Shashwat Sharad

Skin regenerative potentials of curcumin

Curcumin, an active constituent of the spice turmeric, is well known for its chemopreventive properties and is found to be beneficial in treating various disorders including skin diseases. Curcumin protects skin by quenching free radicals and reducing inflammation through the inhibition of nuclear factor‐kappa B. Curcumin also affects other signaling pathways including transforming growth factor‐β and mitogen‐activated protein kinase pathway. Curcumin also modulates the phase II detoxification enzymes which are crucial in detoxification reactions and for protection against oxidative stress. In the present review, the biological mechanisms of the chemopreventive potential of curcumin in various skin diseases like psoriasis, vitiligo, and melanoma is discussed. The application of curcumin in skin regeneration and wound healing is also elucidated. We also explored the recent innovations and advances involved in the development of transdermal delivery systems to enhance the bioavailability of curcumin, particularly in the skin. Recent clinical trials pertaining to the use of curumin in skin diseases establishes its benefits and also the need for additional clinical trials in other diseases are discussed.

Prostate cancer gene expression signature of patients with high body mass index.

The goal of this study was to evaluate prostate cancer gene expression signatures associated with elevated body mass index (BMI). Global gene expression profiles of prostate tumor cells and matching normal epithelial cells were compared between patients with features of normal and high BMI at the time of radical prostatectomy. Knowledge-based analyses revealed an association of high BMI with altered levels of lipid metabolism and cholesterol homeostasis genes, such as stearoyl-CoA desaturase 1 (SCD1) and insulin-induced gene 1 (INSIG1), respectively, in prostate tumor cells. These genes were connected to known pathways of tumorigenesis revealed by the v-maf (musculoaponeurotic fibrosarcoma) oncogene homolog (MAF), notch receptor ligand, jagged 1 (JAG1) and the alanyl aminopeptidase (ANPEP/CD13) genes. This study highlighted that SCD1, a known target of statins, may have a mechanistic role in the recently noted beneficial effects of statin treatment in reducing biochemical recurrence of prostate cancer. An additional finding of our study is that some of the obesity-related genes were upregulated in tumor-matched normal cells within the high BMI group, when compared with normal cells within the normal BMI cohort.

ETS Related Gene mediated Androgen Receptor Aggregation and Endoplasmic Reticulum Stress in Prostate Cancer Development

Mechanistic studies of deregulated ERG in prostate cancer and other cancers continue to enhance its role in cancer biology and its utility as a biomarker and therapeutic target. Here, we show that ERG, through its physical interaction with androgen receptor, induces AR aggregation and endoplasmic reticulum stress in the prostate glands of ERG transgenic mice. Histomorphological alterations and the expression of ER stress sensors Atf6, Ire1α, Perk, their downstream effectors Grp78/BiP and eIF2α in ERG transgenic mouse prostate glands indicate the presence of chronic ER stress. Transient activation of apoptotic cell death during early age correlated well with the differential regulation of ER stress sensors, in particular Perk. Epithelial cells derived from ERG transgenic mouse prostates have increased prostasphere formation with resistance to radiation induced cell death. Continued activation of cell survival factors, Atf6 and Ire1α during chronic ER stress due to presence of ERG in prostate epithelium induces survival pathways and provides a selection pressure in the continuum of ERG dependent neoplastic process. These novel insights will enhance the understanding of the mechanistic functions of ERG in prostate tumor biology and towards development of early targeted therapeutic strategies for prostate cancer.

Loss of miR-449a in ERG-associated prostate cancer promotes the invasive phenotype by inducing SIRT1

Epigenetic regulation by SIRT1, a multifaceted NAD+-dependent protein deacetylase, is one of the most common factors modulating cellular processes in a broad range of diseases, including prostate cancer (CaP). SIRT1 is over-expressed in CaP cells, however the associated mechanism is not well understood. To identify whether specific microRNAs might mediate this linkage, we have screened a miRNA library for differential expression in ERG-associated CaP tissues. Of 20 differentially and significantly expressed miRNAs that distinguish ERG-positive tumors from ERG-negative tumors, we find miR-449a is highly suppressed in ERG-positive tumors. We establish that SIRT1 is a direct target of miR-449a and is also induced by ERG in ERG-associated CaP. Our data suggest that attenuation of miR-449a promotes the invasive phenotype of the ERG-positive CaP in part by inducing the expression of SIRT1 in prostate cancer cells. Furthermore, we also find that suppression of SIRT1 results in a significant reduction in ERG expression in ERG-positive CaP cells, indicating a feed-back regulatory loop associated with ERG, miR-449a and SIRT1. We also report that ERG suppresses p53 acetylation perhaps through miR-449a-SIRT1 axis in CaP cells. Our findings provide new insight into the function of miRNAs in regulating ERG-associated CaP. Thus, miR-449a activation or SIRT1 suppression may represent new therapeutic opportunity for ERG-associated CaP.

ERG Monoclonal Antibody in the Diagnosis and Biological Stratification of Prostate Cancer: Delineation of Minimal Epitope, Critical Residues for Binding, and Molecular Basis of Specificity

Recently we reported the development of a highly specific murine monoclonal antibody (ERG MAb 9FY) against the ERG oncoprotein. ERG is expressed in over half of all prostate cancers (CaP) as a result of specific gene fusions involving ERG and the androgen regulated TMPRSS2 promoter. ERG MAb 9FY has been extensively used in the evaluations of CaP. Increasing use of ERG MAb in CaP has prompted us to characterize the precise ERG epitope it binds to and to define the molecular basis of its specificity to ERG. The 9FY antibody binds to an epitope formed by amino acid residues 42-66 of the ERG protein. To determine the key residues involved in 9FY binding, experiments were carried out using a combination of approaches including overlapping peptides, alanine scanning mutagenesis, ELISA, and immunoblot assays. Analysis of both overlapping and variant peptides harboring truncations of amino acids revealed that a minimal epitope of eight residues (RVPQQDWL) is sufficient for binding to the 9FY antibody. In order to further identify key residues that mediate the binding of the antibody to ERG protein, a 14-residue peptide (P23) with optimal reactivity was subjected to alanine scanning mutagenesis. Alterations to residues QQDW were found to eliminate binding to the antibody, while residues (R50 and L57) were found to contribute to the binding of the antibody. Further experiments showed that peptide P23 competed effectively with ERG protein for binding 9FY. On the other hand, peptides with alanine substitutions for residues Q53 and W56 (P27 and P30, respectively) failed to interfere with binding. These data provide new information about a minimal epitope (RVPQQDWL) within amino acid residues 42-66 of the ERG protein that is recognized by MAb 9FY, which may aid in the diagnosis and also development of antibody based therapeutics against prostate and other cancers showing ERG overexpression.

MP70-13 DEPLETION OF PMEPA1 GENE CONFERS INCREASED CELL PROLIFERATION IN MOUSE PROSTATE EPITHELIUM

expression profiles of PC3CR by microarray to investigate the mechanisms of CBZ resistance. RESULTS: We incubated PC3 cells with gradually increasing concentrations of CBZ for 1.5 years, and established a CBZ-resistant cell line, PC3CR. PC3CR cells underwent cell division with 3 nM CBZ. We compared the cytotoxic effect of CBZ on PC3 and PC3CR cells using a cell viability assay. The half maximal inhibitory concentration (IC50) of CBZ in PC3 and PC3CR cells were 11.0 nM and 3.7 nM, respectively. Mice PC3CR xenograft tumors also had CBZ resistance (Fig. A) Functional annotation clustering (FAC) analysis using microarray data demonstrated cell division (GO: 0051301) and mitotic nuclear division (GO: 0007067) were the most enhanced clusters in PC3CR compared to PC3. These results suggested that enhancement of cell cycle progression signaling was related with CBZ resistance in CRPC. We perform in silico screening for compounds overcoming CBZ resistance by Connectivity Map (CMAP) analysis. Etoposide (VP16) was one of the candidate drugs which reverted gene expression pattern of CBZ-resistant cells into CBZ-sensitive cells. Topoisomerase IIa (TOP2A) is a main target of VP16, so we examined TOP2A expression in PC3CR. Quantitative PCR showed up-regulation of TOP2A in PC3CR. In cell viability assay, 1mM etoposide significantly inhibited PC3CR proliferation (80.3 3.2%). VP16 is ordinary used with platinum-based agents for neuroendocrine tumor. We tested cytotoxic effect of CDDP for CBZ-resistant CPRC. The relative cell viabilities of PC3CR treated with 3mM CDDP was 86.4 1.6%. CDDP was also effective for CBZ-resistant CRPC cells. Next, we tested anti-tumor effect of VP16 and CDDP using PC3CR xenograft tumor model. Both singleagent treatments with VP16 and CDDP significantly inhibited PC3CR xenograft tumors (Fig. B). Moreover, VP16 and CDDP in combination use had a synergic effect for the xenograft tumors. CONCLUSIONS: Etoposide based chemotherapy may be an optimal treatment for CPRC in the post-cabazitaxel setting.

Role of Ionizing Radiation in Neurodegenerative Diseases

Ionizing radiation (IR) from terrestrial sources is continually an unprotected peril to human beings. However, the medical radiation and global radiation background are main contributors to human exposure and causes of radiation sickness. At high-dose exposures acute radiation sickness occurs, whereas chronic effects may persist for a number of years. Radiation can increase many circulatory, age related and neurodegenerative diseases. Neurodegenerative diseases occur a long time after exposure to radiation, as demonstrated in atomic bomb survivors, and are still controversial. This review discuss the role of IR in neurodegenerative diseases and proposes an association between neurodegenerative diseases and exposure to IR.

Abstract B008: PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer

Introduction and Objectives: The dysfunctions of androgen receptor (AR) and TGF-β signaling contribute to prostate tumorigenesis and cancer progression. Our laboratory has identified PMEPA1 as an androgen responsive gene with prostate abundance, as well as PMEPA1/NEDD4 and AR negative feedback loop in the regulation of AR levels in prostate cancer cells. In addition, it has also been established by other groups that PMEPA1 inhibits TGF-β signaling via a similar negative feedback loop as a TGF-β responsive gene. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. This study focuses on understanding of the expression and biologic functions of PMEPA1 isoforms in CaP. Methods: The PMEPA1 isoforms were evaluated for expressions in multiple CaP cell lines, trensfectant derivatives, and prostate tumors. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145, and PC-3 cells individually. Cell proliferation was analyzed by cell counting, cell plating efficiency assay, and soft agar assay. The protein levels of PMEPA1 isoforms, AR, PSA, TGF-β receptor I, Smad2 were detected by immunoblotting, and the transcript levels of KLK3 (PSA), TGF-β responsive genes including THBS1, NEDD9 and COL1A1 were evaluated by QRT-PCR. The TGF-β signaling activity was measured by SMAD reported dual-luciferase assay. The transcript levels of PMEPA1-252, PMEPA3-287-STAG1, AR, PSA in 120 matched human benign and malignant frozen tissue were evaluated with Q-PCR. Results: The expressions of PMEPA1-252 and PMEPA2-344 isoforms were restricted to androgen-responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-287/STAG1, PMEPA4-259 and PMEPA5-237). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1-252 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Only PMEPA1-252 mediated AR protein degradation and inhibited AR signaling. In contrast, PMEPA3-287-STAG1 and PMEPA4-259 inhibited TGF-β signaling luciferase activity and transcript levels of THBS1, NEDD9 and COL1A1. All PMEPA1 isoforms were found to have no effects on protein level of TGF-β receptor I. Moreover, the deletion mutants of N-terminus and transmembrane domains interrupted the isoform specific inhibitory effects on AR/TGF-β signaling. The transcript level of PMEPA1-252 was higher than PMEPA3-287-STAG1 in benign prostate tissue. Different from PMEPA1-252, the expression of PMEPA3-287-STAG1 was not found to decrease in prostate tumor tissue. There was no significant correlation between the expression of PMEPA1-252 and PMEPA3-287-STAG1 in prostate tissue. The transcript of PMEPA1-252 was associated with PSA, but the expression of PMEPA3-287-STAG1 was found not to be associated with PSA or AR in CaP cells. Conclusions: The PMEPA isoforms appear to underscore distinct biologic functions in the context of androgen and TGF-β signaling. Intensively studied PMEPA1-252 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA1 isoforms need to be better defined in prostate cancer and other cancers. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Gyorgy Petrovics, Yongmei Chen, Alagarsamy Srinivasan, Inger Rosner, Albert Dobi, Shiv Srivastava. PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B008.

MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS

The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. METHODS: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center. ERG status was determined in whole mounted prostate specimens by immunohistochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program. RESULTS: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly (<10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G proteincoupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation. CONCLUSIONS: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options.

Abstract 5058: Silencing of NOTCH signaling enhances the sensitivity of ERG positive prostate cancer cells to AR inhibitors

Introduction: Androgen receptor (AR) signaling plays a critical role in all the stages of prostate cancer (CaP) ranging from organ confined to castration-resistant (CRPC) phases. Although androgen deprivation therapy (ADT) remains the mainstay treatment for advanced CaP, the inevitable transition from androgen- sensitive to CRCP presents the most significant challenge in CaP therapy. Androgen dependent expression of oncogenic ETS related gene (ERG) in half of all CaP in western countries plays critical role in the tumorigenesis of CaP through regulation of cancer specific signaling pathways. We found that NOTCH transcription factors are common targets of ERG in ERG positive cancer cells. NOTCH signaling pathway is an important signaling pathway in the development of drug-resistant tumor growth. In the current study we evaluated the combinatorial effects of NOTCH and AR inhibitors in the context of ERG positive prostate cancer cells. Methods: ERG, NOTCH1, NOTCH2 and downstream targets of NOTCH transcription factors were analyzed by Western blot assays. Dose and time kinetics of combining NOTCH inhibitor (γ-Secretase inhibitor 1, GSI-1) and AR inhibitors (Bicalutamide, Enzalutamide, and Abiraterone) were assessed in a panel of ERG positive or ERG negative CaP cells. Trypan blue exclusion, methylthiazole tetrazolium (MTT), or ApoTox-Glo™ Triplex assays were used to asses cell proliferation, apoptosis and drug cytotoxicity. Results: Prostate cancer cell lines with endogenous or ectopic expression of ERG showed upregulation of NOTCH1 and NOTCH2. The NOTCH inhibitor, GSI-1 conferred an increased sensitivity to all tested AR inhibitors (Bicalutamide, Enzalutamide, and Abiraterone) with bicalutamide showing the most robust inhibition of AR, ERG, NOTCH1, NOTCH2, PSA, decreased cell growth and enhanced apoptosis in ERG positive VCaP cells. This observation was not seen in ERG negative LNCaP cells or in ERG positive primary endothelial cells. Conclusions: NOTCH inhibitor enhanced sensitivity of AR inhibitors in ERG positive VCaP cells growth. The combination of the GSI-1 with AR inhibitors has shown synergistic effect when compared to single agent treatment. Taken together, our study suggests that NOTCH inhibitors may enhance the actions of AR inhibitors in the treatment of ERG positive prostate cancers. Inhibition of AR and NOTCH signaling may offer new opportunities in assessing ERG targeted therapy for prostate cancer. Citation Format: Ahmed A. Mohamed, Shyh-Han Tan, Shilpa Katta, Charles P. Xavier, Lakshmi Ravindranath, Wei Huang, Hua Li, Meera Srivastava, Shashwat Sharad, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, Shiv Srivastava. Silencing of NOTCH signaling enhances the sensitivity of ERG positive prostate cancer cells to AR inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2015-5058