Gagan Thangjam

Heat Shock Protein 90 Inhibitors Prevent LPS-Induced Endothelial Barrier Dysfunction by Disrupting RhoA Signaling

Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling.

Novel Role of Androgens in Mitochondrial Fission and Apoptosis

Androgen and androgen receptors (AR) play critical roles in the proliferation of prostate cancer through transcriptional regulation of target genes. Here, we found that androgens upregulated the expression of dynamin-related protein 1 (Drp1), which is involved in the induction of mitochondrial fission, a common event in mitosis and apoptosis. Clinical tissue samples and various prostate cancer cell lines revealed a positive correlation between Drp1 and AR levels. Treatment of androgen-sensitive cells with an AR agonist, R1881, and antagonist, bicalutamide, showed that Drp1 is transcriptionally regulated by androgens, as confirmed by an AR ChIP-seq assay. Live imaging experiments using pAcGFP1-Mito stably transfected LNCaP (mito-green) cells revealed that androgen did not induce significant mitochondrial fission by itself, although Drp1 was upregulated. However, when treated with CGP37157 (CGP), an inhibitor of mitochondrial Ca2+ efflux, these cells exhibited mitochondrial fission, which was further enhanced by pretreatment with R1881, suggesting that androgen-induced Drp1 expression facilitated CGP-induced mitochondrial fission. This enhanced mitochondrial fission was correlated with increased apoptosis. Transfection with dominant-negative (DN-Drp1, K38A) rescued cells from increased apoptosis, confirming the role of androgen-induced Drp1 in the observed apoptosis with combination treatment. Furthermore, we found that CGP reduced the expression of Mfn1, a protein that promotes mitochondrial fusion, a process which opposes fission. We suggest that androgen-increased Drp1 enhanced mitochondrial fission leading to apoptosis. The present study shows a novel role for androgens in the regulation of mitochondrial morphology that could potentially be utilized in prostate cancer therapy. Mol Cancer Res; 9(8); 1067–77. ©2011 AACR.

LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung

Heat shock protein 90 (Hsp90) inhibitors were initially developed as anticancer agents; however, it is becoming increasing clear that they also possess potent anti-inflammatory properties. Posttranslational modifications of Hsp90 have been reported in tumors and have been hypothesized to affect client protein- and inhibitor-binding activities. In the present study we investigated the posttranslational modification of Hsp90 in inflammation. LPS, a prototypical inflammatory agent, induced concentration- and time-dependent tyrosine (Y) phosphorylation of Hsp90α and Hsp90β in bovine pulmonary arterial and human lung microvascular endothelial cells (HLMVEC). Mass spectrometry identified Y309 as a major site of Y phosphorylation on Hsp90α (Y300 of Hsp90β). LPS-induced Hsp90 phosphorylation was prevented by the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin (17-AAG) in vitro as well as in lungs from LPS-treated mice, in vivo. Furthermore, 17-AAG prevented LPS-induced pp60src activation. LPS-induced Hsp90 phosphorylation was also prevented by the pp60src inhibitor PP2. Additionally, Hsp90 phosphorylation was induced by infecting cells with a constitutively active pp60src adenovirus, whereas either a dominant-negative pp60src adenovirus or reduced expression of pp60src by a specific siRNA prevented the LPS-induced Y phosphorylation of Hsp90. Transfection of HLMVEC with the nonphosphorylatable Hsp90β Y300F mutant prevented LPS-induced Hsp90β tyrosine phosphorylation but not pp60src activation. Furthermore, the Hsp90β Y300F mutant showed a reduced ability to bind the Hsp90 client proteins eNOS and pp60src and HLMVEC transfected with the mutant exhibited reduced LPS-induced barrier dysfunction. We conclude that inflammatory stimuli cause posttranslational modifications of Hsp90 that are Hsp90-inhibitor sensitive and may be important to the proinflammatory actions of Hsp90.

Novel Mechanism of Attenuation of LPS-Induced NF-κB Activation by the Heat Shock Protein 90 Inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, in Human Lung Microvascular Endothelial Cells

Heat shock protein (hsp) 90 inhibition attenuates NF-κB activation and blocks inflammation. However, the precise mechanism of NF-κB regulation by hsp90 in the endothelium is not clear. We investigated the mechanisms of hsp90 inhibition by 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) on NF-κB activation by LPS in primary human lung microvascular endothelial cells. Transcriptional activation of NF-κB was measured by luciferase reporter assay, gene expression by real-time RT-PCR, DNA binding of transcription factors by chromatin immunoprecipitation assay, protein-protein interaction by coimmunoprecipitation/immunoblotting, histone deacetylase (HDAC)/histone acetyltransferase enzyme activity by fluorometry, and nucleosome eviction by partial microccocal DNase digestion. In human lung microvascular endothelial cells, 17-AAG-induced degradation of IKBα was accomplished regardless of the phosphorylation/ubiquitination state of the protein. Hence, 17-AAG did not block LPS-induced NF-κB nuclear translocation and DNA binding activity. Instead, 17-AAG blocked the recruitment of the coactivator, cAMP response element binding protein binding protein, and prevented the assembly of a transcriptionally competent RNA polymerase II complex at the κB elements of the IKBα (an NF-κB-responsive gene) promoter. The effect of LPS on IKBα mRNA expression was associated with rapid deacetylation of histone-H3(Lys9) and a dramatic down-regulation of core histone H3 binding. Even though treatment with an HDAC inhibitor produced the same effect as hsp90 inhibition, the effect of 17-AAG was independent of HDAC. We conclude that hsp90 inhibition attenuates NF-κB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.

p53 protects against LPS-Induced Lung Endothelial Barrier Dysfunction

New therapies toward heart and blood vessel disorders may emerge from the development of Hsp90 inhibitors. Several independent studies suggest potent anti-inflammatory activities of those agents in human tissues. The molecular mechanisms responsible for their protective effects in the vasculature remain unclear. The present study demonstrates that the transcription factor p53, an Hsp90 client protein, is crucial for the maintenance of vascular integrity, protects again LPS-induced endothelial barrier dysfunction, and is involved in the mediation of the anti-inflammatory activity of Hsp90 inhibitors in lung tissues. p53 silencing by siRNA decreased transendothelial resistance (a measure of endothelial barrier function). A similar effect was induced by the p53 inhibitor pifithrin, which also potentiated the LPS-induced hyperpermeability in human lung microvascular endothelial cells (HLMVEC). On the other hand, p53 induction by nutlin suppressed the LPS-induced vascular barrier dysfunction. LPS decreased p53 expression in lung tissues and that effect was blocked by pretreatment with Hsp90 inhibitors both in vivo and in vitro. Furthermore, the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin suppressed the LPS-induced overexpression of the p53 negative regulator MDMX as well as p53 and MDM2 (another p53 negative regulator) phosphorylation in HLMVEC. Both negative p53 regulators were downregulated by LPS in vivo. Chemically induced p53 overexpression resulted in the suppression of LPS-induced RhoA activation and MLC2 phosphorylation, whereas p53 suppression caused the opposite effects. These observations reveal new mechanisms for the anti-inflammatory actions of Hsp90 inhibitors, i.e., the induction of the transcription factor p53, which in turn can orchestrate robust vascular anti-inflammatory responses both in vivo and in vitro.

Metabolites of Purine Nucleoside Phosphorylase (NP) in Serum Have the Potential to Delineate Pancreatic Adenocarcinoma

Pancreatic Adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS) and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX). A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7) and Alpha Synuclein (aSyn), both of which are known to be pathognomonic to Parkinsons disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP) which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.

Histone deacetylase inhibitors prevent pulmonary endothelial hyperpermeability and acute lung injury by regulating heat shock protein 90 function

Transendothelial hyperpermeability caused by numerous agonists is dependent on heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores transendothelial permeability. Hyperacetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating lipopolysaccharide (LPS)-induced transendothelial hyperpermeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced transendothelial hyperpermeability, acetylated and suppressed Hsp90 chaperone function, and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor RGFP-966 or the HDAC6-selective inhibitor tubastatin A provided partial protection against LPS-mediated transendothelial hyperpermeability. Similarly, knock down of HDAC3 and HDAC6 by specific small-interfering RNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacological inhibition of both HDAC3 and -6 attenuated the inflammation, capillary permeability, and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased transendothelial hyperpermeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling.

Abstract 1215: Novel role of androgens: Involvement in mitochondrial fission and apoptosis in prostate cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Novel role of androgens: Involvement in mitochondrial fission and apoptosis in prostate cancer cells The role of androgens on prostate cancer growth and proliferation is well known but very little is known about its pro-apoptotic roles. Here, we demonstrate a novel role of androgens in altering the shape and function of the mitochondria. Dynamin-related protein 1 (Drp1) is a key protein necessary for mitochondrial fragmentation or fission (MF). The expression of Drp1 (both protein and mRNA) correlated with the expression of androgen receptor (AR) in prostate cancer cell lines. Interestingly, Drp1 expression was very low in androgen refractory prostate metastatic tissues. Treatment of LNCaP cells with androgen agonist, R1881 increased the expression of Drp1 mRNA and protein. This increase was inhibited by androgen antagonist, bicalutamide. Androgen regulation of Drp1 was confirmed by knock out of AR (using siRNA), which resulted in decreased Drp1 expression. To measure mitochondrial fission (MF), cells were stably transfected with pAcGFP1-Mito, targeting the expression of GFP to mitochondria. Androgen treatment did not induce MF, although Drp1 was upregulated. However, treatment with [CGP37157][1] (CGP), a mitochondrial Na+/Ca2+ channel blocker induced MF. Pre-treatment with R1881 significantly increased CGP-induced MF, suggesting that androgen-induced Drp1 facilitated CGP-induced MF. Androgens increased the proliferation of LNCaP, which was reduced by CGP treatment. Interestingly, treatment with CGP induced apoptosis which was further enhanced by pre-treatment with R1881. Drp1 dominant negative experiments showed that Drp1 was required for enhanced apoptosis, suggesting that androgen-induced Drp1 was responsible for increasing the apoptotic effects of CGP. Experiments to examine the paradoxical effects of androgens showed that induction of proliferation by androgens may be due to differential phosphorylation of Drp1. However, induction of apoptosis by CGP alone or in combination with androgens is due to the reduction of the expression of Mfn1, a protein that promotes mitochondrial fusion (opposite of fission). Use of proteasomal inhibitor, lactacystin, suggested that CGP treatment resulted in proteasome mediated degradation of Mfn1. In summary, our results demonstrated: i) transcriptional regulation of Drp1 by androgens, ii) androgen-induced Drp1 facilitated mitochondrial fission and apoptosis. Significance of these results: Clinical: we suggest that it is not necessary to block the function of androgens, instead administration of drugs (such as calcium channel blockers) that affect mitochondrial function induced death of cancer cells in the presence of androgens. Research: addresses a fundamental question about mechanisms involved in proliferation and apoptosis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1215. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CGP37157&atom=%2Fcanres%2F70%2F8_Supplement%2F1215.atom

Abstract A52: Metabolomic profiling reveals impaired xenobiotic metabolism in bladder cancer

Introduction: Bladder cancer (BCa) is the second most prevalent urological malignancy and the fourth highest cause of cancer-related death in the United States. Earlier studies have linked BCa development to alterations in metabolic pathways. Significant among these are decreased activity of N-acetyl transferases causing a slow-acetylator phenotype leading to inefficient detoxification of aromatic hydrocarbons causal to onset of BCa. Interestingly, Afro-American patients inherently exhibit such slow acetylator phenotype and are known to have a more aggressive form of the tumor compared to Caucasians. This indicates existence of a metabolic niche that governs the racial disparity in BCa, which is not well understood. Also, there is an imminent need to develop non-invasive markers for early detection and prognosis of BCa, since urine cytology which is the current clinical standard is not specific to the tumor. Using mass spectrometry we report metabolic alterations in BCa and delineate bioprocesses that are altered during its progression. Our data for the first-time demonstrates role of methylation in attenuation of xenotbiotic metabolism in BCa. Furthermore, the metabolic profiles seed further analysis to examine the racial disparity in these tumors. Methods: Total metabolome from flash frozen clinically annotated bladder-derived tissues (n=58,31 benign adjacent and 27 BCa, 26 matched pairs) were examined using a combination of Q-TOF (unbiased) and triple-quadrupole (targeted) mass spectrometry. Panel of well-defined standards were used to ensure reproducibility of the profiling process. The metabolites were pre-fractionated using liquid chromatography prior to mass spectrometry in both the positive and negative ionization mode. The unbiased mass spectral data was searched using Metlin library to identify the compounds. The metabolomic profiles thus generated were analyzed to delineate class-specific signatures which were interrogated for altered bioprocesses using Molecular Concept Map (OCM, www.oncomine.org). The altered bioprocesses were validated in cell line models using a combination of Q-PCR, immunoblot analysis and functional assays. Results and discussion: A total of 2019 compounds were detected across the 58 bladder-derived specimens of which, 423 compounds were significantly altered in BCa compared to adjacent benign. 50 of the differential compounds were named and used for developing a classificatory signature and bioprocess mapping. Included among these were polycylic compounds like aniline, catechols, aromatic amino acids, polyamines and S-adenosyl methionine (SAM). Interestingly this BCa-specific metabolic signature in tissues was able to delineate tumor from benign with an accuracy of 75 %. Importantly the functional mapping of the metabolic data revealed enhanced methylation potential in tumors as being one of the factors de-regulating the xenobiotic metabolism. In vitro experiments using bisulfite sequencing and methyltransferase inhibitor 5-Aza-cytidine confirmed this methylation-induced attenuation of phase I/II metabolic genes namely CYP1A1, CYP1B1, EPHX1 and GSTT1 in BCa. In summary, using unbiased metabolomic profiling report metabolic fingerprint for bladder cancer. Importantly our data for the first time reveals methylation-induced silencing of xenobiotic metabolism in bladder tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A52.