Castration-resistant Prostate Cancer Metastases Research Articles

Nuclear receptor TLX functions to promote cancer stemness and EMT in prostate cancer via its direct transactivation of CD44 and stem cell-regulatory transcription factors.

Prostate cancer stem cells (PCSCs) play crucial roles in therapy-resistance and metastasis in castration-resistant prostate cancer (CRPC). Certain functional link between cancer stemness and epithelial-mesenchymal transition (EMT) is involved in CRPC. However, up-stream regulators controlling these two processes in PCSCs are still poorly understood. Recently, we have shown that orphan nuclear receptor TLX can promote tumour initiation and progression in CRPC by repressing androgen receptor and oncogene-induced senescence. PCSCs were isolated from various prostate cancer cell lines and clinical tumour tissues using multiple methods for various in vitro and in vivo oncogenic growth analyses. Direct targets of TLX involved in stemness and EMT regulation were determined by specific reporter gene assays and ligand-driven modulation of TLX activity. PCSCs isolated from various sources exhibited increased expression of TLX. Functional and molecular characterisation showed that TLX could function to promote cancer stemness and EMT in prostate cancer cells via its direct transactivation of CD44, SOX2, POU5F1 and NANOG, which share certain functional crosstalk in these two cellular processes. TLX could act as a key up-stream regulator in transcriptional control of stemness and EMT in PCSCs, which contribute to their tumorigenicity, castration-resistance and metastasis potentials in advanced prostate cancer.

Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs.

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

Abstract 495: Targeting undruggable transcription factor HOXB13 in metastatic prostate cancer by CRISPR/Cas13d-based nanoparticle therapy

Abstract Background: The oncogenic transcription factor HOXB13, which is highly expressed in castration-resistant prostate cancer (CRPC), has been shown to promote CRPC growth and metastasis. However, HOXB13 is considered untargetable by traditional small-molecule-based drug design. Gene therapy is critical alternative strategy with potential to directly target such traditionally undruggable genes. Methods: Selective cell in organ targeting (SCORT) nanoparticles for precise delivery of nanoparticles to metastatic cancer cells in an organ were constructed and characterized by the NanoAssemblr Spark, Zetasizer Nano ZS, and transmission electron microscopy (TEM); Cas13d mRNA with pseudouridine modification was synthesized using in vitro transcription; Hemi-spleen injection of CRPC cells was performed to build CRPC liver metastatic models; RNA-seq was employed to evaluate off-target effects of Cas13d targeting. Results: By incorporating a prostate cancer-specific E3 aptamer to functionalized lipid-like nanoparticles (FTT5 LNP-E3), we have successfully constructed SCORT nanoparticles that enable preferential delivery of mRNAs to metastatic CRPC cells in the liver, as opposed to various normal cells in the surrounding liver tissue. We demonstrated that Cas13d-pre-gHOXB13 mediates highly effective and specific HOXB13 mRNA knockdown in CRPC cells. Significantly, systemic treatment of SCORT nanoparticles carrying Cas13d-pre-gHOXB13 mRNA decreased HOXB13 expression in the metastatic tumors, inhibited metastasis, and prolonged survival of mice bearing androgen receptor (AR)-positive (AR+) or negative (AR-) tumors. Notably, long term (6 and a half weeks) administration of SCORT nanoparticle-Cas13d-pre-gHOXB13 did not significant alter body weight, hepatic and renal function, chemokines and cytokines, and other factors, effectively highlighting its safety. Conclusions: This study is the first to demonstrate that undruggable oncogenic transcription factors can be targeted using nanoparticle-delivered gene therapy based on CRISPR/Cas13 RNA-targeting. The SCORT-CRISPR/Cas13d system is a highly flexible technology that would impact the larger field of translational science by allowing the design of Cas13d-based, cancer cell-specific delivered nanoparticles targeting other previously-undruggable oncogenic transcription factors in metastatic prostate cancer and other solid tumors. Citation Format: Zhifen Cui, Furong Huang, Kun Fang, Jingyue Yan, Yufan Zhou, Jeffrey Everitt, Yue Zhao, William Hankey, Zhong Chen, Hongyan Wang, Victor X. Jin, Yizhou Dong, Qianben Wang. Targeting undruggable transcription factor HOXB13 in metastatic prostate cancer by CRISPR/Cas13d-based nanoparticle therapy [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 495.

Raltitrexed induces apoptosis through activating ROS-mediated ER stress by impeding HSPA8 expression in prostate cancer cells

Prostate cancer is the most common malignant tumor in males, which frequently develops into castration-resistant prostate cancer (CRPC). CRPC metastasis is the main reason for its high mortality rate. At present, it lacks effective treatment for patients with CRPC. Raltitrexed (RTX) has been shown to be effective in the treatment of colorectal cancer. However, the effect of RTX on prostate cancer and the underlying mechanism remain unknown. In the current study, we found that RTX could dose-dependently inhibit proliferation, migration, colony formation and induce apoptosis in DU145 and PC-3 cells. RTX also increased ROS generation in prostate cancer cells. Pretreatment with N-acetyl-L-cysteine (NAC) significantly prevented RTX-induced cell apoptosis and endoplasmic reticulum (ER) stress signaling activation in prostate cancer cells. Additionally, we found RTX-induced ROS generation and ER stress activation depended on the expression of heat shock protein family A member 8 (HSPA8). Over-expression of HSPA8 could alleviate RTX-induced cell apoptosis, ROS generation and ER stress signaling activation. Finally, our study also showed that RTX attenuated the tumor growth of prostate cancer in the DU145 xenograft model and significantly downregulated HSPA8 expression and activated ER stress signaling pathway in tumor tissues. Our study is the first to reveal that RTX induces prostate cancer cells apoptosis through inhibiting the expression of HSPA8 and further inducing ROS-mediated ER stress pathway action. This study suggests that RTX may be a novel promising candidate drug for prostate cancer therapy.

Development and Validation of a New BAG-1L–Specific Antibody to Quantify BAG-1L Protein Expression in Advanced Prostate Cancer

BCL-2–associated athanogene-1L (BAG-1L) is a critical co-regulator that binds to and enhances the transactivation function of the androgen receptor, leading to prostate cancer development and progression. Studies investigating the clinical importance of BAG-1L protein expression in advanced prostate cancer have been limited by the paucity of antibodies that specifically recognize the long isoform. In this study, we developed and validated a new BAG-1L–specific antibody using multiple orthogonal methods across several cell lines with and without genomic manipulation of BAG-1L and all BAG-1 isoforms. Following this, we performed exploratory immunohistochemistry to determine BAG-1L protein expression in normal human, matched castration-sensitive prostate cancer (CSPC) and castration-resistant prostate cancer (CRPC), unmatched primary and metastatic CRPC, and early breast cancer tissues. We demonstrated higher BAG-1L protein expression in CRPC metastases than in unmatched, untreated, castration-sensitive prostatectomies from men who remained recurrence-free for 5 years. In contrast, BAG-1L protein expression did not change between matched, same patient, CSPC and CRPC biopsies, suggesting that BAG-1L protein expression may be associated with more aggressive biology and the development of castration resistance. Finally, in a cohort of patients who universally developed CRPC, there was no association between BAG-1L protein expression at diagnosis and time to CRPC or overall survival, and no association between BAG-1L protein expression at CRPC biopsy and clinical outcome from androgen receptor targeting therapies or docetaxel chemotherapy. The limitations of this study include the requirement to validate the reproducibility of the assay developed, the potential influence of pre-analytical factors, timing of CRPC biopsies, relatively small patient numbers, and heterogenous therapies on BAG-1L protein expression, and the clinical outcome analyses performed. We describe a new BAG-1L–specific antibody that the research community can further develop to elucidate the biological and clinical significance of BAG-1L protein expression in malignant and nonmalignant diseases.

Glucocorticoid receptor-induced non-muscle caldesmon regulates metastasis in castration-resistant prostate cancer

Lethal prostate cancer (PCa) is characterized by the presence of metastases and development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. Here, we report that in vitro downregulation of l-CaD promotes epithelial phenotype and reduces spheroid growth in 3D, which is reflected in vivo in reduced formation of metastases in zebrafish PCa xenografts. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition (EMT) transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets, and GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation.

Abstract A024: Glucocorticoid receptor-induced non-muscle caldesmon regulates growth and metastasis in castration-resistant prostate cancer

Abstract Lethal prostate cancer (PCa) is characterized by the presence of metastases and the development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. The goal of our study was to characterize the potential role of l-CaD in PCa metastases and antiandrogen therapy resistance. In this study, we extracted co-expression data of l-CaD from the largest public PCa patient data sets and identified the common transcripts between the sets. The common co-expression hits were used to recognize biological processes associated with l-CaD in the PCa context. Next, we used in vitro techniques, including 3D culture, cell viability assays, immunofluorescence staining, and Western blotting, to study the role of l-CaD in PCa with a focus on cancer hallmarks and the recognized associated biological processes. Finally, we used in vivo zebrafish and mouse xenograft models to verify the findings observed in silico in PCa patient data and in vitro in PC3, DU145, and VCaP cell lines. Here, we report that in vitro downregulation of l-CaD promotes the epithelial phenotype and reduces the spheroid growth in 3D, which is reflected in vivo in the reduced formation of metastases in a zebrafish PCa xenograft model. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets and that GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation. Citation Format: Verneri Virtanen, Kreetta Paunu, Antti Kukkula, Saana Niva, Ylva Junila, Mervi Toriseva, Terhi Jokilehto, Sari Mäkelä, Riikka Huhtaniemi, Matti Poutanen, Ilkka Paatero, Maria Sundvall. Glucocorticoid receptor-induced non-muscle caldesmon regulates growth and metastasis in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A024.

Genetic profile of bone metabolism and immunity modulators in bone metastasis in castration-resistant prostate cancer

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract 1077: Porcupine inhibition enhances enzalutamide efficacy in drug resistant prostate cancer

Abstract Androgen receptor (AR) signaling continues to participate as a vital component of castration-resistant prostate cancer (CRPC). Subsequently, this has led to the development of Androgen Signaling Inhibitors (ASI), specifically Enzalutamide (ENZ), which is a direct inhibitor of AR, to clinically manage CRPC. Inevitably, ENZ treatment only provides improvement for approximately two months before advancing to an incurable form, ENZ-resistant CRPC. With PCa ranking as the second leading cause of cancer-related deaths in USA males, there is an urgency and necessity for the discovery and development of novel therapeutic approaches for CRPC. Wnt signaling has been extensively documented in its involvement in PCa and the tumor microenvironment (TME), however the mechanism of how the Wnt signaling cascades contribute to ENZ resistance is still ambiguous. Recently we have published that the activation of the canonical Wnt pathway contributes to the progression of ENZ resistance in CRPC and using a combination of β-catenin inhibitor with ENZ resulted in the synergistic inhibition of patient derived xenograft (PDX) tumor growth. Regarding the non-canonical Wnt pathway, we confirmed its contribution to invasion and migration which leads to metastasis in ENZ-resistant CRPC, and when the downstream effector ROCK is depleted or depleted ROCK cells are treated with ENZ, there is a significant hindering of cell migration and invasion. Also, utilizing a combination therapy of ROCK inhibitor with ENZ synergistically inhibited the growth of PDX tumors. Hence the reasoning that by simultaneously inhibiting both the canonical and non-canonical Wnt signaling cascade will result in the inhibition of cell proliferation, migration, and invasion. The goal of this study was to define the mechanism of PORCN in ENZ-resistant CRPC and develop a therapeutic approach to combat this disease. My research has determined that Porcupine (PORCN) is associated with CRPC progression to ENZ-resistance, and that an inhibition or loss of PORCN has resulted in the regain of ENZ sensitivity in ENZ-resistant models. This model has also demonstrated that PORCN and Wnt signaling engage in a paramount role contributing to AR activation, promoting CRPC progression, and the development of ENZ resistance. Citation Format: Katelyn Makayla Jones, Xiaoqi Liu. Porcupine inhibition enhances enzalutamide efficacy in drug resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1077.

Abstract A081: Epigenetic regulation of SPDEF gene in RCC7/T cells, a line of malignant African-American prostate epithelial cells

Abstract Background: Prostate cancer (PCa) in African American (AA) men has an earlier onset, more aggressiveness with higher metastasis and mortality rate than in Caucasian men. SAM-pointed domain-containing Ets-like factor (SPDEF) has been identified as a possible suppressor of metastasis in castration-resistant prostate cancer (CRPC) in earlier investigations. Ongoing studies in our lab are focused on deciphering the role of SPDEF in PCa progression and metastasis with special emphasis on PCa in AA men. We discovered that SPDEF expression is lost in RCC7/T cells, and re-expression of SPDEF limits metastatic properties of these cells, however, the regulatory mechanisms underlying the loss of SPDEF have not been elucidated. In the current study, we investigated the role of epigenetic modulators (DNA methylation and histone modifications) in driving the loss of SPDEF. Methods: We analyzed publicly available data sets from TCGA (GDC TCGA Prostate Cancer, and TCGA Prostate Cancer (PRAD) for DNA methylation using Xena UCSC genome browser (https://xena.ucsc.edu/). We also analyzed histone ChIP-seq data from PCa cell lines using cistrome project (http://cistrome.org/db/#/). RCC7/T cells were grown in DMEM and maintained at 37°C in a humidified incubator. We used bisulfite sequencing (BSP) to examine DNA methylation in SPDEF gene in SPDEF proficient (LNCaP) and deficient (RCC7/T) cells. We also employed the ChIP-qPCR to reveal the active and repressive key histone marks (H3K4me3, H3K27ac, and H3K27me3) across the SPDEF gene. Results: In the clinical cohorts, SPDEF expression showed an inverse correlation with the degree of DNA methylation and expression of the epigenetic writer enzymes such as DNMT1 and EZH2. Our results revealed that the CpG islands in SPDEF gene are hyper-methylated in RCC7/T cells compared to LNCaP cells. Our analysis of the data from the cistrome project revealed an elevated enhancer repressive mark H3K27me3 (marked by EZH2) and a decreased promoter active mark, H3K4me3, in PC3 cells as compared with less metastatic LNCaP cells. Our results from ChIP-qPCR confirmed these findings. Moreover, we observed the methylation profiles in RCC7/T cells were similar to those of PC3 cells. Additionally, combination treatment with 5-aza-2-deoxycytidine (DNMT inhibitor) and GSK-126 (EZH2 inhibitor) increased the PDEF expressions levels and also restricted colony formation, migration and invasion in RCC7/T cells. Conclusion: Overall, these findings reveal an inverse correlation between expression of epigenetic writers (DNMT1 and EZH2) and SPDEF expression in PCa. Our results also suggest that SPDEF expression in PCa is regulated in part by epigenetic modifications, and that combined inhibition of DNMT1 and EZH2 may offer a therapeutic benefit in subsets of PCa patients including in AA men. Acknowledgements: These studies were funded in part by NIH/NCI-7R01CA242839 (HK) and unrestricted funds from the LSU-LCMC Cancer Center, School of Medicine -LSUHSC, New Orleans (HK). Grateful to LCU-LCMC Genomics core for DNA sequencing Citation Format: Mousa Vatanmakanian, Sweaty Koul, Thangavel Chellappagounder, Hari K. Koul. Epigenetic regulation of SPDEF gene in RCC7/T cells, a line of malignant African-American prostate epithelial cells [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A081.

The presence of lymph node metastases and time to castration resistance predict the therapeutic effect of enzalutamide for castration-resistant prostate cancer.

Enzalutamide is effective against castration-resistant prostate cancer (CRPC). However, it is unclear which patients would benefit more from enzalutamide treatment. Here, we analyzed patients who received enzalutamide as first-line therapy for CRPC and evaluated the factors that predict treatment response and prognosis. We retrospectively analyzed 101 patients treated with enzalutamide for CRPC at our institution. As primary endpoints we regarded the prostate-specific antigen (PSA) response rate and PSA-progression-free survival (PSA-PFS) from the start of enzalutamide treatment. Laboratory and imaging data were analyzed to predict treatment efficacy. PSA reductions of ≥ 50% and ≥ 90% were observed in 78 (77%) and 47 (47%) patients, respectively, compared with the baseline. During the follow-up period, 67 (66%) patients showed PSA progression, with a median PSA-PFS of 11months. Moreover, 31 patients (31%) died, with a median overall survival of 64months. On multivariate analysis, lymph node metastases at the start of enzalutamide treatment [odds ratio (OR) 0.0575, 95% confidence interval (CI) 0.0105-0.316, p = 0.0010] and time to CRPC (OR 0.177, 95% CI 0.0428-0.731, p = 0.0167] were associated with ≥ 90% PSA response. Lymph node metastases (hazard ratio [HR] 3.00, 95% CI 1.48-6.09, p = 0.0023) and time to CRPC (HR 1.84, 95% CI 1.02-3.30, p = 0.0419) were also predictors of PSA-PFS on a multivariate model. Time to CRPC and lymph node metastasis were predictors of the PSA response rate and PSA-PFS.

Screening of visceral metastasis in castration-resistant prostate cancer: a cornerstone in personalized patient's care.

Screening of visceral metastasis in castration-resistant prostate cancer: a cornerstone in personalized patient's care.

Emerging Proteins in CRPC: Functional Roles and Clinical Implications.

Prostate cancer (PCa) is the most common cancer in men in the western world, but the lack of specific and sensitive markers often leads to overtreatment of prostate cancer which eventually develops into castration-resistant prostate cancer (CRPC). Novel protein markers for diagnosis and management of CRPC will be promising. In this review, we systematically summarize and discuss the expression pattern of emerging proteins in tissue, cell lines, and serum when castration-sensitive prostate cancer (CSPC) progresses to CRPC; focus on the proteins involved in CRPC growth, invasion, metastasis, metabolism, and immune microenvironment; summarize the current understanding of the regulatory mechanisms of emerging proteins in CSPC progressed to CRPC at the molecular level; and finally summarize the clinical applications of emerging proteins as diagnostic marker, prognostic marker, predictive marker, and therapeutic marker.

Transcriptome analysis identifies networks and key drivers in tumor microenvironment for metastatic castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most cancer-related cause of death in men. The most lethal form of PCa is metastatic castration-resistant prostate cancer (CRPC) that has progressed to the bone. The molecular mechanism underlying PCa progression to bone has not been fully elucidated. Here, we identify gene networks and characterize the cell types that contribute to the heterogeneity and complexity of CRPC associated with bone metastasis. Networks were investigated by using a novel weighted gene network co-expression analysis (WGCNA) method and examining overrepresentation of upstream regulators and signaling pathways within co-expressed transcriptome modules across clinically annotated transcriptomes from mCRPC patients (N=60). Functional Enrichment analysis was used, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analysis to identify biological functions of related hub genes overrepresented in our module of interest. WGCNA identified significant modules that are associated with CRPC bone metastasis. KEGG and GO analysis results revealed genes in these modules were mainly related to collagen formation, cell signaling peptides, and bone regulation processes. Genes positively correlated with bone metastasis exhibited the following biological pathways: PI3K-Akt signaling, ECM-receptor interaction, and protein digestion and absorption pathways. This study provides novel insights into the biological pathways associated with CRPC metastasis to the bone and its tumor immune microenvironment (TIME) niche. The modules associated with bone metastasis and overall survival represent both known and novel pathways. Supported by NIH/NCI Grant: MSM/Tuskegee U/UAB Comp. Cancer Center Partnership Name & Number of Grant Award: NCI - U54CA118638

Spindle pole body component 25 in the androgen-induced regression of castration-resistant prostate cancer.

BackgroundAndrogen plays a critical role in the development and growth of prostate cancer (PCa) by binding to the androgen receptor, a steroid receptor for testosterone and dihydrotestosterone (DHT). Androgen deprivation therapy, a clinical endocrine therapy, has resulted in increases in the occurrence of castration-resistant prostate cancer (CRPC); however, the mechanisms of CRPC have not yet fully been determined. We previously showed that spindle pole body component 25 (SPC25), a component of the NDC80 complex that is critical in kinetochore formation and chromosome segregation during the cell cycle, plays a critical role in PCa tumorigenesis and cancer stemness. However, it is not yet known whether SPC25 plays a role in CRPC; thus, we sought to address this question in the current study.MethodsSPC25 levels were detected in androgen-insensitive PCa cells using the public database and bioinformatics tools. In vitro, SPC25 levels were determined in androgen-sensitive and androgen-insensitive PCa cells treated with or without DHT. The growth of the PCa cells was assessed by the Cell Counting Kit-8 assay. The invasiveness and migratory potential of the PCa cells were assessed by the transwell cell invasive assay and migratory assay, respectively. Gain-of-function and loss-of-function experiments examined the transfection of androgen-sensitive and androgen-insensitive PCa cells by plasmids carrying small-interfering ribonucleic acids for SPC25 or SPC25, respectively.ResultsSPC25 levels were significantly reduced in the androgen-insensitive PCa cells treated with DHT in the Public database. In vitro, PCa cell growth, invasion, and metastasis was reduced in androgen-insensitive PCa cells but increased in androgen-sensitive PCa cells treated with DHT, partially through DHT-regulated expression of SPC25 at transcriptional but not at translational levels.ConclusionsAndrogen treatment reduces CRPC growth, invasion, and metastasis partially through its regulation of SPC25. SPC25 represents a promising target in the treatment of CRPC.

SQLE Mediates Metabolic Reprogramming to Promote LN Metastasis in Castration-Resistant Prostate Cancer.

BackgroundAlmost all metastatic hormone-sensitive prostate cancers (mHSPC) will develop into metastatic castration-resistant prostate cancer (mCRPC) after androgen deprivation therapy (ADT). The expression level of squalene monooxygenase (SQLE) is increased in CRPC cells and regulates cholesterol metabolism. This study verified the biological function and mechanisms of SQLE in CRPC.MethodsThe expression of SQLE in human prostate cancer cells was overexpressed or silenced and its efficacy on cell survival was determined by the MTS test. Energy metabolism phenotype test was evaluated by XF real-time ATP rate assay, XF cell mitochondrial stress test, XF glycolysis stress test and XF mito fuel flex test. Cell migration and invasion were evaluated by colony formation assays and transwell assays; the expression of mRNA and protein was assessed by RT-qPCR and Western blot, respectively. Moreover, BALB/c nude mice model was performed to evaluate the lymph node metastasis.ResultsIn our study, we found that the expression level of SQLE was significantly increased in bicalutamide-resistant-C4-2B cells compared to LNCaP cells. SQLE knockdown partly restored the sensitivity of drug-resistant cells to bicalutamide and reduced lymph node metastasis by inhibiting fatty acid oxidation in mitochondria. We also found that terbinafine, the specific inhibitor of SQLE, can enhance the sensitivity of prostate cancer cells to bicalutamide.ConclusionOur study revealed that SQLE is involved in the progression of castration resistance in CRPC through mediating metabolic reprogramming, presenting SQLE as a new target for the treatment of mCRPC.

Abstract 1458: Targeting fibroblast growth factor receptors in castration-resistant prostate cancer

Abstract Background: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. Although AR signaling inhibitors (ARSI), such as enzalutamide (ENZ) and abiraterone (ABI), extend survival in recurrent and castration-resistant prostate cancer (CRPC), durable complete responses are rare. Bypass mechanisms employed by metastatic CRPC include amplification of AR and AR splice variants in AR-high CRPC (ARPC) and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We showed previously that DNPC bypasses AR-dependence through fibroblast growth factor (FGF) signaling. However, the utility of targeting the FGF pathway in other molecular subtypes of CRPC remains to be determined. Methods: RNASeq was conducted on 98 patient metastases, 18 LuCaP patient-derived xenograft (PDX) models and CRPC cell lines. Sensitivity to ARSI and FGFR inhibitors (FGFRi) alone or in combination in cell lines (C4-2B, VCaP, 22Rv1, DU145 and NCIH660) or ex vivo PDX tumor cells (10 ARPC models, 2 DNPC models, and 6 SCNPC models) was measured through CellTitre Glo assays. On-target effects of inhibitors were assessed through immunoblot and qPCR. In vivo efficacy of FGFRi in ARPC and SCNPC was conducted using LuCaP PDX models. Results: Interrogation of FGF pathway activity and FGFR expression using RNASeq and FGF signature scores in CRPC metastases, LuCaP PDX models and CRPC cell lines revealed upregulated FGF pathway activation in AR-null DNPC and SCNPC and in subsets of ARPC. In vitro/ex vivo analysis of FGFRi (erdafitinib, CH5183284 and rogaratinib) demonstrated robust growth suppression in DNPC and moderate growth suppression in SCNPC. In vivo CH5183284 administration recapitulated the growth suppression observed in vitro for SCNPC LuCaP 93 and LuCaP 173.1 but not for SCNPC LuCaP 49. Furthermore, in vitro/ex vivo analysis of AR-expressing CRPC models determined that combination ARSI (ENZ, ABI or darolutamide) with FGFRi was superior to ARSI or FGFRi monotherapies in a subset of models. Immunoblot and qPCR analyses verified that FGFRi-mediated growth suppression occurs through inactivation of the MAPK/MEK and ERK signal transduction cascade in AR-expressing CRPC. Finally, ARPC LuCaP PDX models treated with combination ARSI and FGFRi showed significant tumor growth inhibition compared to control and monotherapy groups in vivo. Analysis of resistance mechanisms to FGFRi therapy are ongoing. Conclusions: The emergence of bypass mechanisms that subvert AR-dependence in mCRPC highlights a need for novel therapeutic strategies. Our data nominate the FGF pathway as an actionable target in diverse phenotypes of treatment-refractory mCRPC. Citation Format: Mark P. Labrecque, Lisha G. Brown, Ilsa M. Coleman, Bryce Lakely, Holly M. Nguyen, Eva Corey, Peter S. Nelson, Colm Morrissey. Targeting fibroblast growth factor receptors in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1458.

Clinicopathological characteristics of androgen receptor splicing variant 7 (AR-V7) expression in patients with castration resistant prostate cancer: A systematic review and meta‐analysis

Background Studies have shown that AR-V7 may be correlated with the poor prognosis of castration resistant prostate cancer (CRPC), however, clinicopathological characteristics of AR-V7 have not been fully elucidated. ObjectiveThis study aimed at evaluating the clinicopathological features of AR-V7 in CRPC patients. Materials and methodsTo evaluate the clinicopathological features of AR-V7 in CRPC patients. A search of PubMed, Embase, and Web of Science was performed using the keywords prostate cancer, prostate tumor, prostate neoplasm, prostate carcinoma, AR-V7, AR3, androgen receptor splicing variant-7, or androgen receptor-3. Twenty-four trials published by February 2020 were included in this study. ResultsThe proportion of Gleason score ≥ 8 was found to be significantly higher in AR-V7-positive CRPC (69.5%) than negative (54.9%) (OR 1.68, 95% CI 1.25–2.25, p < 0.001), while the rates of T3/T4 stage (OR 1.16, 95% CI 0.60–2.24, p = 0.65) and N1 stage (OR 0.99, 95% CI 0.65–1.51, p = 0.96) were not statistically correlated with AR-V7 status. The AR-V7-positive patients exhibited a significantly higher proportion of any site metastasis (61.3% versus 35.0%; OR 2.19, 95% CI 1.57–3.05, p < 0.001) and bone metastasis (81.7% versus 69.0%; OR 1.97, 95% CI 1.44–2.69, p < 0.001), and a trend close to significance was expected in visceral metastasis (28.8% versus 22.1%; OR 1.29, 95% CI 0.96–1.74, p = 0.09). Incidences of pain in AR-V7-positive CRPC (54.6%) were significantly higher than in negative CRPC (28.1%; OR 4.23, 95% CI 2.52–7.10, p < 0.001), line with worse ECOG performance status (56.7% versus 35.0%, OR 2.18, 95% CI 1.51–3.16, P < 0.001). Limitations of the study include differences in sample sizes and designs, AR-V7 detection assays, as well as disease characteristics of the included studies. ConclusionsAR-V7 positivity is associated with a higher Gleason score, bone or any site metastasis, pain and worse ECOG performance scores in CRPC. However, it is not correlated with tumor stage or lymph node metastasis. More studies are needed to confirm these findings.

Regression of Castration-Resistant Prostate Cancer by a Novel Compound HG122.

Prostate cancer (PCa) is a common aggressive disease worldwide which usually progresses into incurable castration-resistant prostate cancer (CRPC) in most cases after 18–24 months treatment. Androgen receptor (AR) has been considered as a crucial factor involved in CRPC and the study of AR as a potential therapeutic target in CRPC may be helpful in disease control and life-cycle management. In this study, we identified a potent small molecule compound, HG122, that suppressed CRPC cells proliferation and metastasis, and inhibited tumor growth both in subcutaneous and orthotopic tumor model. In addition, HG122 reduced the mRNA expression of PSA and TMPRSS2 which are target genes of AR, resulting in cell growth inhibition and metastasis suppression of CRPC, without affecting the expression of AR mRNA level. Mechanically, HG122 promoted AR protein degradation through the proteasome pathway impairing the AR signaling pathway. In conclusion, HG122 overcomes enzalutamide (ENZ) resistance in CRPC both in vitro and in vivo, thus suggesting HG122 is a potential candidate for the clinical prevention and treatment of CRPC.

Small leucine zipper protein promotes the metastasis of castration-resistant prostate cancer through transcriptional regulation of matrix metalloproteinase-13.

Matrix metalloproteinases (MMPs) function as central modulators of tissue remodeling. Abnormal expression and altered activity of MMPs result in excessive extracellular matrix degradation and increased tumor metastasis in various cancers. Small leucine zipper protein (sLZIP), belonging to the leucine zipper transcription factor family, functions as a transcriptional regulator of genes involved in various cellular processes. However, its role in MMP expression and castration-resistant prostate cancer (CRPC) metastasis remains unclear. In this study, we investigated the role of sLZIP in MMP-13 expression and its involvement in CRPC metastasis. sLZIP increased MMP-13 transcription by directly binding to its promoter in CRPC cells. We found that the expression levels of glucocorticoid receptor (GR), which represses MMP transcription, were elevated in CRPC cells. However, sLZIP suppressed the inhibitory effect of GR and enhanced the secretion of MMP-13 in CRPC cells. sLZIP promoted cell migration and invasion; however, a specific MMP-13 inhibitor blocked sLZIP-induced cell motility. Depletion of sLZIP using the CRISPR/Cas9 system downregulated MMP-13 messenger RNA expression in PC3 cells. Mice injected with sLZIP-depleted PC3 cells showed significantly reduced metastatic tumor volume in the lung compared with mice injected with control PC3 cells. Our findings suggest that sLZIP plays an important role in MMP-13 induction and CRPC metastasis. Therefore, sLZIP inhibition could be a novel therapeutic strategy for metastatic GR-enriched CRPC.