Neuroendocrine Prostate Cancer Progression Research Articles

Proteomic Analysis of Microsomal Proteins Reveals That MVP Is Crucial for the Secretion of GDF-15, Which in Turn Promotes the Neuroendocrine Differentiation of PCa Cells.

Neuroendocrine prostate cancer (NEPC) is an aggressive androgen-independent PCa (AIPC) that tends to resist treatment. Understanding its progression and resistance could improve survival outcomes. Previous studies on PCa cells highlighted microsomal proteins' role in PCa progression, but their role in the progression of NEPC remains unclear. Thus, we investigated microsomal proteins in in vitro differentiated NE-LNCaP cells and their role in NED of PCa. Microsomal proteomics revealed two cancer-associated proteins GDF-15 and MVP as elevated in NE-LNCaP cells with GDF-15 among the top 5 upregulated proteins. MVP is elevated in NE-LNCaP and is also increased in NCI-H660 microsomes compared to LNCaP. GO and protein network analysis showed that different molecular networks are affected by microsomal protein enrichment, and MVP and GDF-15 are mapped to functional subnetworks associated with cancer. Remarkably, GDF-15 and MVP are essential for LNCaP cell differentiation when stimulated with Forskolin. Interestingly, AKT and MAPK/ERK signaling pathways are significantly upregulated in NE-LNCaP and NCI-H660 cells with the direct involvement of GDF-15. In summary, we have uncovered that GDF-15 and MVP are involved in NED, with MVP being essential for GDF-15 secretion, promoting NED in PCa cells. These findings provide insights into NED mechanisms and suggest potential therapeutic targets or biomarkers for NEPC.

Abstract 6268: A study of orphan nuclear receptor SHP (NR0B2) in prostate cancer progression

Abstract Prostate cancer (PCa) is a significant male cancer in many countries accounting for around 14% of new cancer cases globally in 2020 and ranked as 3rd common cancer in males in Hong Kong. The major risk factors include old age, high-fat diet, and genetics. Compared to other cancers, PCa is unique as its initial and advanced growth is androgen dependent. As tumor progresses, it accumulates numerous genetic alterations. Main therapeutic options include surgery, radiotherapy and or active surveillance. Upon treatment failure or when metastasis develops, androgen-deprivation therapy (ADT) with the use of potent androgen receptor (AR) antagonists and androgen biosynthesis inhibitors (e.g. Enzalutamide and abiraterone acetate) aiming to suppress AR signaling axis are the standard treatment options. However, the effect of these treatments is short-lived, in which nearly all patients will inevitably relapse to a hormone-independent and metastatic stage known as metastatic castration-resistant prostate cancer (mCRPC) within few years and further drive progression to a more lethal subtype, described as treatment-induced neuroendocrine prostate cancer (NEPC). The treatment options are limited and therefore resulted in high mortality, representing an emerging clinical problem. Recent studies have illustrated that NEPC progression follows a lineage plasticity model with a phenotypic transition to AR loss and neuroendocrine transdifferentiation. Our preliminary study has identified a nuclear receptor NR0B2 (SHP, small heterodimer partner SHP), which is a unique ligand-independent orphan nuclear receptor without a DNA-binding domain, that displays a significant up-regulation in clinical NEPC and prostate cancer cells with NEPC phenotype. Mechanistic studies revealed that SHP could directly interact with AR utilizing its NR Box1 motif while inducing repression on the AR transactivation via its another NR Box2 motif. Based on this, we conducted in vitro growth studies and revealed that NR0B2 overexpression could promote growth differentially in AR-negative PCa cells. However, AR-positive PCa cells were insensitive to AR blockade, providing supporting evidence that NR0B2 may be involved in repressing AR activity in prostate cancer cells. Multiple key NEPC and oncogenic TGFβ signaling markers showed significant upregulation in gene expression. Based on this, we hypothesize that upregulation of NR0B2 could play an important role in driving the advanced progression of CRPC via its direct interference of AR signaling axis and through other AR-independent signaling pathways, including activation of TGFβ signaling. (Study supported by a CUHK Direct Grant for Research 2022-2023, Project No. 2022.081) Citation Format: Hau Tak Daniel Lam, Franky Leung Chan. A study of orphan nuclear receptor SHP (NR0B2) in prostate cancer progression [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 6268.

Abstract 4567: Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatments. Therefore, there is an urgent need to develop new treatment strategies. N-Myc plays a key role in driving NEPC progression, and Aurora kinase A (AURKA) prevents N-Myc degradation by interact with N-Myc and disallowing the intervention of ubiquitin ligases. The AURKA inhibitor Alisertib inhibits NEPC tumor growth by disrupting N-Myc signaling, but it is failed in a phase II clinical trial. In this study, we tested the synergy of novel HSP70 allosteric inhibitor JG231 and Alisertib in NEPC cells, organoids, and patient derived xenograft (PDX) models, which will provide new therapeutic strategies for the NEPC treatment. Methods: RT-PCR, western blotting, or immunohistochemistry (IHC) were used to determine the expression of N-Myc, neuroendocrine maker (NSE, SYP, CHGA, etc) in different prostate cancer cell lines and PDX tumors. After the knockdown of HSP70 and AURKA by siRNA transfection, cell proliferation and N-Myc expression were assessed by using cell viability assay, RT-PCR and Western blotting respectively. The co-immunoprecipitation assay was performed to detect the N-Myc ubiquitination. RNA-sequencing, gene set enrichment analysis was employed to determine the changes of JG231 on gene programs. The effects of JG231 and Alisertib on the proliferation were examined in NEPC cell lines and PDX organoids models. Results: We determined that PC3, CWR22Rv1, H660 cell lines, LuCaP49, and LuCaP93 PDX tumors had significantly increased the expression of N-Myc and neuroendocrine makers. Knockdown of HSP70 or using the HSP70 inhibitor, JG231, significantly inhibit the growth of H660 and CWR22Rv1 cells and leads to a synergistic effect with Alisertib (p<0.001). JG231 combined with Alisertib also inhibits growth and induces the death of LuCaP93 and H660 tumor derived organoids in a dose-dependent manner (p<0.001). Mechanistically, JG231 inhibits the protein expression of N-Myc through the ubiquitin-proteasome system and promote STUB1 to enter the nucleus to bind to N-Myc. Conclusions: JG231, a novel HSP70 inhibitor, can improves the treatment efficacy of AURKA inhibitor Alisertib against NEPC through the regulation of N-Myc protein homeostasis. Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Shu Ning, Leyi Wang, Christopher Nip, Christopher P. Evans, Marc A. Dall'Era, Allen C. Gao, Jason Gestwicki, Chengfei Liu. Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine 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 4567.

Tumor protein D52 (isoform 3) induces NF-κB – STAT3 mediated EMT driving neuroendocrine differentiation of prostate cancer cells

In prostate cancer (PCa) patients, a proto-oncogene Tumor protein D52 (TPD52) is overexpressed, and it is involved in different cellular functions. In this study, we report that TPD52 expression is positively associated with the emergence of neuroendocrine PCa (NEPC). With overexpression of TPD52 in LNCaP cells, we found neuroendocrine differentiation (NED) of cells in in-vitro and distinct NED features confirmed by NE markers neuron-specific enolase (NSE) and chromogranin A (CHR-A). Further, we investigated the molecular mechanisms involved in TPD52 mediated NED of PCa cells. We found that TPD52 activates the NF- κB – STAT3 axis for the induction of NED in LNCaP cells. Indeed, inhibition of NF-κB – STAT3 attenuated the progression of NED in TPD52 positive LNCaP cells. Importantly, silencing of TPD52 expression or inhibition of NF-κB – STAT3 activity in a neuroendocrine cell line NCI-H660 showed a marked decrease in the expression of NSE and CHR-A, confirming the reversal of the NE properties. Notably, TPD52 overexpression in LNCaP cells induced expression of N-cadherin, Vimentin, ZEB1, and Snail1 indicating that TPD52 positively regulates epithelial to mesenchymal transition (EMT) of PCa cells towards NED. Moreover, silencing of Snail1 in TPD52 positive cells blocked the progression of NED and, in NCI-H660 cells reversed NE properties as expected. Of the few requirements of TPD52, activation of NF-κB – STAT3 is essential for promoting EMT compelling NED of LNCaP cells. Collectively, these results reveal that TPD52 is associated with the progression of NEPC and emphasizes the need for therapeutic targeting of TPD52 in PCa.

MicroRNA-147b induces neuroendocrine differentiation of prostate cancer cells by targeting ribosomal protein RPS15A.

Prostate cancer (PCa) is the leading cause of cancer related deaths in men, often androgen deprivation therapy (ADT) leads to the progression of androgen independent PCa (AIPC) which further leads to Neuroendocrine PCa (NEPC). Identifying the molecular mechanisms which navigate the neuroendocrine differentiation(NED) of PCa cells is clinically relevant. It has been suggested that the micro RNAs (miRNAs) play an important role in the regulation of intrinsic mechanisms relevant to tumor progression, resistance as a result leads to poor prognosis. miR-147b has been transpiring as one of the deregulated miRNAs associated with the occurrence of multiple cancers. The present study has studied the role of miRNA-147b in inducing NEPC. To investigate the functional role of miR-147b in NEPC, we have expressed miRNA mimics or inhibitors in PCa cells and monitored the progression of NEPC along with PCa cell proliferation and survival. The molecular mechanism miRNA-147b follows was studied using western blot and reverse transcriptionpolymerase chain analysis. miRNA target prediction using bioinformatics tools followed by target validation using luciferase reporter assays was performed. In the present study, we found that miR-147b is highly expressed in AIPC cell lines in particular neuroendocrine cells NCI-H660 and NE-LNCaP derived from LNCaP. Mechanistic studies revealed that overexpression of miR-147b or miRNA mimics induced NED in LNCaP cells in in-vitro while its inhibitor reversed the NE features (increased NE markers and reduced prostate specific antigen) of PC3, NCI-H660 and NE-LNCaP cells. In addition, miR-147b reduced the proliferation rate of LNCaP cells via elevated p27kip1 and lowered cyclin D1 for promoting differentiation. In reporter assays, we have identified ribosomal protein S15A (RPS15A) is a direct target of miRNA-147b and RPS15A expression was negatively regulated by miR-147b in PCa cells. Furthermore, we also report that RPS15A is downregulated in NEPC cells and its expression is inversely correlated with NE markers. Targeting the miR-147b - RPS15A axis may overcome the progression of NEPC and serve as a novel therapeutic target to attenuate NED progression of PCa.

Abstract 4760: KDM4A promotes NEPC progression through regulation of MYC expression

Abstract Despite advancements in treatment, prostate cancer (PCa) remains the second leading cause of death among men. Neuroendocrine prostate cancer (NEPC) represents one of the most lethal forms of PCa and lacks life-prolonging treatment. Here we identified histone lysine demethylase KDM4A as a driver in NEPC progression and an effective therapeutic target. KDM4A mRNA and protein are overexpressed in human and mouse NEPC compared to adenocarcinoma. Knockdown or knockout of KDM4A in NEPC cell lines suppressed cancer cell growth in vitro and in vivo. Importantly, the inactivation of Kdm4a in a genetically engineered mouse model of prostate cancer reduces tumor burden, reduces the incidence of NEPC, and prolongs overall survival. Mechanistically, KDM4A directly regulates the transcription of MYC, which is hyper-activated in human and mouse NEPC. Furthermore, a potent pan-KDM4 inhibitor QC6352 significantly reduces NEPC cell growth in vitro and in vivo. Taken together, we demonstrate that KDM4A promotes NEPC progression through regulation of MYC expression and targeting KDM4A can be an effective therapeutic strategy for NEPC. Citation Format: Celia Sze Ling Mak, Ming Zhu, Xin Liang, Feng Wang, Anh G. Hoang, Xinzhi Song, Peter Shepherd, Derek Liang, Jessica Suh, Jiwon Park, Miao Zhang, Eric Metzger, Roland Schule, Abhinav K. Jain, Ellen Karasik, Barbara A. Foster, Min Gyu Lee, Paul Corn, Christopher J. Logothetis, Ana Aparicio, Nora Navone, Patricia Troncoso, Jianhua Zhang, Sue-Hwa Lin, Guocan Wang. KDM4A promotes NEPC progression through regulation of MYC expression. [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 4760.

ZBTB7A as a novel vulnerability in neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer. NEPC is characterized by the loss of androgen receptor (AR) signaling and transdifferentiation toward small-cell neuroendocrine (SCN) phenotypes, which results in resistance to AR-targeted therapy. NEPC resembles other SCN carcinomas clinically, histologically and in gene expression. Here, we leveraged SCN phenotype scores of various cancer cell lines and gene depletion screens from the Cancer Dependency Map (DepMap) to identify vulnerabilities in NEPC. We discovered ZBTB7A, a transcription factor, as a candidate promoting the progression of NEPC. Cancer cells with high SCN phenotype scores showed a strong dependency on RET kinase activity with a high correlation between RET and ZBTB7A dependencies in these cells. Utilizing informatic modeling of whole transcriptome sequencing data from patient samples, we identified distinct gene networking patterns of ZBTB7A in NEPC versus prostate adenocarcinoma. Specifically, we observed a robust association of ZBTB7A with genes promoting cell cycle progression, including apoptosis regulating genes. Silencing ZBTB7A in a NEPC cell line confirmed the dependency on ZBTB7A for cell growth via suppression of the G1/S transition in the cell cycle and induction of apoptosis. Collectively, our results highlight the oncogenic function of ZBTB7A in NEPC and emphasize the value of ZBTB7A as a promising therapeutic strategy for targeting NEPC tumors.

Dependence on MUC1-C in Progression of Neuroendocrine Prostate Cancer.

Castration resistant prostate cancer (CRPC) is responsive to androgen receptor (AR) axis targeted agents; however, patients invariably relapse with resistant disease that often progresses to neuroendocrine prostate cancer (NEPC). Treatment-related NEPC (t-NEPC) is highly aggressive with limited therapeutic options and poor survival outcomes. The molecular basis for NEPC progression remains incompletely understood. The MUC1 gene evolved in mammals to protect barrier tissues from loss of homeostasis. MUC1 encodes the transmembrane MUC1-C subunit, which is activated by inflammation and contributes to wound repair. However, chronic activation of MUC1-C contributes to lineage plasticity and carcinogenesis. Studies in human NEPC cell models have demonstrated that MUC1-C suppresses the AR axis and induces the Yamanaka OSKM pluripotency factors. MUC1-C interacts directly with MYC and activates the expression of the BRN2 neural transcription factor (TF) and other effectors, such as ASCL1, of the NE phenotype. MUC1-C also induces the NOTCH1 stemness TF in promoting the NEPC cancer stem cell (CSC) state. These MUC1-C-driven pathways are coupled with activation of the SWI/SNF embryonic stem BAF (esBAF) and polybromo-BAF (PBAF) chromatin remodeling complexes and global changes in chromatin architecture. The effects of MUC1-C on chromatin accessibility integrate the CSC state with the control of redox balance and induction of self-renewal capacity. Importantly, targeting MUC1-C inhibits NEPC self-renewal, tumorigenicity and therapeutic resistance. This dependence on MUC1-C extends to other NE carcinomas, such as SCLC and MCC, and identify MUC1-C as a target for the treatment of these aggressive malignancies with the anti-MUC1 agents now under clinical and preclinical development.

Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It is characterized by the loss of androgen receptor (AR) signaling in neuroendocrine transdifferentiation, and finally, resistance to AR-targeted therapy. With the application of a new generation of potent AR inhibitors, the incidence of NEPC is gradually increasing. The molecular mechanism of neuroendocrine differentiation (NED) after androgen deprivation therapy (ADT) remains largely unclear. In this study, using NEPC-related genome sequencing database analyses, we screened RACGAP1, a common differentially expressed gene. We investigated RACGAP1 expression in clinical prostate cancer specimens by IHC. Regulated pathways were examined by Western blotting, qRT-PCR, luciferase reporter, chromatin immunoprecipitation, and immunoprecipitation assays. The corresponding function of RACGAP1 in prostate cancer was analyzed by CCK-8 and Transwell assays. The changes of neuroendocrine markers and AR expression in C4-2-R and C4-2B-R cells were detected in vitro. We confirmed that RACGAP1 contributed to NE transdifferentiation of prostate cancer. Patients with high tumor RACGAP1 expression had shorter relapse-free survival time. The expression of RACGAP1 was induced by E2F1. RACGAP1 promoted neuroendocrine transdifferentiation of prostate cancer by stabilizing EZH2 expression in the ubiquitin-proteasome pathway. Moreover, overexpression of RACGAP1 promoted enzalutamide resistance of castration-resistant prostate cancer (CRPC) cells. Our results showed that the upregulation of RACGAP1 by E2F1 increased EZH2 expression, which drove NEPC progression. This study explored the molecular mechanism of NED and may provide novel methods and ideas for targeted therapy of NEPC.

Identification of protein kinase C beta as a therapeutic target for neuroendocrine prostate cancer and development of a nanoparticle-based therapeutic strategy

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of malignant prostate cancer for which there is no effective treatment. Although protein kinases are the most attractive therapeutic targets for cancer treatment, there are no clear kinase-targeted drugs available for NEPC treatment. Herein, we showed that protein kinase C beta (PKCβ) is highly expressed in NEPC and can be transcriptionally activated by c-Myc through the E-box elements present in its promoter region. Furthermore, PKCβ can activate PIM1 kinase by phosphorylating Ser51 and Ser54, which in turn stabilizes and enhances c-Myc function. Three formed a PIM1-c-Myc-PKCβ positive feedback loop to promote the progression of NEPC. Enzastaurin, a specific inhibitor of PKCβ, significantly ameliorated NEPC. However, enzastaurin causes thrombus and other side effects in several clinical trials of other diseases, which greatly limits its clinical application. To address these problems, we developed a functional polymer nanoparticle modified with SSTR2-targeted octreotide and a thrombin-binding aptamer to deliver enzastaurin for NEPC treatment. The tailor-designed nanomedicine efficiently retards tumor growth and the incidence of coagulation abnormalities in vitro and in vivo. Collectively, our findings highlight the key role of PKCβ in NEPC and provide a promising nanoparticle-based therapeutic strategy for the treatment of NEPC.

Sympathetic signaling facilitates progression of neuroendocrine prostate cancer

The 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.

AMPK/SIRT1 signaling through p38MAPK mediates Interleukin-6 induced neuroendocrine differentiation of LNCaP prostate cancer cells

Neuroendocrine Prostate Cancer (NEPC) is an aggressive form of androgen independent prostate cancer (AIPC), correlated with therapeutic resistance. Interleukin (IL)-6 promotes proliferation and neuroendocrine differentiation (NED) of androgen dependent LNCaP cells. We treated LNCaP cells with IL-6 and observed for in vitro NED of cells and also expression of NE markers βIII tubulin, neuron-specific enolase (NSE) and chromogranin A (ChA). Here we investigated the proteins and/or pathways involved in NED of LNCaP cells induced by IL-6 and characterized their role in NED of PCa cells. We found that the altered proteins modulated AMPK signaling pathway in NE cells. Remarkably, IL-6 induces NED of LNCaP cells through activation of AMPK and SIRT1 and also both of these are co-regulated while playing a predominant role in NED of LNCaP cells. Of the few requirements of AMPK-SIRT1 activation, increased eNOS is essential for NED by elevating Nitric oxide (NO) levels. Pleiotropic effects of NO ultimately regulate p38MAPK in IL-6 induced NED. Hence, IL-6 induced AMPK-SIRT1 activation eventually transfers its activation signals through p38MAPK for advancing NED of LNCaP cells. Moreover, inactivation of p38MAPK with specific inhibitor (SB203580) attenuated IL-6 induced NED of LNCaP cells. Therefore, IL-6 promotes NED of PCa cells via AMPK/SIRT1/p38MAPK signaling. Finally, targeting AMPK-SIRT1 or p38MAPK in androgen independent PC3 cells with neuroendocrine features reversed their neuroendocrine characteristics. Taken together these novel findings reveal that targeting p38MAPK mitigated NED of PCa cells, and thus it can be a favorable target to overcome progression of NEPC.

MUC1-C Activates the BAF (mSWI/SNF) Complex in Prostate Cancer Stem Cells.

The Brg/Brahma-associated factor (BAF, mSWI/SNF) chromatin remodeling complex is of importance in development and has been linked to prostate oncogenesis. The oncogenic MUC1-C protein promotes lineage plasticity in the progression of neuroendocrine prostate cancer (NEPC), however, there is no known association between MUC1-C and BAF. We report here that MUC1-C binds directly to the E2F1 transcription factor and that the MUC1-C→E2F1 pathway induces expression of embryonic stem cell-specific BAF (esBAF) components BRG1, ARID1A, BAF60a, BAF155, and BAF170 in castrate-resistant prostate cancer (CRPC) and NEPC cells. In concert with this previously unrecognized pathway, MUC1 was associated with increased expression of E2F1 and esBAF components in NEPC tumors as compared with CRPC, supporting involvement of MUC1-C in activating the E2F1→esBAF pathway with progression to NEPC. MUC1-C formed a nuclear complex with BAF and activated cancer stem cell (CSC) gene signatures and the core pluripotency factor gene network. The MUC1-C→E2F1→BAF pathway was necessary for induction of both the NOTCH1 effector of CSC function and the NANOG pluripotency factor, and collectively, this network drove CSC self-renewal. These findings indicate that MUC1-C promotes NEPC progression by integrating activation of E2F1 and esBAF with induction of NOTCH1, NANOG, and stemness. SIGNIFICANCE: These findings show that MUC1-C, which promotes prostate cancer progression, activates a novel pathway that drives the BAF remodeling complex, induces NOTCH1 and NANOG, and promotes self-renewal of prostate cancer stem cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Nerve growth factor interacts with CHRM4 and promotes neuroendocrine differentiation of prostate cancer and castration resistance

Nerve growth factor (NGF) contributes to the progression of malignancy. However, the functional role and regulatory mechanisms of NGF in the development of neuroendocrine prostate cancer (NEPC) are unclear. Here, we show that an androgen-deprivation therapy (ADT)-stimulated transcription factor, ZBTB46, upregulated NGF via ZBTB46 mediated-transcriptional activation of NGF. NGF regulates NEPC differentiation by physically interacting with a G-protein-coupled receptor, cholinergic receptor muscarinic 4 (CHRM4), after ADT. Pharmacologic NGF blockade and NGF knockdown markedly inhibited CHRM4-mediated NEPC differentiation and AKT-MYCN signaling activation. CHRM4 stimulation was associated with ADT resistance and was significantly correlated with increased NGF in high-grade and small-cell neuroendocrine prostate cancer (SCNC) patient samples. Our results reveal a role of the NGF in the development of NEPC that is linked to ZBTB46 upregulation and CHRM4 accumulation. Our study provides evidence that the NGF-CHRM4 axis has potential to be considered as a therapeutic target to impair NEPC progression.

The Correlation between PSCA Expression and Neuroendocrine Differentiation in Prostate Cancer.

The prostate stem cell antigen (PSCA), as a predominantly prostate-specific marker, is overexpressed in most prostate cancer specimens, is positively correlated with prostate cancer androgen independence, and has the potential to be treated with castration-resistant prostate cancer (CRPC) as a gene therapy target. Using the typical androgen deprivation therapy, most tumors will progress to CRPC, as well as develop into neuroendocrine prostate cancer (NEPC) characterized by the expression of neuroendocrine markers such as enolase 2 (NSE). Our study was aimed at investigating the expressions of PSCA and NSE and the relationship between the two markers, as well as the correlation between the PSCA and NSE expressions and the clinicopathological parameters in prostate cancer specimens from 118 patients by using immunohistochemistry. Our results demonstrated that the PSCA and NSE protein expressions did not correlate with the prostate cancer patients' age or the hormone therapy but showed a significant correlation with the pathological tumor stage of prostate cancer, the Gleason score, and the presence of metastasis. There is a positive association between PSCA and NSE but a negative one between the prostate-specific antigen (PSA) and PSCA or between PSA and NSE. High PSCA and NSE expressions correlated with a poor prognosis in prostate cancer patients. PSCA may play an important role in the progression of neuroendocrine prostate cancer (NEPC).

Abstract 304: Androgen deprivation induces epithelial to mesenchymal transition and neuroendocrine differentiation of prostate cancer via LIF-mediated phosphorylation of Smad2

Abstract The current therapies for prostate cancer, which can either be androgen-sensitive or androgen-insensitive ones, are based on AR-targeting approaches; however, there is no available treatment for neuroendocrine prostate cancer (NEPC). We previously identified the leukemia inhibitory factor (LIF), which is stimulated by androgen deprivation therapy (ADT). Here, we further elucidated the physiological relevance of LIF, whereby the high LIF output is associated with the activation of epithelial-to-mesenchymal transition (EMT), and neuroendocrine differentiation of prostate cancer cells through stimulation of transforming growth factor (TGF)-β signaling. We have elucidated the effect of LIF on inducing the EMT and NEPC progression through the stimulated TGF-β signaling via phosphorylation of Smad2. We also demonstrated that the inverse relationship between inhibition of AR signaling and induction of LIF-Smad2 signaling may predispose prostate cancer to metastasis. Importantly, an established activation in prostate cancer cells was shown between recombinant LIF protein and phosphorylation of Smad2, supporting the connection between LIF and p-Smad2. This was supported by the results obtained in high-LIF samples with elevated expression of p-Smad2 in tissues from high-grade prostate cancer. This study highlights the role of the important crosstalk between the LIF and TGF-β signaling pathways may exist and provide a new direction for investigating NEPC development. Citation Format: Yen-Nien Liu, Shian-Ren Lin, Ntlotlang Mokgautsi, Hsiu-Lien Yeh, Jiaoti Huang. Androgen deprivation induces epithelial to mesenchymal transition and neuroendocrine differentiation of prostate cancer via LIF-mediated phosphorylation of Smad2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 304.

SOX2 has dual functions as a regulator in the progression of neuroendocrine prostate cancer

The mechanisms underlying the lineage switching from prostate adenocarcinoma (AdPC) to lethal neuroendocrine prostate cancer (NEPC) have yet to be completely elucidated. In this study, RNA sequencing data from a unique patient-derived xenograft NEPC model and a clinical NEPC cohort were used to identify the potential genes driving NEPC progression. Enrichr analysis resulted in the identification of SRY-related HMG-box gene 2 (SOX2) as a potential repressor that causes decrease in the expression of AdPC specific genes in NEPC. Assays involving the stable overexpression of SOX2 in LNCaP and CWR22RV1 cells validated this role of SOX2 in vitro. Mechanistic studies showed that the repressor role of SOX2 was attributed to the marked global hypomethylation of histone H3, which was driven by the activation of lysine-specific demethylase 1 (LSD1). Furthermore, Enrichr also predicted SOX2 as a driver gene involved in the upregulation of NEPC specific genes. However, SOX2 alone could only marginally induce the expression of some neuroendocrine markers in vitro, which was consistent with previous reports. Moreover, we also elucidated the molecular features of LNCaP-SOX2 cells that may confer resistance to androgen-deprivation therapy (ADT) and the inclination toward neuroendocrine transdifferentiation. The results of this study reveal a novel mechanism for SOX2 in the progression of NEPC via LSD1-mediated global epigenetic modulation. This discovery suggests that LSD1 may be a selective target for the prevention of NEPC progression.

MUC1-C regulates lineage plasticity driving progression to neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is an aggressive malignancy with no effective targeted therapies. The oncogenic MUC1-C protein is overexpressed in castration-resistant prostate cancer (CRPC) and NEPC, but its specific role is unknown. Here, we demonstrate that upregulation of MUC1-C in androgen-dependent PC cells suppresses androgen receptor (AR) axis signaling and induces the neural BRN2 transcription factor. MUC1-C activates a MYC→BRN2 pathway in association with induction of MYCN, EZH2 and NE differentiation markers (ASCL1, AURKA and SYP) linked to NEPC progression. Moreover, MUC1-C suppresses the p53 pathway, induces the Yamanaka pluripotency factors (OCT4, SOX2, KLF4 and MYC) and drives stemness. Targeting MUC1-C decreases PC self-renewal capacity and tumorigenicity, suggesting a potential therapeutic approach for CRPC and NEPC. In PC tissues, MUC1 expression associates with suppression of AR signaling and increases in BRN2 expression and NEPC score. These results highlight MUC1-C as a master effector of lineage plasticity driving progression to NEPC.

RNA Splicing of the BHC80 Gene Contributes to Neuroendocrine Prostate Cancer Progression

BackgroundProstate adenocarcinoma (AdPC) progression to treatment-induced neuroendocrine prostate cancer (t-NEPC) is associated with poor patient survival. While AdPC and t-NEPC share similar genomes, they possess distinct transcriptomes, suggesting that RNA splicing and epigenetic mechanisms may regulate t-NEPC development. ObjectiveTo characterize the role of alternative RNA splicing of the histone demethylase BHC80 during t-NEPC progression. Design, setting, and participantsThe expression of BHC80 splice variants (BHC80-1 and BHC80-2) were compared between AdPC and t-NEPC patient tumors. Regulatory mechanisms of RNA splicing of the BHC80 gene were studied, and the signal pathways mediated by BHC80 splice variants were investigated in t-NEPC cell and xenograft models. ResultsGlobal transcriptome analyses identified that the BHC80-2 variant is highly expressed in t-NEPC. Compared with the known histone demethylation activities of the BHC80 gene, we discovered a novel nonepigenetic action of BHC80-2, whereby BHC80-2 is localized in the cytoplasm to trigger the MyD88-p38-TTP pathway, which results in increased RNA stability of multiple tumor-promoting cytokines. While BHC80-2 does not induce neuroendocrine differentiation of cancer cells, it stimulates cell proliferation and tumor progression independent of androgen receptor signaling. Blockade of BHC80-2-regulated MyD88 signaling suppresses growth of several t-NEPC cell spheroid and xenograft models. ConclusionsGain of function of BHC80-2 through alternative RNA splicing activates immune responses of cancer cells to promote t-NEPC development. Patient summaryThe main obstacle to develop effective therapies for patients with t-NEPC is the lack of understanding on how t-NEPC is developed. Our study not only identifies a previously unknown BHC80-2-MyD88 signaling pathway that plays an important role during t-NEPC development, but also provides a proof of principle that targeting this signal pathway may offer an avenue to treat t-NEPC.