Neuroendocrine Differentiation Of Prostate Cancer Cells Research Articles

Immunosuppressive role of BDNF in therapy-induced neuroendocrine prostate cancer.

Prostate stromal cells play a crucial role in the promotion of tumor growth and immune evasion in the tumor microenvironment (TME) through intricate molecular alterations in their interaction with prostate cancer (PCa) cells. While the impact of these cells on establishing an immunosuppressive response and influencing PCa aggressiveness remains incompletely understood. Our study shows that the activation of the leukemia inhibitory factor (LIF)/LIF receptor (LIFR) pathway in both prostate tumor and stromal cells, following androgen deprivation therapy (ADT), leads to the development of an immunosuppressive TME. Activation of LIF/LIFR signaling in PCa cells induces neuroendocrine differentiation (NED) and upregulates immune checkpoint expression. Inhibition of LIF/LIFR attenuates these effects, underscoring the crucial role of LIF/LIFR in linking NED to immunosuppression. Prostate stromal cells expressing LIFR contribute to NED and immunosuppressive marker abundance in PCa cells, while LIFR knockdown in prostate stromal cells reverses these effects. ADT-driven LIF/LIFR signaling induces brain-derived neurotrophic factor (BDNF) expression, which, in turn, promotes NED, aggressiveness, and immune evasion in PCa cells. Clinical analyses demonstrate elevated BDNF levels in metastatic castration-resistant PCa (CRPC) and a positive correlation with programmed death-ligand 1 (PDL1) and immunosuppressive signatures. This study shows that the crosstalk between PCa cells and prostate stromal cells enhances LIF/LIFR signaling, contributing to an immunosuppressive TME and NED in PCa cells through the upregulation of BDNF.

The Neuropilin-1/PKC axis promotes neuroendocrine differentiation and drug resistance of prostate cancer

BackgroundNeuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance.MethodsUsing RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression.ResultsWe show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo.ConclusionsThis study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker.

Abstract LB077: Adrenergic signaling induces neuroendocrine differentiation of prostate adenocarcinoma cells

Abstract Prostate cancer is the leading cancer diagnosed among men worldwide. The prostate cancer-associated mortality is due to the development of aggressive neuroendocrine prostate cancer (NEPC). Unfortunately, there is no effective treatment available for NEPC at present. This is due to the lack of proper understanding of the molecular switch that triggers neuroendocrine changes in prostate adenocarcinoma cells. Recent research demonstrated that peripheral nerves promote prostate cancer progression and metastasis. However, there is no clear understanding of the involvement of sympathetic nerves or their signaling in facilitating NEPC. In this work, we examined the effect of the sympathetic neurotransmitter norepinephrine (NE) in inducing neuroendocrine differentiation (NED) of prostate cancer cells in vitro. We used androgen-sensitive LNCaP and androgen-insensitive DU145 cells for the study. We examined the dose and time-dependent effect of NE in inducing NED-related phenotypic alterations in these cells using bright field and fluorescence imaging. NED-related molecular alterations were assessed using real-time PCR, western bot and immunocytochemistry. We found that NE induces characteristic NEPC morphological changes, such as compact cell bodies and neurite-like extensions, in both DU145 and LNCaP cells. At the mRNA level, NE induced the expression of the NED markers chromogranin B, synaptophysin and βIII tubulin in both cell types. Further immunoblot and immunostaining showed the upregulation of chromogranin B in these cells. We repeated the experiments in growth factor deprived conditions by avoiding fetal bovine serum in the cultures to mimic growth deprivation to prostate cancer cells, which they experience during androgen deprivation therapy. Surprisingly, the NE induced NED changes were more pronounced in the growth factor deprived conditions. Overall, our result indicates the potential of NE in inducing NED in prostate cancer cells and point towards a critical role for this sympathetic neurotransmitter in facilitating the development of NEPC. Citation Format: Anand Krishnan, Shubham Dwivedi, Maricris Bautista, Tauqeer Iftikhar. Adrenergic signaling induces neuroendocrine differentiation of prostate adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB077.

Targeting Wnt/EZH2/microRNA-708 signaling pathway inhibits neuroendocrine differentiation in prostate cancer

Prostate cancer (PC) castration resistance has been linked to the differentiation of PC luminal cells into hormone-refractory neuroendocrine (NE) cells. However, the molecular mechanisms controlling the emergence of lethal NE prostate cancer (NEPC) remain unclear. The present study aimed to investigate the mechanisms underlying the transition from prostate adenocarcinoma to NEPC. The microRNA miR-708 was involved in NE differentiation and was downregulated in NEPC cells and tumor specimens. miR-708 targeted Sestrin-3 to inhibit Forkhead Box O1 (FOXO1) phosphorylation, resulting in apoptosis of prostate adenocarcinoma cells and AKT-inactivated NEPC cells, the latter of which was consistent with the progression of tumor xenografts in mice under miR-708 treatment. In silico analysis of PC and NEPC tumor specimens suggested that the polycomb repressive complex subunit Enhancer of zeste homolog 2 (EZH2) was particularly overexpressed in NEPC. Notably, EZH2 bound to the miR-708 promoter and induced its silencing in NEPC. Inhibition of EZH2 prevented NE differentiation of PC cells. EZH2 expression was regulated by both Cyclin Dependent Kinase 1 (CDK1) and Wnt signaling. Silencing transcription factor 4 (TCF4), as a key protein in Wnt signaling, prevented NEPC formation. These results provide a molecular basis for the roles of miR-708 and EZH2 in NE differentiation in PC and highlight a new paradigm in NEPC formation and survival.

Correction: Implications of PI3K/AKT inhibition on REST protein stability and neuroendocrine phenotype acquisition in prostate cancer cells

[This corrects the article DOI: 10.18632/oncotarget.19386.].

Implications of PI3K/AKT inhibition on REST protein stability and neuroendocrine phenotype acquisition in prostate cancer cells.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer (PCa) that arises as a consequence of rigorous androgen receptor (AR) pathway inhibition (ARPI) therapies. While the PI3K/AKT pathway has been investigated as a co-therapeutic target with ARPI for advanced PCa, whether this strategy can prevent tumor progression to t-NEPC remains unknown. Here, we report that PI3K/AKT inhibition alone reduces RE-1 silencing transcription factor (REST) protein expression and induces multiple NE markers in PCa cells. The loss of REST by PI3K/AKT inhibition is through protein degradation mediated by the E3-ubiquitin ligase β-TRCP and REST phosphorylations at the S1024, S1027, and S1030 sites. Since AR inhibition can also deplete REST, the combinational inhibition of PI3K/AKT and AR further aggravated REST protein reduction. We profiled the transcriptomes of AKT and AR inhibitions in the LNCaP cells. The Gene Set Enrichment Analysis (GSEA) showed that these transcriptomes are highly correlated with the REST-regulated gene signature. Co-targeting AKT and AR resulted in a higher correlation comparing to those of single treatment. Comparing these transcriptomes to the t-NEPC gene signature in patients by GSEA, we observed that adding AKT inhibition to AR blockade enhanced the expression of neurogenesis-related genes and resulted in a stronger and broader upregulation of REST-regulated genes specific to t-NEPC. These results indicate that AKT pathway inhibition can induce neuroendocrine differentiation of PCa cells via REST protein degradation. It delineates a potential risk for the AR and PI3K/AKT co-targeting strategy as it may further facilitate t-NEPC development.

Abstract 1577: GRK3 is a direct target of ADT-induced CREB1 activation and it promotes neuroendocrine differentiation of prostate cancer cells

Abstract Treatment-related neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer that is believed to arise through neuroendocrine differentiation (NED) from prostate adenocarcinoma (PAC) upon castration resistance to androgen deprivation treatment (ADT). t-NEPC is highly metastatic with poor prognosis. With the recent introduction of potent ADT drugs in clinic, the incidence of t-NEPC is expected to increase dramatically. No effective therapeutic is available for t-NEPC and its molecular mechanisms remain poorly understood. We reported that GRK3 (G protein-coupled receptor kinase 3) promotes prostate cancer progression. We further demonstrated that the ADT activates CREB1 (cAMP response element binding protein 1), which directly targets and induces GRK3. GRK3 expression is higher in t-NEPC than in PAC cells and mouse models, and it positively correlates with the expression and activity of CREB1 in human prostate cancers. Notably, overexpression of GRK3 in PAC cells increased the expression of NE markers. Conversely, silencing GRK3 blocked CREB1-induced NED in PAC cells, reversed NE phenotypes and inhibited proliferation of t-NEPC cells in culture and in xenograft. Currently, we are investigating the mechanisms underlying NEPC progression promoted by the CREB1/GRK3 axis. Taken together, these results indicate that GRK3 is a new critical activator of NE phenotypes and mediator of CREB1 activation in promoting NED of prostate cancer cells. Citation Format: Dayong Zheng, Mohit Hulsurkar, Meixiang Sang, Songlin Zhang, Jianming Xu, Martin Gleave, Michael Ittmann, Wenliang Li. GRK3 is a direct target of ADT-induced CREB1 activation and it promotes neuroendocrine differentiation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2017-1577

MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells

Autophagy and apoptosis are two well-controlled mechanisms regulating cell fate. An understanding of decision-making between these two pathways is in its infancy. Monoamine oxidase A (MAOA) is a mitochondrial enzyme that is well-known in psychiatric research. Emerging reports showed that overexpression MAOA is associated with prostate cancer (PCa). Here, we show that MAOA is involved in mediating neuroendocrine differentiation of PCa cells, a feature associated with hormone-refractory PCa (HRPC), a lethal type of disease. Following recent reports showing that NED of PCa requires down-regulation of repressor element-1 silencing transcription factor (REST) and activation of autophagy; we observe that MAOA is a novel direct target gene of REST. Reactive oxygen species (ROS) produced by overexpressed MAOA plays an essential role in inhibiting apoptosis and activating autophagy in NED PCa cells. MAOA inhibitors significantly reduced NED and autophagy activation of PCa cells. Our results here show MAOA as a new decision-maker for activating autophagy and MAOA inhibitors may be useful as a potential therapy for neuroendocrine tumors.

GRK3 is a direct target of CREB activation and regulates neuroendocrine differentiation of prostate cancer cells.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that commonly arises through neuroendocrine differentiation (NED) of prostate adenocarcinoma (PAC) after therapy, such as radiation therapy and androgen deprivation treatment (ADT). No effective therapeutic is available for NEPC and its molecular mechanisms remain poorly understood. We have reported that G protein-coupled receptor kinase 3 (GRK3, also called ADRBK2) promotes prostate cancer progression. In this study, we demonstrate that the ADT-activated cAMP response element binding protein (CREB) directly targets and induces GRK3. We show GRK3 expression is higher in NEPC than in PAC cells and mouse models, and it positively correlates with the expression and activity of CREB in human prostate cancers. Notably, overexpression of GRK3 in PAC cells increased the expression of NE markers in a kinase activity dependent manner. Conversely, silencing GRK3 blocked CREB-induced NED in PAC cells, reversed NE phenotypes and inhibited proliferation of NEPC cells. Taken together, these results indicate that GRK3 is a new critical activator of NE phenotypes and mediator of CREB activation in promoting NED of prostate cancer cells.

Posttranscriptional regulation of T-type Ca2+ channel expression by interleukin-6 in prostate cancer cells

BackgroundAt early stages, the growth of prostate cancers is androgen dependent. At later stages, however, the growth of prostate cancers becomes androgen independent, which leads to an increase in mortality. The switch to an androgen-refractory state is associated with neuroendocrine differentiation (NED) of prostate cancer cells. Several factors including interleukin-6 (IL-6) and increased cAMP production promote NED of prostate cancer cells. In this work we investigated whether IL-6 evoked NED of LNCaP cells results in a significant change in T-type Ca2+ channel expression in comparison to non-stimulated LNCaP cells. MethodsT-type Ca2+ channel subunit Cav3.2 expression was studied using PCR analysis, western blot and whole cell recordings. Tubulin IIIβ expression and neurite-like morphology was assessed to investigate the role of T-type Ca2+ channels in the differentiation of prostate cancer cells. ResultsTreatment of LNCaP cells with IL-6 for 4days evokes considerable morphological and biochemical changes consistent with NED. Transcripts of the T-type Ca2+ channel subunit Cav3.2 but not Cav3.1 or Cav3.3 are detected in IL-6 stimulated cells. Real time PCR analysis of IL-6 stimulated cells indicates no significant change in Cav3.2 mRNA expression in comparison to non-stimulated cells. LNCaP cells stimulated with IL-6 show a threefold increase in T-type Ca2+ channel subunit Cav3.2 protein expression, suggesting that channel expression is upregulated by a posttranscriptional mechanism. Electrophysiological recordings reveal that increased Cav3.2 protein expression following IL-6 stimulation of LNCaP cells does not result in increased expression of functional channels in the membrane. Functional expression of Cav3.2 channels in LNCaP cells is facilitated by co-stimulation with IL-6 and the cAMP-stimulating agent, forskolin (FSK). Inhibition of T-type Ca2+ channel activity in IL-6 stimulated LNCaP cells prevents the development of morphological characteristics consistent with NED. ConclusionsThese results indicate that the functional expression of T-type Ca2+ channels is regulated by the interplay of multiple factors in LNCaP cells.

Tissue Chromogranin A Expression during Prostate Cancer Progression: Prediction of Chemosensitivity.

We investigated the clinical significance of chromogranin A (CgA) expression as a neuroendocrine (NE) marker during prostate cancer (PCa) progression, especially as a potential predictor of chemotherapeutic response in castration-resistant PCa (CRPC) patients based on immunohistochemical findings. Sixteen CRPC patients who underwent combination (docetaxel/estramustine/ carboplatin; DEC) chemotherapy were retrospectively studied. Immunostaining of CgA was performed using prostate biopsy samples obtained at the initial PCa diagnosis, during androgen deprivation therapy, at the time of CRPC diagnosis, and after 2 cycles of DEC therapy. The positive rate was expressed as the mean percentage of positively stained tumor cells against the total number of tumor cells. Differences in positive rates among the treatment courses were compared using a Mann-Whitney test. The mean percentage of CgA-positive PCa cells increased in a stepwise manner until CRPC development and then significantly decreased after DEC therapy. Subanalysis of CgA at CRPC diagnosis showed a more evident reduction of CgA expression after DEC therapy in patients who also had a high level of CgA as compared to those with a low CgA level (P = .003). Likewise, longer prostate-specific antigen progression-free survival was related to CRPC and high CgA (P = .028). NE differentiation of PCa cells is accelerated despite androgen deprivation and reaches a peak at the time of CRPC diagnosis. Although further studies using larger samples are needed, CgA expression in CRPC may be a candidate tissue biomarker to reflect the chemotherapy sensitivity of individual PCa cells.

Interleukin-6 induces neuroendocrine differentiation (NED) through suppression of RE-1 silencing transcription factor (REST).

Paracrine interleukin-6 (IL-6) can mediate neuroendocrine (NE) features, including the acquisition of a neurite-like phenotype and growth arrest in prostate cancer cells. However, little is known about the mechanisms underlying neuroendocrine differentiation induced by IL-6. Immunoblotting was performed to determine the status of RE1-silencing transcription factor (REST) and of neuroendocrine markers such as Neuron-specific Enolase (NSE), chromogranin A and synaptophysin in LNCaP cells treated with IL-6. To further study the impact of REST-mediated repression on neuroendocrine differentiation (NED) in LNCaP cells, either wild-type REST or a dominant-positive form of REST, REST-VP16, in which both repressor domains of REST were replaced with the activation domain of the herpes simplex virus protein VP16, was introduced into LNCaP cells. In this study, we show that REST is suppressed in IL-6-induced neuroendocrine differentiation in LNCaP cells. Overexpression of exogenous REST abrogated IL-6-induced NED in prostate cancer cells. Expression of the recombinant REST-VP16 fusion protein activated REST target genes and other neuronal differentiation genes and produced neuronal physiological properties. In addition, REST protein turnover was accelerated in IL-6 induced NE differentiated LNCaP cells via the ubiquitin-proteasome pathway, accompanied by a decrease in the expression of the deubiquitylase HAUSP, indicating that pathway(s) priming REST degradation may be involved in IL-6 induced NE differentiation. These results demonstrate that REST functions as a major switch of IL-6 induced neuroendocrine differentiation in LNCaP cells.

Macrophages induce neuroendocrine differentiation of prostate cancer cells via BMP6‐IL6 Loop

Frequently associated with hormone refractory prostate cancer are neuroendocrine cells. Because these cells do not express androgen receptors and are castration-resistant, further understanding the mechanism of neuroendocrine differentiation (NED) of prostate cancer cells may yield novel intervention methods in hormone refractory prostate cancer. In this regard, the present study investigated the effect of macrophages on prostate cancer NED. THP-1 and LNCaP or RAW264.7 and TRAMP-C2 cell line co-cultures were used to investigate NED-macrophage interactions. Also interleukin-6 (IL-6) knockout mice and macrophage-depleted mice were used to test NED in vivo. We found that co-culturing with THP-1 human monocytic cell line and RAW 264.7 murine macrophage cell line led to the NED of LNCaP and TRAMP-C2 prostate cancer cells, respectively. Specifically, the conditioned media of activated macrophages stimulated the expression of parathyroid hormone-related peptide (PTHrP), a marker of NED, in both LNCaP and TRAMP-C2 cells. Mechanistically, bone morphogenetic protein-6 (BMP-6) derived from prostate cancer cells increased the expression of IL-6 in macrophages. Subsequently, IL-6 induced the NED of prostate cancer cells. When this feedback loop was disrupted with neutralizing antibodies to either BMP-6 or IL-6, NED was no longer observed. In human prostate cancer tissues, neuroendocrine cells frequently co-localized with macrophages and BMP-6. In mice, the removal of IL-6 or macrophages blocked the BMP-6-induced NED of prostate cancer cells. Therefore, we propose that BMP-6 secreted by prostate cancer cells induces IL-6 expression in macrophages; IL-6, in turn, stimulates the NED of prostate cancer cells.