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

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

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.

Dimethylarginine Dimethylaminohydrolase - 1 expression is increased under tBHP-induced oxidative stress regulates nitric oxide production in PCa cells attenuates mitochondrial ROS-mediated apoptosis.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression is frequently elevated in different cancers including prostate cancer (PCa) and enhances nitric oxide (NO) production in tumor cells by metabolising endogenous nitric oxide synthase (NOS) inhibitors. DDAH1 protects the PCa cells from cell death and promotes survival. In this study, we have investigated the cytoprotective role of DDAH1 and determined the mechanism of DDAH1 in protecting the cells in tumor microenvironment. Proteomic analysis of PCa cells with stable overexpression of DDAH1 has identified that oxidative stress-related activity is altered. Oxidative stress promotes cancer cell proliferation, survival and causes chemoresistance. A known inducer of oxidative stress, tert-Butyl Hydroperoxide (tBHP) treatment to PCa cells led to elevated DDAH1 level that is actively involved in protecting the PCa cells from oxidative stress induced cell damage. In PC3-DDAH1- cells, tBHP treatment led to higher mROS levels indicating that the loss of DDAH1 increases the oxidative stress and eventually leads to cell death. Under oxidative stress, nuclear Nrf2 controlled by SIRT1 positively regulates DDAH1 expression in PC3 cells. In PC3-DDAH1+ cells, tBHP induced DNA damage is well tolerated compared to wild-type cells while PC3-DDAH1- became sensitive to tBHP. In PC3 cells, tBHPexposure has increased the production of NO and GSH which may be acting as an antioxidant defence to overcome oxidative stress. Furthermore, in tBHP treated PCa cells, DDAH1 is controlling the expression of Bcl2, active PARP and caspase 3. Taken together, these results confirm that DDAH1 is involved in the antioxidant defence system and promotes cell survival.

N-end rule pathway inhibitor sensitizes cancer cells to antineoplastic agents by regulating XIAP and RAD21 protein expression.

Anticancer drugs exert their effects on cancer cells by deregulating many pathways linked to cell cycle, apoptosis, etc. but cancer cells gradually become resistive against anticancer drugs, thereby necessitating the development of newer generation anticancer molecules. N-end rule pathway has been shown to be involved in the degradation of many cell cycle and apoptosis-related proteins. However, the involvements of this pathway in cancer are not well established. Recently, we developed a non-peptide-based N-end rule pathway inhibitor, RF-C11 for type 1 and 2 recognition domains of E3 ubiquitin ligases. The inhibitor significantly increased the half-life of potential N-degrons leading to significant physiological changes in vivo. We hypothesized RF-C11 may be used to decipher the N-end rule pathway's role in cancer towards the development of anticancer therapeutics. In this study, we showed that RF-C11, barring noncancer cells, significantly sensitizes cancer cells towards different anticancer agents tested. We further find that the profound cellular sensitization to anticancer drugs was affected by (a) downregulation of X-linked inhibitor of apoptosis protein, an antiapoptotic protein and (b) by stabilization of RAD21, and thereby inhibiting metaphase to anaphase promotion. The study shows that RF-C11 or its analogs may be used as a novel additive in combination therapy against cancer.

Corticosteroids protect infected cells against mycobacterial killing in vitro.

The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 µM. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling.

Synthesis and evaluation of a novel quinoline-triazole analogs for antitubercular properties via molecular hybridization approach.

Towards a quest for establishing new antitubercular agents, we have designed new quinoline-triazole hybrid analogs in a six-step reaction sequence involving versatile reactions like Vilsmeier-Haack and click reaction protocol. The design is based on the structural modification of bedaquiline moiety and involves molecular hybridization approach. The structure of the synthesized product was elucidated by single crystal X-ray diffraction study. The synthesized target compounds were screened for their antitubercular activity against Mycobacterium bovis. Interestingly, two compounds of the series (8d and 8m) showed significant inhibition with MIC of 31.5 and 34.8 µM. Compounds bearing 3-fluoro phenyl and n-octyl groups on the 1,2,3-triazole ring emerged as the most potent leads among the compounds tested. Further these hit compounds were also screened for their cytotoxic effect on human embryonic kindey 293 (HEK293) cells and other cancer cell lines such as HeLa (Cervical), PC3 (Prostate), Panc-1 (Pancreatic) and SKOV3 (Ovarian) indicating to be safer with the minimal cytotoxicity.

Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.

Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well-understood. Surprisingly, here we found occupancy of the metastasis suppressor non-metastatic 2 (NME2) within the hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells and NME2-mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in up-regulated hTERT expression. Mechanistically, additional results indicated that the RE1-silencing transcription factor (REST)-lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in an NME2-dependent way and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G-quadruplex-binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G-quadruplex promoter motif, which potentially can be targeted by tailored small molecules.

Interleukin-6 induced overexpression of valosin-containing protein (VCP)/p97 is associated with androgen-independent prostate cancer (AIPC) progression.

Though Androgen deprivation therapy (ADT) is effective initially, numerous patients become resistant to it and develop castration resistant PCa (CRPC). Cytokines promotes ligand independent activation of AR. Interleukin-6 (IL-6) levels are elevated in CRPC patients and regulate AR activity. However, progression to CRPC is not fully understood. In this study, we analyzed differential protein expression in LNCaP cells treated with IL-6 using proteomics. Results revealed altered expression of 27 proteins and Valosin-containing protein (VCP)/p97 plays a predominant role in co-regulation of altered proteins. Interestingly, IL-6 induced VCP expression through Pim-1 via STAT3 is AR independent there by suggesting a role for VCP in CRPC. Transfection of LNCaP cells for VCP overexpression showed an increased cell proliferation, migration, and invasion where as its inhibition by NMS-873 showed the reverse effect causing cell death. Mechanistic studies demonstrate that cell death occurs due to apoptosis by endoplasmic reticulum (ER) stress, elevated cell cycle inhibitors p21, p27kip1, and active PARP and reduced Bcl-2. VCP promotes cell invasion and migration by altering E-cadherin and Vimentin levels inversely triggering EMT of PCa cells. VCP immunostaining revealed no staining in BPH but strong staining in PCa. This study determines VCP may play an important role in progression to CRPC and it can be a favorable target with to develop new therapies to treat ADT resistant prostate cancer.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is frequently upregulated in prostate cancer, and its overexpression conveys tumor growth and angiogenesis by metabolizing asymmetric dimethylarginine (ADMA).

Tissue microarray analysis confirmed higher dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression in prostate cancer (PCa) compared to benign and normal prostate tissues. DDAH1 regulates nitric oxide (NO) production by degrading endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA). This study examined whether DDAH1 has any physiological role in PCa progression. Using overexpression of DDAH1 in PCa (PC3 and LNCaP) cell lines, we found that DDAH1 promotes cell proliferation, migration and invasion by lowering ADMA levels, as well as increasing NO production. VEGF, HIF-1α and iNOS were upregulated in DDAH1 expressing cells as result of elevated NO. DDAH1 increased secretion of pro-angiogenic signals bFGF and IL-8, into conditioned media. Treatment of DDAH1-positive PCa cells with NOS inhibitors (L-NAME and 1400 W) attenuated DDAH1 activity to promote cell growth. Xenografts derived from these cells grew significantly faster (> twofold) than those derived from control cells. Proliferation rate of cells stably expressing mutant DDAH1 was same as control cells unlike wild-type DDAH1-positive PCa cells. Xenograft tumors derived from mutant-positive cells did not differ from control tumors. VEGF, HIF-1α and iNOS expression did not differ in DDAH1 mutant-positive tumors compared to control tumors, but was upregulated in wild-type DDAH1 overexpressing tumors. Furthermore, CD31 immunostaining on xenograft tissues demonstrated that DDAH1 tumors had high endothelial content than mutant DDAH1 tumors. These data suggest that DDAH1 is an important mediator of PCa progression and NO/DDAH pathway needs to be considered in developing therapeutic strategies targeted at PCa.

All-Trans-Retinoic Acid Stimulates Overexpression of Tumor Protein D52 (TPD52, Isoform 3) and Neuronal Differentiation of IMR-32 Cells.

Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration, and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (ß III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. J. Cell. Biochem. 118: 4358-4369, 2017. © 2017 Wiley Periodicals, Inc.

Tumor protein D52 (isoform 3) contributes to prostate cancer cell growth via targeting nuclear factor-κB transactivation in LNCaP cells.

Our previous study showed that TPD52 overexpression could increase migration and proliferation of LNCaP cells contributing to the development of prostate cancer. However, mechanism of TPD52 in prostate cancer initiation and progression remains elusive. In this study, we investigated the possible underlying mechanism of TPD52 in prostate cancer progression. In LNCaP cells, TPD52 expression was altered by transfecting with either EGFP-TPD52 or specific short hairpin RNA. Overexpression of TPD52 protected LNCaP cells from apoptosis through elevated anti-apoptotic proteins XIAP, Bcl-2, and Cyclin D1, whereas Bax was downregulated. Mechanistically, we found that TPD52 confers transactivation of nuclear factor-κB, thereby enhancing its target gene expression in LNCaP cells. TPD52 promotes LNCaP cell invasion probably via increased matrix metalloproteinase 9 expression and its activity while tissue inhibitor of metalloproteinase expression is significantly downregulated. Notably, TPD52 might be involved in cell adhesion, promoting tumor metastasis by inducing loss of E-cadherin, expression of vimentin and vascular cell adhesion molecule, and additionally activation of focal adhesion kinase. Furthermore, TPD52 directly interacts with nuclear factor-κB p65 (RelA) and promotes accumulation of phosphorylated nuclear factor-κB (p65)S536 that is directly linked with nuclear factor-κB transactivation. Indeed, depletion of TPD52 or inhibition of nuclear factor-κB in TPD52-positive cells inhibited secretion of tumor-related cytokines and contributes to the activation of STAT3, nuclear factor-κB, and Akt. Interestingly, in TPD52 overexpressing LNCaP cells, nuclear factor-κB inhibition prevented the autocrine/paracrine activation of STAT3. TPD52 activates STAT3 through ascertaining a cross talk between the nuclear factor-κB and the STAT3 signaling systems. Collectively, these results reveal mechanism by which TPD52 is associated with prostate cancer progression and highlight the approach for therapeutic targeting of TPD52 in prostate cancer.

Expanding the tetrahydroquinoline pharmacophore.

Tetrahydroquinoline is a privileged scaffold with a large number of biological applications. The tetrahydroquinoline pharmacophore has been expanded to yield 34 compounds. Biological screening of these compounds led to the identification of tetrahydroquinoline as neurotropic agents not reported earlier.

Solution Nuclear Magnetic Resonance Studies of Sterol Carrier Protein 2 Like 2 (SCP2L2) Reveal the Insecticide Specific Structural Characteristics of SCP2 Proteins in Aedes aegypti Mosquitoes.

Sterol carrier protein 2 like 2 from Aedes aegypti (AeSCP2L2) plays an important role in lipid transport in mosquitoes for its routine metabolic processes. Repeated unsuccessful attempts to crystallize ligand free SCP2L2 prompted us to undertake nuclear magnetic resonance (NMR) spectroscopy to determine its three-dimensional structure. We report here the three-dimensional structures and dynamics of apo-AeSCP2L2 and its complex with palmitate. The (15)N heteronuclear single-quantum coherence spectrum of apo-AeSCP2L2 displayed multiple peaks for some of the amide resonances, implying the presence of multiple conformations in solution, which are transformed to a single conformation upon formation of the complex with plamitate. The three-dimensional structures of apo-AeSCP2L2 and palmitated AeSCP2L2 reveal an α/ß mixed fold, with five ß-strands and four α-helices, very similar to the other SCP2 protein structures. Unlike the crystal structure of palmitated AeSCP2L2, both solution structures are monomeric. It is further confirmed by the rotational correlation times determined by NMR relaxation times (T1 and T2) of the amide protons. In addition, the palmitated AeSCP2L2 structure contains two palmitate ligands, bound in the binding pocket, unlike the three palmitates bound in the dimeric form of AeSCP2L2 in the crystals. The relaxation experiments revealed that complex formation significantly reduces the dynamics of the protein in solution.

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

Evaluation of reverse phase protein array (RPPA)-based pathway-activation profiling in 84 non-small cell lung cancer (NSCLC) cell lines as platform for cancer proteomics and biomarker discovery.

The reverse phase protein array (RPPA) approach was employed for a quantitative analysis of 71 cancer-relevant proteins and phosphoproteins in 84 non-small cell lung cancer (NSCLC) cell lines and by monitoring the activation state of selected receptor tyrosine kinases, PI3K/AKT and MEK/ERK1/2 signaling, cell cycle control, apoptosis, and DNA damage. Additional information on NSCLC cell lines such as that of transcriptomic data, genomic aberrations, and drug sensitivity was analyzed in the context of proteomic data using supervised and non-supervised approaches for data analysis. First, the unsupervised analysis of proteomic data indicated that proteins clustering closely together reflect well-known signaling modules, e.g. PI3K/AKT- and RAS/RAF/ERK-signaling, cell cycle regulation, and apoptosis. However, mutations of EGFR, ERBB2, RAF, RAS, TP53, and PI3K were found dispersed across different signaling pathway clusters. Merely cell lines with an amplification of EGFR and/or ERBB2 clustered closely together on the proteomic, but not on the transcriptomic level. Secondly, supervised data analysis revealed that sensitivity towards anti-EGFR drugs generally correlated better with high level EGFR phosphorylation than with EGFR abundance itself. High level phosphorylation of RB and high abundance of AURKA were identified as candidates that can potentially predict sensitivity towards the aurora kinase inhibitor VX680. Examples shown demonstrate that the RPPA approach presents a useful platform for targeted proteomics with high potential for biomarker discovery. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Synthesis and evaluation of novel fluorinated pyrazolo-1,2,3-triazole hybrids as antimycobacterial agents.

A library of novel 3-trifluoromethyl pyrazolo-1,2,3-triazole hybrids (5-7) were accomplished starting from 5-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-amine (1) via key intermediate 2-azido-N-(5-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)acetamide (3) through click chemistry approach. Thus obtained compounds in 5-7 series were evaluated for in vitro antimycobacterial activity against Mycobacterium smegmatis (MC(2) 155) and also verified the cytotoxicity. These studies engendered promising lead compounds 5q, 7b and 7c with MIC (µg/mL) values 15.34, 16.18 and 16.60, respectively. Amongst these three compounds, 2-(4-(4-methoxybenzoyl)-1H-1,2,3-triazol-1-yl)-N-(5-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl) acetamide (5q) emerged as the most promising antitubercular agent with lowest cytotoxicity against the A549 cancer cell line. This is the first report to demonstrate the pyrazolo triazole hybrids as potential antimycobacterial agents.

Synthesis, in vitro anticancer and antimycobacterial evaluation of new 5-(2,5-dimethoxyphenyl)-1,3,4-thiadiazole-2-amino derivatives.

A series of 2,5-disubstituted-1,3,4-thiadiazole derivatives 5a-5l, 7a-7e and 9 have been synthesised and screened for in vitro antimycobacterial activity against Mycobacterium smegmatis MC-155. In addition these compounds have also been screened for cytotoxic activity against cancer cell lines HT-29, MDA-MB-231 by MTT colorimetric assay. The compounds are well characterized by spectral analysis viz. (1)H NMR, (13)C NMR, FT-IR, mass and HRMS. Screening results indicate that compounds 5g, 7a possess good antitubercular activity with MIC value 65.74 and 40.86, respectively, compounds 5g, 7a, 7b, 7d, 7e and 9 displayed promising cytotoxic activity against the cell lines tested. 5g and 7a stand out to be potent antimycobacterial and anticancer agents among the tested series. Further the title compounds were also tested on human normal cells HEK293T and are found to be safer with lesser cytotoxicity. It is interesting to observe that compound 5g has come out to be safer, potent anticancer and antimycobacterial agent.

5-epi-Torrubiellutin C shows antiproliferative activity on DU145 prostate cancer cells through inactivation of the AKT/mTOR pathway.

Cell-based assays for evaluation of the anticancer potential of a focused small molecule library have identified a few potential hit molecules. Among the hits identified, Torrubiellutins (3a) showed good anticancer potential across the cells used in screening assays. Torrubiellutins are isolated from fungal insects Torrubiella luteorostrata and diverse pharmacological effects for these have been reported. However, it is not known as to how Torrubiellutins act through signaling pathways inhibiting the growth of eukaryotic cells. The current study aimed to determine the anticancer potential of Torrubiellutins by defining the molecular mechanism of cytotoxicity using DU145 cells. The results showed that the inhibition of prostate cancer cell growth by 3a was associated with inhibition of anchorage-independent growth, cell migration, and, to a small extent, apoptosis-mediated cell death by caspase activation. The growth-inhibitory effects of 3a are supported by inactivation of prosurvival pathways. Immunoblot analysis showed that the treatment of DU145 cells with 3a resulted in specific downregulation of AKT/mammalian target of rapamycin (mTOR) and its downstream effector proteins p70S6K, GSK3ß, and STAT3. On the basis of these findings, we propose that the changes observed in the AKT/mTOR signaling axis are new targets of 3a that are involved in its inhibitory activity on the proliferation of prostate cancer cells, suggesting its potential for further investigation as a promising anticancer agent.

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.