Human Castration-resistant Prostate Cancer Research Articles

Co-targeting SKP2 and KDM5B inhibits prostate cancer progression by abrogating AKT signaling with induction of senescence and apoptosis.

Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated β-galactosidase (SA-β-Gal) staining. Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.

Blocking lipid synthesis induces DNA damage in prostate cancer and increases cell death caused by PARP inhibition.

Androgen deprivation therapy (ADT) is the primary treatment for prostate cancer; however, resistance to ADT invariably develops, leading to castration-resistant prostate cancer (CRPC). Prostate cancer progression is marked by increased de novo synthesis of fatty acids due to overexpression of fatty acid synthase (FASN), making this enzyme a therapeutic target for prostate cancer. Inhibition of FASN results in increased intracellular amounts of ceramides and sphingomyelin, leading to DNA damage through the formation of DNA double-strand breaks and cell death. We found that combining a FASNi with the poly-ADP ribose polymerase (PARP) inhibitor olaparib, which induces cell death by blocking DNA damage repair, resulted in a more pronounced reduction in cell growth than that caused by either drug alone. Human CRPC organoids treated with a combination of PARP and FASNi were smaller, had decreased cell proliferation, and showed increased apoptosis and necrosis. Together, these data indicate that targeting FASN increases the therapeutic efficacy of PARP inhibitors by impairing DNA damage repair, suggesting that combination therapies should be explored for CRPC.

Abstract LB217: Novel organoid models of lineage plasticity in castration resistant prostate cancer

Abstract The development of next-generation AR-targeted drugs, such as abiraterone and enzalutamide, has improved survival of patients with castration-resistant prostate cancer (CRPC). However, many patients eventually progress to therapy-resistant lethal prostate cancer. CRPC exhibits variant cellular states, including AR-pathway active prostate cancer (ARPC), AR activity-low prostate cancer (ARLPC), neuroendocrine prostate cancer (NEPC), amphicrine prostate cancer co-expressing AR and neuroendocrine genes (AMPC), and double-negative prostate cancer (DNPC) lacking both AR expression and neuroendocrine differentiation, indicating that lineage plasticity facilitates therapeutic resistance. While molecular characteristics of ARPC and NEPC are well described, other common forms of CRPC, such as DNPC which occurs in 20-25% of CRPC patients, remain poorly understood. Therefore, characterization of pre-clinical models of DNPC and identification of novel regulators of lineage plasticity in DNPC are urgent necessities for the development of effective treatment for lethal prostate cancer.In our studies, we have generated a new mouse model of CRPC in which Pten and Smad4 are deleted in distal luminal cells upon tamoxifen administration (Nkx3.1CreERT2/+; Ptenfl/fl; Smad4fl/fl; R26R-eYFP/+, NPS). We observed a molecular transition from ARPC to ARLPC and DNPC in the prostate tumors derived from NPS mice during tumor progression. Moreover, a combination of castration and enzalutamide treatment promoted a partial epithelial-mesenchymal transition (EMT) in DNPC. To identify the evolutionary trajectories of cancer progression and drivers of lineage plasticity in CRPC, we have established organoid lines from the prostate tumor cells of NPS mice. Single-cell RNA-sequencing (scRNA-seq) and marker validation analyses shown that these organoid lines display overt lineage plasticity, corresponding to ARPC, ARLPC, partial EMT-absent DNPC, and partial EMT-present DNPC. Orthotopic grafts derived from the established NPS organoid lines show consistent molecular phenotypes and confirm that AR-targeted therapies facilitate partial EMT in vivo. Furthermore, cross-species bioinformatic analyses using scRNA-seq datasets indicate that the established NPS organoid lines partially recapitulate the spectrum of lineage plasticity in human CRPC.In summary, we have established prostate cancer organoid lines from a novel mouse model of CRPC, which not only provide large and reproducible sources of two distinct forms of DNPC (partial EMT-absent DNPC and partial EMT-present DNPC) for molecular analyses, but also can capture the regulatory complexity of lineage plasticity in human CRPC. Therefore, NPS mice provide an excellent pre-clinical model of CRPC, especially DNPC, to identify novel regulators that drive lineage plasticity and to pursue assays for development of candidate drugs that can inhibit or even possibly convert therapy-resistant forms of CRPC to therapy-sensitive ones. Citation Format: Jinqiu Lu, Michael M. Shen. Novel organoid models of lineage plasticity in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB217.

Abstract 466: microRNA regulators of neuroendocrine differentiation in metastatic castration-resistant prostate cancer

Abstract Therapy-induced neuroendocrine prostate cancer (NEPC), an extremely aggressive variant of castration-resistant prostate cancer (CRPC), is increasing in incidence with the widespread use of highly potent androgen receptor (AR)-pathway inhibitors (APIs) via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED). The molecular basis of NED is not completely understood leading to a lack of effective molecular markers for its diagnosis. We hypothesized that alterations in key miRNAs drive NED, leading to lineage switching and emergence of NEPC. The aims of our study are: (i) identify key miRNA alterations that occur as PCa cells undergo neuroendocrine differentiation; (ii) develop a miRNA-based classifier for predicting/diagnosing NEPC; (iii) understand the functional role of key miRNA alterations. To systematically investigate the key miRNAs alterations driving therapy induced NED, we performed small RNA-NGS in a retrospective cohort of human metastatic CRPC clinical samples + PDX models with adenocarcinoma features (CRPC-adeno) vs those with neuroendocrine features (CRPC-NE). Further, with the application of machine learning algorithms to sequencing data, we trained a ‘miRNA classifier’ that could robustly classify ‘CRPC-NE’ from ‘CRPC-Adeno’ cases. The performance of classifier was validated in additional cohorts of mCRPC patients acquired from independent sites by employing single model prediction algorithm. We further sought to understand the functional role of these miRNAs by overexpessing in PCa cell lines followed by functional assays. In vivo xenograft studies were performed using PC3 cells overexpressing miR-28/control miRNA. We demonstrate for the first time, that lineage switching to NE states is accompanied by key miRNA alterations. Our analysis, measured using receiver operating characteristic (ROC) curves, demonstrated an area under the curve (AUC) value of 0.8318, confirming the robustness of our classifier in distinguishing CRPC-NE from CRPC-Adeno. Furthermore, we identified a select group of 5 miRNAs that were pivotal in distinguishing between these two prostate cancer subtypes. We focused on miR-28-3p, little studied miRNA in prostate cancer. Our in vitro and in vivo data with prostate cancer cell lines and xenograft model suggests that miR-28-3p acts as a tumor suppressor miRNA in advanced stage prostate cancer. We further found mesenchymal gene Vimentin to be a direct miR-28-3p target. We propose that loss of miR-28-3p promotes epithelial-to-mesenchymal transition (EMT), thereby favoring NED. Our study has important clinical implications and transformative potential as our 'miRNA classifier' can be used as a molecular tool to stratify mCRPC patients into those with/without NED and guide treatment decisions. Further, we identify novel miRNA NED drivers that can be exploited for NEPC therapeutic targeting. Citation Format: Diana Asante, Sandip Nathani, Tae Jin Lee, Ashok Sharma, Sharanjot Saini. microRNA regulators of neuroendocrine differentiation in metastatic castration-resistant 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 466.

Abstract 6545: Reversing lineage plasticity and drug resistance in lethal prostate cancer

Abstract Although major mechanisms of resistance to targeted therapies include mutation of the drug target, recent evidence suggests cell-adaptive mechanisms as alternative avenues for escaping treatment. In several cancers, tumor cells become plastic in response to target inhibition, changing their identity and molecular programs without acquiring further mutations. In the case of castration-resistant prostate cancer (CRPC) treated with standard-of-care, next-generation anti-androgens enzalutamide, tumor cells often switch lineage to survive drug pressure, acquiring neuroendocrine (NE) features and thereby transforming into neuroendocrine prostate cancer (CRPC-NE). Existing targeted therapies for CRPC and CRPC-NE provide limited benefit, representing an unmet clinical need. Hence, the elucidation of tumor plasticity and consequent heterogeneity that contribute to drug resistance is of paramount importance to identify novel therapeutic opportunities. We have recently developed a network-based precision cancer medicine framework to characterize molecular and drug sensitivity profiles of ~100 Genetically-Engineered Mouse Models (GEMM)-derived tumors of prostate cancer (Vasciaveo, et al. 2023). This study revealed that tumors derived from GEMMs with the loss of function of both Pten and Trp53 (NPp53) align with CRPC patients and exhibit high heterogeneity in regulatory programs and histopathologic features, including NE differentiation. We then adopted the NPp53 model to generate several 3D organoid lines in order to study tumor plasticity and heterogeneity. To elucidate their tumors’ regulatory programs, we reverse-engineered genome-wide regulatory networks using multimodal single-cell RNASeq and ATACSeq data to identify Master Regulator (MR) proteins implementing tumor cell identities and characterize their epigenetic landscape. Our network-based regulatory analysis identified several distinct cell populations, including cells activating epithelial-to-mesenchymal (EMT) programs and NE differentiation, all resistant to enzalutamide treatment. Cross-species analysis aligned these cells to human CRPC and CRPC-NE tumors. In particular, our analyses have shed light into the roles of NSD2, a known MR of lethal prostate cancer, whose ablation restored sensitivity to enzalutamide. In summary, we leveraged single-cell, multimodal data from CRPC and CRPC-NE 3D mouse organoids to elucidate regulatory programs of drug resistant, lethal prostate cancer and characterize its heterogeneity. Importantly, we also identified a mechanism that can be exploited for therapeutic intervention to restore tumor cells’ sensitivity Citation Format: Alessandro Vasciaveo, Jia J. Li, Dimitrios Karagiannis, Xiao Chen, Andrea Califano, Chao Lu, Michael M. Shen. Reversing lineage plasticity and drug resistance in lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6545.

Abstract C034: First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors

Abstract Background: ATRN-119 is an oral and highly selective nanomolar inhibitor of Ataxia Telangiectasia and Rad3- related (ATR) kinase, an enzyme that is activated by replication stress and DNA damage and prevents the collapse of DNA replication forks into double strand breaks. ATRN-119 is being studied in advanced solid tumors with dysregulated components of the DNA damage response (DDR) based on the hypothesis that these tumors will be more susceptible to ATR inhibition. Single agent ATRN-119 demonstrates increased cytotoxicity in vitro against a broad spectrum of human colon, breast, pancreatic, sarcoma, ovarian and prostate cancer cell lines that harbor genomic alterations of DDR genes. In vivo, ATRN-119 has demonstrated tumor growth inhibition in human castration-resistant prostate cancer and human colon cancer cell line-derived xenografts (CDX) mice models, and in BRCA mutant high grade serous ovarian cancer patient derived xenografts (PDXs). Trial design: This is a first-in-human Phase 1/2a open-label study (NCT04905914) to evaluate safety, tolerability, pharmacokinetics (PK), and preliminary e!cacy of oral daily ATRN-119 in subjects with advanced solid tumors, conducted in 2 parts. Part 1 consists of a 3+3 dose escalation design at up to 6 dose levels to evaluate the tolerability, PK, dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of repeated daily doses of ATRN-119. Enrollment in Part 1 is ongoing. Subjects must be ≥12 years old with advanced solid tumor, have documented DDR mutations, or advanced Merkel cell carcinoma, must have failed at least one prior standard treatment, have ECOG performance status of 0 or 1, with a life expectancy &amp;gt; 3 months. Primary objective is safety/tolerability and secondary objective is efficacy. After determination of the MTD or maximum feasible dose (MFD) in Part 1, Part 2 will enroll up to 30 additional subjects. The trial is currently recruiting across 4 locations in the United States. Clinical trial identification: ClinicalTrials.gov Identifier: NCT04905914 Citation Format: Fiona Simpkins, Reva Schneider, Amit Mahipal, Patricia LoRusso, Crystal Miller, Eric Brown, Mike Carleton, Joachim Gullbo, Nadeem Q Mirza. First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C034.

Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer.

In castration-resistant prostate cancer (CRPC), increased glucocorticoid receptor (GR) expression and ensuing transcriptional activity have been proposed as an oncogenic "bypass" mechanism in response to androgen receptor (AR) signaling inhibition (ARSi). Here, we report that GR transcriptional activity acquired following ARSi is associated with the upregulation of cyclic adenosine monophosphate (cAMP)-associated gene expression pathways in both model systems and metastatic prostate cancer patient samples. In the context of ARSi, the expression of GR-mediated genes encoding cAMP signaling pathway-associated proteins can be inhibited by treatment with selective GR modulators (SGRMs). For example, in the context of ARSi, we found that GR activation resulted in upregulation of protein kinase inhibitor beta (PKIB) mRNA and protein levels, leading to nuclear accumulation of the cAMP-dependent protein kinase A catalytic subunit (PKA-c). Increased PKA-c, in turn, is associated with increased cAMP response element-binding protein phosphorylation and activity. Furthermore, enzalutamide and SGRM combination therapy in mice bearing CRPC xenografts delayed CRPC progression compared with enzalutamide therapy alone, and reduced tumor PKIB mRNA expression. Supporting the clinical importance of GR/PKA signaling activation in CRPC, we found a significant enrichment of both cAMP pathway signaling-associated gene expression and high NR3C1 (GR) activity in patient-derived xenograft models and metastatic human CRPC samples. These findings suggest a novel mechanism linking CRPC-induced GR transcriptional activity with increased cAMP signaling in AR-antagonized CRPC. Furthermore, our findings suggest that GR-specific modulation in addition to AR antagonism may delay GR+ CRPC time to recurrence, at least in part, by inhibiting tumor cAMP/PKA pathways.

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells.

Background: Transient receptor potential vanilloid subfamily member 6 (TRPV6), a highly calcium-selective channel, has been shown to play a significant role in calcium homeostasis and to participate both in vitro and in vivo in growth, cell survival, and drug resistance of prostate cancer. Its role and the corresponding calcium-dependent pathways were mainly studied in hormone-dependent human prostate cancer cell lines, often used as a model of early-stage prostate cancers. The goal of the present study was to describe the TRPV6-specific phenotype and signaling pathways it is involved in, using castration-resistant prostate cancer cell lines. Methods: RNA sequencing (RNA-seq) was used to study the gene expression impacted by TRPV6 using PC3Mtrpv6-/- versus PC3Mtrpv6+/+ and its derivative PC3M-luc-C6trpv6+/+ cell line in its native and TRPV6 overexpressed form. In addition to the whole-cell RNA sequencing, immunoblotting, quantitative PCR, and calcium imaging were used to validate trpv6 gene status and functional consequences, in both trpv6 -/- and TRPV6 overexpression cell lines. Results: trpv6 -/- status was validated using both immunoblotting and quantitative PCR, and the functional consequences of either trpv6 gene deletion or TRPV6 overexpression were shown using calcium imaging. RNA-seq analysis demonstrated that the calcium channel TRPV6, being a crucial player of calcium signaling, significantly impacts the expression of genes involved in cancer progression, such as cell cycle regulation, chemotaxis, migration, invasion, apoptosis, ferroptosis as well as drug resistance, and extracellular matrix (ECM) re-organization. Conclusion: Our data suggest that the trpv6 gene is involved in and regulates multiple pathways related to tumor progression and drug resistance in castration-resistant prostate cancer cells.

Abstract A060: Genetic alterations induce distinct histone post-translational modifications during the transition to castration-resistant prostate cancer

Abstract Background: Epigenetic alterations can impact gene expression and have been implicated in tumorigenesis. One major process is the post-translational modification (PTM) of histone proteins governed by histone-modifying enzyme (HME) activity. There is growing evidence that the dysregulation of HMEs plays an essential role in cancer initiation and progression. In this study, we hypothesized that alterations in histone PTMs and their associated enzymes drive the development of castration-resistant prostate cancer (CRPC) from hormone-sensitive prostate cancer (HSPC). To address this, we performed an unbiased screen of histone PTMs in a series of prostate cancer (PC) PDXs before and after the transition to CRPC. Methods: Histone PTM levels were analyzed in 10 paired HSPC and CRPC PDXs using mass spectrometry. Histone PTM patterns were subcategorized based on common genetic alterations found in PC. RNA-sequencing data for the PDXs and publicly available human PC datasets were used to further characterize HMEs. Co-expression of CHD1, NSD2, and other HMEs was examined in human PC tissue microarrays (TMAs). CHD1 KO clones were generated using CRISPR and levels of histone PTMs and HMEs were assessed and quantified. Gene set enrichment analysis (GSEA) was performed to determine the pathways altered with CHD1 in CRPC PDXs. Results: A comparison of all PDX tumors revealed increased H3K14 and H4 acetylation in CRPC tumors relative to paired HSPC derivatives. We also observed distinct histone PTM patterns associated with common genetic alterations. When compared to tumors with CHD1 WT, tumors with CHD1 loss develop reduced histone PTM H3.3K36me2. CHD1 is a chromatin remodeler commonly deleted in ~ 15% of primary PCs. Screening the differential expression and activity of HMEs in these PDXs reveals that HME NSD2 correlates with CHD1. The human CRPC SU2C/PCF dataset shows a strong positive correlation between CHD1 and NSD2 mRNA (r=0.44). Human CRPC TMAs show decreased protein expression of NSD2 and its histone mark H3K36me2 is associated with reduced CHD1 levels. CHD1 KO in DU145 results in a loss of H3.3K36me2 and a 0.5-fold decrease in NSD2 expression. GSEA revealed that a reduction in the DNA repair pathway is associated with CHD1 loss. The CRPC PDXs (n=24) and human dataset SU2C/PCF 2019 showed a strong positive correlation between CHD1, NSD2, and DNA damage repair genes. Conclusions: We have identified for the first time that genetic alterations alter histone marks during the transition to castration-resistant cancer. Reduced histone H3.3K36 methylation is associated with the genetic loss of CHD1 during the transition to CRPC. The expression of NSD2 is altered in the CRPC state in a CHD1 status-dependent manner. The CHD1-NSD2 phenotype may be the target of novel therapeutic drug combinations – the subject of ongoing research. Citation Format: Tanaya A. Purohit, Joseph Gawdzik, Bing Yang, Eric A. Armstrong, Peter W. Lewis, John M. Denu, David F. Jarrard. Genetic alterations induce distinct histone post-translational modifications during the transition to 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 A060.

Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors.

Androgen deprivation therapy (ADT) is a cornerstone of prostate cancer (PCa) management. Although tumors initially regress, many progress to a hormone-independent state termed castration-resistant PCa (CRPC), for which treatment options are limited. We here report that the major luminal cell population in tumors of Pten(i)pe-/- mice, generated by luminal epithelial cell-specific deletion of the tumor suppressor PTEN after puberty, is castration-resistant and that the expression of inflammation and stemness markers is enhanced in persistent luminal cells. In addition, hypoxia-inducible factor 1 (HIF1) signaling, which we have previously demonstrated to be induced in luminal cells of Pten(i)pe-/- mice and to promote malignant progression, is further activated. Importantly, we show that genetic and pharmacological inhibition of HIF1A sensitizes Pten-deficient prostatic tumors to castration and provides durable therapeutic responses. Furthermore, HIF1A inhibition induces apoptotic signaling in human CRPC cell lines. Therefore, our data demonstrate that HIF1A in prostatic tumor cells is a critical factor that enables their survival after ADT, and identify it as a target for CRPC management.

PD16-01 SYNERGISTIC EFFECTS OF CURCUMIN AND LACTOFERRIN TO INHIBIT THE PROSTATIC CANCER CELLS GROWTH AND MIGRATION ABILITY

You have accessJournal of UrologyCME1 Apr 2023PD16-01 SYNERGISTIC EFFECTS OF CURCUMIN AND LACTOFERRIN TO INHIBIT THE PROSTATIC CANCER CELLS GROWTH AND MIGRATION ABILITY Erica Costantini, Michele Marchioni, Lisa Aielli, Simone Ferretti, Antonio De Palma, Davide Ciavarella, Giulio Litterio, Marcella Reale, Marta Di Nicola, and Luigi Schips Erica CostantiniErica Costantini More articles by this author , Michele MarchioniMichele Marchioni More articles by this author , Lisa AielliLisa Aielli More articles by this author , Simone FerrettiSimone Ferretti More articles by this author , Antonio De PalmaAntonio De Palma More articles by this author , Davide CiavarellaDavide Ciavarella More articles by this author , Giulio LitterioGiulio Litterio More articles by this author , Marcella RealeMarcella Reale More articles by this author , Marta Di NicolaMarta Di Nicola More articles by this author , and Luigi SchipsLuigi Schips More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003271.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Castration resistant prostate cancer (CRPC) represents a challenge in urologic oncology. Several treatments are available for this tumor, but their efficacy tends to reduce in time. In recent years, several natural compounds have been tested as possible adjuvant treatment in CRPC patients. Among the other curcumin (CU) showed interesting bioactive properties, but low penetration within cells. We hypothesized that the combination with lactoferrin (LF) might exert a synergistic effect on prostatic cancer cells as anti-cancer. Their effects have been evaluated in PC3 and DU145 prostate cancer cells, that are in vitro models of CRPC. METHODS: Human CRPC cell lines (DU-145, PC3) were cultured in medium added with or without LF (175 µM) ± CU (2.5 µg/ml and 5 µg/ml). Cell viability was determined using a standard colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) reduction assay. To test ability of cell migration observation at 6 and 24 h after scratch wounds were made. Quantitative Real-time PCR was performed to evaluate gene expression of death receptors (DR) and integrins (α3/β1). The human annexin V ELISA assay was used for the quantitative detection of annexin V, involved in apoptosis regulation. RESULTS: In PC3 cells, the proliferation rate is reduced by treatments alone or in association, with a significant reduction in presence of CU, both at 2.5 (p=0.007) and 5 ug/ml (p=0.001), in a dose-dependent manner. In DU145 cells, the inhibitory effect of CU is more significant in presence of LF. In PC3 migration, LF significantly weakened invasion capacity with an increase of the cell-free area of 24.9%, 31.8%, and 24% respectively in presence of LF alone, CU2.5+LF and CU5+LF, after 6 h, with similar results in DU145 cells. In accord the integrins α3/β1, showed a significant reduction in both prostate cell lines, in presence of CU2.5, CU5, and LF175 and their combination (p<0.005). Moreover, in PC3 and DU145 an upregulation of pro-apoptotic DR4 and DR5 gene expression have been observed. Finally, in both PC3 and DU145 cells line an increased concentration of human annexin V, a marker of apoptosis, have been recorded (p<0.005). CONCLUSIONS: Our results showed that the association of CU and LF might led to beneficial effect on CRPC prostate cancer reducing the proliferative activity of cancerous cells and their ability to migrate and produce metastases. In addition, we can suggest a potential pro-apoptotic effect of CU and LF leading to a potential application as an adjuvant treatment for the treatment of CRPC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e489 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Erica Costantini More articles by this author Michele Marchioni More articles by this author Lisa Aielli More articles by this author Simone Ferretti More articles by this author Antonio De Palma More articles by this author Davide Ciavarella More articles by this author Giulio Litterio More articles by this author Marcella Reale More articles by this author Marta Di Nicola More articles by this author Luigi Schips More articles by this author Expand All Advertisement PDF downloadLoading ...

Design, Synthesis, and Anti-Cancer Evaluation of Novel Cyclic Phosphate Prodrug of Gemcitabine.

ProTide and cyclic phosphate ester are two successful prodrug technologies to overcome the limitations of nucleoside drugs, among which the cyclic phosphate ester strategy has not been widely used in the optimization of gemcitabine. Herein, we designed a series of novel ProTide and cyclic phosphate ester prodrugs of gemcitabine. Cyclic phosphate ester derivative 18c exhibits much higher anti-proliferative activity than positive control NUC-1031 with IC50s of 3.6-19.2 nM on multiple cancer cells. The metabolic pathway of 18c demonstrates that 18c's bioactive metabolites prolong its anti-tumor activity. More importantly, we separated the two P chiral diastereomers of gemcitabine cyclic phosphate ester prodrugs for the first time, revealing their similar cytotoxic potency and metabolic profile. 18c displays significant in vivo anti-tumor activity in both 22Rv1 and BxPC-3 xenograft tumor models. These results suggest that compound 18c is a promising anti-tumor candidate for treating human castration-resistant prostate and pancreatic cancer.

Role of HIKESHI on Hyperthermia for Castration-Resistant Prostate Cancer and Application of a Novel Magnetic Nanoparticle with Carbon Nanohorn for Magnetic Hyperthermia.

The prognosis of castration-resistant prostate cancer (CRPC) is technically scarce; therefore, a novel treatment for CRPC remains warranted. To this end, hyperthermia (HT) was investigated as an alternative therapy. In this study, the analysis focused on the association between CRPC and heat shock protein nuclear import factor "hikeshi (HIKESHI)", a factor of heat tolerance. Silencing the HIKESHI expression of 22Rv1 cells (human CRPC cell line) treated with siRNAs inhibited the translocation of heat shock protein 70 from the cytoplasm to the nucleus under heat shock and enhanced the effect of hyperthermia. Moreover, a novel magnetic nanoparticle was developed via binding carbon nanohorn (CNH) and iron oxide nanoparticle (IONP) with 3-aminopropylsilyl (APS). Tumor-bearing model mice implanted with 22 Rv1 cells were examined to determine the effect of magnetic HT (mHT). We locally injected CNH-APS-IONP into the tumor, which was set under an alternative magnetic field and showed that tumor growth in the treatment group was significantly suppressed compared with other groups. This study suggests that HIKESHI silencing enhances the sensitivity of 22Rv1 cells to HT, and CNH-APTES-IONP deserves consideration for mHT.

Immunoproteasome inhibition prevents progression of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment. This study aims to explore the effect and underlying mechanisms of immunoproteasome inhibition,a novel immunotherapy, on the progression of CRPC. The immunoproteasome subunit LMP7 was silenced by using gene knockout or inhibited by theepoxyketone inhibitor ONX 0914 in a mouse CRPC tumour graft model and in interferon-γ-pretreated human CRPC cell lines in vitro. CRPC tissues reveal a significant "tumour-elicited" Th17-type inflammatory response which induces immunoproteasome subunit expression. LMP7 deficiency in host mice or in CRPC tumour grafts had no effect on the "tumour-elicited" Th17-type inflammatory response and tumour progression. However, the selective LMP7 inhibitor ONX 0914 strongly suppressed the "tumour-elicited" Th17-type inflammatory response and CRPC tumour progression. Treatment of wild-type mice receiving LMP7-deficient CRPC tumour grafts with ONX 0914 further suggested that immunoproteasome inhibition prevents CRPC progression through suppressing IL-17-induced angiogenesis and epithelial-mesenchymal transition via inactivation of COX-2/VEGF-A signalling and β-catenin/Snail signalling. Treatment of LMP7-deficient mice receiving wild-type CRPC tumour grafts with ONX 0914 and inhibition of LMP7 in PC3 and 22Rv.1 cells with ONX 0914 showed that immunoproteasome inhibition also prevents CRPC progression through inducing CRPC cell apoptosis via activation of the unfolded protein response. We define a critical role of the immunoproteasome in CRPC and propose immunoproteasome inhibition as a promising therapeutic approach to suppress CRPC progression.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Acetyl-CoA carboxylase 1 depletion suppresses de novo fatty acid synthesis and mitochondrial β-oxidation in castration-resistant prostate cancer cells

Cancer cells, including those of prostate cancer (PCa), often hijack intrinsic cell signaling to reprogram their metabolism. Part of this reprogramming includes the activation of de novo synthesis of fatty acids that not only serve as building blocks for membrane synthesis but also as energy sources for cell proliferation. However, how de novo fatty acid synthesis contributes to PCa progression is still poorly understood. Herein, by mining public datasets, we discovered that the expression of acetyl-CoA carboxylase alpha (ACACA), which encodes acetyl-CoA carboxylase 1 (ACC1), was highly expressed in human PCa. In addition, patients with high ACACA expression had a short disease-free survival time. We also reported that depletion of ACACA reduced de novo fatty acid synthesis and PI3K/AKT signaling in the human castration-resistant PCa (CRPC) cell lines DU145 and PC3. Furthermore, depletion of ACACA downregulates mitochondrial beta-oxidation, resulting in mitochondrial dysfunction, a reduction in ATP production, an imbalanced NADP+/NADPhydrogen(H) ratio, increased reactive oxygen species, and therefore apoptosis. Reduced exogenous fatty acids by depleting lipid or lowering serum supplementation exacerbated both shRNA depletion and pharmacological inhibition of ACACA-induced apoptosis invitro. Collectively, our results suggest that inhibition of ectopic ACACA, together with suppression of exogenous fatty acid uptake, can be a novel strategy for treating currently incurable CRPC.

The ERα-NRF2 signalling axis promotes bicalutamide resistance in prostate cancer

BackgroundBicalutamide is a nonsteroidal antiandrogen widely used as a first-line clinical treatment for advanced prostate cancer (PCa). Although patients initially show effective responses to bicalutamide treatment, resistance to bicalutamide frequently occurs and leads to the development of castration-resistant PCa (CRPC). This research investigated the roles of the oestrogen receptor α (ERα)-nuclear factor E2-related factor 2 (NRF2) signalling pathway in bicalutamide resistance in PCa cells.MethodsWe performed bioinformatic analysis and immunohistochemical staining on normal and cancerous prostate tissue to evaluate ERα and NRF2 expression and their correlation. Gene expression and localization in PCa cell lines were further investigated using real-time reverse transcription PCR/Western blotting and immunofluorescence staining. We treated PCa cells with the ER inhibitor tamoxifen and performed luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays to understand ERα-dependent NRF2 expression. Overexpression and knockdown of ERα and NRF2 were used to explore the potential role of the ERα-NRF2 signalling axis in bicalutamide resistance in PCa cells.ResultsWe found that the expression of ERα and NRF2 was positively correlated and was higher in human CRPC tissues than in primary PCa tissues. Treatment with oestrogen or bicalutamide increased the expression of ERα and NRF2 as well as NRF2 target genes in PCa cell lines. These effects were blocked by pretreatment with tamoxifen. ChIP assays demonstrated that ERα directly binds to the oestrogen response element (ERE) in the NRF2 promoter. This binding led to increased transcriptional activity of NRF2 in a luciferase reporter assay. Activation of the ERα-NRF2 signalling axis increased the expression of bicalutamide resistance-related genes. Inhibition of this signalling axis by knockdown of ERα or NRF2 downregulated the expression of bicalutamide resistance-related genes and inhibited the proliferation and migration of PCa cells.ConclusionsWe demonstrated the transcriptional interaction between ERα and NRF2 in CRPC tissues and cell lines by showing the direct binding of ERα to the ERE in the NRF2 promoter under oestrogen treatment. Activation of the ERα-NRF2 signalling axis contributes to bicalutamide resistance in PCa cells, suggesting that the ERα-NRF2 signalling axis is a potential therapeutic target for CRPC.CxbrQyr1MPyN3X1_gKxrKxVideo

Metformin regulates multiple signaling pathways within castration-resistant human prostate cancer cells

BackgroundThe biguanide metformin has been shown to not only reduce circulating glucose levels but also suppress in vitro and in vivo growth of prostate cancer. However, the mechanisms underlying the anti-tumor effects of metformin in advanced prostate cancers are not fully understood. The goal of the present study was to define the signaling pathways regulated by metformin in androgen-receptor (AR) positive, castration-resistant prostate cancers.MethodsOur group used RNA sequencing (RNA-seq) to examine genes regulated by metformin within the C4–2 human prostate cancer cell line. Western blot analysis and quantitative RT-PCR were used to confirm alterations in gene expression and further explore regulation of protein expression by metformin.ResultsData from the RNA-seq analysis revealed that metformin alters the expression of genes products involved in metabolic pathways, the spliceosome, RNA transport, and protein processing within the endoplasmic reticulum. Gene products involved in ErbB, insulin, mTOR, TGF-β, MAPK, and Wnt signaling pathways are also regulated by metformin. A subset of metformin-regulated gene products were genes known to be direct transcriptional targets of p53 or AR. Western blot analyses and quantitative RT-PCR indicated these alterations in gene expression are due in part to metformin-induced reductions in AR mRNA and protein levels.ConclusionsTogether, our results suggest metformin regulates multiple pathways linked to tumor growth and progression within advanced prostate cancer cells.

Abstract 2635: Inhibiting deoxycytidine kinase significantly inhibits tumor growth in xenograft models of castration resistant prostate cancer

Abstract Mammalian cells synthesis deoxyribonucleoside triphosphate (dNTP) precursors for DNA replication via the de novo pathway (DNP), and to a lesser extent, via the nucleoside salvage pathway (NSP). Evidence suggests that rapid tumor cell growth associated with increased dNTP needs results in elevated NSP activity. As deoxycytidine kinase (dCK) is the rate-limiting enzyme of the NSP, and the only enzyme that can supply cells with all dNTP precursors for DNA replication, dCK has been implicated as a promising therapeutic target. Data from the Cancer Cell Line Encyclopedia demonstrated that most human cancer cell lines have elevated dCK gene expression (2- to 17-fold) therefore suggesting selective susceptibility of tumor cells to dCK inhibition. Accordingly, a potent first-in-class oral dCK inhibitor, TRE-515, was evaluated as a single agent in two human castration-resistant prostate cancer (CRPC) models. In the first CRPC model, C4-2, TRE-515 was administered intraperitoneally once a day for eight consecutive days (50 mg/kg, QDx8). TRE-515 significantly inhibited tumor growth (50.7%, p=0.021, relative to vehicle control), with 50% of the TRE-15 treated tumors regressing during the eight-day treatment window. In a repeat C4-2 study, TRE-515 monotherapy trended toward tumor growth inhibition, (53.7%, p=0.061, relative to vehicle control). A second CRPC model, 22Rv1, was then selected as it has been shown to carry DNP mutations, suggesting enhanced sensitivity to dCK inhibition. In this study, TRE-515 was dosed orally two times a day for 28 days (75 mg/kg, BIDx28) and plasma deoxycytidine (dC) concentrations (a substrate for dCK) were measured 7 hours after the morning dose on day 21. TRE-515 alone significantly inhibited tumor growth (49.9%, p=0.0004, relative to vehicle control), and growth inhibition was associated with a 77% increase in plasma dC levels (p&amp;lt;0.0001), relative to vehicle control, consistent with TRE-515 inhibitory activities on dCK. These data are in agreement with a previously reported study on target enzyme inhibition by TRE-515 which employed a clinically validated PET probe imaging of dCK. Taking together these results support the therapeutic potential for TRE-515 to selectively inhibit cancer cells that rely on dCK for DNA synthesis and rapid malignant proliferation. As such, TRE-515 is currently being evaluated in a phase 1 open-label, dose escalation study in solid tumors (NCT #05055609). Citation Format: Caius G. Radu, Kenneth A. Schultz, Katharina Lückerath, H Michael Shepard, Johannes Czernin. Inhibiting deoxycytidine kinase significantly inhibits tumor growth in xenograft models of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2635.

Abstract 1513: FASN inhibition-induced BRCAness as a therapeutic option for castration-resistant prostate cancer (CRPC)

Abstract Androgen deprivation therapy (ADT) is the primary treatment for prostate cancer. However, resistance to ADT invariably develops, leading to castration-resistant prostate cancer (CRPC). Prostate cancer progression is marked by overexpression of fatty acid synthase (FASN) and increased de novo synthesis of fatty acids. Importantly, the majority of normal tissues have low expression of FASN and rely mostly on exogenous lipids, making FASN an interesting therapeutic target for prostate cancer. Previously our group evaluated the effects of inhibiting FASN in prostate cancer, with especially promising results in CRPC models. We observed that FASN inhibition decreases DNA damage repair capability by transcriptionally modulating homologous recombination (BRCAness) and non-homologous end joining repair pathways, which leads to increased DNA damage and apoptosis. Key enzymes involved in DNA repair pathways are reduced at the protein level, and the addition of exogenous palmitate, the product of FASN, is able to rescue their expression to normal levels. Interestingly, genes involved in the synthesis of ceramide are upregulated after FASN blockade, with consequent increased cellular levels of ceramides, dihydroceramides and sphingomyelin. Additionally, we observed increased acyl chain unsaturation levels in these lipid species, in line with the uptake of polyunsaturated fatty acids (PUFA) observed when de novo lipogenesis is blocked. The inhibition of ceramide synthesis, both through genetic knock-down with siRNA or pharmacologically with an inhibitor of serine palmitoyltransferase, is capable of partially rescuing the DNA damage induced by FASN inhibition, suggesting a role of ceramides in DNA damage response modulation. Next, we investigated the synergistic effect of combining FASN inhibitor with PARP inhibitor and observed a higher inhibition of cell growth in prostate cancer cell lines in comparison to either drug alone. Human CRPC organoids when treated with PARP and FASN inhibitor in combination showed reduced diameter, as well as reduced cell proliferation, when compared with each agent alone. Overall, our data demonstrate that targeting de novo lipogenesis can increase the therapeutic efficacy of PARP inhibitors and benefit prostate cancer patients that do not harbor BRCA mutations, by pharmacologically downregulating DNA damage repair pathways, particularly HR. Citation Format: Caroline Fidalgo Ribeiro, Silvia D. Rodrigues, Debora C. Bastos, Hubert Pakula, Giorgia Zadra, Marco Foiani, Massimo Loda. FASN inhibition-induced BRCAness as a therapeutic option for castration-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1513.