Prostate Tumor Tissue Research Articles

Abstract B019: Androgen receptor-regulated lncRNA PRCAT71 promotes AR signaling through the interaction with RBPs in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer death in American men. PCa patients usually receive androgen deprivation therapy but eventually, they will relapse and tumors become castration-resistant PCa (CRPC) without effective treatments, highlighting the urgent need to develop better therapeutics to target advanced PCa. The main mechanism identified leading to castration resistance is aberrant androgen receptor (AR) reactivation, including AR overexpression. Approximately 80% of CRPC patients have a marked increase in AR mRNA and protein expression. Emerging evidence suggests genomic and transcriptomic changes insufficiently predict biology and mRNA and protein expression levels frequently do not correlate. Posttranscriptional regulation of AR mRNA can lead to AR overexpression. RNA binding proteins (RBPs) and non-coding RNAs (ncRNAs), such as microRNAs, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), can control gene expression via direct binding to mRNAs and regulating mRNA processing, modification, and translation. Here in this study, we identified a novel PCa-specific lncRNA, PRCAT71, which is higher expressed in prostate tumor tissues than in benign tissues. Silencing PRCAT71 suppressed the cancerous properties of PCa cells, and reduced AR mRNA and protein expression levels, leading to inhibited AR signaling. We further uncovered that PRCAT71 formed an RNA-RNA duplex with AR mRNA, which enhanced the stability of AR mRNA. RNA pulldown assay identified PRCAT71- interacted RBPs. Silencing PRCAT71 reduced the interaction between key RBPs and AR mRNA, which is related to PRCAT71 regulating AR mRNA stability. Interestingly, PRCAT71 is transcriptionally regulated by AR-driven enhancers, thus forming a positive regulatory loop between AR and PRCAT71 in PCa. The high expression of PRCAT71 and key RBPs in PCa tumors are positively correlated with PSA level, AR activity and indicate worse patient overall survival and metastasis-free survival. Our study revealed a novel regulatory mechanism by which lncRNA PRCAT71 enhances the interaction of key RBPs with AR mRNA, stabilizes AR mRNA and promotes AR expression and PCa progression. Targeting PRCAT71-RBPs is a promising approach to treating CRPC. Citation Format: Yongyong Yang, Ting-You Wang, Qianru Li, Adam B Weiner, Jiawen Lu, Yanan Ren, Qingxiang Guo, Chaehyun Yum, Qi Liu, Rui Wang, Zhe Ji, Tamara L Lotan, Edward M Schaeffer, Rendong Yang, Qi Cao. Androgen receptor-regulated lncRNA PRCAT71 promotes AR signaling through the interaction with RBPs in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B019.

Sialylated glycoproteins suppress immune cell killing by binding to Siglec-7 and Siglec-9 in prostate cancer.

Prostate cancer is the second leading cause of male cancer death in the U.S. Current immune checkpoint inhibitor-based immunotherapies have improved survival for many malignancies; however, they have failed to prolong survival for prostate cancer. Siglecs (sialic acid-binding immunoglobulin-like lectins) are expressed on immune cells and regulate immune responses and function. Siglec-7 and Siglec-9 contribute to immune evasion by interacting with their ligands. However, the role of Siglec-7/9 receptors and their ligands in prostate cancer remains poorly understood. Here, we find that Siglec-7 and Siglec-9 are associated with poor prognosis in prostate cancer patients, and are highly expressed in myeloid cells, including macrophages, in prostate tumor tissues. Siglecs-7 and -9 ligands were expressed in prostate cancer cells and human prostate tumor tissues. Blocking the interactions between Siglec-7/9 and sialic acids inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice in vivo. Using a CRISPRi screen and mass spectrometry, we identified CD59 as a candidate Siglec-9 ligand in prostate cancer. The identification of Siglecs-7 and -9 as potential therapeutic targets, including CD59/Siglec-9 axis, opens up opportunities for immune-based interventions in prostate cancer.

Evaluating the Expression Levels of Human Endogenous Retrovirus-K 10 (HERV-K10) Gag as a Biomarker in Prostate Cancer Tissue.

Prostate cancer is one of the most common major health problems. Several risk factors are potentially involved in its development. Therefore, a biomarker capable of early diagnosis is necessary to facilitate the early detection and treatment of prostate cancer. Human endogenous retroviruses (HERVs) are abnormally expressed in various diseases. Our study aims to evaluate the specific role of HERV K-10 gag expressions in the progression of prostate cancer. For this, we collected a set of 50 prostate tumor tissue samples as well as 50 healthy tissue samples. After extracting RNA from the prostate samples, we analyzed the expression of HERV-K gag using quantitative real-time PCR (qRT-PCR). The resulting data revealed a significant correlation of HERV-K gag expression in malignant regions of the prostate in men with prostate cancer than in men without prostate cancer (p < 0.05). The presence of the HERV-K gag protein was detected in 10 of 50 tumor samples (20%), while no healthy samples presented this protein. These results suggest that increased HERV-K gag RNA and protein expression could serve as a sensitive and specific biomarker of prostate malignancy in this cohort of prostate carcinoma patients, further supporting its potential as a promising clinical marker.

Coffee, Phosphoinositide 3-Kinase Signaling Pathway, and Prostate Cancer: A Prospective Study in the Health Professionals Follow-Up Study

BackgroundHigher coffee intake has been associated with reduced risk of prostate cancer, particularly aggressive forms. The activation of the phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in prostate carcinogenesis. ObjectiveTo evaluate associations between prediagnostic coffee intake and a PI3K activation score, the expression/presence of PI3K regulators, and downstream effectors in tumor tissue from men with prostate cancer in the Health Professionals Follow-Up Study, a prospective cohort study conducted in the United States. DesignA case-only study design was applied. Coffee intake was assessed using validated food frequency questionnaires completed in 1986 and every 4 years thereafter until prostate cancer diagnosis. Participants settingStudy participants comprised 1242 men diagnosed with prostate cancer from 1986 to 2009 and with tumor markers assessed from tissue microarrays constructed from tumor specimens. Main outcome measuresThe outcomes include the PI3K activation score; expression of insulin receptor and insulin-like growth factor 1 receptor; angiogenesis markers; and presence of the tumor suppressor phosphatase and tensin homolog, chronic and acute inflammation, simple atrophy, and post-atrophic hyperplasia. Statistical analyses performedMultivariable linear or logistic regression was conducted to estimate associations between coffee intake and tumor marker expression/presence. ResultsAmong coffee drinkers (86.6% of the population), median (25th, 75th percentile) coffee intake was 2 c/day (1, 3 c/day). The associations between coffee consumption and the tumor markers of interest were generally weak with modest precision. When comparing men who drank >3 c/day coffee with nondrinkers, the absolute percent difference in the PI3K activation score and angiogenesis markers ranged from 0.6% to 3.6%. The odds ratios for phosphatase and tensin homolog loss, insulin-like growth factor 1 receptor and insulin receptor expression, and presence of chronic and acute inflammation, simple atrophy, and postatrophic hyperplasia also were not statistically significant, were imprecise, and ranged from 0.82 to 1.58. ConclusionsCoffee intake was not observed to be associated with PI3K activation, related regulators, and several effectors in prostate tumor tissue. Studies exploring alternative pathways or earlier steps in carcinogenesis are needed to investigate the underlying mechanisms of the coffee and prostate cancer association.

Identifying proteomic risk factors for overall, aggressive, and early onset prostate cancer using Mendelian Randomisation and tumour spatial transcriptomics

Understanding the role of circulating proteins in prostate cancer risk can reveal key biological pathways and identify novel targets for cancer prevention. We investigated the association of 2002 genetically predicted circulating protein levels with risk of prostate cancer overall, and of aggressive and early onset disease, using cis-pQTL Mendelian randomisation (MR) and colocalisation. Findings for proteins with support from both MR, after correction for multiple-testing, and colocalisation were replicated using two independent cancer GWAS, one of European and one of African ancestry. Proteins with evidence of prostate-specific tissue expression were additionally investigated using spatial transcriptomic data in prostate tumour tissue to assess their role in tumour aggressiveness. Finally, we mapped risk proteins to drug and ongoing clinical trials targets. We identified 20 proteins genetically linked to prostate cancer risk (14 for overall [8 specific], 7 for aggressive [3 specific], and 8 for early onset disease [2 specific]), of which the majority replicated where data were available. Among these were proteins associated with aggressive disease, such as PPA2 [Odds Ratio (OR) per 1 SD increment=2.13, 95% CI: 1.54-2.93], PYY [OR=1.87, 95% CI: 1.43-2.44] and PRSS3 [OR=0.80, 95% CI: 0.73-0.89], and those associated with early onset disease, including EHPB1 [OR=2.89, 95% CI: 1.99-4.21], POGLUT3 [OR=0.76, 95% CI: 0.67-0.86] and TPM3 [OR=0.47, 95% CI: 0.34-0.64]. We confirmed an inverse association of MSMB with prostate cancer overall [OR=0.81, 95% CI: 0.80-0.82], and also found an inverse association with both aggressive [OR=0.84, 95% CI: 0.82-0.86] and early onset disease [OR=0.71, 95% CI: 0.68-0.74]. Using spatial transcriptomics data, we identified MSMB as the genome-wide top-most predictive gene to distinguish benign regions from high grade cancer regions that comparatively had five-fold lower MSMB expression. Additionally, ten proteins that were associated with prostate cancer risk also mapped to existing therapeutic interventions. Our findings emphasise the importance of proteomics for improving our understanding of prostate cancer aetiology and of opportunities for novel therapeutic interventions. Additionally, we demonstrate the added benefit of in-depth functional analyses to triangulate the role of risk proteins in the clinical aggressiveness of prostate tumours. Using these integrated methods, we identify a subset of risk proteins associated with aggressive and early onset disease as priorities for investigation for the future prevention and treatment of prostate cancer. This work was supported by Cancer Research UK (grant no. C8221/A29017).

Interaction of pRb and beta-catenin in cancer and normal tissue in the human prostate

Prostate cancer (PCa) is one of the most common oncological diseases, which goes through two stages in its development. The first stage, localized prostate cancer, can proceed indefinitely in a dormant form that does not require active medical intervention, or suddenly turn into an aggressive metastatic form with lethal outcome. The pathogenesis of the transition of the dormant form of PCa to the metastatic form remains not fully understood. The signaling pathways of the tumor suppressor pRb and the proto-oncogene β-catenin are probably the most involved in the pathogenesis of PCa but the role of their interaction in the pathogenesis of prostate cancer has not been studied. The publication on the pathogenesis of tumors in other tissues suggests that pRb may lose some properties of a tumor suppressor at the initial stage of PCa development due to its interaction with β-catenin that enables tumor cells to gain competitive advantages for reproduction. In this work, we have shown that the RB and β-catenin (CTNNB1) genes are well expressed in tumor and normal prostate tissue. Unlike β-catenin, pRb is not detected by immunoblotting in tumor and normal prostate tissue, but is easily determined in this way in extracts of control T98G cells. Co-immunoprecipitation with antibodies to pRb from extracts of tumor and normal prostate tissue makes it possible to detect this protein and β-catenin by subsequent immunoblotting, which indicates the physical interaction of these proteins in prostate tissue. On the other hand, immunoprecipitation of β-catenin with antibodies to its C-terminal fragment does not detect this protein in prostate extracts by subsequent immunoblotting using the same antibody. In contrast to prostate tissue, β-catenin is readily detected by immunoprecipitation combined with immunoblotting in T98G control cell extracts. The obtained data suggest that pRb and β-catenin physically interact with each other in cells of different tissue specificity. In T98G cells, this interaction probably occurs through the C-terminal fragment of β-catenin, but in prostate cells it occurs in a different way, since the C-fragment of β-catenin is shielded from such interaction, possibly due to its physical association with pRb.

LGALS3 cfDNA methylation in seminal fluid as a novel prostate cancer biomarker outperforming PSA.

The unmet challenge in prostate cancer (PCa) management is to discriminate it from benign prostate hyperplasia (BPH) due to the lack of specific diagnostic biomarkers. Contemporary research on potential PCa biomarkers is directed towardmethylated cell-free DNA (cfDNA) from liquid biopsies since epigenetic mechanisms are strongly involved in PCa development. In the present research, cfDNA methylation of the LGALS3 gene in blood and seminal plasma of PCa and BPH patients was assessed using pyrosequencing, as well as LGALS3 DNA methylation in tissue biopsies. Liquid biopsy samples were taken from patients with clinical suspicion of PCa, who were subsequently divided into two groups, that is, 42 with PCa and 55 with BPH, according to the histopathological analysis. Statistically significant higher cfDNA methylation of LGALS3 in seminal plasma of BPH than in PCa patients was detected by pyrosequencing. ROC curve analysis showed that it could distinguish PCa and BPH patients with 56.4% sensitivity and 70.4% specificity, while PSA did not differ between the two patient groups. In contrast, there was no statistically significant difference in LGALS3 cfDNA methylation in blood plasma between the two patient groups. In prostate tumor tissue, there was a statistically significant DNA hypermethylation of LGALS3 compared to surrounding nontumor tissue and BPH tissue. The DNA hypermethylation of the LGALS3 gene represents an event specific to PCa development. In conclusion, LGALS3 cfDNA methylation in seminal fluid discriminates early PCa and BPH presenting itself as a powerful novel PCa biomarker highly outperforming PSA.

Using Lithium-6 Filter for Study of Dose Distribution with Max. and Min. Displacement of Prostate Inside the Body by BNCT Method

Physical dose assessment of prostate tumor and adjacent healthy tissues in ORNL, adult male (AM) and KTMAN-2 phantoms was performed using the Monte Carlo transport code. Two different spectra of MIT reactor (without and with lithium filter) were considered. To evaluate the effectiveness of this method in destroying cancer cells for different depths of the prostate, the prostates were moved three times with a distance of 3 cm towards the surface of the body. In addition, the effects of using a lithium filter with different thicknesses on the physical doses of the prostate and adjacent healthy tissues were investigated. It has been observed that decreasing the depth of the prostate increases the physical dose received by the tumor. When the prostate is placed at the maximum depth inside the body, the tissues located in the path of the beam receive the maximum amount of dose. However, the use of a lithium filter reduces damage to healthy tissues, so that when the prostate is near the surface of the body, the lithium filter increases the amount of dose delivered to tumors. In these conditions, the average energy of the penetration distance of epithermal neutrons and neutrons in tissues increases. By changing the location of the prostate gland inside the body, the physical doses of normal tissues did not change, but it was observed that increasing the thickness of the lithium filter significantly reduced the dose of normal tissues.

386 The Analysis of N-glycans and Collagen to Predict Prostate Adenocarcinoma Outcome

OBJECTIVES/GOALS: Distinguishing indolent from aggressive prostate cancer and early identification of men at risk of developing aggressive, metastatic disease is of great importance. We aim to explore the relationship between N-glycan and collagen composition in prostate tumor tissue and the long-term outcome of the disease. METHODS/STUDY POPULATION: Matrix assisted laser desorption/ionization mass spectrometry can be utilized to characterize N-glycan profiles in formalin fixed paraffin embedded tissues. Collagen may also be characterized using ECM-targeted collagenase MALDI imaging. These approaches were used to analyze prostatectomy samples with different clinical outcomes. Tissue microarrays containing tissues from 75 non-progressors (no evidence of disease; NED) and 50 metastatic cases (MET) were examined. From a combined list of 90 N-glycans and 500 collagenase peptides, the average AUC intensity value for each glycan and collagen peptide was extracted and assessed as a predictor of metastatic progression. Ancestral informative markers were analyzed and polygenic hazard risk scores were generated for samples as well. RESULTS/ANTICIPATED RESULTS: Three N-glycans and three collagen peptides were found to discriminate between NED and MET cases with statistical significance. The best performing N-glycan was Hex6HexNAc6Fuc1 with an AUC of 0.77 (p&lt;0.001). While the best performing collagen peptide was COL1A2 with an AUC of C 0.77 (p&lt;0.001). DISCUSSION/SIGNIFICANCE: Both a collagen peptide and N-glycan were discovered as promising biomarkers to predict metastasis. Future validation studies are needed to confirm biomarker potential and to determine if the addition of these biomarkers can strengthen current genomic classifier’s ability to predict metastatic prostate cancer.

RNA m6a Methylation Regulator Expression in Castration-Resistant Prostate Cancer Progression and Its Genetic Associations.

N6-methyladenosine (m6A) methylation, a prevalent epitranscriptomic modification, plays a crucial role in regulating mRNA expression, stability, and translation in mammals. M6A regulators have gained attention for their potential implications in tumorigenesis and clinical applications, such as cancer diagnosis and therapeutics. The existing literature predominantly addresses m6A regulators in the context of primary prostate cancer (PCa). However, a notable gap in the knowledge emerges regarding the dynamic expression patterns of these regulators as PCa progresses towards the castration-resistant stage (CRPC). Employing sequential window acquisition of all theoretical mass spectra (SWATH-MS) and RNAseq analysis, we comprehensively profiled the expression of 27 m6A regulators in hormone/androgen-dependent and -independent PCa cell lines, revealing distinct clustering between tumor and adjacent normal prostate tissues. High-grade PCa tumors demonstrated the upregulation of METTL3, RBM15B, and HNRNAPA2B1 and the downregulation of ZC3H13, NUDT21, and FTO. Notably, we identified six m6A regulators associated with PCa survival. Additionally, association analysis of the PCa-associated risk loci in the cancer genome atlas program (TCGA) data unveiled genetic variations near the WTAP, HNRNPA2B1, and FTO genes as significant expression quantitative trait loci. In summary, our study unraveled abnormalities in m6A regulator expression in PCa progression, elucidating their association with PCa risk loci. Considering the heterogeneity within the PCa phenotypes and treatment responses, our findings suggest that prognostic stratification based on m6A regulator expression could enhance PCa diagnosis and prognosis.

Abstract 7320: Expression of circadian rhythm genes and lethal prostate cancer

Abstract Background: The circadian rhythm regulates diverse physiological and oncologic processes. We have previously shown in epidemiology studies that disruption of the circadian rhythm may be associated with increased risk of aggressive prostate cancer, including germline polymorphisms in certain circadian rhythm genes. This study sought to investigate the relationship between tumor circadian rhythm gene expression and lethal prostate cancer. Methods: We studied patients with primary prostate cancer in the Health Professionals Follow-up Study (HPFS) and Physicians’ Health Study (PHS). We created a composite score of mRNA expression using principal components that incorporated 11 circadian rhythm genes: AANAT, ARNTL, CLOCK, CRY1, CSNK1E, MTNR1A, MTNR1B, NPAS2, PER1, PER2, PER3. An age-stratified analysis was performed, and the mean differences of the score both by Gleason score and compared to normal tissue were calculated. The relationship between the score in prostate tumor tissue and lethal prostate cancer (metastases/prostate cancer-specific death) vs. indolent disease (no metastases after &amp;gt;8 years of follow-up) was assessed using logistic regression. The contribution of each gene was evaluated by leave-one-out analysis, generating scores leaving out one gene and assessing the relationship between the modified score and lethal disease. Results: Among 404 cancer patients in HPFS/PHS, there were 119 lethal events over 30 years of follow-up (median: 17 years). The circadian rhythm gene score was lower in tumor tissue compared to tumor-adjacent, histologically normal tissue (mean difference in units of standard deviation [SD] of gene score: -0.31, 95% confidence interval [CI]: -0.48 to -0.15). In addition, the score was lower in Gleason 9-10 compared to Gleason 6 tumors (mean difference in SD units: -1.05, 95% CI: -1.55 to -0.54). The three PER genes were the most strongly correlated with each other and with the score (Spearman correlation coefficients from 0.52-0.88 for the score and 0.31-0.63 between PER genes). Higher circadian rhythm gene scores were inversely associated with lethal prostate cancer (odds ratio [OR] for highest vs. lowest quartile: 0.43, 95% CI: 0.20 to 0.90) in a dose-response fashion and after adjusting for clinical and pathologic factors. The association appeared to be stronger in patients aged ≥65 years (OR for highest vs. lowest quartile: 0.21, 95% CI: 0.08 to 0.47) compared to &amp;lt;65 years at diagnosis (OR: 0.78, 95% CI: 0.29, 2.17). Results were consistent in the leave-one-out analysis for all genes except PER2 (OR for highest vs. lowest quartile: 1.00, 95% CI: 0.55 to 1.83) and PER1 (OR: 0.55, 95% CI: 0.30 to 1). Conclusion: This study examining intratumoral circadian rhythm gene expression contributes further evidence in support of the relationship between circadian rhythm disruption and aggressive prostate cancer. Tumor expression of PER1 and PER2 may be a predominant factor in the association. Citation Format: Jane Bailey Vaselkiv, Sarah C. Markt, Hannah E. Guard, Anqi Wang, Kathryn L. Penney, Konrad H. Stopsack, Lorelei A. Mucci. Expression of circadian rhythm genes and lethal prostate cancer [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 7320.

Abstract 7418: LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data

Abstract Gene fusion resulted from genomic translocation plays important oncogenic roles including but not limited to gene activation and changing protein coding sequences. Historically, fusion gene analysis has been limited to bulk levels resulting in mixed oncogene activation models. Single cell Nanopore RNA sequencing (scNanoRNAseq) has the unique strength in detecting full-length chimeric transcripts in thousands of single cells in parallel. However, robust computational tools for detecting fusion genes in single cells from scNanoRNAseq data are missing. Here, we developed LongFuse, a fast and accurate tool, to detect fusion genes and their splicing isoforms in single cells from scNanoRNAseq data. LongFuse implements an XOR logic gate algorithm to speed up fusion candidate searching and stringent criteria to eliminate false discoveries due to trans-splicing events and randomly ligated chimeras. We applied LongFUSE onto scNanoRNAseq data of both human prostate cancer cell line, VCaP, and tumor tissues with known TMPRSS2-ERG fusion. Our results showed that both cancer cell line and prostate tumors harbored a mixture of fusion positive and fusion negative tumor cells. Gene expression analysis revealed transcriptional differences between fusion positive and negative cells including genes involved in cancer cell proliferation and invasion programs such as ELL2, ZBTB16, IGF1R, ANK3, MYPOP and PLPP1 genes. LongFUSE also calculates the splicing isoforms of fusion gene transcripts, allowing prediction of protein sequences of fusion gene transcripts. Taken together, our data demonstrated that LongFUSE is a robust computational tool for detecting gene fusions and their splicing isoforms at single cell level from scNanoRNAseq data. Citation Format: Cheng-Kai Shiau, Yueying He, Hsiao-Yun Lin, Lina Lu, Xiao-Dong Lu, Jindan Yu, Ruli Gao. LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data [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 7418.

Abstract 4386: Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1

Abstract Prostate cancer represents a major health concern for men in the United States and globally, disproportionately affecting men of African descent. Despite recent advances, the precise etiology and molecular mechanisms underpinning fatal prostate cancer in humans remain inadequately understood. RNA binding proteins (RBPs) regulate various aspects of RNA biology, and when their regulation is disrupted, they can lead to human diseases, including cancer. However, the comprehensive alterations in RBP-mRNA interactions in prostate cancer have not been explored. In this study, we investigated the dynamics of RBPs and their interactions with polyadenylated mRNA in the castration-sensitive LNCaP and castration-resistant C4-2 prostate cancer cell lines. Using an enhanced RNA interactome capture assay in combination with mass spectrometry, we discovered that LNCaP and C4-2 cells showed distinct RBP-mRNA interaction patterns in steady-state growth conditions and in response to androgen treatment. Additionally, we investigated the interplay between the oncogene Yes-associated protein 1 (YAP1) and the RNA-binding protein nucleophosmin (NPM1). We identified NPM1 as a component of the YAP1 protein complex through mass spectrometry-based proteomics. Our proximity ligation assay demonstrated that androgen signaling could regulate the interaction between YAP1 and NPM1. Also, our GST-pulldown revealed that NPM1 binds to the proline-rich domain of YAP1 in LNCaP cells, while the WW/SH3 domain of YAP1 mediates the interaction with NPM1 in C4-2 cells. Genetic and pharmacological inhibition of NPM1 activity reduced prostate cancer cell growth in culture by modulating cell cycle progression. Furthermore, our computational and immunological analysis of prostate tumor tissues indicated that the upregulation of NPM1 might be associated with the clinical advancement of human prostate cancer. These findings underscore the critical role of the molecular functional connection between YAP1 and RBPs and the global changes in RBP and RNA interaction patterns, in the progression and recurrence of prostate cancer. Citation Format: Abdulrahman M. Dwead, Marwah M. Al-Mathkour, Bekir Cinar. Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1 [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 4386.

Abstract 7028: Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers

Abstract Prostate cancer is the second leading cause of cancer related deaths among men in the United States. Health disparities in men diagnosed with prostate cancer are observed between patients of African and those of European ancestry. To elucidate molecular mechanisms behind prostate tumorigenesis and racial disparities, researchers have identified genetic alterations such as gene fusions, deletions, and mutations that occur at varying frequencies among different ethnic groups. However, results are inconsistent across studies, suggesting that racial disparities may be multifactorial. Our approach to further understand the mechanisms underlying racial disparities is to compare DNA methylation profiles between cancers from different ethnic groups using formalin-fixed paraffin-embedded (FFPE) prostate cancer tissue samples. For this study, FFPE prostate cancer specimens from 32 Black men and 30 age-matched White men were obtained in the form of tissue sections on glass slides. To profile DNA methylation in these samples, tumor and normal tissue regions (in a subset of cases) were outlined on a hematoxylin &amp; eosin (H&amp;E) reference slide and the respective regions were macro-dissected off the corresponding unstained slides from the same block. The areas of macro-dissected regions were measured using ImageJ, and the number of slides were taken to normalize DNA yield. Next, we compared magnetic bead and column based DNA extraction methods. We evaluated them on their relative DNA yield per tissue size, and identified that the bead-based extraction method had both higher and more consistent DNA yield. In total, we used 95 unique tissue regions to profile global DNA methylation levels using Illumina Epic array. We found that a subset of samples harbored an aberrant DNA methylation pattern. Therefore, we extracted DNA from additional FFPE tissue slides using the bead-based method and repeated the assay with a larger amount of DNA, successfully rescuing the majority of the aberrant samples. Next, we will evaluate associations between DNA methylation levels in different ethnic groups, clinicopathological features, and follow-up data to help identify potential DNA methylation biomarkers and therapeutic targets linked to prostate cancer from different ethnicities. Citation Format: Colton Duran Stensrud, Leonardo Gonzalez-Smith, Claire Stevens, Huan Cao, Jenaye Mack, Olumide Arigbede, Daniel Weisenberger, Sarah G. Buxbaum, Sara M. Falzarano, Suhn Rhie. Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers [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 7028.

Abstract 7524: Targeting siglec-7/9 glyco-immune checkpoints in prostate cancer for enhanced immune responses and tumor suppression

Abstract Prostate cancer is one of the leading causes of death in men worldwide. Despite significant advancements in the fight against cancer, immune checkpoint inhibitors have limited effectiveness in treating prostate cancer. Evidence shows that disrupting the interactions between Siglec-7/9 and sialic acids can enhance immune responses and effectively inhibit tumor growth in several cancers. However, the understanding of Siglec-7/9-sialic acid interactions in prostate cancer remains limited. Here, we found that tumor-infiltrating immune cells derived from prostate cancer patients exhibited high levels of Siglec-7 and Siglec-9. TCGA data analysis suggests that high levels of Siglec-7/9 are correlated to worse patient survival. Elevated levels of Siglec-7/9 ligands were found in prostate tumor tissues compared to adjacent normal tissues. Prostate cancer cell lines exhibited significant expression levels of Siglec-7 and Siglec-9 ligands, and cell surface sialic acids. Proteomic analysis suggests that increased sialylation was observed in tumor tissues in comparison to adjacent normal tissues. Disrupting interactions between Siglec-7/9 and sialoglycans enhanced T cell-mediated cytotoxicity against prostate cancer cells. Treatment with anti-Siglec-7/9 antibodies effectively suppressed the growth of PC3 and 22Rv1 tumors in a humanized mouse model. Immunohistochemistry analysis of tumor tissues showed that PC3 and 22Rv1 tumors treated with anti-Siglec-7/9 exhibited reduced proliferation, decreased vascularization, increased apoptosis, and enhanced immune cell infiltration. The CRISPR screen and mass spectrometry analysis suggested CD59 as a potential ligand for Siglec-9. Knockout of CD59 in prostate cancer cells decreased their binding activity of Siglec-9-Fc chimera proteins and promoted T cell-mediated killing ability. These findings provide valuable insights into the potential therapeutic strategy of targeting Siglec-7/9 and their ligands for prostate cancer treatment. Citation Format: Ru Wen, G Edward Marti, Jessica Stark, Fernando Jose Garcia Marques, Nick Riley, Abel Bermudez, Hongjuan Zhao, Carolyn Bertozzi, Sharon Pitteri, James Brooks. Targeting siglec-7/9 glyco-immune checkpoints in prostate cancer for enhanced immune responses and tumor suppression [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 7524.

Abstract 7330: Identifying proteomic risk factors for overall, aggressive and early onset prostate cancer using mendelian randomization and tumor spatial transcriptomics

Abstract Background: Understanding the role of circulating proteins in prostate cancer risk can reveal key biological pathways and identify novel targets for cancer prevention. Methods: We investigated the association of 2,002 genetically predicted circulating protein levels with risk of prostate cancer overall, and of aggressive and early onset disease, using cis-pQTL Mendelian randomization (MR) and colocalization. Findings for proteins with support from both MR, after correction for multiple-testing, and colocalization were replicated using two independent cancer GWAS, one of European and one of African ancestry. Proteins with evidence of prostate-specific tissue expression were additionally investigated using spatial transcriptomic data in prostate tumor tissue to assess their role in tumor aggressiveness. Finally, we mapped risk proteins to drug and ongoing clinical trials targets. Results: We identified 20 proteins genetically linked to prostate cancer risk (14 for overall [8 specific], 7 for aggressive [3 specific], and 8 for early onset disease [2 specific]), of which a majority were novel and replicated. Among these were proteins associated with aggressive disease, such as PPA2 [Odds Ratio (OR) per 1 SD increment = 2.13, 95% CI: 1.54-2.93], PYY [OR = 1.87, 95% CI: 1.43-2.44] and PRSS3 [OR = 0.80, 95% CI: 0.73-0.89], and those associated with early onset disease, including EHPB1 [OR = 2.89, 95% CI: 1.99-4.21], POGLUT3 [OR = 0.76, 95% CI: 0.67-0.86] and TPM3 [OR = 0.47, 95% CI: 0.34-0.64]. We confirm an inverse association of MSMB with prostate cancer overall [OR = 0.81, 95% CI: 0.80-0.82], and also find an inverse association with both aggressive [OR = 0.84, 95% CI: 0.82-0.86] and early onset disease [OR = 0.71, 95% CI: 0.68-0.74]. Using spatial transcriptomics data, we identified MSMB as the genome-wide top-most predictive gene to distinguish benign regions from high grade cancer regions that had five-fold lower MSMB expression. Additionally, ten proteins that were associated with prostate cancer risk mapped to existing therapeutic interventions. Conclusion: Our findings emphasize the importance of proteomics for improving our understanding of prostate cancer etiology and of opportunities for novel therapeutic interventions. Additionally, we demonstrate the added benefit of in-depth functional analyses to triangulate the role of risk proteins in the clinical aggressiveness of prostate tumors. Using these integrated methods, we identify a subset of risk proteins associated with aggressive and early onset disease as priorities for investigation for the future prevention and treatment of prostate cancer. Citation Format: Trishna A. Desai, Asa K. Hedman, Marios Dimitriou, Mine Koprulu, Sandy Figiel, Wencheng Yin, Matthias Johansson, Eleanor L. Watts, Joshua R. Atkins, Aleksandr V. Sokolov, Helgi B. Schioth, Marc J. Gunter, Konstantinos K. Tsilidis, Richard M. Martin, Maik Pietzner, Claudia Langenberg, Ian G. Mills, Alastair D. Lamb, Anders Malarstig, Tim J. Key, The PRACTICAL Consortium, Ruth C. Travis, Karl Smith-Byrne. Identifying proteomic risk factors for overall, aggressive and early onset prostate cancer using mendelian randomization and tumor spatial transcriptomics [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 7330.

Tumor microenvironment deconvolution identifies cell-type-independent aberrant DNA methylation and gene expression in prostate cancer

BackgroundAmong men, prostate cancer (PCa) is the second most common cancer and the second leading cause of cancer death. Etiologic factors associated with both prostate carcinogenesis and somatic alterations in tumors are incompletely understood. While genetic variants associated with PCa have been identified, epigenetic alterations in PCa are relatively understudied. To date, DNA methylation (DNAm) and gene expression (GE) in PCa have been investigated; however, these studies did not correct for cell-type proportions of the tumor microenvironment (TME), which could confound results.MethodsThe data (GSE183040) consisted of DNAm and GE data from both tumor and adjacent non-tumor prostate tissue of 56 patients who underwent radical prostatectomies prior to any treatment. This study builds upon previous studies that examined methylation patterns and GE in PCa patients by using a novel tumor deconvolution approach to identify and correct for cell-type proportions of the TME in its epigenome-wide association study (EWAS) and differential expression analysis (DEA).ResultsThe inclusion of cell-type proportions in EWASs and DEAs reduced the scope of significant alterations associated with PCa. We identified 2,093 significantly differentially methylated CpGs (DMC), and 51 genes associated with PCa, including PCA3, SPINK1, and AMACR.ConclusionsThis work illustrates the importance of correcting for cell types of the TME when performing EWASs and DEAs on PCa samples, and establishes a more confounding-adverse methodology. We identified a more tumor-cell-specific set of altered genes and epigenetic marks that can be further investigated as potential biomarkers of disease or potential therapeutic targets.

Association between copy number alterations estimated using low-pass whole genome sequencing of formalin-fixed paraffin-embedded prostate tumor tissue and cancer-specific clinical parameters

Copy number alterations (CNAs) are frequently observed in early-stage prostate cancer and are associated with disease recurrence and tumor aggressiveness. Cost-effective assessment of CNAs could enhance clinical utility of CNAs. Here, we combined the cost-effectiveness of low-pass (low coverage) whole genome sequencing (LPWGS) and the routine availability of formalin-fixed paraffin-embedded (FFPE) tumor tissue for assessing CNAs in a cohort of 187 men with early-stage localised prostate cancer. We detected well known CNAs in 8p, 8q, 13q and 16q and recurrent gains of the oncogene MYC and losses of the tumor suppressor genes NKX3-1, PTEN and RB1, indicating assay reliability. The estimated burden of CNAs was significantly associated with Gleason score, pathological T stage, surgical margin status and biochemical recurrence. Further, genomic losses or gains in specific chromosomal arms were significantly associated with worse BCR-free survival. Copy number signatures extracted from the LPWGS data showed potential for risk stratifying patients, where signatures S1 and S2 showed significant association to worse BCR-free survival compared to S3. Our study provides clinical validation of the associations between CNAs and tumor aggressiveness in an independent and representative RP cohort, while demonstrating the feasibility of performing LPWGS of FFPE tumor tissue for cost-effective assessment of CNAs.

Abstract C059: Optimization of DNA extraction methods from FFPE prostate tumor tissues of Black men to identify DNA methylation biomarkers

Abstract Prostate cancer is the second leading cause of cancer related deaths among men in the United States, and health disparities in those diagnosed with prostate cancer are observed between those of African and European ancestry. Our approach to understand the mechanisms underlying these differences is to compare DNA methylation profiles between samples using formalin-fixed parafin-embedded (FFPE) prostate tumor tissues. To profile DNA methylation from FFPE prostate tumor tissues, we tested magnetic bead and column based DNA extraction methods, and evaluated them on their total DNA yield. Additionally, we assessed the relationship between DNA yield and clinical features. Prostate specimens for this study were obtained from the University of Florida in the form of FFPE tissue slides. Both tumor and normal tissue regions were outlined on an H&amp;E reference slide and the respective regions were macro-dissected from unstained slides of the same block. The areas of macro-dissected regions were measured using ImageJ, and the number of slides were taken to further normalize DNA yield. Magnetic bead and column based FFPE DNA extraction kits were used to isolate DNA, and the relative amounts of the isolated DNA per tissue size were evaluated. In addition, various clinical features of the samples were compared to determine if their presence influences DNA yield. Preliminary data suggests that total DNA extracted from both bead and column based methods are relatively comparable, but bead based methods yielded better quality of DNA. We observed minimal correlation between DNA yield and grade group. We detected a potential association between cribriform pattern and DNA yield. We are in the process of profiling DNA methylation using the isolated DNA. Successful completion of this study will identify DNA methylation biomarkers and further develop diagnostic and therapeutic tools for prostate cancer patients from African ancestry. Citation Format: Colton Stensrud, Claire Stevens, Leonardo Gonzalez-Smith, Huan Cao, Sarah Buxbaum, Sara Falzarano, Suhn Rhie. Optimization of DNA extraction methods from FFPE prostate tumor tissues of Black men to identify DNA methylation biomarkers [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C059.

Abstract PR007: Neighborhood socioeconomic deprivation, racial segregation, and prostate tumor RNA expression of stress-related genes among African American and European American men

Abstract INTRODUCTION: African American (AA) men experience greater prostate cancer incidence and mortality compared to European American (EA) men. Growing literature supports associations of neighborhood social factors (NSF) including neighborhood socioeconomic deprivation and residential segregation with advanced or aggressive prostate cancer, and AA men may experience these factors to a greater extent than EA men. Here we tested associations of NSF with prostate tumor RNA expression of stress-related genes, hypothesizing that these factors would be related and contribute to prostate tumor aggressiveness. METHODS: We leveraged available transcriptomic data from prostate tumor tissue for AA and EA men with prostate cancer who received radical prostatectomy surgery at the University of Maryland Medical Center. We geocoded each participant’s address at diagnosis, determined the corresponding census tract, and assigned tract-based Area Deprivation Index (ADI) and Racial Isolation Index (RI) scores to each participant. Based on data availability, ADI analyses included men diagnosed in 2005 or later (118 AA men and 43 EA men), and RI analyses included those diagnosed in 2009 or later (110 AA men and 37 EA men). We evaluated 105 stress-related genes, including those in the Conserved Transcriptional Response to Adversity, among others. We fit separate linear regression models for expression of each gene in relation to ADI or RI, respectively. Models were adjusted for race and age and year at surgery. We obtained q-values (p-values adjusted for multiple comparisons) using the Benjamini-Hochberg method. RESULTS: Median (interquartile range) ADI scores were 116 (101-131) for AA men and 91 (83-103) for EA men, and RI scores were 0.68 (0.38-0.87) for AA men and 0.10 (0.05-0.14) for EA men, indicating greater neighborhood deprivation and Black residential segregation among AA participants. The greatest values for these scores were concentrated in central and west Baltimore. ADI scores were positively and significantly (p&amp;lt;0.05) associated with expression for 11 genes. One gene, HTR6 (serotonin pathway), remained significant after multiple comparison adjustment (beta=0.0029, 95% confidence interval: 0.0014-0.0043; p&amp;lt;0.001; q=0.02). RI scores were positively and significantly associated with expression for 7 genes (p&amp;lt;0.05), but findings did not remain significant after multiple comparison adjustment. Four genes, including HTR6, IFIT2 and MX2 (roles in Type I IFN response), and IGLL1 (antibody synthesis) were significantly associated with both ADI and RI (p&amp;lt;0.05). Top findings among AA men only were similar to the overall results (AA and EA men combined). CONCLUSIONS: We identified several genes in stress-related pathways whose expression in prostate tumor tissue was higher among men with higher neighborhood deprivation or higher racial segregation. Additional analyses will consider other neighborhood measures, including historical redlining, to further investigate the role of NSF in prostate tumor RNA expression, tumor aggressiveness, and prostate cancer disparities. Citation Format: Joseph Boyle, Jessica Yau, Jimmie L. Slade, Derrick Butts, Yuji Zhang, Teklu B. Legesse, Ashley Cellini, Kimberly Clark, Jessica Wimbush, Nicholas Ambulos Jr., Jing Yin, Arif Hussain, Eberechukwu Onukwugha, Cheryl L. Knott, David C. Wheeler, Kathryn Hughes Barry. Neighborhood socioeconomic deprivation, racial segregation, and prostate tumor RNA expression of stress-related genes among African American and European American men [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr PR007.