Event In Prostate Carcinogenesis Research Articles

Abstract 1528: Flowcytometric based approach to investigate prostate tumor microenvironment

Abstract Prostate cancer (PC) and its tumor microenvironment (TME) are heterogenous and evolving along with the treatment patients are exposed to. Modelling of the TME represents an attractive approach to understand how PC evolves and spreads. Flow cytometry is a technique for quantitative measurement of single cell profiling based on the cell surface as well as intracellular markers. We designed a flowcytometric based approach to investigate the interplay between the PC cells and immune cells in a direct coculture. Cells are fluorescently tagged with a cell tracer and the analysis is performed separately on the adherent and non-adherent cells. To optimize the readout method, we investigate the interplay between Jurkat cells and PC cells (LNCaP) as well as immortalized prostate epithelial cell models modified to mimic early events in prostate carcinogenesis (RWPE-1 cells overexpressing androgen receptor, RWPE-1-AR, and having PTEN deletion, RWPE-1-AR-PTENdel).Jurkat (suspension culture) cocultured with LNCaP (adherent culture) exhibited 40% decrease in viability but only 30% of LNCaP dying cell in the floating population compared to LNcaP only which is 60%. When Jurkat were activated using Phorbol 12-myristate 13-acetate (PMA), we observed an increase in LNCaP cell death. Jurkat viability was not impacted in presence of RWPE-1 neither when exposed to RWPE1-AR-PTENdel. In summary our Flowcytometric-based approach will help to understand the interplay between immune cells and prostate cancer cells. Citation Format: karishma sajnani, Sini Eerola, Konsta Kukkonen, Hanna Rauhala, Tapio Visakorpi, Alfonso Urbanucci. Flowcytometric based approach to investigate prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1528.

PiR-31470 epigenetically suppresses the expression of glutathione S-transferase pi 1 in prostate cancer via DNA methylation

Inactivation of glutathione S-transferase pi 1 (GSTP1) via hypermethylation is an early and common event in prostate carcinogenesis. Functional inactivation of GSTP1 increases the susceptibility to oxidative stress and enhance progression risk of the prostatic carcinoma. In this study, we hypothesized that the Piwi-interacting RNA (piRNA) could be a sequence-recognition and guidance molecule for induction of promoter methylation of GSTP1 facilitating prostate carcinogenesis. We found that piR-31470 was highly expressed in prostate cancer cells, and piR-31470 could bind to piwi-like RNA-mediated gene silencing 4 (PIWIL4) to form the PIWIL4/piR-31470 complex. This complex could bind to the nascent RNA transcripts of GSTP1, and recruit DNA methyltransferase 1, DNA methyltransferase 3 alpha and methyl-CpG binding domain protein 2 to initiate and maintain the hypermethylation and inactivation of GSTP1. Our data demonstrated that the overexpression of piR-31470 inhibited the levels of GSTP1 and increased vulnerability to oxidative stress and DNA damage in human prostate epithelial RWPE1 cells. In conclusion, this study characterized the roles of the PIWIL4/piR-31470 complex in the regulation of the transcription of GSTP1 by methylating the CpG island of GSTP1. This discovery may provide a novel therapeutic strategy by targeting piRNAs for the epigenetic treatment of prostate cancer.

Expression of human leukocyte antigen-a and b2-microglobulin in prostate cancer.

e16550 Background: Prostate cancer (PC) is generally resistant to current immune checkpoint blockade. Reduced expression of human leukocyte antigen-A (HLA-A) and b2-microglobulin (B2M) of cancer cells can cause deficit immune recognition and attack by cytotoxic T cells, and may be the major mechanism of intrinsic resistance to immune checkpoint blockade in PC patients. Methods: Formalin-fixed paraffin-embedded tissue of human localized or metastatic PC was used for evaluation of expression of HLA-A and B2M by Immunohistochemical staining. The expression was scored as: 0, no tumor cells stained; 1+, < 10% of tumor cells stained; 2+, 10-50% of tumor cells stained; and 3+, > 50% of tumor cells stained. Differences in HLA-A or B2M expression among groups were assessed by Chi-Square or Fisher’s exact test. Results: A total of 134 PC specimens [69 localized PC (LPC), 32 metastatic castration-naïve PC (mCNPC), and 33 metastatic castration-resistant PC (mCRPC)] from 115 patients were assessed. Significant loss (0 or 1+) of HLA-A was found in 56 (81.2%) LPCs, 30 (93.8 %) mCNPCs and 29 (87.9%) mCRPCs; while significant loss of B2M was found in 22 (32.6%) LPC, 11 (34.4%) mCNPCs, and 17 (51.2%) mCRPCs. In localized PCs, the expression of HLA-A or B2M was similar between high-grade prostate intraepithelial neoplasia foci and corresponding malignant counterpart, and did not significantly correlate to biochemical failure. In 14 patients with paired CN and CR PC specimens, significant loss of HLA-A was found in all tumors. B2M expression varied between CN and CR PCs, and progressive loss of B2M was found in 7 (50%) patients. Among 3 patients receiving immune checkpoint blockade, 1 patient with intact expression of HLA-A and B2M in pre-treatment tumor specimen achieved partial response; whereas the others with reduced expression of HLA-A (0) and B2M (1+) did not. Conclusions: Reduced expression of HLA-A or B2M is an early event in prostate carcinogenesis.

Effect of PTEN loss on metabolic reprogramming in prostate cancer cells.

The tumor suppressor gene PTEN is one of the most often deleted genes in human prostate cancer. Loss of PTEN is an important event in prostate carcinogenesis. Metabolic reprogramming induced by PTEN loss fuels malignant growth and proliferation of prostate cancer cells. Targeted metabolomics analysis was used to investigate the effects of PTEN loss on intracellular metabolic pathways in prostate cancer cells. DU-145 cells were transfected with PTEN siRNAs (siRNA-1 and siRNA-2) for 48 h, and endogenous PTEN expression was monitored by western blotting. Changes in intracellular metabolites were determined by liquid chromatography-tandem mass chromatography (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS). Most intracellular metabolites involved in glycolysis and glutaminolysis were increased in PTEN knockdown prostate cancer cells. In addition, most intracellular metabolites involved in fatty acid de novo synthesis, fatty acid beta oxidation and branched chain amino acid catabolism were also increased in PTEN knockdown prostate cancer cells. These results revealed that PTEN loss induced the metabolic reprogramming of prostate cancer cells and promoted the malignant proliferation of prostate cancer cells. The present metabolomics analysis indicates that tumor suppressor gene PTEN mutation or deletion can induce metabolic reprogramming in prostate cancer cells and tumorigenesis by altering the metabolic flux of glycolysis, glutaminolysis, fatty acid metabolism and branched chain amino acid catabolism pathways. Metabolic reprogramming is one of the contributors to PTEN-loss driven prostate cancer.

Global analysis of H3K27me3 as an epigenetic marker in prostate cancer progression

BackgroundH3K27me3 histone marks shape the inhibition of gene transcription. In prostate cancer, the deregulation of H3K27me3 marks might play a role in prostate tumor progression.MethodsWe investigated genome-wide H3K27me3 histone methylation profile using chromatin immunoprecipitation (ChIP) and 2X400K promoter microarrays to identify differentially-enriched regions in biopsy samples from prostate cancer patients. H3K27me3 marks were assessed in 34 prostate tumors: 11 with Gleason score > 7 (GS > 7), 10 with Gleason score ≤ 7 (GS ≤ 7), and 13 morphologically normal prostate samples.ResultsHere, H3K27me3 profiling identified an average of 386 enriched-genes on promoter regions in healthy control group versus 545 genes in GS ≤ 7 and 748 genes in GS > 7 group. We then ran a factorial discriminant analysis (FDA) and compared the enriched genes in prostate-tumor biopsies and normal biopsies using ANOVA to identify significantly differentially-enriched genes. The analysis identified ALG5, EXOSC8, CBX1, GRID2, GRIN3B, ING3, MYO1D, NPHP3-AS1, MSH6, FBXO11, SND1, SPATS2, TENM4 and TRA2A genes. These genes are possibly associated with prostate cancer. Notably, the H3K27me3 histone mark emerged as a novel regulatory mechanism in poor-prognosis prostate cancer.ConclusionsOur findings point to epigenetic mark H3K27me3 as an important event in prostate carcinogenesis and progression. The results reported here provide new molecular insights into the pathogenesis of prostate cancer.

Highlights of This Issue

The TMPRSS2:ERG gene fusion is the most common somatic event in prostate cancer. There is intriguing evidence that TMPRSS2:ERG represents a unique molecular subtype. Penney and colleagues examined the association of 39 prostate cancer genetic risk variants with TMPRSS2:ERG fusion positive and negative disease. Six of the variants were significantly differentially associated with fusion-positive versus fusion-negative disease. These findings suggest that the molecular events in prostate carcinogenesis may be distinct for men depending on their underlying genetic predisposition. When examining other risk factors for prostate cancer, the integration of molecular subtypes may improve understanding of the etiology of this disease.It is important to develop risk prediction models for individuals with Barrett's esophagus (BE) who are at increased risk of esophageal adenocarcinoma (EAC). Sun and colleagues developed a risk prediction model using singly ascertained BE pedigrees and multiplex BE pedigrees and incorporating family history and clinical risk factors. This study indicates that family information is valuable in predicting BE risk, and this new model will assist in both identifying high-risk individuals for BE screening and potentially reducing mortality from EAC.Little is known about the serostability of polyomaviruses (PyV) over time, or associations with cutaneous squamous cell carcinoma (SCC). In a case–control study, Gossai and colleagues measured antibody response against BK and JC. Among controls, BK and JC seroreactivity was stable over time, and there was little evidence of seroconversion following SCC diagnosis. JC seropositivity prior to diagnosis was associated with an elevated risk of SCC. A single measure of PyV seroreactivity appears a reliable indicator of long-term antibody status, and seropositivity to certain PyVs may be related to subsequent occurrence of SCC.Johnson and colleagues examined the effects of residential segregation and deprivation on treatment and survival in non–small cell lung cancer patients living in the state of Georgia. Living in areas with high levels of segregation was associated with decreased odds of receipt of surgery. Black patients living in high residential segregation/high deprivation were 31% more likely to die, even after surgery was controlled for. For white patients, economic deprivation was associated with decreased odds of surgery but not survival, and residential segregation had no effect. This study suggests differences in how black and white individuals experience segregation and poverty lead to differences in adverse health outcomes.

The NF-κB subunit RelB regulates the migration and invasion abilities and the radio-sensitivity of prostate cancer cells.

NF-κB subunits play important roles in carcinogenesis of a variety of human malignancies and response to cancer therapy; however, the contribution of an individual subunit has not been thoroughly defined. Constitutive activation of the canonical NF-κB subunit is a critical event in prostate carcinogenesis. Recent findings point out that RelB, which contributes to the non-canonical NF-κB activity, functions importantly in the prostate cancer progression. Here, we investigated systemically the functional roles of RelB in prostate cancer and examine its significance as a therapeutic target. Targeting RelB using short hairpin RNA approach in androgen-independent DU145 prostate cancer cells interfered with various biological behaviors of cells. We observed that RelB knockdown inhibited prostate cancer cell growth, migration, and invasion, and enhanced proteasome inhibitor sensitivity. The altered expression of anti-apoptotic gene Bcl-2 played critical roles in regulating both spontaneous and radiation-induced apoptosis in the presence of RelB knockdown. For the first time, we showed that RelB knockdown significantly attenuated the migration and invasion of DU145 prostate cancer cells, due to the reduction of integrin β-1. Collectively, we provided evidence that RelB functioned as an oncogene in prostate cancer. Developing a RelB-targeted therapeutic intervention, is valuable in treating advanced, metastatic prostate cancer.

Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38

Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To gain insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in an STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients.

Inflammation and prostate cancer: friends or foe?

Prostate cancer is the most common non-cutaneous malignancy diagnosed in men. Moving from histological observations since a long time, it has been recognized that innate and adaptive immunity actively participates in the pathogenesis, surveillance, and progression of prostate cancer. A PubMed and Web of Science databases search was performed for studies providing evidence on the roles of the innate and adaptive immunity during the development and progression of prostate cancer. There are growing evidences that chronic inflammation is involved in the regulation of cellular events in prostate carcinogenesis, including disruption of the immune response and regulation of the tumor microenvironment. This review discusses the role played by the innate and adaptive immune system in the local progression of prostate cancer, and the prognostic information that we can currently understand and exploit.

Hypoxia-Induced Signaling Promotes Prostate Cancer Progression: Exosomes Role as Messenger of Hypoxic Response in Tumor Microenvironment.

Prostate cancer (PCA) is the leading malignancy in men and the second leading cause of cancer-related deaths. Hypoxia (low O2 condition) is considered an early event in prostate carcinogenesis associated with an aggressive phenotype. In fact, clinically, hypoxia and hypoxia-related biomarkers are associated with treatment failure and disease progression. Hypoxia-inducible factor 1 (HIF-1) is the key factor that is activated under hypoxia, and mediates adaptation of cells to hypoxic conditions through regulating the expression of genes associated with angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, survival, proliferation, metabolism, sternness, hormone-refractory progression, and therapeutic resistance. Besides HIF-1, several other signaling pathways including PI3K/Akt/mTOR, NADPH oxidase (NOX), Wnt/b-catenin, and Hedgehog are activated in cancer cells under hypoxic conditions, and also contribute in hypoxia-induced biological effects in HIF-1-dependent and -independent manners. Hypoxic cancer cells cause extensive changes in the tumor microenvironment both local and distant, and recent studies have provided ample evidence supporting the crucial role of nanosized vesicles "exosomes" in mediating hypoxia-induced tumor microenvironment remodeling. Exosomes' role has been reported in hypoxia-induced angiogenesis, sternness, activation of cancer-associated fibroblasts (CAFs), and EMT. Together, existing literature suggests that hypoxia plays a predominant role in PCA growth and progression, and PCA could be effectively prevented and treated via targeting hypoxia/hypoxia-related signaling pathways.

EpCAM is overexpressed in local and metastatic prostate cancer, suppressed by chemotherapy and modulated by MET-associated miRNA-200c/205

Background:Expression of epithelial cell adhesion molecule (EpCAM) is deregulated in epithelial malignancies. Beside its role in cell adhesion, EpCAM acts as signalling molecule with tumour-promoting functions. Thus, EpCAM is part of the molecular network of oncogenic receptors and considered an interesting therapeutic target.Methods:Here, we thoroughly characterised EpCAM expression on mRNA and protein level in comprehensive tissue studies including non-cancerous prostate specimens, primary tumours of different grades and stages, metastatic lesions, and therapy-treated tumour specimens, as well as in prostate cancer cell lines.Results:Epithelial cell adhesion molecule was overexpressed at mRNA and at protein level in prostate cancer tissues and cell lines. Altered EpCAM expression was an early event in prostate carcinogenesis with an upregulation in low-grade cancers and further induction in high-grade tumours and metastatic lesions. Interestingly, EpCAM was repressed upon induction of epithelial-to-mesenchymal transition (EMT) following chemotherapeutic treatment with docetaxel. Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells. Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro.Conclusions:In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression. Epithelial cell adhesion molecule displays a dynamic, heterogeneous expression and associates with epithelial cells rather than mesenchymal, chemoresistant cells along with processes of EMT and MET.

Inflammation and prostate cancer: the role of interleukin 6 (IL‐6)

Environmental and genetic aspects are reflected in the development of prostate cancer. In this context, there is growing evidence that chronic inflammation is involved in the regulation of cellular events in prostate carcinogenesis, including disruption of the immune response and regulation of the tumour microenvironment. One of the best surrogates of chronic inflammation in prostate cancer is interleukin 6 (IL-6). Serum IL-6 levels are elevated in patients with untreated metastatic or castration-resistant prostate cancer (CRPC) and correlate negatively with tumour survival and response to chemotherapy. Via multiple signal pathways including the Janus tyrosine family kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, the extracellular signal-regulated kinase 1 and 2 (ERK1/2)-mitogen activated protein kinase (MAPK) pathway, and the phosphoinositide 3-kinase (PI3-K) pathway, IL-6 is able to promote prostate cancer cell proliferation and inhibit apoptosis in vitro and in vivo. IL-6 is associated with aggressive prostate cancer phenotype and may be involved in the metastatic process through regulation of epithelial-mesenchymal transition (EMT) and homing of cancer cells to the bone. A substantial body of evidence suggests that IL-6 plays a major role in the transition from hormone-dependent to CRPC, most notably through accessory activation of the androgen receptor. Collectively, these data have stimulated the development of agents targeting IL-6 signalling pathways. A chimeric anti-IL-6 monoclonal antibody has been tested in clinical trials, with mixed results.

Recent insights into NF‐κB signalling pathways and the link between inflammation and prostate cancer

Inflammation is involved in regulation of cellular events in prostate carcinogenesis through control of the tumour micro-environment. A variety of bone marrow-derived cells, including CD4+ lymphocytes, macrophages and myeloid-derived suppressor cells, are integral components of the tumour micro-environment. On activation by inflammatory cytokines, NF-κB complexes are capable of promoting tumour cell survival through anti-apoptotic signalling in prostate cancer (PCa). Positive feedback loops are able to maintain NF-κB activation. NF-κB activation is also associated with the metastatic phenotype and PCa progression to castration-resistant prostate cancer (CRPC). A novel role for inhibitor of NF-κB kinase (IKK)-α in NF-κB-independent PCa progression to metastasis and CRPC has recently been uncovered, providing a new mechanistic link between inflammation and PCa. Expansion of PCa progenitors by IKK-α may be involved in this process. In this review, we offer the latest evidence regarding the role of the NF-κB pathway in PCa and discuss therapeutic attempts to target the NF-κB pathways. We point out the need to further dissect inflammatory pathways in PCa in order to develop appropriate preventive measures and design novel therapeutic strategies.

Methylation of APC and GSTP1 in Non-Neoplastic Tissue Adjacent to Prostate Tumour and Mortality from Prostate Cancer

BackgroundMarkers that can discriminate between indolent and aggressive prostate tumours are needed. We studied gene methylation in non-neoplastic tissue adjacent to prostate tumour (NTAT) in association with prostate cancer mortality.MethodsFrom two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostate tumour tissues and used probe real-time PCR to analyse methylation of the glutathione S-transferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters.ResultsPrevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostate tumour tissue for the same two genes as well as with a high Gleason score. The hazard ratio (HR) of prostate cancer mortality was 2.38 (95% confidence interval: 1.23–4.61) for APC methylation, and 2.92 (1.49–5.74) for GSTP1 methylation in NTAT. It changed to 1.91 (1.03–3.56) and 1.60 (0.80–3.19) after adjusting for Gleason score and methylation in prostate tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 4.30 (2.00–9.22), which decreased to 2.40 (1.15–5.01) after adjustment. Results were stronger in the first 5 years of follow-up (adjusted HR: 3.29, 95% CI: 1.27–8.52).ConclusionsChanges in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker to be evaluated in clinical studies.

Modelling synergistic interactions between HER2, Sprouty2 and PTEN in driving prostate carcinogenesis

Prostate cancer remains a major global health issue and a major cause of morbidity and mortality in men worldwide. Activation of androgen receptor and inactivation of the tumour suppressor gene phosphatase and tensin homologue (PTEN) represent two major events in prostate carcinogenesis. Using a range of clinical resources, in vitro and in vivo models, we explored potential complex interactions among receptor tyrosine kinases (such as HER2/3 and EGFR) and tumour suppressor genes, namely, Sprouty2 (SPRY2) and PTEN. The impacts on their downstream effectors (including PI3K and MAPK) to result in fine regulation of the signalling networks were also considered, which may represent important targets for developing treatment in the context of personalized medicine.

Abstract 691: Methylation of APC and GSTP1 in non-neoplastic tissue adjacent to prostate tumour and mortality from prostate cancer.

Abstract Prostate cancer is the most common tumour in men in many populations, with a crude incidence of 120-130 per 105 in Europe and the United States. The incidence of prostate cancer has increased dramatically since the introduction of prostate-specific antigen (PSA) testing at the end of the 1980s; opportunistic PSA-based screening for prostate cancer is widespread, although its ability to decrease mortality is still being debated. We studied gene methylation in non-neoplastic tissue adjacent to prostate tumours (NTAT) in association with prostate cancer mortality, as a marker to discriminate between indolent and aggressive prostate tumours. From two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostatic tumour tissues and used probe real-time PCR to analyse methylation of the glutathione S-transferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters. Prevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostatic tumour tissue for the same two genes as well as with high Gleason. The hazard ratio (HR) of prostate cancer mortality was 2.16 (1.13-4.13) for APC methylation, and 2.83 (1.42-5.65) for GSTP1 methylation in NTAT. It changed to 1.75 (0.95-3.22) and 1.61 (0.80-3.22) after adjusting for Gleason score and methylation in prostatic tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 3.84 (1.83-8.03), which decreased to 2.12 (1.03-4.37) after adjustment. Results were stronger in the first 5 years of follow-up (adjusted HR: 3.03, 95% CI: 1.20-7.67) and in the biopsy sub-cohort (HR: 5.91, 95% CI: 1.00-35.0). This results show that changes in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker. Citation Format: Chiara Celestina Grasso, Lorenzo Richiardi, Valentina Fiano, Daniela Zugna, Anna Gillio Tos, Luisa Delsedime, Franco Merletti. Methylation of APC and GSTP1 in non-neoplastic tissue adjacent to prostate tumour and mortality from prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2013-691

Diagnostic value of DNA alteration: loss of heterozygosity or allelic imbalance—promising for molecular staging of prostate cancers

The biological behavior of prostate cancer is uncertain, and therefore, search for molecular prognostic markers associated with disease progression seems to be essential. We performed microsatellite allelotyping of DNA isolated from primary prostate tumors biopsies (prostate adenocarcinoma, PCa). We evaluated the frequency of allelic imbalance (AI), including loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) as well as the association of these DNA alterations with clinicopathological variables. We assessed the significance of LOH/MSI alterations in selected imprinted and non-imprinted chromosomal regions (IR and NIR) in PCa. A total of 50 biopsies of prostate tumor (containing >75 % tumor cells) were histologically examined confirming prostate carcinoma. Microsatellite allelotyping using 16 microsatellite markers linked to the following chromosomal regions: 1p31.2, 3p21.3–25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1 was performed. The incidence of LOH/MSI alterations in prostate tumor cells was the highest for chromosomal regions 7q32.2 and 16q22.1 (31.25 and 26.60 %, respectively), followed by 1p31.2 and 3p21.3–25.3 (26.50 and 17.40 %, respectively). Statistically significant increase in LOH/MSI alterations has been observed for markers: D1S2137 (1p region; p = 0.00032), D9S974 (9p region; p = 0.0017), and D16S3025 (16q region; p = 0.0017). Statistically significant differences in frequency of LOH/MSI alterations in particular chromosomal regions have been found for 1p31.2, 7q32.2 and 16q22.1 (p = 0.027, p = 0.012 and p = 0.031, respectively). We documented statistically significant association between Fractional Allele Loss (FAL) index and advanced tumor stage (p < 0.05). We suggest that genomic instability of LOH/MSI type is a frequent event in prostate carcinogenesis and assessed as FAL index has clinical value for the molecular staging of prostate cancer in (TRUS)-guided prostate biopsy material.

Novel, gross chromosomal alterations involving PTEN cooperate with allelic loss in prostate cancer

AbstractThere is increasing evidence that multiple chromosomal rearrangements occur in prostate cancer. PTEN loss is considered to be a key event in prostate carcinogenesis but the mechanisms of loss remain to be fully elucidated. We hypothesised that gross rearrangements may exist that cause disruption of the PTEN gene in the absence of genomic deletion. We therefore designed a novel fluorescence in situ hybridisation (FISH) assay with probes overlying regions 3′ and 5′ of PTEN and a third probe overlying the gene. We aimed to identify both genomic deletions and gross rearrangements of PTEN that would be overlooked by previously reported single-probe FISH assays. We proceeded to evaluate a tissue microarray with radical prostatectomy and trans-urethral resection of the prostate specimens from 187 patients. We identified PTEN genomic loss in 45/150 (30%) radical prostatectomy patients and 16/37 (43%) trans-urethral resection of the prostate patients. Importantly, our assay detected novel chromosomal alterations in the PTEN gene (characterised by splitting of FISH signals) in 13 tumours (6.9% of all prostate cancers; 21% of PTEN-lost cancers). All PTEN-rearranged tumours had genomic loss at the other allele and had no expression of PTEN by immunohistochemistry. PTEN-rearranged tumours were significantly more likely to have an underlying ERG rearrangement. Our assay differentiated loss of the probe overlying PTEN in isolation or in combination with either one of or both the probes overlying the 3′ and 5′ regions. This gave an indication of the size of genomic loss and we observed considerable inter-tumoural heterogeneity in the extent of genomic loss in PTEN-lost tumours. In summary, gross rearrangements of the PTEN locus occur in prostate cancer and can be detected by a ‘break-apart' FISH assay. This observation could explain the absence of PTEN protein expression in a subgroup of tumours previously classified as having heterozygous genomic loss using single-probe traditional FISH assays.

Silencing of GSTP1, a Prostate Cancer Prognostic Gene, by the Estrogen Receptor-β and Endothelial Nitric Oxide Synthase Complex

We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-β and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ERβ/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ERβ antagonist or its natural ligand 5α-androstane-3β,17β-diol, eNOS inhibitors or ERβ small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ERβ/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ERβ/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ERβ/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ERβ/eNOS function by 3β-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications.

Abstract 4831: Monoclonal origin of multifocal cancer and the step-wise genetic events of prostate carcinogenesis

Abstract Prostate cancer is commonly detected as multiple foci in a single prostate gland and it is of etiological and clinical importance to identify the origin of these multifocal cancer lesions. Due to the limited number of markers analyzed, previous studies to address this issue are inconclusive. A high-resolution genome-wide study should address this issue better. Therefore, we have performed a genome-wide analysis using Affymetrix Array 6.0 of individually laser-capture microdissected foci to compare the genetic similarities and/or differences of these foci within the same prostate gland. Fluorescence in situ hybridization was used to confirm SNP array results. A total of 43 cancer and high-grade prostatic intraepithelial neoplasia (PIN) foci from 16 prostate cancer cases were analyzed for genomic copy number changes. Copy number changes from same-case foci were compared to determine the genetic relationship between the foci. In many cases, we found extensive genetic differences between different foci isolated from the same case. However, omniclonal copy number gains and losses were detected in 11/11 informative case-matched multifocal tumor cases and 8/8 informative case-matched PIN and tumor cases, suggesting that multiple cancer foci arise from a common prostate cancer precursor clone. In addition, a higher degree of similarity was observed between PIN and adjacent cancer foci, as compared to distant lesions. The strong genetic similarities between PIN and prostate cancer foci from the same region suggest clonal expansion of separate PIN lesions to invasive cancer. Based on the monoclonal model, we examined the step-wise accumulation of these genomic copy number changes. Loss of 10q23.2-10q23.31 and 16q were commonly shared in same-case tumor foci and, in many cases, also matched PIN foci, suggesting that they are early events in prostate carcinogenesis. Other common genomic changes, such as loss of 6q, 8p and 21q22.2-21q22.3, were found less frequently in all same-case foci and rarely in PIN lesions, suggesting these genetic events occur at a relatively later stage. In conclusion, using a high-density SNP array, we reveal a monoclonal origin for the majority of multifocal prostate cancer samples analyzed. From these SNP array data, we have also begun to elucidate the step-wise genetic events in prostate carcinogenesis, including early events such as loss of 10q23.2-10q23.31 and 16q Taken together, these findings should significantly enhance our understanding of prostate carcinogenesis and potentially affect the clinical management of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4831. doi:10.1158/1538-7445.AM2011-4831