Jay Sharma

Resveratrol Inhibits Pancreatic Cancer Stem Cell Characteristics in Human and KrasG12D Transgenic Mice by Inhibiting Pluripotency Maintaining Factors and Epithelial-Mesenchymal Transition

Background Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and KrasG12D transgenic mice. Methodology/Principal Findings Human pancreatic CSCs (CD133+CD44+CD24+ESA+) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from KrasG12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in KrasG12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and KrasG12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSCs migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail). Conclusions/Significance These data suggest that resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. In conclusion, resveratrol can be used for the management of pancreatic cancer.

Functional analysis of microRNAs in human hepatocellular cancer stem cells

MicroRNAs are endogenous small non‐coding RNAs that regulate gene expression and cancer development. A rare population of hepatocellular cancer stem cells (HSCs) holds the extensive proliferative and self‐renewal potential necessary to form a liver tumour. We postulated that specific transcriptional factors might regulate the expression of microRNAs and subsequently modulate the expression of gene products involved in phenotypic characteristics of HSCs. We evaluated the expression of microRNA in human HSCs by microarray profiling, and defined the target genes and functional effects of two groups of microRNA regulated by IL‐6 and transcriptional factor Twist. A subset of highly chemoresistant and invasive HSCs was screened with aberrant expressions of cytokine IL‐6 and Twist. We demonstrated that conserved let‐7 and miR‐181 family members were up‐regulated in HSCs by global microarray‐based microRNA profiling followed by validation with real‐time polymerase chain reaction. Importantly, inhibition of let‐7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR‐181 led to a reduction in HSCs motility and invasion. Knocking down IL‐6 and Twist in HSCs significantly reduced let‐7 and miR‐181 expression and subsequently inhibited chemoresistance and cell invasion. We showed that let‐7 directly targets SOCS‐1 and caspase‐3, whereas miR‐181 directly targets RASSF1A, TIMP3 as well as nemo‐like kinase (NLK). In conclusion, alterations of IL‐6‐ and Twist‐regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy. Our results define a novel regulatory mechanism of let‐7/miR‐181s suggesting that let‐7 and miR‐181 may be molecular targets for eradication of hepatocellular malignancies.

GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSCs viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma(null) mice were used to examine the effects of GANT-61 on CSCs tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and metastasis. It is evident from growing reports that PI3K/Akt/mTOR and Sonic Hedgehog (Shh) signaling pathways are aberrantly reactivated in pancreatic CSCs. Here, we examined the efficacy of combining NVP-LDE-225 (PI3K/mTOR inhibitor) and NVP-BEZ-235 (Smoothened inhibitor) on pancreatic CSCs characteristics, microRNA regulatory network, and tumor growth. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting pancreatic CSCs characteristics and tumor growth in mice by acting at the level of Gli. Combination of NVP-LDE-225 and NVP-BEZ-235 inhibited self-renewal capacity of CSCs by suppressing the expression of pluripotency maintaining factors Nanog, Oct-4, Sox-2 and c-Myc, and transcription of Gli. NVP-LDE-225 co-operated with NVP-BEZ-235 to inhibit Lin28/Let7a/Kras axis in pancreatic CSCs. Furthermore, a superior interaction of these drugs was observed on spheroid formation by pancreatic CSCs isolated from Pankras/p53 mice. The combination of these drugs also showed superior effects on the expression of proteins involved in cell proliferation, survival and apoptosis. In addition, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting EMT through modulation of cadherin, vimentin and transcription factors Snail, Slug and Zeb1. In conclusion, these data suggest that the combined inhibition of PI3K/Akt/mTOR and Shh pathways may be beneficial for the treatment of pancreatic cancer.

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Abstract 4408: Targeting cancer stem cells as potential new therapy for pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer mortality in the US, despite significant improvements in diagnostic imaging and operative modalities. The 5-year survival rate remains less than 6% because of microscopic or gross metastatic disease at time of diagnosis. Although the treatment of pancreatic cancer remains a huge challenge, it is entering a new era with the development of new strategies and trial designs. Because there is an increasing number of novel therapeutic agents and potential combinations available to test in patients with pancreatic cancer, the identification of robust prognostic and predictive markers and of new targets and relevant pathways is a top priority as well as the design of adequate trials incorporating molecular-driven hypothesis. Over the past decade, increasing evidence suggested that stem cells play a crucial role not only in the generation of complex multicellular organisms, but also in the development and progression of malignant diseases. Most tumors have been shown to contain a subset of distinct cancer cells that is responsible for tumor initiation and propagation. These cells are termed cancer stem cells or tumor-initiating cells and they are highly resistant to chemotherapeutic agents. Here, we examined the efficacy of combined treatments of CEP1101 and gemcitabine in human pancreatic cancer cells, and pancreatic CSCs (CD133, CD44, CXCR4, SSEA3/4, Oct4, ALDH, Telomerase & Nestin) from the same donors. CEP1101 inhibited the growth of CSCs, while gemcitabine suppressed the viability of non-CSCs. In vivo studies showed that CEP1101 combined with gemcitabine eliminate the engraftment of human pancreatic cancer and CSCs, more effectively than the individual agents. These data demonstrate that administration of CEP1101, which targets CSCs, may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate pancreatic cancer. This study shows potential molecular therapeutic targets to eradicate the tumor - and metastasis-initiating cells and their progenies for the evaluation of new effective combination therapies against locally advanced and metastatic pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4408. doi:1538-7445.AM2012-4408

Abstract 5545: Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for pancreatic cancer stem cell.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumors and to decrease the use of experimental animals. The induction of chemotherapy or concomitant chemoradiotherapy has been used to increasingly to improve survival, and organ preservation. This approach encounters significant morbidity and mortality. Therefore reliable chemosensitivity assays are needed to accurately predict the response to chemotherapy and guide the selection and treatment of cancer patients. The purpose of this study is to examine and evaluate optimum drug candidates in vitro chemosensitivity on patient tumor tissues directly in culture and on their Cancer stem cell cultures. The tumor samples obtained after surgery or biopsy, were placed immediately in Celprogen Tumor Transportation Media and shipped at 4-8 OC for processing. Tissues were washed with 1X PBS solution and aseptically cut into 0.5mm sections and cultured in 6 well tissue culture plates with an insert pre-coated with ECM. All cancer cell types remain viable and maintain their native architecture for at least 14 days and incorporated DNA measured by adding EdU(5-ethynyl-2’-deoxyuridine) to the culture. The efficacy of various therapeutic agents targeting major pathways (wnt,Notch,PI3K,MAPK,STAT) and chemotherapy agents were tested using DNA uptake and TUNNEL assay anti-cancer agents was calculated according to the inhibition index. The same compounds were tested for utilizing the patients Pancreatic Cancer Stem Cell Cultures established with Celprogens Media and ECM. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, β-catenin and vimentin were quantified by qPCR or immunocytochemistry per cell culture. The epithelial-mesenchymal transition (EMT) is linked to induction of a stem-cell like phenotype. We cultured the cells in low oxygen since Tumor hypoxia induces EMT, which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Among the 600 compounds tested Gemcitabine, Taxol, Fluorouracil, Leucovorin, Irinotecan, and Oxaliptin were not effective against Pancreatic Cancer Stem cell (CSC) but were effective on tumor cells (differentiated CSCs). We were able to show 6 compounds that were effective against Pancreatic CSC targeting selected pathways. Citation Format: John P. Clery, Esteban Gomez, Michael Sharma, Aabha khemani, Cristian Sharma, Rubio Punzalan, miriam navel, Natalee Amezcua, Jitesh Jani, Jay Sharma. Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for pancreatic cancer stem cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5545. doi:10.1158/1538-7445.AM2013-5545

Abstract 3306: Screening potential drug candidates for treatment of glioblastoma patients utilizing an in-vivo mouse/ rat model system

Cancer stem cells (CSCs) are thought to be critical for engraftment and long-term growth of many tumors, including glioblastoma multiforme (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. Human glioblastoma multiforme is a heterogenous tumor composed from tumor cells and small portion of cancer stem cells, which have a high tumorigenic potential and a low proliferation rate. Glioblastoma cancer stem cells are phenotypically similar to the normal stem cells, they express CD133 gene and other genes characteristic of neural stem cells and posses self-renewal potential. The CD133+ GBM CSCs have been isolated with Celprogen Media and ECM and characterized as chemo-/radio-resistant tumor-initiating cells which are responsible as one of the many factors involved in post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM CSCs that resist treatment, we isolated CD133+ GBM CSCs from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM CSCs sorted from the CD133+ GBM CSC spheres express SOX2,CD44 and are capable of clonal self-renewal. We generated in-vivo model systems in mice and rats to screen potential drug agents for the treatment of GBM patients. We were capable of generating brain tumors in mice and rats within 20 days by subcutaneously injecting 1000 GBM CSCs at the hind limbs of the rodents. Within day 5 the GBM CSCs were already present in the brain9s of the rodents. At days 10, 15 & 20 the GBM CSCs labeled with GFP were located in the brain. We tested various compound candidates in this model system and we were able to show decrease in the migration of the GBM CSCs in the brain when we started the treatment at day 10 compared to non-treatment groups. We were able to demonstrate all the gene signatures associated with GBM CSCs in this model system. We do see a down regulation of certain cancer promotes genes in the treatment group when compared to non-treatment groups. The drug candidates were tested for TGFβ and STAT pathways. The chemotherapeutic agents tested were Among the 300 compounds tested Adria, Taxol, DTIC, Thalidomide, Paclitaxel, Abraxane, Ertibux and Velcade. We were able to show 4 compounds that were effective against GBM CSC targeting selected pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2011-3306

Abstract 3319: Human triple-negative breast cancer stem cells utilized for drug discovery therapeutics for triple-negative breast cancer patients

Breast cancer is the most common malignancy among women in developed countries, affecting more than a million women per year worldwide. Of these, triple negative breast carcinoma represents 10-17 %. Triple negative breast carcinomas, characterized by estrogen, progesterone and HER2 receptor negativity are very aggressive tumors with poor prognosis. Breast Cancer Stem derived from triple negative parental Breast Cancer tumors, are a subpopulation of cells within the parental breast cancer population within the individual which are positive for the following markers: CD133+CD44+CD24+ESA+SSEA-1+TRA-1-61+& TRA1-81+ and Oct ¾ these Breast Cancer Stem cells are highly tumorigenic and possess the stem cell-like properties of self-renewal and the ability to produce differentiated progeny. Breast Cancer Stem cells also demonstrate up regulation of SSEA3+, SSEA4+ upon differentiation into parental cancer phenotype. Individualized treatment (tailored therapy) based on molecular biology markers of tumor and patient is the trend in clinical practice these days. However, molecular targets and predictors for the treatment of triple negative breast carcinoma do not currently exist. With the identification and characterization of Breast Cancer Stem Cells from parental triple negative tumors, enables one to screen novel drug candidates for potential development of therapeutics for triple negative Breast Cancer Patients. In this study we have utilized Breast Cancer Stem Cells from triple negative Tumors to screen potential drug candidates. The Breast Cancer Stem Cell based assay system may provide novel therapeutic approaches into treatment of triple negative breast cancer patients, which are resistant to standard chemotherapy and radiation. 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 3319.

Inhibition of sonic hedgehog and PI3K/Akt/mTOR pathways cooperate in suppressing survival, self-renewal and tumorigenic potential of glioblastoma-initiating cells

Since PI3K/Akt/mTOR and sonic hedgehog (SHH) signaling pathways are highly activated in glioblastoma-initiating cells (GICs), we examined the effects of inhibiting these pathways on GIC characteristics and tumor growth in mice. NVP-LDE-225 (inhibitor of Smoothened) inhibited the expression of Gli1, Gli2, Smoothened, Patched1, and Patched2, and induced the expression of SuFu, whereas NVP-BEZ-235 (dual inhibitor of PI3K and mTOR) inhibited the expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting the self-renewal capacity of GICs, expression of pluripotency maintaining factors (Nanog, c-Myc, Oct4, and Sox2), Musashi1, cyclin D1, and Bcl-2, and transcription and expression of Gli, and in inducing the expression of cleaved caspase-3, cleaved PARP and Bim. Additionally, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting epithelial-mesenchymal transition. Finally, the combination of NVP-LDE-225 and NVP-BEZ-235 was superior in inhibiting tumor growth, regulating the expression of pluripotency promoting factors, stem cell markers, cell cycle, and cell proliferation, and modulating EMT compared to single agent alone. In conclusion, the combined inhibition of PI3K/Akt/mTOR and SHH pathways was superior to single pathway inhibition in suppressing glioblastoma growth by targeting GICs.