Differentiation Of Prostate Cancer Cells Research Articles

Targeting the αVβ3/NgR2 pathway in neuroendocrine prostate cancer

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVβ3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVβ3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVβ3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVβ3 ligands. Moreover, we describe for the first time co-fractionation of αVβ3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients’ plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as “adhesion competent”. Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

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.

Yin Yang 1 promotes the neuroendocrine differentiation of prostate cancer cells via the non-canonical WNT pathway (FYN/STAT3).

A growing number of studies have shown that Yin Yang 1 (YY1) promotes the development of multiple tumours. The purpose of the current study was to determine the mechanism by which YY1 mediates neuroendocrine differentiation of prostate cancer (NEPC) cells undergoing cellular plasticity. Using the Cancer Genome Atlas and Gene Expression Omnibus (GEO) databases, we bioinformatically analyzed YY1 expression in prostate cancer (PCa). Aberrant YY1 expression was validated in different PCa tissues and cell lines via quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemistry. In vivo and in vitro functional assays verified the oncogenicity of YY1 in PCa. Further functional assays showed that ectopic expression of YY1 promoted cellular plasticity in PCa cells via epithelial-mesenchymal transition induction and neuroendocrine differentiation. Androgen deprivation therapy induced a decrease in YY1 protein ubiquitination, enhanced its stability, and thus enhanced the transcriptional activity of FZD8. Castration enhanced FZD8 binding to Wnt9A and mediated cellular plasticity by activating the non-canonical Wnt (FZD8/FYN/STAT3) pathway. We identified YY1 as a novel dysregulated transcription factor that plays an important role in NEPC progression in this study. We believe that an in-depth investigation of the mechanism underlying YY1-mediated disease may lead to improved NEPC therapies.

Abstract A037: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2

Abstract Background: Treatment-related neuroendocrine prostate cancer (NEPC) is an aggressive subset of castration-resistant prostate cancer (CRPC), found in ~20% of lethal CRPC. The mechanisms underlying the progression of prostate cancer to NEPC are largely unclear, and new drug targets are desperately needed. NEPC is known to be highly vascularized. Elevated expression of NE markers and increased angiogenesis are two prominent phenotypes of NEPC, and thus are expected to be linked. However, direct molecular links between these two phenotypes are still elusive, whose elucidation will substantially expand our knowledge in NEPC and enable the development of effective treatments for NEPC. Through RNAi & cDNA screening and functional validations, we previously discovered that GPCR-kinase 3 (GRK3) is essential preferentially for highly metastatic cancer cells as compared to lowly metastatic cancer cells. The mechanisms of GRK3 in prostate cancer progression were mostly unknown. Methods: We assessed GRK3’s expression in patient samples through IHC staining on TMA and data mining on public RNA-seq datasets with large cohorts. Using molecular and cell biology methods, we determined the impacts of GRK3 and HDAC2 overexpression and silencing on NE marker expression and angiogenesis in prostate cancer cells. Through biochemistry, we investigated the nature of GRK3-HDAC2 relation. By ChIP-PCR, we measured the impacts of GRK3 genetic and pharmacological modulations on HDAC2’s epigenetic activity. Through compound library screening and biochemical characterization, we searched for novel GRK3 inhibitors to study GRK3 biology and to evaluate whether GRK3 is a suitable drug target for prostate cancer. Results: GRK3 is significantly overexpressed in metastatic prostate tumors from patients, especially in NEPC. GRK3 promotes both angiogenesis and neuroendocrine differentiation in prostate cancer cells, indicating that it is a key missing link for these two phenotypes. Mechanistically, GRK3 enhances the epigenetic repressor activity of histone deacetylase 2 (HDAC2) to suppress key repressors of angiogenesis or NE phenotype. We have identified several compounds that block kinase activity of GRK3 much more potently than that of GRK2, the closest-related kinase to GRK3. Of note, our GRK3 inhibitors could substantially reduce angiogenesis and NE marker expression, as well as significantly inhibit NEPC cell growth in culture and in mouse xenografts. Conclusion: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression. Its mechanism of actions is at least in part through enhancing HDAC2’s epigenetic activity. Results based on our novel GRK3 inhibitors suggest that GRK3 is a valuable new drug target for aggressive prostate cancer. Citation Format: Samira Naderinezhad, Zheng Wang, Guoliang Zhang, Michael Ittmann, Martin Gleave, Wenliang Li. Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2 [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 A037.

CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

Current treatment options for prostate cancer focus on targeting androgen receptor (AR) signaling. Inhibiting effects of AR may activate neuroendocrine differentiation and lineage plasticity pathways, thereby promoting the development of neuroendocrine prostate cancer (NEPC). Understanding the regulatory mechanisms of AR has important clinical implications for this most aggressive type of prostate cancer. Here, we demonstrated the tumor-suppressive role of the AR and found that activated AR could directly bind to the regulatory sequence of muscarinic acetylcholine receptor 4 (CHRM4) and downregulate its expression. CHRM4 was highly expressed in prostate cancer cells after androgen-deprivation therapy (ADT). CHRM4 overexpression may drive neuroendocrine differentiation of prostate cancer cells and is associated with immunosuppressive cytokine responses in the tumor microenvironment (TME) of prostate cancer. Mechanistically, CHRM4-driven AKT/MYCN signaling upregulated the interferon alpha 17 (IFNA17) cytokine in the prostate cancer TME after ADT. IFNA17 mediates a feedback mechanism in the TME by activating the CHRM4/AKT/MYCN signaling-driven immune checkpoint pathway and neuroendocrine differentiation of prostate cancer cells. We explored the therapeutic efficacy of targeting CHRM4 as a potential treatment for NEPC and evaluated IFNA17 secretion in the TME as a possible predictive prognostic biomarker for NEPC.

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 LB032: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2

Abstract Treatment-related neuroendocrine prostate cancer (t-NEPC, here as NEPC) is an aggressive subset of castration-resistant prostate cancer (CRPC), found in ~20% of lethal CRPC. The mechanisms underlying the progression of prostate cancer to NEPC are largely unclear, and new drug targets are desperately needed. NEPC is known to be highly vascularized. Elevated expression of NE markers and increased angiogenesis are two prominent phenotypes of NEPC, and thus are expected to be linked. However, direct molecular links between these two phenotypes are still elusive, whose elucidation will substantially expand our knowledge in NEPC and enable the development of effective treatments for NEPC. Through RNAi & cDNA screening and functional validations, we previously discovered that GPCR-kinase 3 (GRK3) is essential preferentially for highly metastatic cancer cells as compared to lowly metastatic cancer cells. The mechanisms of GRK3 in prostate cancer progression were mostly unknown. Our new data indicate that GRK3 is significantly overexpressed in metastatic prostate tumors from patients, especially in NEPC. GRK3 promotes both angiogenesis and neuroendocrine differentiation in prostate cancer cells, indicating that it is a key missing link for these two phenotypes. Mechanistically, GRK3 enhances the epigenetic repressor activity of histone deacetylase 2 (HDAC2) to suppress key repressors of angiogenesis or NE phenotype. Through compound library screening, we have identified several compounds that block kinase activity of GRK3 much more potently than that of GRK2, the closest-related kinase to GRK3. Of note, our GRK3 inhibitors could substantially reduce angiogenesis and NE marker expression, as well as significantly inhibit NEPC cell growth in culture and in mouse xenografts. In summary, kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression. Its mechanism of actions is at least in part through enhancing HDAC2’s epigenetic activity. Results based on our novel GRK3 inhibitors suggest that GRK3 is a valuable new drug target for aggressive prostate cancer. Citation Format: Samira Naderinezhad, Guoliang Zhang, Zheng Wang, Dayong Zheng, Junwei Lian, Wenliang Li. Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB032.

Abstract 1698: Novel role of Tribbles 2 driving treatment-emergent neuroendocrine differentiation in prostate cancer

Abstract Background: Prostate cancer initially responds to androgen receptor (AR) targeted therapies, such as enzalutamide. However, therapy-resistant, incurable disease invariably develops which remains as the major cause for mortality among prostate cancer patients. Molecular mechanism underlying emergence of resistant prostate cancer is yet to be fully characterized. Methods: We developed an in vitro cell culture model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. These cells were transcriptionally profiled for gene expression analysis by Illumina Hi-Seq whole genome gene-expression array. Hits were validated by RT-qPCR and Western blot. For clinical correlation, enzalutamide treated prostate PDX models and surgical tumors (warm autopsy) from prostate cancer patients were comprehensively analyzed. Regulation and role of new hits were determined by treating cells and xenograft tumors with lentiviral-based shRNAs followed by analysis of cell viability, apoptosis, and effects on downstream targets. Pro-cancer effects were analyzed by cell proliferation, invasion, and soft-agar colony formation assays. Results: We identified that the Tribbles 2 (TRIB2) pseudokinase is overexpressed in enzalutamide-resistant prostate cancer (ERPC) cells. Elevated level of TRIB2 was also observed in enzalutamide treated PDX tumors, and in prostate tumors from patients who developed enzalutamide resistant metastatic disease. Interestingly, TRIB2 protein level is especially higher in prostate cancer cells and tumors, where the AR activity is low or absent due to genetic changes. Transfection and overexpression of TRIB2 results in aggressive prostate cancer cell growth and resistance to enzalutamide, apalutamide, darolutamide, and abiraterone. Furthermore, TRIB2 overexpression results in trans-differentiation in prostate cancer cells including neurite outgrowth and adeno-to-neuroendocrine lineage transition. Curiously, we found that TRIB2 downregulates luminal markers (AR, CK8), but upregulates the neuroendocrine markers (CHGA, NSE, SYP). Knockdown of TRIB2 triggers loss of neuroendocrine features, reduces viability of ERPC cells, and sensitize them back to enzalutamide, suggesting that TRIB2 plays a direct role in neuroendocrine differentiation and therapeutic resistance. We also found that TRIB2 upregulates the master neuronal transcription factor, BRN2, and the stemness factor, SOX2, and inhibition of either BRN2 or SOX2 re-sensitizes ERPC cells to enzalutamide. Conclusion: These findings reveal that TRIB2 is a novel driver for lineage plasticity and neuroendocrine differentiation in prostate cancer cells and greatly contributes to enzalutamide resistance. Citation Format: Jitender Monga, Craig Rogers, Shirish Gadgeel, Dhananjay Chitale, Jagadananda Ghosh. Novel role of Tribbles 2 driving treatment-emergent neuroendocrine differentiation in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1698.

The Role of Epigenetic Change in Therapy-Induced Neuroendocrine Prostate Cancer Lineage Plasticity.

The androgen receptor (AR) signaling pathway is critical for growth and differentiation of prostate cancer cells. For that reason, androgen deprivation therapy with medical or surgical castration is the principal treatment for metastatic prostate cancer. More recently, new potent AR signaling inhibitors (ARSIs) have been developed. These drugs improve survival for men with metastatic castration-resistant prostate cancer (CRPC), the lethal form of the disease. However, ARSI resistance is nearly universal. One recently appreciated resistance mechanism is lineage plasticity or switch from an AR-driven, luminal differentiation program to an alternate differentiation program. Importantly, lineage plasticity appears to be increasing in incidence in the era of new ARSIs, strongly implicating AR suppression in this process. Lineage plasticity and shift from AR-driven tumors occur on a continuum, ranging from AR-expressing tumors with low AR activity to AR-null tumors that have activation of alternate differentiation programs versus the canonical luminal program found in AR-driven tumors. In many cases, AR loss coincides with the activation of a neuronal program, most commonly exemplified as therapy-induced neuroendocrine prostate cancer (t-NEPC). While genetic events clearly contribute to prostate cancer lineage plasticity, it is also clear that epigenetic events—including chromatin modifications and DNA methylation—play a major role. Many epigenetic factors are now targetable with drugs, establishing the importance of clarifying critical epigenetic factors that promote lineage plasticity. Furthermore, epigenetic marks are readily measurable, demonstrating the importance of clarifying which measurements will help to identify tumors that have undergone or are at risk of undergoing lineage plasticity. In this review, we discuss the role of AR pathway loss and activation of a neuronal differentiation program as key contributors to t-NEPC lineage plasticity. We also discuss new epigenetic therapeutic strategies to reverse lineage plasticity, including those that have recently entered clinical trials.

Alternative isoforms of RNA polymerase III impact the non-coding RNA transcriptome, viability, proliferation and differentiation of prostate cancer cells

RNA polymerase III (pol III) synthesizes short noncoding RNA (ncRNA) exclusively and is unique in having alternative paralogues of one of its subunits, POLR3G and POLR3GL. Although most pol III target loci can be transcribed by either isoform, exceptions have been found. For example, depletion of POLR3G curtails the production of BC200 and snaR ncRNAs that are implicated in cancer progression. Furthermore, POLR3G may protect pol III against repression by MAF1, a key physiological regulator. Expression of POLR3G is promoted selectively by MYC, NANOG and OCT4A, master regulators of stem cell pluripotency, resulting in its preferential accumulation in undifferentiated cells. Indeed, differentiation of prostate cancer cells is suppressed by a positive feedback mechanism between POLR3G and NANOG, involving the control of NANOG mRNA degradation by ncRNAs. Specific knockdown of POLR3G inhibits proliferation and induces differentiation of prostate cancer cells, but this response is not seen following comparable depletion of its POLR3GL paralogue. ML-60218 is a cell-permeable small molecule pol III inhibitor that triggers the replacement of POLR3G with POLR3GL. Proliferation and viability of primary prostate cancer cells are suppressed by ML-60218, whereas differentiation is induced, effects that mimic POLR3G depletion. Transient exposure to ML-60218 reduced tumour initiating activity in a xenograft model. Untransformed prostate cells are much less sensitive to these treatments, raising the possibility of therapeutic benefit.

Increased Expression of AKT3 in Neuroendocrine Differentiated Prostate Cancer Cells Alters the Response Towards Anti-Androgen Treatment.

Simple SummaryMetastatic castration-resistant prostate cancer is still untreatable, and patients have a very short life expectancy after diagnosis. One factor that makes metastatic castration-resistant prostate cancer so aggressive and difficult to treat is neuroendocrine differentiation of prostate carcinoma cells. We already showed that neuroendocrine differentiation of LNCaP cells results in increased AKT3 expression. The aim of this study was to demonstrate the role of AKT3 in neuroendocrine differentiation. Therefore, the previously obtained in vitro data were validated and extended to tissue from patient with neuroendocrine prostate cancer, where we show the presence of AKT3 in neuroendocrine cells. Furthermore, we demonstrate the oncogenic consequences of an increased AKT3 expression including inactivation of apoptotic proteins and a potential role of some miR-17 family members, negatively regulating AKT3, in neuroendocrine differentiation.Patients with advanced prostate carcinoma are often treated with an androgen deprivation therapy but long-term treatment can result in a metastatic castration-resistant prostate cancer. This is a more aggressive, untreatable tumor recurrence often containing areas of neuroendocrine differentiated prostate cancer cells. Using an in vitro model of NE-like cancer cells, it could previously be shown that neuroendocrine differentiation of LNCaP cells leads to a strong deregulation of mRNA and miRNA expression. We observe elevated RNA and protein levels of AKT Serine/Threonine Kinase 3 (AKT3) in neuroendocrine-like LNCaP cells. We used prostate resections from patients with neuroendocrine prostate cancer to validate these results and detect a co-localization of neuroendocrine marker genes with AKT3. Analysis of downstream target genes FOXO3A and GSK3 strengthens the assumption AKT3 may play a role in neuroendocrine differentiation. Overexpression of AKT3 shows an increased survival rate of LNCaP cells after apoptosis induction, which in turn reflects the significance in vivo or for treatment. Furthermore, miR-17, −20b and −106b, which are decreased in neuroendocrine-like LNCaP cells, negatively regulate AKT3 biosynthesis. Our findings demonstrate AKT3 as a potential therapeutic target and diagnostic tool in advanced neuroendocrine prostate cancer and a new mRNA–miRNA interaction with a potential role in neuroendocrine differentiation of prostate cancer.

Galectin-3 in Prostate Cancer Stem-Like Cells Is Immunosuppressive and Drives Early Metastasis.

Galectin-3 (Gal-3) is an extracellular matrix glycan-binding protein with several immunosuppressive and pro-tumor functions. The role of Galectin-3 in cancer stem-like cells (CSCs) is poorly investigated. Here, we show that prostate CSCs also colonizing prostate-draining lymph nodes of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice overexpress Gal-3. Gal-3 contributes to prostate CSC-mediated immune suppression because either Gal-3 silencing in CSCs, or co-culture of CSCs and T cells in the presence of the Gal-3 inhibitor N-Acetyl-D-lactosamine rescued T cell proliferation. N-Acetyl-D-lactosamine also rescued the proliferation of T cells in prostate-draining lymph nodes of TRAMP mice affected by prostate intraepithelial neoplasia. Additionally, Gal-3 impacted prostate CSC tumorigenic and metastatic potential in vivo, as Gal-3 silencing in prostate CSCs reduced both primary tumor growth and secondary invasion. Gal-3 was also found expressed in more differentiated prostate cancer cells, but with different intracellular distribution as compared to CSCs, which suggests different functions of Gal-3 in the two cell populations. In fact, the prevalent nuclear and cytoplasmic distribution of Gal-3 in prostate CSCs made them less susceptible to apoptosis, when compared to more differentiated prostate cancer cells, in which Gal-3 was predominantly intra-cytoplasmic. Finally, we found Gal-3 expressed in human and mouse prostate intraepithelial neoplasia lesions and in metastatic lymph nodes. All together, these findings identify Gal-3 as a key molecule and a potential therapeutic target already in the early phases of prostate cancer progression and metastasis.

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.

Stimulation of neuroendocrine differentiation in prostate cancer cells by GHRH and its blockade by GHRH antagonists.

Prostate cancer is the second leading cause of cancer-related deaths among men in developed countries. Neuroendocrine prostate cancer, in particular, is associated with an aggressive phenotype and a poor prognosis. Neuroendocrine cells produce and secrete peptide hormones and growth factors in a paracrine/autocrine manner which promote the progression of the disease. Recent studies have demonstrated that extracellular vesicles or exosomes are released by prostate cancer cells, supporting the spread of prostate cancer. Hence, the aim of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) on neuroendocrine differentiation (NED) in the androgen-dependent prostate cancer cell line LNCaP and the molecular mechanisms underlying these effects. GHRH induced an increase in the percentage of neurite-bearing cells and in the protein levels of Neuron-Specific Enolase. Both effects were blocked by the GHRH receptor antagonist MIA-690. In addition, pretreatment of these cells with the calcium chelator BAPTA, the EGFR inhibitor AG-1478 or the HER2 inhibitor AG-825 reduced the effectof GHRH, suggesting that the GHRH-induced stimulation of NED involves calcium channel activation and EGFR/HER2 transactivation. Finally, PC3-derived exosomes led to an increase in NED, cell proliferation and cell adhesion. Altogether, these findings suggest that GHRH antagonists should be considered for in the management of neuroendocrine prostate cancer.

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

The anticancer potential of metformin on prostate cancer.

Prostate cancer (PCa) is characterized as the most frequent type of cancer in males. Recent research has suggested patients who have diabetes mellitus taking metformin (MF) have a lower risk of PCa. MF has antineoplastic effects such as adenosine monophosphate-activated protein kinase (AMPK)-dependent and independent mechanisms, suppression of androgen signaling pathway, and alterations of insulin-like growth factor-1 (IGF-1) signaling pathways that cause the growth and proliferation of PCa. Based on epidemiological factors, patients with diabetes mellitus may have a protective effect on PCa. A literature search on MEDLINE® was conducted using a combined query of "prostate cancer" and "metformin" to yield publications unveiling the mechanisms of action, biological effects, epidemiological evidence, and research advances of MF with respect to PCa. Evidence has shown that MF has multiple antineoplastic effects through AMPK-dependent and independent mechanisms, the alteration of IGF-1 signaling pathways, suppression of the androgen receptor pathway, inhibition of the mTOR pathway, and lipogenesis. Conduction of meta-analysis suggests mortality benefit to patients who exhibit PCa when taking MF. Clinical trials have shown evidence, demonstrating MF to improving significantly. Herewith we review the literature regarding the numerous mechanisms of action of MF on PCa in order to decrease or repress the growth, proliferation, and differentiation of PCa cells. We analyze the molecular impacts of MF as well as adjunct therapies such as androgen deprivation therapy, aspirin, statin, or chemotherapy, proposing that MF may have a future role in the treatment protocol of PCa whether as a monotherapy or in combination with other drugs.

Androgen deprivation-induced ZBTB46-PTGS1 signaling promotes neuroendocrine differentiation of prostate cancer

Androgen receptor (AR) targeting is an important therapeutic strategy for treating prostate cancer. Most tumors progress to castration-resistant prostate cancer (CRPC) and develop the neuroendocrine (NE) phenotype under androgen deprivation therapy (ADT). The molecular basis for NE transdifferentiation after ADT remains incompletely understood. Herein, we show that an immunocyte expression protein, ZBTB46, induces inflammatory response gene expression and contributes to NE differentiation of prostate cancer cells. We demonstrated a molecular mechanism whereby ZBTB46 can be regulated by the androgen-responsive gene, SPDEF, and is associated with NE prostate cancer (NEPC) differentiation. In addition, ZBTB46 acts as a transcriptional coactivator that binds to the promoter of prostaglandin-endoperoxide synthase 1 (PTGS1) and transcriptionally regulated PTGS1 levels. Overexpression of ZBTB46 decreases the sensitivity of the combination of enzalutamide and a PTGS1 inhibitor; however, knockdown of ZBTB46 sensitizes the PTGS1 inhibitor and reduces tumor malignancy. ZBTB46 is inversely correlated with SPDEF and is increased in higher tumor grades and small-cell NE prostate cancer (SCNC) patients, which are positively associated with PTGS1. Our findings suggest that the induction of ZBTB46 results in increased PTGS1 expression, which is associated with NEPC progression and linked to the dysregulation of the AR-SPDEF pathway.

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.].

Abstract 2141: Stromal SPARC deficiency skews prostate cancer toward neuroendocrine differentiation

Abstract Tumor progression is a multifaceted process in which, complex interactions between tumor and different types of stromal cells and extracellular matrix components, actively contribute to its phenotypic heterogeneity. Among extracellular matrix proteins, secreted protein acidic and rich in cysteine (SPARC) has been deeply studied since conflicting reports have described its expression to be either increased or decreased in different cancer settings, also depending on whether it is produced by the neoplasm or by the neighboring stroma. Nevertheless, the different contribution of tumor- or stromal-derived SPARC in prostate tumor microenvironment has not been addressed at least for tumor tissue histotype. Given this evidence, we aimed at providing new insights into the mechanism by which SPARC modulation influences prostate cancer development and progression. We modeled human disease using TRAMP mice, which spontaneously develop autochthonous prostate tumors following the onset of puberty. Crossing TRAMP mice with Sparc-/- mice, we found the appearance of focal areas of neuroendocrine differentiation within adenocarcinoma. In patients this phenomenon commonly results after androgen ablation therapy and correlates with poor prognosis. Interestingly, areas of neuroendocrine differentiation in Sparc-/- TRAMP mice were both positive for cytokeratin 8 and synaptophysin (usually expressed by luminal proliferating cells within adenocarcinoma or neuroendocrine cells, respectively), further suggesting a differentiation of adenocarcinoma cells to a neuroendocrine-like phenotype. Moreover, immunohistochemistry showed SPARC positivity not only in scattered tumor cells but also in fibroblasts and myeloid cells infiltrating TRAMP prostate. Accordingly, in vitro experiments suggested that stromal-derived SPARC limits neuroendocrine differentiation of prostate cancer cells, while they excluded a role of endogenous SPARC in this phenomenon. Indeed, prostate cancer cell lines co-cultured in presence of Sparc-deficient fibroblasts increased or acquired neuroendocrine features. This likely occurs through the effect of IL-6, a cytokine recently discovered to induce neuroendocrine differentiation, and that we found to be released by Sparc-deficient, but not sufficient, fibroblasts. Data collected so far indicate that stromal SPARC deficiency skews prostate carcinogenesis toward neuroendocrine differentiation. A deeper understanding of the molecular mechanisms governing the balance between prostate adenocarcinoma and neuroendocrine tumors according to extracellular matrix composition will provide important insights for the development of new prognostic and therapeutic strategies. Citation Format: Claudia Enriquez, Valeria Cancila, Ivano Arioli, Claudio Tripodo, Mario Paolo Colombo, Elena Jachetti. Stromal SPARC deficiency skews prostate cancer toward neuroendocrine differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2141.

Abstract 5901: The role of POU3F2 in the aggressive behavior of neuroendocrine prostate cancer

Abstract Neuroendocrine and Small Cell Prostate Cancer (NEPC) is a lethal disease emerging from Castrate Resistant Prostate Cancer (CRPC) after treatment with next-generation anti-androgen therapies such as abiraterone and enzalutamide. The mechanisms by which treatment resistant prostate epithelial cancer cells trans-differentiate to poorly differentiated small cell cancer are not well understood. We and others found that Nervous System-Specific Octamer-Binding Transcription Factor (POU3F2) expression is induced in NEPC. POU3F2 is a reprogramming transcription factor that controls cellular differentiation and can induce stem-like cells from differentiated glioblastoma. POU3F2 also plays a critical role in melanoma progression and in small cell carcinoma of the lung. NEPC shares similarities with small cell carcinoma from other organs. We hypothesized that POU3F2 may be a key transcription factor that is required for transdifferentiation and the survival of NEPC cells. We further hypothesize that the underlying mechanism of therapy resistance in NEPC is that NEPC consists of stem-like cells that were reprogrammed from differentiated prostate cancer cells under the selective pressure of androgen receptor (AR) pathway-targeted therapy. We elucidated the functional role of POU3F2 in the aggressive cancer behavior of NEPC —including stem-cell properties, tumorigenesis, invasion, and chemo-resistance. We also characterized the mechanisms that regulate POU3F2 expression in therapy-resistant NEPC. The study provides a rational for targeted therapy for treatment of NEPC. It could also be used to design a strategy to halt the emergence of NEPC from CRPC. Citation Format: Naoko Kobayashi, Dai Hong, Chun Wang, Joyce Yamashiro, Johnny Guan, Robert E. Reiter. The role of POU3F2 in the aggressive behavior of neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5901.