Radiation therapy effectively treats prostate cancer, but tumor recurrence remains a clinical challenge, highlighting the need for strategies to enhance radiosensitivity. In this study, we developed a simple, high-throughput drug repurposing screening platform to find radiosensitizers from compounds approved by the U.S. Food and Drug Administration. A library of 1134 compounds was systematically screened at two concentrations (0.2 and 2 μM) using LNCaP cells stably expressing Metridia luciferase, allowing highly sensitive and quantitative assessment of cell viability through luciferase activity in culture supernatants. In the primary screening, 8 and 12 candidate radiosensitizers were identified at 0.2 μM and 2 μM, respectively. In the secondary screening, 19 compounds were evaluated at two radiation doses (4 Gy and 6 Gy) and six drug concentrations, identifying 5 radiation-sensitizing candidate compounds. Through this stepwise screening approach, cladribine was identified as the most potent radiosensitizer. Cladribine increased radiation-induced cytotoxicity in multiple prostate cancer cell lines (22Rv1, DU145, and PC3), with dose-modifying factors of 1.46, 1.55, and 1.43, respectively, based on the radiation dose needed to achieve 90% cell death. Mechanistically, cladribine prevented the repair of radiation-induced DNA double-strand breaks, shown by increased γH2AX levels. Importantly, its radiosensitizing effect was further confirmed in vivo using 22Rv1 and DU145 xenograft models. This study demonstrates that a luciferase-based high-throughput drug repurposing platform is useful for identifying clinically relevant radiosensitizers, revealing that cladribine is a promising candidate for further translational research in prostate cancer radiotherapy.

MicroRNAs (miRNAs) are central regulators of gene expression, and rare pathogenic mutations in miRNA genes can cause Mendelian disease. However, the contribution of germline miRNA variants to prostate cancer (PCa) risk and clinical outcomes remains unclear, in part due to limited coverage of miRNA genes in previous exome, GWAS, and population sequencing datasets. We performed ultra-deep, targeted germline sequencing and rigorous variant calling of over 500 miRNA genes (including flanking regions) in 1,502 PCa patients from hereditary and sporadic cohorts, representing both high- and low-grade disease. Variants were systematically annotated for functional region (stem, seed, mature, loop) and predicted impact on pre-miRNA folding (ΔG). Frequencies of recurrent variants (>2 carriers) were compared to gnomAD whole-genome sequencing (WGS) and whole-exome sequencing (WES) populations using Fisher’s exact test within ancestry-matched groups. Results were cross-referenced with miRNASNP-v4, miRNA-seq data from prostate cancer cell lines, and TCGA-PRAD expression/outcome data. Per-gene miRNA burdens and individual variants were assessed for association with clinical features (family history, age, grade, metastasis, progression, PSA, cause of death). We generated a comprehensive, high-confidence annotation resource of miRNA gene variants in germline DNA from prostate cancer patients, identifying 48 recurrent miRNA gene variants significantly enriched in PCa cases versus gnomAD (p < 1×10^−8), including both known and novel polymorphisms. A subset displayed significant pre-miRNA folding change (ΔG > 2 or > 5 kcal/mol) and/or mapped to seed or mature regions. Several variants overlapped miRNA genes robustly detected in PCa cell lines and differentially expressed in TCGA-PRAD, with recurrent hits in known risk regions such as 8q24 (notably enriched among African/African American patients). This study establishes the deepest and most extensive annotation of miRNA gene variants in prostate cancer to date, integrating germline miRNA variation, functional prediction, and population ancestry. We observed significant discrepancies in miRNA variant allele frequencies when comparing gnomAD WGS to WES data, underscoring the critical need to use WGS datasets for reliable estimation of miRNA gene variant frequencies. Our results identify multiple miRNA gene variants that are enriched in our PCa cohort compared to gnomAD WGS reference populations and are predicted to impact miRNA biogenesis, function, and/or PCa cell biology. While these findings are novel and present strong candidates for further study, they remain preliminary and should be validated in independent cohorts for association with PCa risk and biological effects. Shawn E. Lupold, Jun Wei, Siqun Zheng, Jun Luo, William Isaacs, Jianfeng Xu. Comprehensive Evaluation of Recurrent microRNA Gene Variants in Prostate Cancer: Integrative Association with Risk, Predicted Function, and Clinical Outcome [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A041.

Androgen deprivation therapy or Androgen receptor-targeted therapies are used to treat metastatic Prostate Cancer (PCa). However, nearly all cases eventually progress to castration-resistant prostate cancer (CRPC). The molecular pathways associated with CRPC development and progression remain incompletely defined. This study aims to identify new drivers and delineate the underlying mechanisms in CRPC. Three isogenic castration-sensitive and castration-resistant prostate cancer cell lines were analyzed by miRNA and RNA sequencing. Gene transcripts associated with castration resistance were further characterized by RT-PCR and bioinformatic analyses. Gene knockout and knockdown in CRPC cell lines were carried out using the CRISPR/Cas9 technology and RNA interference (RNAi) technique. In addition, the HOXC gene cluster was overexpressed in castration-sensitive prostate cancer cell lines using predesigned synthetic sgRNAs and the CRISPR activation (CRISPRa) system. Cell growth and survival were assessed using the IncuCyte®-SX5 live cell analysis system. Differential gene expression analysis identified significantly elevated miR-196a expression in CRPC cell lines. Visualization of RNA sequencing read density peaks in IGV revealed pri-miR-196a-2 as the source of elevated miR-196a expression in CRPC cells. MIR196A2 is located within the HOXC gene cluster. Transcriptional analyses demonstrate that miR-196a-2 is an intronic miRNA co-transcribed within HOXC gene transcripts. Multiple HOXC gene transcripts were more active in CRPC cell lines. Bioinformatic analyses demonstrated significantly higher HOXC gene expression in a subset of metastatic and CRPC tumors. The elevated expression was further validated in clinical samples by RT-qPCR. HOXC8 gene over-expression using plasmid gene expression vectors and CRISPRa enhanced prostate cancer cell proliferation, and HOXC8 gene knockout attenuated CRPC cell proliferation. HOXC gene cluster is activated in in a subset of metastatic and castration-resistant prostate cancer with HOXC8 playing a crucial role in regulating cell proliferation. Isaacson B. Adelani, Akira Kurozumi, Tobiloba Owojuyigbe, Sanchit Sanyal, Su Mi Choi, Srinivasan Yegnasubramanian, Shawn E. Lupold. Elevated HOXC gene expression in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1441.

MicroRNA (miRNA) dysregulation is implicated in testicular germ cell tumor (TGCT) pathogenesis. Here, we characterized miRNA expression profiles across TGCT histologic subtypes using miRNA-sequencing on 43 formalin-fixed paraffin-embedded (FFPE) tissue samples (31 primary, 12 metastases) from 29 patients to identify diagnostic markers and their regulatory functions. From 20 seminomas (SEM), 14 non-seminomatous germ cell tumors (N-SEM), and 9 teratomas, we profiled a total of 2606 miRNAs. Compared to teratomas, 154 miRNAs (targeting 657 genes) were enriched in SEM, and 141 miRNAs (targeting 358 genes) in N-SEM. miR-200-3p, targeting the DNA methyltransferase DNMT3B, was enriched in N-SEM versus SEM. Our findings showed high concordance with The Cancer Genome Atlas (TCGA)-TGCT data (Pearson R > 0.66, p < 1e-10). miRNA expression was largely similar between primary and metastatic tissues and between chemotherapy-treated and untreated teratomas, reflecting teratoma chemo-resistance. Using novel candidates, miRNA-based logistic regression classifiers distinguished viable GCT (SEM/N-SEM) from teratoma (Area Under the Curve [AUC] > 0.96) and SEM from N-SEM (AUC = 0.81), outperforming well-known miRNA markers. Target gene analysis implicated FOXO and RUNX1 regulation, somatotroph signaling, and height-related pathways. Overall, our comprehensive tissue-level miRNA profiling in TGCTs identified potential diagnostic biomarkers for histologic subtypes, offering insights into miRNA-mediated transcriptional dysregulation.

Aberrant microRNA expression is common in cancer, yet cell-type-specific microRNA activity in the tumor microenvironment (TME) remains poorly understood. Here, we show that germline deletion of miR-21 significantly attenuated the progression of MYC-driven prostate cancer (PCa), reducing prostate weight, tumor burden, and proliferation index in Hi-Myc mice. In situ hybridization revealed elevated miR-21 expression in multiple cell types during disease progression. Inflammatory and premalignant lesions in mouse and human prostate showed increased miR-21 in both stroma and epithelium, with further enrichment in the stroma of invasive adenocarcinoma. In Hi-Myc mice, single cell RNA-sequencing revealed miR-21 gene regulation in neoplastic, stromal, and immune cells in a cell-type-specific manner, impacting both direct and indirect targets. Notably, miR-21 deletion reduced immune infiltration into the prostate TME, particularly Trem2 -expressing macrophages and regulatory T cells. The Timp1-fibroblast gene signature in MYC-driven PCa was suppressed in miR-21 knockout prostates. Cell-cell communication analysis showed that miR-21 suppressed TGF-beta signaling in the TME, partially through Ski and Smad7 suppression in cancer-associated fibroblasts. These findings underscore the crucial role of miR-21 in PCa and provide some of the first in situ insights into cell-type-specific miRNA activity in solid tumors.

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

BackgroundMicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. These epigenetic regulators play a crucial role in T-cell activation and differentiation. MicroRNA-21 (miR-21) is abundantly expressed in mammalian cells that promotes tissue inflammation and autoimmunity by regulating the balance among Th1, Th2 and Th17 responses. It is also an important modifier of T-cell immunity via alteration of antigen presenting cells in the context of inflammation and cancer. We hypothesize that miR-21 regulates T-cell effector genes that limit inflammatory responses after allogeneic HCT.ObjectivesWe sought to determine the role of miR-21 in regulating T-cell responses following allogeneic HCT using well-established animal models of GVHD and graft-vs-tumor GVT activity. Understanding pathways that distinguish desirable GVT effects from those that promote GVHD is critical to improve HCT outcomes.MethodsmiR-21 deficient mice (miR-21−/−) were used as donors in murine HCT models to characterize the impact of miR-21 loss on donor T-cell activation and GVHD severity. HCT recipients were followed for the development of systemic GVHD. In parallel experiments, splenic T-cells were harvested on days 7 and 14 and analyzed by multicolor flow cytometry to examine T-cell expansion, cytokine expression, and markers of cytotoxicity. Serum cytokine and chemokines were quantified using Luminex bead-based multiplex assays. Peripheral blood counts were also measured.ResultsCompared to recipients of WT cells, allo-HCT recipients of miR-21 KO donors exhibit a distinct phenotype characterized by accelerated, severe, GVHD, as measured by survival and clinical GVHD score. This phenotype was reproduced in replicate experiments in two strain combinations reflecting haploidentical and full MHC mismatched models (Fig 1), and the impact on target organ GVHD is under investigation. Graft failure was ruled-out as a contributor to early mortality. Allo-HCT recipients of miR-21 KO donors exhibited increased proinflammatory cytokine production and enhanced cytotoxicity markers, both on a per-cell basis as demonstrated by flow-cytometry and in the serum on days 7 and 14 after HCT (Fig 2).ConclusionsOur results reveal a critical role for miR-21 in regulating T-cell alloreactivity after HCT. Experiments are planned to interrogate the contribution of miR21 in the donor myeloid component to GVHD induction and in strain combinations wherein GVHD is induced specifically by CD4+ or CD8+ T-cells. Single cell RNAseq on splenic T-cell populations will uncover molecular pathways that control T-cell tolerance. Finally, the impact of miR21 on GVT activity will be determined. Insights generated from this line of investigation will illuminate targets for regulating donor immunity to mitigate GVHD and enhance GVT activity and improve outcomes after HCT.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (PD09)1 May 2024PD09-01 REGULATORY EFFECTS OF MIR-21 ON THE PROSTATE WITH OR WITHOUT TUMOR-BEARING CONDITIONS: A NOVEL PERSPECTIVE BASED ON EXTRACELLULAR VESICLES Cong Hu, Polina Sysa-Shah, Xinrui Wu, Sarah R. Amend, Wei Xue, Shawn E. Lupold, Kenneth J. Pienta, and Liang Dong Cong HuCong Hu , Polina Sysa-ShahPolina Sysa-Shah , Xinrui WuXinrui Wu , Sarah R. AmendSarah R. Amend , Wei XueWei Xue , Shawn E. LupoldShawn E. Lupold , Kenneth J. PientaKenneth J. Pienta , and Liang DongLiang Dong View All Author Informationhttps://doi.org/10.1097/01.JU.0001008572.33286.63.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Most studies have shown a negative correlation between miR-21 and the prognosis of prostate cancer (PCa) patients. Extracellular vesicles (EVs) play a pivotal role as a means of cell-to-cell communication at all stages of disease. Whether miR-21 can regulate extracellular vesicular contents to influence disease turnover is not elucidated. METHODS: In this study we separated EVs from ex vivo prostate tissue and urine of four mice models and performed mRNA profiling using the NanoString nCounter. Wild-type (WT) WT with miR-21 knockout (WT-miR21 KO), Hi-Myc transgenic (TG) model of PCa (Hi-Myc TG) and Hi-Myc TG with miR-21 knockout (Hi-Myc TG-miR21 KO) were used to investigate the regulation of EVs-containing mRNAs by miR-21 in physiological and pathological conditions of the prostate. RESULTS: Overall, tissue-derived EVs are more responsive to alterations mediated by miR-21 than urine EVs. More proliferation-associated mRNAs were enriched in EVs from Hi-Myc TG than WT. We found that miR-21 is capable of exerting indispensable effects on gland development and morphological maintenance of prostate with or without tumor-bearing conditions. More differentially carried transcripts in EVs were found when comparing Hi-Myc TG with Hi-Myc TG-miR21 KO, versus the comparison between WT and WT-miR21 KO, demonstrating miR-21 may play a more important role in PCa than in normal prostate, including but not limited to the modulation of drug resistance related to platinum and EGFR tyrosine kinase inhibitor. EV-derived Fen1 was identified to be a potential target to block the miR21-mediated cancer proliferation. CONCLUSIONS: Our results elucidate the effects exerted by miR-21 in prostate with or without tumor-bearing conditions based on EV mRNA profiling for the first time. Download PPT Source of Funding: NCI grants U54CA143803, CA163124, CA093900, and CA143055, and the Prostate Cancer Foundation © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e179 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Cong Hu More articles by this author Polina Sysa-Shah More articles by this author Xinrui Wu More articles by this author Sarah R. Amend More articles by this author Wei Xue More articles by this author Shawn E. Lupold More articles by this author Kenneth J. Pienta More articles by this author Liang Dong More articles by this author Expand All Advertisement PDF downloadLoading ...

Supplementary Figures S1-S2. S1. Schematic of the LimiNIR{trade mark, serif} System S2. PSMA expression in mouse epididymis and RT-PCR to validate the presence of PSMA mRNA

Supplementary Movie S2. The intensity of the signal is proportional to the power of the laser used. During the surgery we intentionally changed the intensity of the laser to observe the change in intensity of the signal from the kidney.

Supplementary Table S1: siRNA and shRNA Vectors Supplementary Table S2: The sequence of primers used in this study Supplementary Figure S1. miR-21 suppresses PDCD4 expression in normal prostate epithelial cells. Supplementary Figure S2. Androgen signaling and miR-21 suppresses PDCD4 mRNA expression. Supplementary Figure S3. Transient PDCD4 over-expression and PDCD4 knockdown. Supplementary Figure S4. Transient PDCD4-overexpression induces apoptosis of LAPC4, but not LNCaP, cells. Supplementary Figure S5. PDCD4 expression regulates cell proliferation and apoptosis.

Supplementary Movie S3. A PSMA positive xenograft pre-stained with YC-27 was placed behind the peritoneum of a pig during laparoscopic surgery. The xenograft could easily be detected with the help of the LumiNIR{trade mark, serif} system and removed with the NIR guidance.

Supplementary Movie S1. To model human laparoscopic tumor resection, athymic Nu/Nu mice harboring PSMA-positive xenografts were administered 19.1 μg/kg (HED of 100 μg for a 70 Kg male) YC-27 via tail vein injection. The mice were placed in a laparoscopic training box, and the PSMA-positive xenografts were resected 10 hours after injection of YC-27 with the guidance of the LumiNIR{trade mark, serif} system. Imaging was done using a 830nm laser to visualize the whole animal and 780nm laser to identify the PSMA-positive xenograft.

Supplementary Methods, Figures 1-5, Tables 1-2 from miR-21: An Androgen Receptor–Regulated MicroRNA that Promotes Hormone-Dependent and Hormone-Independent Prostate Cancer Growth

Table S1: Weight of the tumors;Figure S1: Correlation of in vivo NIR imaging and ex vivo biodistribution analysis; Figure S2: Evaluation of injected J591-conjugates; Figure S3: Assessment of tumor vascular permeability; Figure S4: Passive dye uptake in PSMA-positive and PSMA-negative tumors; Figure S5: Ex vivo tumor image analysis of LMD/PSMA xenograft model; Figure S6: Ex vivo biodistribution analysis of J591-conjugates in LMD tumor model; Figure S7: Ex vivo tumor image analysis of PC3/PSMA xenograft model; Figure S8: PSMA expression and J591 conjugate distribution

As a prototypical nuclear hormone receptor, the androgen receptor (AR) signals via a sequential cascade triggered by binding to androgenic ligands such as testosterone and dihydrotestosterone (DHT). This cascade includes dimerization of the ligand-receptor complex, nuclear translocation, chromatin binding to response elements, recruitment of TOP2B and co-activator complexes, and induction of an effector transcriptional program. In prostate cancers, this AR signaling cascade is an essential driver of growth and survival, yet its activity confers potential vulnerabilities through transient TOP2B-mediated DNA double strand breaks. We investigated the ability of non-steroidal AR ligands to activate initial steps of the AR signaling cascade up to the point of AR- and TOP2B-mediated double strand breaks, with subsequent arrest of the signaling cascade to prevent induction of pro-growth/survival transcriptional programs in prostate cancer cells. We identified hydroxyflutamide (FLU) as such an androgen receptor arrested agonist; in androgen-deprived conditions, FLU induced AR nuclear translocation, chromatin binding, and TOP2B-mediated double strand breaks, but failed to induce AR target gene expression and prostate cancer cell growth. The FLU-mediated arrest in the signaling cascade could be attributed to the inability of FLU to allow association of AR with SMARCD2, a critical component of the BAF chromatin remodeling complex required for androgen induced AR co-activation. Interestingly, the FLU-induced, AR- and TOP2B-mediated double strand breaks could be used to selectively sensitize AR-positive prostate cancer cells to ionizing radiation in vitro and in vivo . These findings support a novel arrested agonist paradigm for selective radiosensitization of prostate cancer cells without inducing AR-mediated pro-growth and survival transcriptional programs.