Androgen Receptor Signaling Pathway Research Articles

PSTAT3 Expression is Increased in Advanced Prostate Cancer in Post-Initiation of Androgen Deprivation Therapy.

The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) plays a role in carcinogenesis and is involved in processes, such as proliferation, differentiation, drug resistance and immunosuppression. STAT3 can be activated by phosphorylation of tyrosine at position 705 (pSTAT3Tyr705) or serine at 727 (pSTAT3Ser727). High expression levels of pSTAT3 are implicated in advanced stages of prostate cancer (PCa) and are known to interact with the androgen receptor signaling pathway. However, not much is known about how androgen deprivation therapy (ADT) in advanced disease affects pSTAT3 expression. The aim of this study was to determine the influence of ADT on pSTAT3 expression in PCa tissue. The study cohort came from a PCa tissue microarray resource containing prostate specimens from patients before and post-initiation of ADT. Tissue samples from 111 patients were immunostained for pSTAT3Tyr705 and pSTAT3Ser727. H-score was used to evaluate the intensity and the percentage of pSTAT3 expression in malignant epithelial and stromal compartments. Univariate and multivariable Cox regression analyses were used to assess pSTAT3Tyr705 and pSTAT3Ser727 as biomarkers of oncological outcome in patients undergoing ADT. Post-ADT PCa samples demonstrated increased nuclear and cytoplasmic levels of pSTAT3Ser727 in the stroma compared to pre-ADT samples, whereas pSTAT3Tyr705 expression was increased significantly in both stromal and malignant epithelial compartments except for stromal cytoplasm. High cytoplasmic pSTAT3Ser727 in stromal compartments correlated with reduced overall survival, shorter time to castration-resistant PCa development, and decreased metastasis-free survival. An increase in nuclear and cytoplasmic pSTAT3Ser727 expression within the stromal compartment of post-ADT samples corresponded to a shorter time to CRPC development, which was not observed for pSTAT3Tyr705. Multivariable survival analysis using Cox's regression identified that high cytoplasmic pSTAT3Ser727 expression in the stroma of post-ADT samples and pT3 or pT4-stage were associated with worse overall survival and 5-year metastasis-free survival (MFS). This study presents novel insights into the impact of ADT on the expression levels of pSTAT3Tyr705 and pSTAT3Ser727 in PCa. Cytoplasmic pSTAT3Ser727 status of cancer-associated stromal cells in post-ADT samples may serve as an independent prognostic marker for OS and 5-year MFS, identifying prostate cancer patients prone to developing resistance to ADT.

The impact of androgen-induced translation in modulating androgen receptor activity.

Dysregulated androgen receptor (AR) activity is central to various diseases, particularly prostate cancer (PCa), in which it drives tumour initiation and progression. Consequently, antagonising AR activity via anti-androgens is an indispensable treatment option for metastatic PCa. However, despite initial tumour remission, drug resistance occurs. Therefore, the AR signalling pathway has been intensively investigated. However, the role of AR protein stability in AR signalling and therapy resistance has not yet been deciphered. Therefore, this study aimed to investigate the role of AR protein changes in transactivity and assess its mechanism as a possible target in PCa. LNCaP, C4-2, and 22Rv1 cells were treated with R1881, enzalutamide, cycloheximide, and Rocaglamide. Mass spectrometry analyses were performed on LNCaP cells to identify the pathways enriched by the treatments. Western blotting was performed to investigate AR protein levels and localisation changes. Changes in AR transactivity were determined by qPCR. Mass spectrometry analyses were performed on LNCaP cells to decipher the molecular mechanisms underlying androgen- and antiandrogen-induced alterations in the AR protein. Pathway analysis revealed the enrichment of proteins involved in different pathways that regulate translation. Translational and proteasome inhibitor experiments revealed that these AR protein changes were attributable to modifications in translational activity. Interestingly, the effects on AR protein levels in castration-resistant PCa (CRPC) cells C4-2 or enzalutamide-resistant cells 22Rv1 were less prominent and non-existent. This outcome was similarly observed in the alteration of AR transactivation, which was suppressed in hormone-sensitive prostate cancer (HSPC) LNCaP cells by translational inhibition, akin to the effect of enzalutamide. In contrast, treatment-resistant cell lines showed only a slight change in AR transcription. This study suggests that in HSPC, AR activation triggers a signalling cascade that increases AR protein levels by enhancing its translation rate, thereby amplifying AR activity. However, this mechanism appears to be dysregulated in castration-resistant PCa cells.

Loss of PI5P4Kα slows the progression of a Pten mutant basal cell model of prostate cancer.

While early prostate cancer (PCa) depends on the androgen receptor (AR) signaling pathway, which is predominant in luminal cells, there is much to be understood about the contribution of epithelial basal cells in cancer progression. Herein, we observe cell-type specific differences in the importance of the metabolic enzyme phosphatidylinositol 5-phosphate 4-kinase alpha (PI5P4Kα β ; gene name PIP4K2A) in the prostate epithelium. We report the development of a basal-cell-specific genetically engineered mouse model (GEMM) targeting Pip4k2a alone or in combination with the tumor suppressor phosphatase and tensin homolog (Pten). PI5P4Kα is enriched in basal cells, and no major histopathological changes were detectable following gene deletion. Notably, the combined loss of Pip4k2a slowed the development of Pten mutant mouse prostatic intraepithelial neoplasia (mPIN). Through the inclusion of a lineage tracing reporter, we utilize single-cell RNA sequencing to evaluate changes resulting from in vivo downregulation of Pip4k2a and characterize cell populations influenced in the established Probasin-Cre and Cytokeratin 5 (CK5)-Cre driven GEMMs. Transcriptomic pathway analysis points towards the disruption of lipid metabolism as a mechanism for reduced tumor progression. This was functionally supported by shifts of carnitine lipids in LNCaP PCa cells treated with siPIP4K2A. Overall, these data nominate PI5P4Kα as a target for PTEN mutant PCa. Implications: PI5P4Kα is enriched in prostate basal cells and its targeted loss slows the progression of a model of advanced PCa.

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

MYBL2 Drives Prostate Cancer Plasticity: Inhibiting Its Transcriptional Target CDK2 for RB1-Deficient Neuroendocrine Prostate Cancer.

Prostate cancers that escape therapy targeting the androgen receptor signaling pathways via phenotypic plasticity are currently untreatable. Our study identifies MYBL2 as a MR-TF in phenotypic plastic prostate cancer and implicates CDK2 inhibition as a novel therapeutic target for this most lethal subtype of prostate cancer.

Interaction of Bisphenol A and Its Analogs with Estrogen and Androgen Receptor from Atlantic Cod (Gadus morhua).

The widespread use of bisphenol A (BPA) in polycarbonate plastics and epoxy resins has made it a prevalent environmental pollutant in aquatic ecosystems. BPA poses a significant threat to marine and freshwater wildlife due to its documented endocrine-disrupting effects on various species. Manufacturers are increasingly turning to other bisphenol compounds as supposedly safer alternatives. In this study, we employed in vitro reporter gene assays and ex vivo precision-cut liver slices from Atlantic cod (Gadus morhua) to investigate whether BPA and 11 BPA analogs exhibit estrogenic, antiestrogenic, androgenic, or antiandrogenic effects by influencing estrogen or androgen receptor signaling pathways. Most bisphenols, including BPA, displayed estrogenic properties by activating the Atlantic cod estrogen receptor alpha (gmEra). BPB, BPE, and BPF exhibited efficacy similar to or higher than that of BPA, with BPB and BPAF being more potent agonists. Additionally, some bisphenols, like BPG, induced estrogenic effects in ex vivo liver slices despite not activating gmEra in vitro, suggesting structural modifications by hepatic biotransformation enzymes. While only BPC2 and BPAF activated the Atlantic cod androgen receptor alpha (gmAra), several bisphenols exhibited antiandrogenic effects by inhibiting gmAra activity. This study underscores the endocrine-disrupting impact of bisphenols on aquatic organisms, emphasizing that substitutes for BPA may pose equal or greater risks to both the environment and human health.

Exploring the interplay between circadian rhythms and prostate cancer: insights into androgen receptor signaling and therapeutic opportunities.

Circadian rhythm disruption is closely related to increased incidence of prostate cancer. Incorporating circadian rhythms into the study of prostate cancer pathogenesis can provide a more comprehensive understanding of the causes of cancer and offer new options for precise treatment. Therefore, this article comprehensively summarizes the epidemiology of prostate cancer, expounds the contradictory relationship between circadian rhythm disorders and prostate cancer risk, and elucidates the relationship between circadian rhythm regulators and the incidence of prostate cancer. Importantly, this article also focuses on the correlation between circadian rhythms and androgen receptor signaling pathways, as well as the applicability of time therapy in prostate cancer. This may prove significant in enhancing the clinical treatment of prostate cancer.

Testosterone regulates thymic remodeling by activating glucocorticoid receptor signaling pathway to accelerate thymocyte apoptosis in male rats

Thymic atrophy affects T cell generation and migration to the periphery, thereby affecting T cell pool diversity. However, the mechanisms underlying thymic atrophy have not been fully elucidated. Here, gonadotropin-releasing hormone (GnRH) immunization and surgical castration did not affect thymocyte proliferation, but significantly reduced the apoptosis and increased the survival rate of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes. Following testosterone supplementation in rats subjected to GnRH immunization and surgical castration, thymocyte proliferation remained unchange, but the apoptosis of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes significantly increased. Transcriptome analyses of the thymus after GnRH immunization and surgical castration showed a significant reduction in the thymus’s response to corticosterone. Cholesterol metabolism and the synthesis and secretion of corticosterone were significantly reduced. Analysis of the enzyme levels involved in the corticosterone synthesis pathway revealed that corticosterone synthesis in thymocytes was significantly reduced after GnRH immunization and surgical castration, whereas exogenous testosterone supplementation relieved this process. Testosterone promoted thymocyte apoptosis in a concentration-dependent manner, and induced corticosterone secretion in vitro. Blocking the intracellular androgen receptor (AR) signaling pathway did not significantly affect thymocyte apoptosis, but blocking the glucocorticoid receptor (GR) signaling pathway significantly reduced it. Our findings indicate that testosterone regulates thymus remodeling by affecting corticosterone synthesis in thymocytes, which activates GR signal transduction and promotes thymocyte apoptosis.

Reciprocal regulation between RACGAP1 and AR contributes to endocrine therapy resistance in prostate cancer

BackgroundEndocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors.MethodsBioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth.ResultsRACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa.ConclusionIn summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.

Abstract PS15-07: Proxalutamide plus Endocrine Therapies in Women with HR+/HER2-/AR+ Metastatic Breast Cancer: A Phase Ic Study

Abstract Abstract Background: Resistance to endocrine therapy (ET) and cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) is a major obstacle to the management of hormone-receptor (HR)+/human epidermal growth factor receptor 2 (HER2)- metastatic breast cancer (mBC). Targeting the AR signaling pathway has been demonstrated with promising results in this population. This phase Ic study aimed to assess the safety, efficacy, pharmacokinetic (PK), and pharmacodynamic (PD) characteristics of the combination of proxalutamide, a high-affinity AR antagonist, with ETs in androgen receptor (AR)+/HR+/HER2- mBC. Methods: Patients in part 1 regimen-finding phase received proxalutamide plus specific ETs (letrozole [cohort A], exemestane [cohort B], or fulvestrant [cohort C]) and were assessed for dose-limiting toxicity (DLT), PK, PD, and anti-tumor activity. Part 2 expansion phase evaluated the safety and efficacy of proxalutamide plus fulvestrant. The primary endpoint was safety and tolerability. Results: Between June 18, 2019, and Sep 15, 2022, 38 (18 in part 1 and 20 in part 2) patients were treated. Efficacy analysis indicated that the ORR was 16.7% (2/12, 95% CI, 2.1%-48.4%) for cohort C and 15.0% (3/20; 95% CI, 3.2%-37.9%) for part 2. Patients achieved a median progression-free survival (PFS) of 6.4 months (95% CI, 2.7-19.3) in cohort C while an mPFS of 11.0 months (95% CI, 5.5- not estimable) in part 2. In part 1, no DLTs were reported. Grade 3 or more treatment-emergent adverse events (TEAEs) were observed in 11 (29.7%) patients (7 [18.9%] in part 1 and 4 [10.8%] in part 2) and mainly included neutrophil count decreased (8.1%) and platelet count decreased (5.4%). Seven (18.9%) had serious AEs (4 [10.8%] in part 1 and 3 [8.1%] in part 2)[1]. PD results showed a greater reduction of estradiol in cohort C compared with that in cohort B. In addition, proxalutamide was absorbed rapidly into the body with the plasma concentrations of proxalutamide and metabolites reaching a nearly steady state on day 29. Patients with AR/ER of ≤1 seemed to achieve longer PFS over those of >1 (8.4 months vs. 4.1 months). Conclusions: These findings suggested favorable clinical outcomes and safety profiles of the combination of proxalutamide and fulvestrant in AR+/HR+/HER2- mBC patients who have progressed on the first-line therapy, and maybe with better efficacy in patients with lower AR/ER ratio. Trial registration: NCT20191063 Citation Format: Hui-Ping Li, Guohong Song, Hanfang Jiang, Xu Liang, Xiaojia Wang, Hua Yang, Lili Zhang, Youzhi Tong. Proxalutamide plus Endocrine Therapies in Women with HR+/HER2-/AR+ Metastatic Breast Cancer: A Phase Ic Study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS15-07.

MicroRNA (miRNA) PROFILING IN PROSTATE CANCER CARCINOGENESIS: EXPLORATORY RESEARCH

Future research will continue to be carried out in terms of looking for molecular markers involved in the early processes of carcinogenesis, metastasis and therapeutic targets in prostate cancer patients. MicroRNAs (miRNAs) are small noncoding RNAs that contribute to prostate cancer progression by controlling genes involved in the androgen receptor (AR) signaling pathway, ectopic expression of proteins involved in the cell cycle and apoptosis, epithelial-mesenchymal transition (EMT) and metastasis of cancer stem cells (CSCs). This study was an exploratory study, the aim was to assess miRNA expression in low histopathological grade prostate cancer compared with high histopathological grade prostate cancer. The novelty of this research is to assess the 25 most increased (up-regulated) and 25 lowest (down-regulated) miRNAs from 800 types of miRNA. Next, we will analyze the mechanisms and confirm the carcinogenesis of prostate cancer. The research results can be used for further research data, especially for targeted therapy in prostate cancer patients.

Exploring key genes and pathways associated with sex differences in autism spectrum disorder: integrated bioinformatic analysis.

Autism spectrum disorder (ASD) is a heterogenous neurodevelopmental disorder marked by functional abnormalities in brain that causes social and linguistic difficulties. The incidence of ASD is more prevalent in males compared to females, but the underlying mechanism, as well as molecular indications for identifying sex-specific differences in ASD symptoms remain unknown. Thus, impacting the development of personalized strategy towards pharmacotherapy of ASD. The current study employs an integrated bioinformatic approach to investigate the genes and pathways uniquely associated with sex specific differences in autistic individuals. Based on microarray dataset (GSE6575) extracted from the gene expression omnibus, the dysregulated genes between the autistic and the neurotypical individuals for both sexes were identified. Gene set enrichment analysis was performed to ascertain biological activities linked to the dysregulated genes. Protein-protein interaction network analysis was carried out to identify hub genes. The identified hub genes were examined to determine their functions and involvement in the associated pathways using Enrichr. Additionally, hub genes were validated from autism-associated databases and the potential small molecules targeting the hub genes were identified. The present study utilized whole blood transcriptomic gene expression analysis data and identified 2211 and 958 differentially expressed unique genes in males and females respectively. The functional enrichment analysis revealed that male hub genes were functionally associated with RNA polymerase II mediated transcriptional regulation whereas female hub genes were involved in intracellular signal transduction and cell migration. The top male hub genes exhibited functional enrichment in tyrosine kinase signalling pathway. The pathway enrichment analysis of male hub genes indicates the enrichment of papillomavirus infection. Female hub genes were enriched in androgen receptor signalling pathway and functionally enriched in focal adhesion specific excision repair. Identified drug like candidates targeting these genes may serve as a potential sex specific therapeutics. Wortmannin for males, 5-Fluorouracil for females had the highest scores. Targeted and sex-specific pharmacotherapies may be created for the management of ASD. The current investigation identifies sex-specific molecular signatures derived from whole blood which may serve as a potential peripheral sex-specific biomarkers for ASD. The study also uncovers the possible pharmacological interventions against the selected genes/pathway, providing support in development of therapeutic strategies to mitigate ASD. However, experimental proofs on biological systems are warranted.

Abstract 3266: Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer

Abstract The androgen receptor (AR) signaling pathway plays critical role in the development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC). As a result, androgen signaling inhibitors (ASIs) like abiraterone and enzalutamide (ENZ) have emerged as the first-line treatment for CRPC. Nevertheless, the limited success of ASIs underscores the urgency of developing new approaches for managing CRPC patients who have become unresponsive to ASIs. Programmed cell death 4 (Pdcd4) is a tumor suppressor, which is frequently down-regulated during PCa progression. It has been shown that reduced Pdcd4 expression promotes both AR-dependent and AR-independent growth in PCa. In this study, we found that down-regulation of Pdcd4 led to acquisition of ENZ resistance in PCa. We also observed that down-regulation of Pdcd4 resulted in activation of mTORC2 activity. Additionally, knockdown of Rictor, a component of mTORC2 complex, enhanced sensitivity to ENZ treatment in PCa. These findings suggested that activation of mTORC2 due to Pdcd4 loss contributes to ASI resistance in PCa. To understand how Pdcd4 is down-regulated in the PCa, we identified that polo-like kinase (Plk1) phosphorylated Pdcd4 at Ser239. Phosphorylation of Pdcd4 by Plk1 led to ubiquitin-mediated proteasomal degradation of Pdcd4. Over-expression of the phosphomimic Pdcd4(S239D) in 22Rv1 cells promoted tumor cell growth in cultured cells and mice. Taken together, our data suggest that targeting Plk1/Pdcd4/mTORC2 signaling axis holds promise as a strategy to overcome ENZ resistance in PCa. Citation Format: Qing Wang, Qiongsi Zhang, Yanquan Zhang, Xiaoqi Liu, Hsin-Sheng Yang. Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in 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 3266.

Abstract 2147: Genetic ancestry associations with prostate adenocarcinoma mutational profiles: New Insights from a diverse 5,959-patient real-world cohort

Abstract In prostate cancer, Black men face higher incidence rates, more advanced diagnoses and worse outcomes than White men. These disparities partially stem from environmental, socioeconomic, and healthcare access factors. Recent data highlight racial differences in prostate cancer molecular profiles, with tumors from Black men showing increased SPOP and decreased PIK3CA mutations and amplified androgen receptor signaling and inflammatory pathway activations. Thus, somatic mutation patterns may also contribute to these disparities. We sought to identify associations of mutational profiles in prostate adenocarcinoma (PRAD) with genetic ancestry instead of race and ethnicity categories, analyzing a de-identified cohort of 5,959 patients who underwent the Tempus xT 648-gene NGS tumor profiling test. We used 654 ancestry-informative markers to estimate genetic ancestry by calculating similarity to reference populations for five regions: Africa (AFR), Americas (AMR), East Asia (EAS), Europe (EUR), and South Asia (SAS). Associations between genetic ancestry proportions and somatic variants were assessed for 67 PRAD-related genes using logistic regression models. Analyses included copy number alterations (CNAs), oncogenic gene fusions, small splice and non-synonymous mutations (NS), OncoKB L1/2 & R1, and small missense driver somatic mutations predicted with boostDM. Likelihood ratio tests were employed to compare models with and without ancestry terms. The Benjamini-Hochberg method was utilized to adjust p-values and control the FDR at 5%. We confirmed reported associations in PRAD between AFR ancestry and SPOP NS mutations (OR=1.03 per doubling of AFR ancestry proportion, p=0.03) and decreased AFR with PIK3CA OncoKB and driver mutations (OR=0.94, p=0.02), PTEN CNAs (OR=0.95, p=0.0001), and TMPRSS2-ERG fusions (OR=0.95, p<0.0001). However, we also observed negative associations between AFR and AR CNAs (OR=0.95, p=0.004), EAS and PTEN CNAs (OR=0.96, p=0.01), and EAS and TMPRSS2-ERG fusions (OR=0.97, p=0.02). Additionally, a negative association was found between EAS and TP53 driver NS mutations (OR=0.96, p=0.04), while positive associations with driver mutations in FOXA1 (OR=1.06, p=0.03) and NS mutations in the PI3K/AKT/mTOR pathway genes (OR=1.04, p=0.01) were noted. Lastly, we identified novel positive associations between AMR and driver mutations in PIK3C3A (OR=1.12, p=0.0003) as well as small NS mutations or CNAs in the PI3K/AKT/mTOR pathway genes (OR=1.03, p=0.04). By analyzing a large, diverse real-world cohort and leveraging NGS-inferred genetic ancestry, our study confirms known associations between somatic alterations in PRAD cancer genes and race and ethnicity, while unveiling novel associations in understudied populations of potential significance for understanding disparities in disease outcomes. Citation Format: Brooke Rhead, Yannick Pouliot, Francisco M. De La Vega, David W. Craig, John Carpten. Genetic ancestry associations with prostate adenocarcinoma mutational profiles: New Insights from a diverse 5,959-patient real-world cohort [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 2147.

Abstract 7525: Investigating B7-H3 as a checkpoint and antibody drug conjugate target across prostate cancer variants- both androgen receptor dependent and independent

Abstract Background: CD276 (B7-H3) has recently emerged as a promising presumptive immune checkpoint inhibitor (ICI) and antibody-drug conjugate (ADC) target for PCa. Unfortunately, current immunotherapy trials have yielded few objective responses in PCa and hormonal therapy seldom cures necessitating the need to develop new immunotherapy or targeted antibody-drug conjugate (ADC) treatment options for PCa, including B7-H3 targeted approaches. Method: In this work, we evaluated important ADC targets and immune checkpoints (PD-1, PD-L1, PD-L2, LAG3, TIGIT, OX40, 4-1BB, CTLA4, STEAP1, STEAP2, PSMA, NECTIN1, TROP-2, DLL3 and B7-H3) in various PCa cell lines and multiple patients spanning various subtypes of PCa using publicly available bulk RNA sequencing data and single-cell RNA sequencing (scRNA-seq) data respectively. The results were validated at RNA and protein levels in listed cell lines. Further, the impact of androgen receptor (AR) signaling on B7-H3 expression was quantified using charcoal stripped media, AR agonist (R1881) and antagonist (enzalutamide) using real-time PCR and flow cytometry at RNA and protein levels respectively. Results: We found that B7-H3 shows high expression and very low variability compared to other ADC targets and immune checkpoints in AR-positive (LnCAP), hormone-refractory (VCAP), Castrate resistant (22RVI), AR negative (PC3, DU145), and neuroendocrine (H660) cell lines, as well as in—PCa cells and tumor microenvironment (TME) myeloid cells—of multiple patients spanning various subtypes of PCa. Western blotting confirms the ubiquitous presence of B7-H3 in all these cell lines, even when PSMA is absent. Furthermore, B7-H3 expression was modifiable via androgen depletion, R1881 supplementation, or addition of the AR antagonist enzalutamide. These changes were significant in AR-positive LnCAP cells compared to AR-negative DU145 cells further endorsing the crosstalk between AR and B7-H3 signaling pathways, which will be presented in greater detail. Conclusion: This work explores the therapeutic relevance of important ADC targets and immune checkpoints for Prostate Cancer (PCa), highlighting B7-H3 as a unique therapeutic ADC and ICI candidate across the continuum of PCa—androgen sensitive, castration resistant, and neuroendocrine—due to its stable expression across various subtypes and on TME myeloid cells. The relationship between AR and B7-H3 opens the possibility of future combination therapies based on this interaction. Citation Format: Nikita Mundhara, Shivang Sharma, Emirhan Tekoglu, Ezra G. Baraban, Nathan A. Lack, Eugene Shenderov. Investigating B7-H3 as a checkpoint and antibody drug conjugate target across prostate cancer variants- both androgen receptor dependent and independent [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 7525.

Structure-Activity Relationship (SAR) Studies of Novel Monovalent AR/AR-V7 Dual Degraders with Potent Efficacy against Advanced Prostate Cancer.

Androgen receptor (AR) has been extensively established as a potential therapeutic target for nearly all stages of prostate cancer (PCa). However, acquired resistance to AR-targeted drugs inevitably develops and severely limits their clinical efficacy. Particularly, there currently exists no efficient treatment for patients expressing the constitutively active AR splice variants, such as AR-V7. Herein, we report the structure-activity relationship studies of 55 N-heterocycle-substituted hydantoins, which identified the structural motifs required for AR/AR-V7 degradation. Among them, the most potent compound 27c exhibited selective AR/AR-V7 degradation over other hormone receptors and excellent antiproliferative activities in LNCaP and 22RV1 cells. RNA sequence analysis confirmed that 27c effectively suppressed transcriptional activity of the AR signaling pathway. Importantly, 27c demonstrated potent antitumor efficacy in an enzalutamide-resistant 22RV1 xenograft model. These results highlight the potential of 27c as a promising dual AR/AR-V7 degrader for overcoming drug resistance in advanced PCa expressing AR splice variants.

AKR1C3 in carcinomas: from multifaceted roles to therapeutic strategies.

Aldo-Keto Reductase Family 1 Member C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5) or prostaglandin F (PGF) synthase, functions as a pivotal enzyme in androgen biosynthesis. It catalyzes the conversion of weak androgens, estrone (a weak estrogen), and PGD2 into potent androgens (testosterone and 5α-dihydrotestosterone), 17β-estradiol (a potent estrogen), and 11β-PGF2α, respectively. Elevated levels of AKR1C3 activate androgen receptor (AR) signaling pathway, contributing to tumor recurrence and imparting resistance to cancer therapies. The overexpression of AKR1C3 serves as an oncogenic factor, promoting carcinoma cell proliferation, invasion, and metastasis, and is correlated with unfavorable prognosis and overall survival in carcinoma patients. Inhibiting AKR1C3 has demonstrated potent efficacy in suppressing tumor progression and overcoming treatment resistance. As a result, the development and design of AKR1C3 inhibitors have garnered increasing interest among researchers, with significant progress witnessed in recent years. Novel AKR1C3 inhibitors, including natural products and analogues of existing drugs designed based on their structures and frameworks, continue to be discovered and developed in laboratories worldwide. The AKR1C3 enzyme has emerged as a key player in carcinoma progression and therapeutic resistance, posing challenges in cancer treatment. This review aims to provide a comprehensive analysis of AKR1C3's role in carcinoma development, its implications in therapeutic resistance, and recent advancements in the development of AKR1C3 inhibitors for tumor therapies.

Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Increased PI3K pathway activity is associated with recurrent breast cancer in patients with low and intermediate 21-gene recurrence score

AimsWe investigated key signalling pathways’ activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence.MethodsThis is...