NCL1, A Highly Selective Lysine-Specific Demethylase 1 Inhibitor, Suppresses Castration-Resistant Prostate Cancer Growth via Regulation of Apoptosis and Autophagy

Recent studies have shown that epigenetic alterations lead to oncogenic activation, thus indicating that these are therapeutic targets. Herein, we analyzed the efficacy and therapeutic potential of our developed histone lysine demethylase 1 (LSD1) inhibitor, NCL1, in castration-resistant prostate cancer (CRPC). The CRPC cell lines 22Rv1, PC3, and PCai1CS were treated with NCL1, and LSD1 expression and cell viability were assessed. The epigenetic effects and mechanisms of NCL1 were also evaluated. CRPC cells showed strong LSD1 expression, and cell viability was decreased by NCL1 in a dose-dependent manner. Chromatin immunoprecipitation analysis indicated that NCL1 induced histone H3 lysine 9 dimethylation accumulation at promoters of P21. As shown by Western blot and flow cytometry analyses, NCL1 also dose-dependently induced caspase-dependent apoptosis. The stimulation of autophagy was observed in NCL1-treated 22Rv1 cells by transmission electron microscopy and LysoTracker analysis. Furthermore, WST-8 assay revealed that the anti-tumor effect of NCL1 was reinforced when autophagy was inhibited by chloroquine in 22Rv1 cells. Combination index analysis revealed that a concurrent use of these drugs had a synergistic effect. In ex vivo analysis, castrated nude mice were injected subcutaneously with PCai1 cells and intraperitoneally with NCL1. Tumor volume was found to be reduced with no adverse effects in NCL1-treated mice compared with controls. Finally, immunohistochemical analysis using consecutive human specimens in pre- and post-androgen deprivation therapy demonstrated that LSD1 expression levels in CRPC, including neuroendocrine differentiation cases, were very high, and identical to levels observed in previously examined prostate biopsy specimens. NCL1 effectively suppressed prostate cancer growth in vitro and ex vivo without adverse events via the regulation of apoptosis and autophagy, suggesting that NCL1 is a potential therapeutic agent for CRPC.

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SLX4IP-mediated telomere maintenance is essential for androgen receptor-independent castration-resistant prostate cancer

10.1101/2020.03.31.018846 ◽

2020 ◽

Author(s):

Tawna L. Mangosh ◽

Wisam N. Awadallah ◽

Magdalena M. Grabowska ◽

Derek J. Taylor

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Neuroendocrine Differentiation ◽

Telomere Maintenance ◽

Dependent Manner ◽

Cancer Resistance ◽

Castration Resistant Prostate Cancer ◽

Castration Resistant ◽

Alternative Lengthening

ABSTRACTIn advanced prostate cancer, resistance to androgen deprivation therapy is achieved through numerous mechanisms, including loss of the androgen receptor (AR) allowing for AR-independent growth. Therapeutic options are limited for AR-independent castration-resistant prostate cancer, and defining mechanisms critical for its survival is of utmost importance for targeting this lethal disease. Our studies have focused on defining the telomere maintenance mechanism (TMM) required for castration-resistant prostate cancer (CRPC) cell survival. TMMs are responsible for telomere elongation to instill replicative immortality and prevent senescence, with the two TMM pathways available being telomerase and alternative lengthening of telomeres (ALT). Here, we show that AR-independent CRPC exhibits ALT hallmarks and limited telomerase expression and activity, whereas AR-dependent models use telomerase for telomere maintenance. AR-independent CRPC exhibited elevated levels of SLX4IP, a protein implicated in TMM switching. SLX4IP overexpression in AR-dependent CRPC C4-2B cells promoted ALT hallmarks in vitro. SLX4IP knockdown in AR-independent CRPC cells (DU145 and PC-3) led to the loss of ALT hallmarks, dramatic telomere shortening, induction of senescence, and reduced tumor volume. Using an in vitro model of CRPC progression, induction of neuroendocrine differentiation in AR-dependent CRPC cells promoted ALT hallmarks in an SLX4IP-dependent manner. Lack of sufficient SLX4IP expression prevented ALT hallmarks rendering a TMM deficient environment, thus inducing senescence. This study demonstrates a unique reliance of AR-independent CRPC on SLX4IP-mediated ALT. Furthermore, ALT hallmark inhibition via SLX4IP induces senescence, thereby abolishing the replicative immortality of AR-independent CRPC.

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MicroRNA-1205 Regulation of FRYL in Prostate Cancer

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.647485 ◽

2021 ◽

Vol 9 ◽

Author(s):

Michelle Naidoo ◽

Fayola Levine ◽

Tamara Gillot ◽

Akintunde T. Orunmuyi ◽

E. Oluwabunmi Olapade-Olaopa ◽

...

Keyword(s):

Prostate Cancer ◽

Neuroendocrine Differentiation ◽

Castration Resistant Prostate Cancer ◽

Chromosomal Locus ◽

Protein Coding ◽

Castration Resistant ◽

Dendritic Branching ◽

Very High

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (CRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). The 8q24 chromosomal locus is a region of very high PCa susceptibility that carries genetic variants associated with high risk of PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes a cluster of microRNAs including, microRNA-1205 (miR-1205), which are largely understudied. Herein, we demonstrate that miR-1205 is underexpressed in PCa cells and tissues and suppresses CRPC tumors in vivo. To characterize the molecular pathway, we identified and validated fry-like (FRYL) as a direct molecular target of miR-1205 and observed its overexpression in PCa cells and tissues. FRYL is predicted to regulate dendritic branching, which led to the investigation of FRYL in neuroendocrine PCa (NEPC). Resistance toward ADT leads to the progression of treatment related NEPC often characterized by PCa neuroendocrine differentiation (NED), however, this mechanism is poorly understood. Underexpression of miR-1205 is observed when NED is induced in vitro and inhibition of miR-1205 leads to increased expression of NED markers. However, while FRYL is overexpressed during NED, FRYL knockdown did not reduce NED, therefore revealing that miR-1205 induces NED independently of FRYL.

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IL1B loss is associated with increased AR activity in castration-resistant prostate cancer

10.1101/2021.08.31.458406 ◽

2021 ◽

Author(s):

Wisam N. Awadallah ◽

Jagpreet S. Nanda ◽

Sarah E. Kohrt ◽

Magdalena M Grabowska

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Cancer Cells ◽

Marker Gene ◽

Neuroendocrine Differentiation ◽

Castration Resistant Prostate Cancer ◽

Prostate Cancer Cells ◽

Castration Resistant ◽

Expression And Activity

Castration-resistant prostate cancer represents a continuum of phenotypes, including tumors with high levels of androgen receptor (AR) expression and activity and those which do not express AR and rely on alternative pathways for survival. The process by which AR-positive prostate cancer cells and tumors lose AR expression and acquire neuroendocrine features is referred to as neuroendocrine differentiation. Numerous therapies and exposures have been demonstrated to induce neuroendocrine differentiation in vitro, including the pro-inflammatory cytokine, interleukin 1 beta (IL-1β), encoded by the gene IL1B. The purpose of our studies was to determine the relationship between the expression and activity of AR in relationship to IL-1β and IL1B in prostate cancer. We performed analysis of de-identified human clinical data and generated prostate cancer cell lines with overexpression or knockout of IL1B. In primary prostate cancer, higher expression of IL1B predicts longer time to biochemical recurrence. In metastatic castration-resistant prostate cancer, IL1B expression is decreased and inversely correlates with AR and AR-target gene expression and AR activity, while positively correlating with the neuroendocrine prostate cancer (NEPC) score and neuroendocrine marker gene expression. In vitro, we report that AR-positive castration-resistant prostate cancer cells (C4-2B, 22Rv1) secrete IL-1β, and knockout of IL1B in these cells results in increased AR activity, in the presence and absence of dihydrotestosterone (DHT). Importantly, knockout of IL1B prevented AR attrition during androgen-deprivation. Taken together, our studies demonstrate that loss of IL1B in AR-positive castration-resistant prostate cancer cells can increase and maintain AR activity in the absence of androgens, suggesting another potential mechanism of high AR activity in castration-resistant prostate cancer.

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Sympathetic signaling facilitates progression of neuroendocrine prostate cancer

Cell Death Discovery ◽

10.1038/s41420-021-00752-1 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Shubham Dwivedi ◽

Maricris Bautista ◽

Sanskriti Shrestha ◽

Hussain Elhasasna ◽

Tanaya Chaphekar ◽

...

Keyword(s):

Prostate Cancer ◽

Neuroendocrine Differentiation ◽

Castration Resistant Prostate Cancer ◽

Castration Resistant ◽

Neuroendocrine Prostate Cancer ◽

Intervention Point ◽

High Concentrations ◽

Potential Therapeutic Intervention

AbstractThe progression of prostate cancer (PC) into neuroendocrine prostate cancer (NEPC) is a major challenge in treating PC. In NEPC, the PC cells undergo neuroendocrine differentiation (NED); however, the exact molecular mechanism that triggers NED is unknown. Peripheral nerves are recently shown to promote PC. However, their contribution to NEPC was not studied well. In this study, we explored whether sympathetic neurosignaling contributes to NED. We found that human prostate tumors from patients that later developed metastases and castration-resistant prostate cancer (CRPC), a stage preceding to NEPC, have high sympathetic innervations. Our work revealed that high concentrations of the sympathetic neurotransmitter norepinephrine (NE) induces NED-like changes in PC cells in vitro, evident by their characteristic cellular and molecular changes. The NE-mediated NED was effectively inhibited by the Adrβ2 blocker propranolol. Strikingly, propranolol along with castration also significantly inhibited the development and progression of NEPC in vivo in an orthotopic NEPC model. Altogether, our results indicate that the NE-Adrβ2 axis is a potential therapeutic intervention point for NEPC.

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Molecular and genomic characterization of invasive circulating tumor cells (iCTCs) from men with metastatic castration-resistant prostate cancer (mCRPC).

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.120 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 120-120

Author(s):

Terence W. Friedlander ◽

Gayatri Premasekharan ◽

Vy Ngo ◽

Shaun Doty ◽

Anna Harris ◽

...

Keyword(s):

Prostate Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Ex Vivo ◽

Genomic Analysis ◽

Comparative Genomic ◽

Castration Resistant Prostate Cancer ◽

Culture Methods ◽

Castration Resistant

120 Background: Identification, enumeration, and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration-resistant prostate cancer (mCRPC). The Vitatex VitaAssay platform captures invasive CTCs (iCTCs) in a cell surface marker-independent fashion based on their ability to invade a fluorescently-labeled cell-adhesion matrix (CAM), allowing for the analysis of multiple CTC subpopulations. Here we sought to estimate epithelial, mesenchymal, and stem-like iCTC subpopulation diversity in men with CRPC starting abiraterone acetate therapy, to compare the genomic profiles of iCTCs to matched metastatic biopsies, and to explore the potential for 2D and 3D CTC culture. Methods: iCTCs were isolated from men with mCRPC using the CAM platform, and paired metastatic biopsies were performed. iCTCs were defined as CAM+/CD45-/CD14-/DAPI+, mesenchymal iCTCs as vimentin+/CAM+/CD45-/CD14-/DAPI+, and stem-like iCTCs as CD44+/CAM+/CD45-/CD14-/DAPI+. iCTCs were enumerated and purified using FACS. Agilent array comparative genomic hybridization (aCGH) of iCTCs and paired biopsies was performed, and to explore the potential for ex-vivo cell expansion and spheroid formation, iCTCs were cultured separately in CAM and in matrigel for up to 10 days. Results: iCTCs were isolated using the CAM platform from 29 men, of whom seven have undergone paired metastatic biopsy. The median pre-FACS purity was 1.06% (range 0.11%-10.16%). Post-FACS purity was increased to greater than 90%, and a median of 60 (range 2 to 1,314) iCTCs/7.5ml were detected by FACS. Both vimentin+ and CD44+ iCTCs are detectable, and compromise between 10 to 50% of total iCTCs. iCTC aCGH profiles resemble paired soft tissue biopsy, in vitro iCTCs culture is feasible, and iCTC spheroids were observed. Conclusions: Multiple CRPC iCTC subpopulations are identifiable from men starting abiraterone therapy, and cell sorting techniques increase iCTC purity. iCTCs resemble metastatic CRPC tissue and can be expanded in culture. Further enumeration, genomic profiling, and clinical correlation of paired iCTCs taken from men with abiraterone-resistant CRPC is underway, and may shed light on mechanisms of abiraterone resistance.

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Zeb1 and SK3 Channel Are Up-Regulated in Castration-Resistant Prostate Cancer and Promote Neuroendocrine Differentiation

Cancers ◽

10.3390/cancers13122947 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2947

Author(s):

Fanny Bery ◽

Mathilde Cancel ◽

Maxime Guéguinou ◽

Marie Potier-Cartereau ◽

Christophe Vandier ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Epithelial To Mesenchymal Transition ◽

Neuroendocrine Differentiation ◽

Calcium Signal ◽

Castration Resistant Prostate Cancer ◽

Mesenchymal Transition ◽

Castration Resistant

Therapeutic strategies for metastatic castration-resistant prostate cancer aim to target androgen receptor signaling. Despite initial survival benefits, treatment resistance invariably occurs, leading to lethal disease. Therapies targeting the androgen receptor can induce the emergence of a neuroendocrine phenotype and reactivate embryonic programs associated with epithelial to mesenchymal transition. We recently reported that dysregulation of the calcium signal can induce the transcription factor Zeb1, a key determinant of cell plasticity during tumor progression. The aim of this study was to determine whether the androgen receptor-targeted treatment Enzalutamide could induce dysregulation of the calcium signal involved in the progression toward epithelial to mesenchymal transition and neuroendocrine differentiation, contributing to therapeutic escape. Our results show that Zeb1 and the SK3 potassium channel are overexpressed in vivo in neuroendocrine castration-resistant prostate cancer and in vitro in LNCaP cells neurodifferentiated after Enzalutamide treatment. Moreover, the neuroendocrine phenotype is associated with a deregulation of the expression of Orai calcium channels. We showed that Zeb1 and SK3 are critical drivers of neuroendocrine differentiation. Interestingly, Ohmline, an SK3 inhibitor, can prevent the expression of Zeb1 and neuroendocrine markers induced by Enzalutamide. This study offers new perspectives to increase hormone therapy efficacy and improve clinical outcomes.

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Computational modeling identifies multitargeted kinase inhibitors as effective therapies for metastatic, castration-resistant prostate cancer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103623118 ◽

2021 ◽

Vol 118 (40) ◽

pp. e2103623118

Author(s):

Thomas Bello ◽

Claudia Paindelli ◽

Luis A. Diaz-Gomez ◽

Anthony Melchiorri ◽

Antonios G. Mikos ◽

...

Keyword(s):

Prostate Cancer ◽

Computational Modeling ◽

Late Stage ◽

Kinase Inhibitors ◽

Ex Vivo ◽

Growth Factor Signaling ◽

Castration Resistant Prostate Cancer ◽

Castration Resistant

Castration-resistant prostate cancer (CRPC) is an advanced subtype of prostate cancer with limited therapeutic options. Here, we applied a systems-based modeling approach called kinome regularization (KiR) to identify multitargeted kinase inhibitors (KIs) that abrogate CRPC growth. Two predicted KIs, PP121 and SC-1, suppressed CRPC growth in two-dimensional in vitro experiments and in vivo subcutaneous xenografts. An ex vivo bone mimetic environment and in vivo tibia xenografts revealed resistance to these KIs in bone. Combining PP121 or SC-1 with docetaxel, standard-of-care chemotherapy for late-stage CRPC, significantly reduced tibia tumor growth in vivo, decreased growth factor signaling, and vastly extended overall survival, compared to either docetaxel monotherapy. These results highlight the utility of computational modeling in forming physiologically relevant predictions and provide evidence for the role of multitargeted KIs as chemosensitizers for late-stage, metastatic CRPC.

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Novel, non-invasive markers for detecting therapy induced neuroendocrine differentiation in castration-resistant prostate cancer patients

Scientific Reports ◽

10.1038/s41598-021-87441-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Divya Bhagirath ◽

Michael Liston ◽

Theresa Akoto ◽

Byron Lui ◽

Barbara A. Bensing ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Patients ◽

Neuroendocrine Differentiation ◽

Extracellular Vesicle ◽

Machine Learning Algorithms ◽

Castration Resistant Prostate Cancer ◽

Cellular Models ◽

Non Invasive ◽

Castration Resistant ◽

Neuroendocrine Prostate Cancer

AbstractNeuroendocrine prostate cancer (NEPC), a highly aggressive variant of castration-resistant prostate cancer (CRPC), often emerges upon treatment with androgen pathway inhibitors, via neuroendocrine differentiation. Currently, NEPC diagnosis is challenging as available markers are not sufficiently specific. Our objective was to identify novel, extracellular vesicles (EV)-based biomarkers for diagnosing NEPC. Towards this, we performed small RNA next generation sequencing in serum EVs isolated from a cohort of CRPC patients with adenocarcinoma characteristics (CRPC-Adeno) vs CRPC-NE and identified significant dysregulation of 182 known and 4 novel miRNAs. We employed machine learning algorithms to develop an ‘EV-miRNA classifier’ that could robustly stratify ‘CRPC-NE’ from ‘CRPC-Adeno’. Examination of protein repertoire of exosomes from NEPC cellular models by mass spectrometry identified thrombospondin 1 (TSP1) as a specific biomarker. In view of our results, we propose that a miRNA panel and TSP1 can be used as novel, non-invasive tools to identify NEPC and guide treatment decisions. In conclusion, our study identifies for the first time, novel non-invasive exosomal/extracellular vesicle based biomarkers for detecting neuroendocrine differentiation in advanced castration resistant prostate cancer patients with important translational implications in clinical management of these patients that is currently extremely challenging.

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In Vitro Anticancer Properties of Table Grape Powder Extract (GPE) in Prostate Cancer

Nutrients ◽

10.3390/nu10111804 ◽

2018 ◽

Vol 10 (11) ◽

pp. 1804 ◽

Cited By ~ 7

Author(s):

Avinash Kumar ◽

Melinee D’silva ◽

Kshiti Dholakia ◽

Anait Levenson

Keyword(s):

Prostate Cancer ◽

Cell Viability ◽

Epidemiological Data ◽

Grape Seed ◽

Dependent Manner ◽

Anticancer Activities ◽

Anticancer Properties ◽

Vegetables And Fruits ◽

Powder Extract

Although the link between diet and cancer is complex, epidemiological data confirm that diet is a risk factor for prostate cancer and indicate a reduced prostate cancer incidence associated with a diet rich in vegetables and fruits. Because of the known protective effect of grape seed extract (GSE) against prostate cancer, we evaluated the effects of grape powder extract (GPE) on cell viability, proliferation, and metastatic capability. Importantly, we explored the possible novel mechanism of GPE through metastasis-associated protein 1 (MTA1) downregulation in prostate cancer, since our previous studies indicated resveratrol (Res)- and pterostilbene (Pter)-induced MTA1-mediated anticancer activities in prostate cancer. We found that GPE inhibited the cell viability and growth of prostate cancer cells only at high 100 μg/mL concentrations. However, at low 1.5–15 μg/mL concentrations, GPE significantly reduced the colony formation and wound healing capabilities of both DU145 and PC3M cells. Moreover, we found that GPE inhibited MTA1 in a dose-dependent manner in these cells, albeit with considerably less potency than Res and Pter. These results indicate that stilbenes such as Res and Pter specifically and potently inhibit MTA1 and MTA1-associated proteins compared to GPE, which contains low concentrations of Res and mainly consists of other flavonoids and anthocyanidins. Our findings support continued interest in GPE as a chemopreventive and anti-cancer agent against prostate cancer but also emphasize the unique and specific properties of stilbenes on MTA1-mediated anticancer effects on prostate cancer.

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