Neuroendocrine Differentiation In Prostate Cancer Research Articles

Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models

BackgroundNKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1’s levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date.MethodsA chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells.ResultsWe uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it.ConclusionsNKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.

Neuroendocrine Differentiation of Prostate Cancer : A Case Report

A 79-year-old man underwent a transrectal prostate needle biopsy with a prostate-specific antigen (PSA) level of 12.0 ng/ml. He was diagnosed with adenocarcinoma (Gleason score 4＋3, cT3aN0M0) and underwent radiation therapy. Eight months later, he was administered hormone therapy because of a rise in PSA level to 8.4 ng/ml. One year and 5 months later, he experienced back pain, and computed tomography revealed multiple lymphadenopathies and irregular prostate enlargement. The PSA level was 0.097 ng/ml. Re-biopsy of the prostate and biopsy of the lymph node were performed. Pathological examination revealed neuroendocrine differentiation of the prostate. The disease progressed rapidly, and the patient died 4 months after the biopsy. Neuroendocrine differentiation of prostate cancer is rare, and its development may not be consistent with PSA levels. Therefore, periodic imaging examinations should be conducted, even when PSA levels are low.

MicroRNA-1205 Regulation of FRYL in Prostate Cancer.

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

HOXB5 Overexpression Is Associated with Neuroendocrine Differentiation and Poor Prognosis in Prostate Cancer.

Homeobox genes function as master regulatory transcription factors during embryogenesis. HOXB5 is known to play an important role in several cancers. However, the biological role of HOXB5 in prostate cancer (PCa) is not fully elucidated. This study aimed to analyze the expression and function of HOXB5 and involvement of HOXB5 in neuroendocrine differentiation in PCa. Immunohistochemistry showed that 56 (43.8%) of 128 cases of localized PCa were positive for HOXB5. HOXB5-positive cases were associated with poor prostate-specific antigen recurrence-free survival after prostatectomy. Among 74 cases of metastatic PCa, 43 (58.1%) were positive for HOXB5. HOXB5 expression was higher in metastatic PCa than that in localized PCa. HOXB5 knockdown suppressed cell growth and invasion, but HOXB5 overexpression increased cell growth and invasion in PCa cell lines. Furthermore, HOXB5 regulated RET expression. Gene set enrichment analysis revealed that Nelson androgen response gene set was enriched in low HOXB5 expression group. RB1 knockout increased HOXB5 expression. Of note, additional p53 knockdown further increased HOXB5 expression in RB1 knockout cells. In silico analysis showed that HOXB5 expression was increased in neuroendocrine PCa (NEPC). These results suggest that HOXB5 may be a promising prognostic marker after prostatectomy and is involved in progression to NEPC.

Neuropilin-2 Promotes the Development of Neuroendocrine Prostate Cancer

Neuroendocrine prostate cancer (NEPC), a lethal subset of the disease, is characterized by loss of AR signaling and resulting resistance to AR-targeted therapy during neuroendocrine transdifferentiation, for which the molecular mechanisms remain unclear. Here, we report that neuropilin 2 (NRP2) is upregulated in both de novo and therapy-induced NEPC, which induces neuroendocrine markers, neuroendocrine cell morphology and NEPC cell aggressive behaviors. NRP2 silencing reduced or even eliminated NEPC tumor xenograft growth. Mechanistically, NRP2 engages in reciprocal crosstalk with AR, where NRP2 is a direct transcriptional target inhibited by AR, and in turn, suppresses AR signaling by downregulating the AR transcriptional program and confers resistance to enzalutamide. Moreover, NRP2 physically interacts with VEGFR2 through the intracellular SEA domain to activate STAT3 phosphorylation and subsequently SOX2, thus driving NEPC differentiation and growth. Together these results characterize NRP2 as a driver of NEPC and suggest NRP2 as a potential therapeutic target in NEPC.

Abstract 2363: MicroRNA-1205 modulates FRYL/Aurora A kinase protein-protein interaction in prostate cancer cells

Abstract High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (mCRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). Resistance to second generation ADTs leads to the progression to AR-independent treatment related-neuroendocrine PCa (t-NEPC), which is observed in nearly 1 in 5 men with mCRPC and is associated with very poor outcomes. PCa neuroendocrine differentiation (PCND) occurs in t-NEPC that is characterized by amplification of Aurora A kinase (AurA). However, the mechanisms that drive t-NEPC are poorly understood. Further understanding PCND in ADT resistance may prevent poorer outcomes. The 8q24 chromosomal locus is an important PCa susceptibility region containing genetic variants associated with increased PCa incidence and aggressiveness. PVT1 is a gene located within this region that encodes microRNA-1205 (miR-1205), whose function is largely unknown. We previously reported miR-1205 underexpression in a cohort of histologically confirmed PCa tissue when compared to normal tissue and that exogenous miR-1205 significantly inhibited tumor growth in CRPC in vivo, suggesting that miR-1205 is a tumor suppressor. To understand the molecular mechanism of miR-1205, we first identfied Fry-like (FRYL) as a direct molecular target of miR-1205. FRYL is predicted to regulate dendritic branching which led us to hypothesize that it is involved in PCND. A Weill Cornell Medicine cohort of 29 benign prostate, 66 localized PCa, 73 CRPC, and 36 NEPC samples whose transcriptome was profiled by RNA-seq, we observed significant overexpression of FRYL in NEPC when compared to localized PCa and benign prostate. After validating FRYL as a target, we examined miR-1205 regulation of FRYL in PCND by culturing LNCaP androgen sensitive PCa cells under androgen deprivation conditions. We observed FRYL mRNA overexpression and significant underexpression of miR-1205 in LNCaP-PCND cells when compared to undifferentiated LNCaP cells. To further characterize this mechanism, miR-1205 was inhibited in RWPE-1 non-tumorigenic and LNCaP cells. As a result, FRYL and AurA protein expression was increased along with NEPC marker expression (Neuron specific enolase 2) when compared to cells transfected with a negative control, suggesting that miR-1205 targets FRYL and AurA to promote PCND. However, RNA pulldown analysis showed that AurA is not a direct target of miR-1205. We observed that FRYL and AurA interaction occurs in LNCaP but not in RWPE-1 cells via co-immunoprecipitation. Interestingly, transfection with miR-1205 mimic inhibits FRYL expression, but not AurA in LNCaP. However, miR-1205 mimic inhibits AurA binding to FRYL. Altogether, these data show that miR-1205 modulates FRYL:AurA protein interaction suggesting that miR-1205 regulation of FRYL:AurA interactions may be important in PCND and t-NEPC. Citation Format: Michelle K. Naidoo, Fayola Levine, Princesca Dorsaint, Andrea Sboner, Olorunseun Ogunwobi. MicroRNA-1205 modulates FRYL/Aurora A kinase protein-protein interaction in prostate cancer 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 2363.

Abstract 2152: EHMT1 activity to repress neuroendocrine differentiation of prostate cancer is mediated by dual lysine-methylation

Abstract Background: Lysine methylation is an important post-translational regulation on histones, but its molecular functions on non-histone proteins remain largely unknown. In a pilot study, we identified novel lysine methylations at non-histone proteins in prostate cancer (PCa) cells through an affinity pull-down assay. EHMT1/GLP, containing monomethylated K450 and K451, was one of these proteins, and it was known to repress transcription through methylating histone 3 lysine 9 (H3K9). EHMT1 and EHMT2/G9a are members of the REST (NRSF) repressor complex, that includes LSD1/CoREST/HDACs and represses neuronal genes in non-neuronal cells. Loss of function for this complex has been indicated as a mechanism to drive neuroendocrine (NE) transition of PCa. We hypothesize that the K450/451 methylations may inhibit the activity of EHMT1 and that LSD1 and other lysine demethylase(s) may sequentially demethylate these lysines and thus induce EHMT1 activity to silence the expression of NE related genes. Methods: LNCaP stable cell lines overexpressing doxycycline-regulated V5-tagged EHMT1-WT, K450R, K451R, and K450/451R were generated. We performed ChIP-seq analyses of V5 and H3K9me2, and RNA-seq analyses in all four cell lines treated with/out doxycycline. We also conducted in vitro demethylation assays to examine whether LSD1 directly demethylates EHMT1. Moreover, we performed co-immunoprecipitation assays to determine the effect of LSD1 inhibition on the interaction of LSD1 with EHMT1. Neuronal target gene expression was examined through ChIP and mRNA analysis. Results: Using mass-spectrometry analysis, we confirmed K450 and K451 methylations of EHMT1 in LNCaP PCa cell line. The demethylation assays indicated that methylated K450 but not K451 is a potential substrate of LSD1. Moreover, LSD1 interacts with K450/451R mutant but not EHMT1-WT. Importantly, only K450/451R mutant had a significant increase of chromatin binding in V5 and elevated levels of H3K9me2 compared to the weak chromatin binding seen in WT, K450R and K451R. Analyses in RNA-Seq revealed that the K450/K451R mutant repressed genes enriched for neuronal pathways, which was not seen in WT, K450R or K451R. Conclusions: Our studies suggest that both K450 and K451 of EHMT1 are methylated in PCa cells. K450-methylation of EHMT1 can be specifically demethylated by LSD1, but this alone is not sufficient to induce the chromatin recruitment of EHMT1. However, demethylation at both K450/K451 can dramatically increase the chromatin binding of EHMT1, which subsequently represses neuronal pathways. This mechanism may be particularly important for maintaining the cell lineage of adenocarcinoma of PCa and loss of this demethylation might lead to the emergence of NE-like PCa. Citation Format: Anna Besschetnova, Wanting Han, Mingyu Liu, Dong Han, Shuai Gao, Changmeng Cai. EHMT1 activity to repress neuroendocrine differentiation of prostate cancer is mediated by dual lysine-methylation [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 2152.

Abstract 725: LIFR promotes SUCLG2-dependent castration resistance and neuroendocrine differentiation of prostate cancer

Abstract Neuroendocrine (NE) differentiation is a well-recognized phenotypic change of prostate cancer after androgen-deprivation therapy (ADT) that ultimately develops an aggressive subtype. An increasing number of patients with NE histologic type prostate cancer present with metabolic genes disorder. However, the metabolic pathways that influence the malignant transformation and the NE differentiation of prostate cancer remain unclear. In this study, we found that ADT induced the upregulation of leukemia inhibitory factor receptor (LIFR), which contributes to the expression of succinate-CoA ligase GDP-forming beta subunit (SUCLG2), thus activating metabolic reprogramming to enable NE differentiation and increase aggressiveness. Activated LIF-LIFR-STAT3 axis stimulates SUCLG2 expression, subsequently upregulating glucose metabolism and the NE differentiation of prostate cancer. SUCLG2 knockdown markedly reduced mitochondrial DNA content and inhibited the enzymatic activity of succinyl-CoA synthetase and nucleoside diphosphate kinase (NDPK) in prostate cancer cells. SUCLG2 overexpression promoted androgen-independent proliferation; however, SUCLG2 knockdown suppressed NE differentiation and the ADT resistance of prostate cancer cells. Analysis of prostate tissue samples showed an increased intensity of LIFR associated with SUCLG2 in castration-resistant prostate cancer tumors. Our study identifies SUCLG2 as a potential prognostic marker of ADT-resistant prostate cancer and suggests it could be a target for prostate cancer treatment. Citation Format: Hsiu-Lien Yeh, Shian-Ren Lin, Kuo-Ching Jiang, Wei-Hao Chen, Yen-Nien Liu. LIFR promotes SUCLG2-dependent castration resistance and neuroendocrine differentiation of prostate cancer [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 725.

PARP and CDK4/6 Inhibitor Combination Therapy Induces Apoptosis and Suppresses Neuroendocrine Differentiation in Prostate Cancer.

We analyzed the efficacy and mechanistic interactions of PARP inhibition (PARPi; olaparib) and CDK4/6 inhibition (CDK4/6i; palbociclib or abemaciclib) combination therapy in castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) models. We demonstrated that combined olaparib and palbociblib or abemaciclib treatment resulted in synergistic suppression of the p-Rb1-E2F1 signaling axis at the transcriptional and posttranslational levels, leading to disruption of cell-cycle progression and inhibition of E2F1 gene targets, including genes involved in DDR signaling/damage repair, antiapoptotic BCL-2 family members (BCL-2 and MCL-1), CDK1, and neuroendocrine differentiation (NED) markers in vitro and in vivo In addition, olaparib + palbociclib or olaparib + abemaciclib combination treatment resulted in significantly greater growth inhibition and apoptosis than either single agent alone. We further showed that PARPi and CDK4/6i combination treatment-induced CDK1 inhibition suppressed p-S70-BCL-2 and increased caspase cleavage, while CDK1 overexpression effectively prevented the downregulation of p-S70-BCL-2 and largely rescued the combination treatment-induced cytotoxicity. Our study defines a novel combination treatment strategy for CRPC and NEPC and demonstrates that combination PARPi and CDK4/6i synergistically promotes suppression of the p-Rb1-E2F1 axis and E2F1 target genes, including CDK1 and NED proteins, leading to growth inhibition and increased apoptosis in vitro and in vivo Taken together, our results provide a molecular rationale for PARPi and CDK4/6i combination therapy and reveal mechanism-based clinical trial opportunities for men with NEPC.

Castration-Induced Downregulation of SPARC in Stromal Cells Drives Neuroendocrine Differentiation of Prostate Cancer.

Fatal neuroendocrine differentiation (NED) of castration-resistant prostate cancer is a recurrent mechanism of resistance to androgen deprivation therapies (ADT) and antiandrogen receptor pathway inhibitors (ARPI) in patients. The design of effective therapies for neuroendocrine prostate cancer (NEPC) is complicated by limited knowledge of the molecular mechanisms governing NED. The paucity of acquired genomic alterations and the deregulation of epigenetic and transcription factors suggest a potential contribution from the microenvironment. In this context, whether ADT/ARPI induces stromal cells to release NED-promoting molecules and the underlying molecular networks are unestablished. Here, we utilized transgenic and transplantable mouse models and coculture experiments to unveil a novel tumor-stroma cross-talk that is able to induce NED under the pressure of androgen deprivation. Castration induced upregulation of GRP78 in tumor cells, which triggers miR29-b-mediated downregulation of the matricellular protein SPARC in the nearby stroma. SPARC downregulation enabled stromal cells to release IL6, a known inducer of NED. A drug that targets GRP78 blocked NED in castrated mice. A public, human NEPC gene expression dataset showed that Hspa5 (encoding for GRP78) positively correlates with hallmarks of NED. Finally, prostate cancer specimens from patients developing local NED after ADT showed GRP78 upregulation in tumor cells and SPARC downregulation in the stroma. These results point to GRP78 as a potential therapeutic target and to SPARC downregulation in stromal cells as a potential early biomarker of tumors undergoing NED. SIGNIFICANCE: Tumor-stroma cross-talk promotes neuroendocrine differentiation in prostate cancer in response to hormone therapy via a GRP78/SPARC/IL6 axis, providing potential therapeutic targets and biomarkers for neuroendocrine prostate cancer.

A Potent, Selective CBX2 Chromodomain Ligand and Its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation.

Polycomb group (PcG) proteins are epigenetic regulators that facilitate both embryonic development and cancer progression. PcG proteins form Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 lysine 27 (H3K27me3), a histone mark recognized by the N-terminal chromodomain (ChD) of the CBX subunit of canonical PRC1. There are five PcG CBX paralogs in humans. CBX2 in particular is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable; however, high structural similarity among the CBX ChDs has been challenging for developing selective CBX ChD inhibitors. Here, we utilize selections of focused DNA encoded libraries (DELs) for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a Kd of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs in vitro. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.

Neuroendocrine Differentiation of Prostate Cancer Is Not Systematically Associated with Increased 18F-FDG Uptake.

Neuroendocrine differentiation (NED) of prostate cancer represents an acknowledged predictor of resistant and more aggressive disease. NED can be functionally exploited in vivo using PET/CT imaging with somatostatin analogs radiolabeled with 68Ga. Many previous reports have shown that 18F-FDG PET/CT should also be used in cases such as guiding management, as NED is systematically associated with increased glycolysis. We hereby discuss the case of a metastatic prostate cancer patient in which 68Ga-Dotatoc PET/CT revealed the occurrence of NED with low FDG-avidity.

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.

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.

Abstract PO-136: MicroRNA-1205 directly binds to FRYL and suppresses aggressive prostate cancer

Abstract High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (mCRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). Resistance to second generation ADTs leads to the progression to AR-independent neuroendocrine PCa (NEPC), which is observed in nearly 1 in 5 men with mCRPC and is associated with very poor outcome. PCa neuroendocrine differentiation (PCND) is observed in treatment-related NEPC, however, this mechanism is poorly understood. The 8q24 chromosomal locus is an important prostate cancer (PCa) susceptibility region containing genetic variants associated with increased PCa incidence and aggressiveness. PVT1 is a gene located within this region that encodes microRNA- 1205 (miR-1205), whose function is largely unknown. We have previously reported that miR-1205 is underexpressed in a cohort of histologically confirmed PCa tissue, when compared to normal tissue and is also underexpressed in vitro in CRPC cells, when compared to non-CRPC cells. We also demonstrated that exogenous miR-1205 significantly inhibited tumor volume in CRPC-derived xenograft male NOD/SCID gamma mice, suggesting that miR-1205 is a tumor suppressor. To understand the molecular mechanism of miR-1205, we demonstrated that miR-1205 directly targets Fry-like (FRYL) by performing an RNA pulldown assay. FRYL is predicted to regulate dendritic branching leading to the hypothesis that FRYL plays a role in PCND. To examine miR-1205 regulation of FRYL in PCND in vitro, LNCaP cells were cultured under androgen deprivation conditions for fourteen days. We observed FRYL mRNA overexpression and a significant reduction of miR-1205 expression in LNCaP-PCND cells when compared to undifferentiated LNCaP cells. To further determine whether this mechanism drives PCND, miR-1205 was inhibited in RWPE-1 and LNCaP androgen-sensitive cells. As a result, FRYL protein expression was increased along with neuroendocrine marker expression when compared to cells transfected with a negative control. To determine whether PCND is driven directly through FRYL activity, FRYL was silenced using an siRNA in PC-3 cells (model of NEPC). Interestingly, knockdown of FRYL did not reveal a decrease of neuroendocrine markers, suggesting that miR-1205 may induce PCND independently of FRYL. Further understanding this molecular mechanism may provide novel insights into overcoming ADT resistance in aggressive PCa. Citation Format: Xavier Graña-Amat, Olorunseun Ogunwobi, Michelle K. Naidoo, Fayola Levine. MicroRNA-1205 directly binds to FRYL and suppresses aggressive prostate cancer [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-136.

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).

EGFR-upregulated LIFR promotes SUCLG2-dependent castration resistance and neuroendocrine differentiation of prostate cancer.

Neuroendocrine (NE) differentiation is a well-recognized phenotypic change of prostate cancer after androgen deprivation therapy (ADT), and it ultimately develops into an aggressive subset of this disease. However, the contribution of signaling pathways that lead to metabolic disorders and NE differentiation of prostate cancer remains unclear. In this study, we identified that ADT induced upregulation of the succinate-CoA ligase GDP-forming beta subunit (SUCLG2), which regulates succinate metabolism and NE differentiation of prostate cancer. We demonstrated a connection that upregulation of epidermal growth factor receptor (EGFR)-leukemia inhibitory factor receptor (LIFR) signaling induced SUCLG2 expression in prostate cancer cells. The LIFR is upregulated by nuclear EGFR, which acts as a transcriptional regulator, directly binds to the LIFR promoter, and drives NE differentiation and glycolysis of prostate cancer. LIFR upregulation is associated with SUCLG2, which increased succinate synthesis and enzymatic activities of mitochondrial nucleoside diphosphate kinase (NDPK) in prostate cancer cells. Knockdown of SUCLG2 suppressed NE differentiation in cultured cells and reduced prostate tumor growth in a xenograft model. Analysis of prostate tissue samples showed increased intensity of nuclear EGFR associated with the LIFR and SUCLG2 in castration-resistant prostate cancer tumors. Our study provides a mechanism whereby ADT upregulates EGFR-LIFR signaling that activates SUCLG2, which subsequently stimulates the metabolic changes associated with NE differentiation and aggressive prostate cancer phenotype.

Abstract 2532: MicroRNA-1205 directly binds to FRYL and suppresses aggressive prostate cancer

Abstract High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (mCRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). Resistance to second generation ADTs leads to the progression to AR-independent neuroendocrine PCa (NEPC), which is observed in nearly 1 in 5 men with mCRPC and is associated with very poor outcome. PCa neuroendocrine differentiation (PCND) is observed in treatment related NEPC, however, this mechanism is poorly understood. Therefore, further understanding of PCND in ADT resistance may prevent poorer outcomes. The 8q24 chromosomal locus is an important prostate cancer (PCa) susceptibility region containing genetic variants associated with increased PCa incidence and aggressiveness. PVT1 is a gene located within this region that encodes microRNA-1205 (miR-1205), whose function is largely unknown. We have previously reported that miR-1205 is underexpressed in a cohort of histologically confirmed PCa tissue obtained from moAA, when compared to normal tissue and is also underexpressed in vitro in CRPC cells, when compared to non-CRPC cells. We also demonstrated that exogenous miR-1205 significantly inhibited tumor volume in CRPC-derived xenograft male NOD/SCID gamma mice, suggesting that miR-1205 is a tumor suppressor. To understand the molecular mechanism of miR-1205, we demonstrated that miR-1205 directly targets Fry-like (FRYL) by performing an RNA pulldown assay. FRYL is predicted to regulate dendritic branching leading to the hypothesis that FRYL plays a role in PCND. To examine miR-1205 regulation of FRYL in PCND in vitro, LNCaP cells were cultured under androgen deprivation conditions for fourteen days. We observed FRYL mRNA overexpresssion and a significant reduction of miR-1205 expression in LNCaP-PCND cells when compared to undifferentiated LNCaP cells. To further determine whether this mechanism drives PCND, miR-1205 was inhibited in RWPE-1 and LNCaP androgen-sensitive cells. As a result, FRYL protein expression was increased along with neuroendocrine marker expression (Neuron specific enolase 2) when compared to cells transfected with a negative control. To determine whether PCND is driven directly through FRYL activity, FRYL was silenced using an siRNA in PC-3 cells (model of NEPC). Interestingly, knockdown of FRYL did not reveal a decrease of neuroendocrine markers, suggesting that miR-1205 may induce PCND independently of FRYL. Further investigation into the role of miR-1205 and FRYL in PCND is required. Further understanding this molecular mechanism may provide novel insights into overcoming ADT resistance in aggressive PCa. Citation Format: Michelle K. Naidoo, Fayola Levine, Xavier Graña, Olorunseun Ogunwobi. MicroRNA-1205 directly binds to FRYL and suppresses aggressive prostate cancer [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 2532.

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.

Abstract B090: Molecular mechanisms of microRNA-1205 in aggressive prostate cancer in men of African ancestry

Abstract High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (mCRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). Resistance towards second generation ADTs leads to the progression of AR-independent neuroendocrine PCa (NEPC) phenotypes, which are observed in nearly 1 in 5 men with mCRPC and is associated with short survival rates. Moreover, men of African ancestry (moAA) have a higher NEPC incidence and mortality rates, compared to Caucasian males. PCa neuroendocrine differentiation (PCND) is observed in treatment related NEPC, however, this mechanism is poorly understood. Therefore, further understanding of PCND in ADT resistance may prevent poorer outcomes. The 8q24 chromosomal locus is an important PCa susceptibility region containing genetic variants associated with increased PCa incidence among moAA. PVT1 is a gene located within this region that encodes microRNA-1205 (miR-1205), whose function is largely unknown. We have previously reported that miR-1205 is underexpressed in a cohort of histologically confirmed PCa tissue obtained from moAA, when compared to normal tissue and is also underexpressed in vitro in CRPC cells, when compared to non-CRPC cells. We also demonstrated that exogenous miR-1205 significantly inhibited tumor volume in CRPC-derived xenograft male NOD/SCID gamma mice, suggesting that miR-1205 is a tumor suppressor. To understand the molecular mechanism of miR-1205, we demonstrated that miR-1205 directly targets Fry-like (FRYL). FRYL is predicted to regulate dendritic branching leading to the hypothesis that FRYL plays a role in PCND. To examine miR-1205 regulation of FRYL in NEPC, we examined RNA sequencing data from a cohort of histologically confirmed NEPC tissues (n=14) and PCa adenocarcinoma tissues (n=30) obtained from TCGA using the cBioPortal platform. We observed increased FRYL mRNA expression in NEPC tissues when compared to PCa adenocarcinoma tissues. In vitro data revealed that FRYL mRNA was overexpressed and miR-1205 was significantly underexpressed after fourteen days of induced PCND differentiation using LNCaP cells when compared to undifferentiated LNCaP cells. Lastly, we performed RNA pulldown, and subsequent RNA sequencing to identify the molecular targets of miR-1205. Interestingly, we observed that in addition to FRYL, the AURKA gene, which is amplified during PCND, was significantly enriched. In conclusion, these data suggest that miR-1205 regulation of FRYL may be a critical mechanism in NEPC. Further understanding this molecular mechanism may provide novel insights into overcoming ADT resistance in aggressive PCa that disproportionally affects moAA. Citation Format: Michelle K Naidoo, Fayola Levine, Xavier Graña-Amat, Olorunseun Ogunwobi. Molecular mechanisms of microRNA-1205 in aggressive prostate cancer in men of African ancestry [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B090.