPA1-a and PMEPA1-b) with disparities within N-terminus protein sequences navigate different androgen/TGF-β signaling regulations. In this study, the roles of PMEPA1 isoforms in disease progressions were investigated in solid tumors of prostate (CaP), breast, lung and colon. Methods: RNA seq data from total 2479 solid tumor samples in the TCGA dataset were used to study the correlation between expressions of PMEPA1 isoforms and disease progression including Gleason score, pathology stages, progression free survival rate (PFS) and overall survival rate (OS). The cohort is composed of 482 prostate, 1049 breast, 499 lung and 449 colon cancer patients. Results: In CaP, the TCGA data analysis showed that lower transcript level of PMEPA1-b isoform associated with higher Gleason scores and lower progression free survival rate (PFS) (P = 0.014) and worse overall survival rate (OS) (P &lt; 0.01). The ratio of mRNA levels of PMEPA1-a versus PMEPA-b indicated higher Gleason score, lower PFS rate (P = 0.0063) and worse OS rate (P = 0.0042). In contrast, higher expression of both PMEPA1-a and PMEPA- b associated with lower PFS (P = 0.023 and 0.028, respectively) in breast cancer. And the enhanced ratio of PMEPA1-a/ b was also found to indicate lower PFS (P = 0.016) and worse OS (P = 0.016) in breast cancer. Similarly, the increased transcript levels of PMEPA1-a and PMEPA1-b isoforms significantly associated with lower PFS and worse OS rates in lung and colon cancer. The expression of PMEPA1 isoforms was not found to associate with pathology stages of diseases. Conclusions: Our data establish the biomarker potential of PMEPA1 gene isoforms ( a and b) indicating more aggressive disease progressions in 4 solid tumors, further underscoring the PMEPA1 isoform specific biological functions to differentiate regulation of androgen and TGF-β signaling in cancer cells.

Common ELF1 deletion in prostate cancer bolsters oncogenic ETS function, inhibits senescence and promotes docetaxel resistance.

ETS family transcription factors play major roles in prostate tumorigenesis with some acting as oncogenes and others as tumor suppressors. ETS factors can compete for binding at some cis-regulatory sequences, but display specific binding at others. Therefore, changes in expression of ETS family members during tumorigenesis can have complex, multimodal effects. Here we show that ELF1 was the most commonly down-regulated ETS factor in primary prostate tumors, and expression decreased further in metastatic disease. Genome-wide mapping in cell lines indicated that ELF1 has two distinct tumor suppressive roles mediated by distinct cis-regulatory sequences. First, ELF1 inhibited cell migration and epithelial-mesenchymal transition by interfering with oncogenic ETS functions at ETS/AP-1 cis-regulatory motifs. Second, ELF1 uniquely targeted and activated genes that promote senescence. Furthermore, knockdown of ELF1 increased docetaxel resistance, indicating that the genomic deletions found in metastatic prostate tumors may promote therapeutic resistance through loss of both RB1 and ELF1

Cooperation of loss of NKX3.1 and inflammation in prostate cancer initiation.

ABSTRACTAlthough it is known that inflammation plays a critical role in prostate tumorigenesis, the underlying processes are not well understood. Based on analysis of genetically engineered mouse models combined with correlative analysis of expression profiling data from human prostate tumors, we demonstrate a reciprocal relationship between inflammation and the status of the NKX3.1 homeobox gene associated with prostate cancer initiation. We find that cancer initiation in aged Nkx3.1 mutant mice correlates with enrichment of specific immune populations and increased expression of immunoregulatory genes. Furthermore, expression of these immunoregulatory genes is similarly increased in human prostate tumors having low levels of NKX3.1 expression. We further show that induction of prostatitis in Nkx3.1 mutant mice accelerates prostate cancer initiation, which is coincident with aberrant cellular plasticity and differentiation. Correspondingly, human prostate tumors having low levels of NKX3.1 have de-regulated expression of genes associated with these cellular processes. We propose that loss of function of NKX3.1 accelerates inflammation-driven prostate cancer initiation potentially via aberrant cellular plasticity and impairment of cellular differentiation.This article has an associated First Person interview with the first author of the paper.

RAD9A promotes metastatic phenotypes through transcriptional regulation of anterior gradient 2 (AGR2)

RAD9A plays an important role in prostate tumorigenesis and metastasis-related phenotypes. The protein classically functions as part of the RAD9A-HUS1-RAD1 complex but can also act independently. RAD9A can selectively transactivate multiple genes, including CDKN1A and NEIL1 by binding p53-consensus sequences in or near promoters. RAD9A is overexpressed in human prostate cancer specimens and cell lines; its expression correlates with tumor progression. Silencing RAD9A in prostate cancer cells impairs their ability to form tumors in vivo and migrate as well as grow anchorage independently in vitro. We demonstrate herein that RAD9A transcriptionally controls AGR2, a gene aberrantly overexpressed in patients with metastatic prostate cancer. Transient or stable knockdown of RAD9A in PC-3 cells caused downregulation of AGR2 protein abundance. Reduced AGR2 protein levels were due to lower abundance of AGR2 mRNA. The AGR2 genomic region upstream of the coding initiation site contains several p53 consensus sequences. RAD9A bound specifically to the 5'-untranslated region of AGR2 in PC-3 cells at a partial p53 consensus sequence at position +3136 downstream from the transcription start site, determined by chromatin immunoprecipitation, followed by PCR amplification. Binding of RAD9A to the p53 consensus sequence was sufficient to drive AGR2 gene transcription, shown by a luciferase reporter assay. In contrast, when the RAD9A-binding sequence on the AGR2 was mutated, no luciferase activity was detected. Knockdown of RAD9A in PC-3 cells impaired cell migration and anchorage-independent growth. However, ectopically expressed AGR2 in RAD9A-depleted PC-3 cells restored these phenotypes. Our results suggest RAD9A drives metastasis by controlling AGR2 abundance.

Association Analysis of a Microsatellite Repeat in the TRIB1 Gene With Prostate Cancer Risk, Aggressiveness and Survival.

With an estimated 1.1 million men worldwide diagnosed with prostate cancer yearly, effective and more specific biomarkers for early diagnosis could lead to better patient outcome. As such, novel genetic markers are sought for this purpose. The tribbles homologue 1 gene (TRIB1) has recently shown to have a role in prostate tumorigenesis and data-mining of prostate cancer expression data confirmed clinical significance of TRIB1 in prostate cancer. For the first time, a polymorphic microsatellite in this gene was studied for its potential association with prostate cancer risk and aggressiveness. Genomic DNA was extracted from a cohort of 1,152 prostate cancer patients and 1,196 cancer-free controls and the TTTTG-TRIB1 microsatellite was genotyped. The socio-demographic and clinical characteristics were analyzed using the non-parametric t-test and two-way ANOVA. Association of the TTTTG-TRIB1 microsatellite and prostate cancer risk and aggressiveness were analyzed by binary logistic regression and confirmed by bootstrapping. Total and prostate cancer mortality was analyzed using the Kaplan Meier test. Genotype and allele correlation with TRIB1 mRNA levels was analyzed using the non-parametric Kolmogorov–Smirnov test. To predict the effect that the TTTTG-TRIB1 polymorphisms had on the mRNA structure, the in silico RNA folding predictor tool, mfold, was used. By analyzing the publicly available data, we confirmed a significant over-expression of TRIB1 in prostate cancer compared to other cancer types, and an over-expression in prostate cancerous tissue compared to adjacent benign. Three alleles (three–five repeats) were observed for TTTTG-TRIB1. The three-repeat allele was associated with prostate cancer risk at the allele (OR = 1.16; P = 0.044) and genotypic levels (OR = 1.70; P = 0.006) and this association was age-independent. The four-repeat allele was inversely associated with prosatet cancer risk (OR = 0.57; P < 0.0001). TRIB1 expression was upregulated in tumors when compared to adjacent cancer-free tissue but was not allele specific. In silico analysis suggested that the TTTTG-TRIB1 alleles may alter TRIB1 mRNA structure. In summary, the three-repeat allele was significantly associated with prostate cancer risk, suggesting a biomarker potential for this microsatellite to predict prostate cancer. Further studies are needed to elucidate the functional role of this microsatellite in regulating TRIB1 expression, perhaps by affecting the TRIB1 mRNA structure and stability.

Abstract A006: Skp2 associates with Slug and androgen receptor in aggressive prostate cancer

Abstract Introduction: The Skp2 F-box protein is the substrate recruiting component of the SCF (Skp1-Cullin 1-F-box) type of E3 ubiquitin-ligase complexes. Skp2 plays an important role in prostate tumorigenesis (e.g., via recently reported stabilization of EZH2 or Twist1), which needs further elucidation. Patients and Methods: Prostate cancer patient cohort (N=101) was analyzed by immunohistochemistry for the following proteins: Skp2, AR, Ki-67, Slug, E-cadherin, MdmX, Mdm2, and p53. The indicated proteins were also analyzed in seminal vesicles and lymph node metastases (SV=35 and LN=18 patients, respectively), as well as in benign prostatic hyperplasia (BPH=34). Colocalization analysis was performed using Perkin Elmer Opal Multiplex kit, Vectra 3.0 imaging system, and confocal microscope Carl Zeiss LSM 780. Independent prostate cancer cohort (N=30) was evaluated for TMPRSS2-ERG fusion by a triple-color deletion assay and assessed for Skp2, ERG, and NQO1 protein expression. Cox regression analysis and Kaplan-Meier curves were used to assess importance of Skp2, Slug, EZH2, and Twist1 for biochemical relapse in the TCGA prostate cancer dataset (N=488, RNA seq). Results: Expression of E-cadherin was lower in primary prostate cancer and seminal vesicles compared to BPH and LN, which may be linked to epithelial-mesenchymal transition. Opposite expression profile was observed for Skp2, MdmX, and Slug. High Gleason score (≥8, N=30) was significantly associated with higher Skp2 and lower E-cadherin expression (p&amp;lt;0.001 and 0.011, respectively). Skp2 slightly correlated with AR and Slug in the whole patient cohort (Rs 0.37 and 0.322, respectively). These correlations were stronger in patients with high Gleason score (Rs 0.53 and 0.56, respectively). In patients with metastasis into lymph nodes, Skp2 similarly correlated with Slug and AR (Rs 0.56 and 0.37, respectively) while androgen receptor further correlated with Ki-67 (Rs 0.50). Confocal microscopy revealed colocalization of Skp2 and Slug in prostate cancer cells. In an independent cohort, Skp2 correlated with expression of ERG, NQO1, and percentage of cancer cells positive for TMPRSS2-ERG (Rs 0.503, 0.387 and 0.615, respectively). In the TCGA dataset, high mRNA levels of Skp2 and EZH2 (but not Slug or Twist1) associated with shorter time to biochemical relapse, compared to low mRNA levels [HR 3.62 (2.1 – 6.23), p&amp;lt;0.001]. Conclusion: Immunohistochemistry and colocalization studies revealed association between Skp2 and Slug in aggressive prostate cancer. Other factors such as TMPRSS2-ERG fusion or oxidative stress may contribute to prostate cancer progression. Citation Format: Jan Bouchal, Gvantsa Kharaishvili, Alena Mickova, Mariam Gachechiladze, Alena Burdova, Martin Mistrik, Milan Kral, Balazs Gyorffy, Zdenek Kolar. Skp2 associates with Slug and androgen receptor in aggressive prostate cancer [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 A006.