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

BACKGROUND: 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. METHODS: 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 transcription polymerase chain analysis. miRNA target prediction using bioinformatics tools followed by target validation using luciferase reporter assays was performed. RESULTS: 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. CONCLUSION: 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.

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.

AMPK targets a proto-oncogene TPD52 (isoform 3) expression and its interaction with LKB1 suppress AMPK-GSK3ß signaling axis in prostate cancer.

Tumor protein D52 (TPD52) is a proto-oncogene overexpressed in prostate cancer (PCa) due to gene amplification and it is involved in the cancer progression of many cancers including PCa. However, the molecular mechanisms underlying the role of TPD52 in cancer progression are still under investigation. In this study, we report that the activation of AMP-activated protein kinase (AMPK) by AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) inhibited the LNCaP and VCaP cells growth by silencing TPD52 expression. Activation of AMPK inhibited the proliferation and migration of LNCaP and VCaP cells. Interestingly, AICAR treatment to LNCaP and VCaP cells led to the downregulation of TPD52 via activation of GSK3ß by a decrease of inactive phosphorylation at Ser9. Moreover, in AICAR treated LNCaP cells, inhibition of GSK3ß by LiCl attenuated downregulation of TPD52 indicating that AICAR acts via GSK3ß. Furthermore, we found that TPD52 interacts with serine/threonine kinase 11 or Liver kinase B1 (LKB1) a known tumor suppressor and an upstream kinase for AMPK. The molecular modeling and MD simulations indicates that the interaction between TPD52 and LKB1 leads to inhibition of the kinase activity of LKB1 as its auto-phosphorylation sites were masked in the complex. Consequently, TPD52-LKB1 interaction may lead to inactivation of AMPK. Moreover, overexpression of TPD52 is found to be responsible for the reduction of pLKB1 (Ser428) and pAMPK (Thr172). Therefore, TPD52 may be playing its oncogenic role via suppressing the AMPK activation. Altogether, our results revealed a new mechanism of PCa progression in which TPD52 overexpression inhibits AMPK activation by interacting with LKB1. These results support that the use of AMPK activators and/or small molecules that could disrupt the TPD52-LKB1 interaction might be useful to suppress PCa cell growth. TPD52 interacts LKB1 and interfere with activation of AMPK in PCa cells.

HIF-1α and Nrf2 regulates hypoxia induced overexpression of DDAH1 through promoter activation in prostate cancer.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is overexpressed in prostate cancer (PCa) and promotes PCa progression in in vivo through the ADMA-NO pathway by degrading nitric oxide synthase (NOS) inhibitors such as asymmetric dimethylarginine (ADMA) and monomethylamine arginine (L-NMMA). In this study, we investigated the molecular mechanism involved in the overexpression of DDAH1 in PCa and examined its potential role as a therapeutic target. We observed that DDAH1expression is elevated in PCa (PC3, LNCaP, and DU145) cell lines under hypoxia. ChIP and reporter assay results confirmed that DDAH1 expression is positively regulated by HIF-1α through directly binding to the hypoxia response elements (HRE) located within the promoter region between - 1242/- 1238 upstream of its transcription start site (TSS). Under hypoxia, HIF-1α is translocated into the nucleus and activates its target gene expression in PC3 cells. Interestingly, in the event of HIF-1α inhibition or siRNA-mediated knockdown, an alternative transcription factor Nrf2 promotes DDAH1 expression through antioxidant response elements (AREs) on its promoter. ChIP assay results showed that Nrf2 binds to AREs located between -1016 / -1008 bp from the TSS of DDAH1. Furthermore, knockdown of PCa therapeutic target HSP90, an essential co-factor for both HIF-1α and Nrf2 causes attenuation of hypoxia induced DDAH1 overexpression in PCa cells. These results demonstrate that hypoxia induced upregulation of DDAH1 expression is positively regulated by HIF-1α and Nrf2 in association with HSP90. Therefore, targeting tumor angiogenesis promoting DDAH1 along with standard androgen receptor (AR) targeted therapy may offer an effective strategy to prevent PCa progression.

Activation of TGF-ß - SMAD2 signaling by IL-6 drives neuroendocrine differentiation of prostate cancer through p38MAPK.

Neuroendocrine prostate cancer (NEPC) is an aggressive, androgen independent PCa and it is detected in patients undergoing androgen deprivation therapy (ADT). Interleukin-6 (IL-6) is a pleiotropic cytokine elevated in PCa patients promotes neuroendocrine differentiation (NED). In this study, PCa cells were differentiated with IL-6 in in-vitro to identify novel targets or signaling pathways associated with emergence of NEPC on deprivation of androgens. From the results, we observed an activation of TGF-ß signaling pathway is altered through multiple proteins in differentiated LNCaP cells. Hence, we investigated the role of TGF-ß axis in PCa cells differentiation. LNCaP cells treated with IL-6 in androgens deprived media release excess TGF-ß ligand and this as conditioned media added to cells stimulated NED of PCa cells. TGF-ß released by IL-6 stimulated cells activate p38MAPK through SMAD2 thereby promote NED. Inhibition of TGF-ßRI and TGF-ßRII signaling activation in LNCaP cells treated with IL-6 did not reversed the NED of cells, possibly due to the reason that the inhibition of TGF-ß axis is further activating p38MAPK through SMAD independent manner in PCa cells. However, siRNA mediated knock down or inhibition p38MAPK inactivated TGF-ß - SMAD axis in differentiating cells and attenuated NED of LNCaP cells. This result suggests that p38MAPK is the central node for receiving IL-6 signals and promotes NED of LNCaP cells in androgens free media. Remarkably, downregulation or inhibition of p38MAPK in NCI-H660 reversed NED characteristics as well as markers along with inactivation of SMAD2 whereas no effect observed in WPMY-1 normal prostate cells. Taken together these findings unveil that p38MAPK and its upstream regulators are potential targets to overcome the progression of NED of PCa and develop novel therapeutic measures along ADT for effective treatment of PCa.

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

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

Tumor protein D52 (isoform 3) interacts with and promotes peroxidase activity of Peroxiredoxin 1 in prostate cancer cells implicated in cell growth and migration.

Tumor protein D52 (TPD52) is overexpressed in multiple cancers including prostate cancer due to gene amplification and investigations to understand its role in the pathophysiology of different cancers are continuing. GST pull-down assays and Tandem affinity purification of TPD52 as bait identified novel prey Peroxiredoxin 1 (PRDX1) in prostate cancer (PCa) cells. PRDX1 interaction with TPD52 was confirmed in immunoprecipitation and affinity interaction assays. Mapping of interaction domain indicated that PRDX1 interacts with C-terminal region of TPD52 containing PEST domain between 152 and 179 amino acids, a new binding region of TPD52. Here we show that TPD52 interaction with PRDX1 increased its peroxidase activity and ectopic expression of TPD52 induced dimerization of PRDX1 in PCa cells. Moreover, H2O2 exposure evoked the interaction between TPD52 and PRDX1 while depletion of both proteins led to the accumulation of H2O2 suggesting peroxidase activity is important to maintain oxidative capacity in PCa cells. We also observed that overexpression or downregulation of TPD52 and PRDX1 individually or together affecting PCa cells growth, survival, and migration. Altogether, our results show a novel interaction partner of TPD52 providing new insights of its functions and ascertain the role of TPD52-PRDX1 interaction in PCa progression.

Novel Cellularly Active Inhibitor Regresses DDAH1 Induced Prostate Tumor Growth by Restraining Tumor Angiogenesis through Targeting DDAH1/ADMA/NOS Pathway.

Dimethylarginine dimethylaminohydrolase1 (DDAH1) inhibitors are important therapeutics by virtue of their ability to control nitric oxide (NO) production by elevating asymmetric dimethylarginine (ADMA) levels. In a screening campaign, we identified that DD1E5 (3-amino-6- tert-butyl-N-(1,3-thiazol-2-yl)-4-(trifluoromethyl)thieno[2,3- b]pyridine-2- carboxamide) inhibits the DDAH1 activity both in vitro and in cultured cells. Mechanistic studies found that DD1E5 is a competitive inhibitor (dissociation constant ( Ki) of 2.05 ± 0.15 µM). Enzyme kinetic assays showed time and concentration dependent inhibition of DDAH1 with DD1E5, which shows tight binding with an inactivation rate constant of 0.2756 ± 0.015 M-1 S-1. Treatment of cancer cells with DDAH1 inhibitors shows inhibition of cell proliferation and a subsequent decrease in NO production with ADMA accumulation. DD1E5 reversed the elevated VEGF, c-Myc, HIF-1α, and iNOS levels induced by exogenous DDAH1 overexpression in PCa cells. Moreover, DD1E5 significantly increased intracellular levels of ADMA and reduced NO production, suggesting its therapeutic potential for cancers in which DDAH1 is upregulated. In in vitro assays, DD1E5 abrogated the secretion of angiogenic factors (bFGF and IL-8) into conditional media, indicating its antiangiogenic potential. DD1E5 inhibited in vivo growth of xenograft tumors derived from PCa cells with DDAH1 overexpression, by reducing tumor endothelial content represented with low CD31 expression. VEGF, HIF-1α, and iNOS expression were reversed in DD1E5 treated tumors compared to respective control tumors. In this work, integrating multiple approaches shows DD1E5 is a promising tool for the study of methylarginine-mediated NO control and a potential therapeutic lead compound against pathological conditions with elevated NO production such as cancers and other diseases.