Parthasarathy Rangarajan

Crocetinic acid inhibits hedgehog signaling to inhibit pancreatic cancer stem cells

Pancreatic cancer is the fourth leading cause of cancer deaths in the US and no significant treatment is currently available. Here, we describe the effect of crocetinic acid, which we purified from commercial saffron compound crocetin using high performance liquid chromatography. Crocetinic acid inhibits proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. In addition, it induced apoptosis. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, crocetinic acid decreased the number and size of the pancospheres in a dose-dependent manner, and suppressed the expression of the marker protein DCLK-1 (Doublecortin Calcium/Calmodulin-Dependent Kinase-1) suggesting that crocetinic acid targets cancer stem cells (CSC). To understand the mechanism of CSC inhibition, the signaling pathways affected by purified crocetinic acid were dissected. Sonic hedgehog (Shh) upon binding to its cognate receptor patched, allows smoothened to accumulate and activate Gli transcription factor. Crocetinic acid inhibited the expression of both Shh and smoothened. Finally, these data were confirmed in vivo where the compound at a dose of 0.5 mg/Kg bw suppressed growth of tumor xenografts. Collectively, these data suggest that purified crocetinic acid inhibits pancreatic CSC, thereby inhibiting pancreatic tumorigenesis.

Tandutinib Inhibits the Akt/mTOR Signaling Pathway to Inhibit Colon Cancer Growth

The c-Kit receptor can activate distinct signaling pathways including phosphoinositide 3-kinase (PI3K)/Akt and mTOR. Aberrant c-Kit activation protects cells from apoptosis and enhances invasion of colon carcinoma cells. Tandutinib is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases including c-Kit. We determined the effect of tandutinib on colon cancer growth and identified a mechanism of action. Tandutinib inhibited phosphorylation of c-Kit, Akt, mTOR, and p70S6 kinase. In addition, tandutinib significantly inhibited the proliferation and colony formation ability of colon cancer cell lines but did not affect normal colonic epithelial cells. There were increased levels of activated caspase-3 and Bax/Bcl2 ratio, coupled with a reduction in cyclin D1, suggesting apoptosis. There was also a downregulation of COX-2, VEGF, and interleukin-8 expression, suggesting effects on cancer-promoting genes. In addition, overexpressing constitutively active Akt partially suppressed tandutinib-mediated colon cancer cell growth. In vivo, intraperitoneal administration of tandutinib significantly suppressed growth of colon cancer tumor xenografts. There was a reduction in CD31-positive blood vessels, suggesting that there was an effect on angiogenesis. Tandutinib treatment also inhibited the expression of cancer-promoting genes COX-2 and VEGF and suppressed the activation of Akt/mTOR signaling proteins in the xenograft tissues. Together, these data suggest that tandutinib is a novel potent therapeutic agent that can target the Akt/mTOR/p70S6K signaling pathway to inhibit tumor growth and angiogenesis. Mol Cancer Ther; 12(5); 598–609. ©2013 AACR.

Abstract 3314: Targeting Hsp90 affects stem cell signaling in triple negative breast cancer

Background: Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. Many studies have also suggested that breast cancer9s ability to proliferate, progress, and spread is due to the presence of a rare subpopulation of cancer stem cells. The aim of this study is to investigate the efficacy of HSP90 inhibitors celastrol and triptolide (from the Chinese herb “Thunder God of Vine” (Tripterygium wilfordii) on stem cells as a novel therapy for TNBC. Methods: We performed in vitro studies using the TNBC cell lines BT20 and MDA-MB-231. We performed mammosphere assays to determine self-renewal capacity. For in vivo, we injected BT20 cells into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively. Results: Both celastrol and triptolide significantly suppressed mammosphere size and number. Furthermore, expression of breast cancer stem cell markers DCLK1, ALDH1 and CD133 were significantly reduced in the two cell lines following treatment. Flow cytometry also suggested a significant reduction in DCLK1+, ALDH+ and CD133+ cells. Recently, Notch signaling has been shown to be critical for self-renewal of cancer stem cells. In cells treated with either celastrol or triptolide, there was a significant reduction in activated Notch intracellullar domain (NICD), and its downstream target Hes-1. However, in cells where we ectopically overexpressed NICD, neither compound was as potent as control, vector transfected cells in reducing 2D cell proliferation or 3D mammosphere formation, suggesting the direct role for inhibiting Notch activation as a mechanism of action for the two compounds. Furthermore, inhibition of Notch signaling pathway by using Υ-secretase inhibitor DAPT shows further reduction in mammosphere formation and Notch and its downstream target gene. We confirmed these finding in vivo using BT20 tumor xenografts grown in athymic nude mice. There was a reduction in the size and weight of tumors in mice treated with celastrol or triptolide. Western blot data showed that there is a decrease in activated Notch-1 protein and stem cell marker, DCLK1+ and ALDH+ in celastrol and triptolide treated xenograft tissue. Conclusion: Taken together these data suggest that both celastrol and triptolide affect cancer stem cells in TNBC, in part through inhibition of Notch signaling. Citation Format: Prabhu Ramamoorthy, Parthasarathy Rangarajan, Roy Jensen, Shrikant Anant. Targeting Hsp90 affects stem cell signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3314.

Abstract 4632: Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC

Background: Breast cancer is the second leading cause of death for woman. Within breast cancer, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. The aim of this study was to elucidate whether the two natural inhibitors of Hsp90, celastrol and triptolide inhibit triple negative breast cancer growth. Both these compounds are terpenoids and were obtained from the Chinese herb “Thunder God of Vine” (Tripterygium wilfordii). Methods: BT20, BT549, MDA-MB-157 and MDA-MB-231 cells (all TNBC cells), and immortalized human mammary epithelial cells (HMLE) were grown in DMEM containing 10% FBS as per ATCC recommendations. Cell proliferation was assessed by hexoseaminidase activity, and IC50 values calculated using GraphPad Prism5. For clonogenicity, 500 cells were incubated with IC50 concentrations of each compound for 24 h, after which they were allowed to grow and form colonies. For in vivo, BT20 cells were injected into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively. Results: Celastrol and triptolide treatment suppressed proliferation and colony forming ability of all four TNBC cell lines, but not that of the immortalized HMLE cells. The compounds increased apoptotic cell death, based on increased Annexin V staining. Moreover, there was increased expression of the pro-apoptotic protein Bax but decreased expression of the anti-apoptotic protein, Bcl2 and BclXL. Immunoprecipitation-coupled western blots also showed that the compounds inhibit HSP90/CDC37 complex formation. Interestingly, the coupled immunoprecipitation-western blot analyses showed increased HSP90-BRCA1 interaction after treatment with the compounds. Coupled to this, western blot and immunostaining assays showed increased cytosolic levels and reduced nuclear levels of BRCA1 protein. Similar results were obtained in vivo with BT20 xenografts. In addition to decreased tumor size in response to treatment with celastrol or triptolide, the xenograft tissues showed an increase in cytoplasmic BRCA1 levels following treatment. In addition, there was increased in HSP90-BRCA1 complex formation in the treated xenograft tissues. Finally, knockdown of BRCA1 using specific silencer RNA resulted in partial inhibition in cell growth reduction after celastrol and triptolide treatment in both BT20 and MDA-MB-231 cells. Conclusion: Taken together, these data suggest that both celastrol and triptolide suppress TNBC cell growth, in part through increasing cytosolic HSP90/BRCA1 complex formation. Citation Format: Prabhu Ramamoorthy, Parthasarathy Rangarajan, Ossama Tawfik, Shrikant Anant, Roy A. Jensen. Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4632.

Abstract 1895: Ciclopirox prodrug for the prevention and therapy of non-muscle invasive bladder cancer

Objective: Ciclopirox prodrug for the prevention of Non-Muscle Invasive Bladder Cancer (NMIBC) represents a potential breakthrough in the management of high-grade, NMIBC. If successful, this represents the first systemic approach to manage NMIBC. Ciclopirox olamine, the active pharmaceutical ingredient in several topical antifungal drug products, has demonstrated anticancer activity in blood and solid tumors. We developed a phosphoryloxymethyl prodrug of ciclopirox (CPX) for efficient intravenous delivery, thereby avoiding dose-limiting gastrointestinal toxicities and first-pass effect associated with oral administration. Methods: In vitro anticancer activity of CPX was performed in both NMIBC (T24) and MIBC (253-JBV) cell lines. Cell proliferation was determined by hexoseaminidase assay. Stemness was determined by the Spheroid assay. Flow cytometric analyses was performed for cell cycle analysis. RT-PCR array analysis was performed to identify signaling pathways. Real Time PCR, western blot and immunofluorescent studies were performed for determining gene expression. Results: Pharmacokinetic studies conducted in mice, rats and dogs demonstrated that the drug is rapidly and completely metabolized and eliminated via the urine. In vitro, CPX inhibited growth of T24 and 253-JBV cells at 4 and 2 μM, respectively coupled with S-phase cell cycle arrest. FITC conjugated Annexin V-coupled flow cytometry studies showed that CPX induces apoptotic cell death. CPX also suppressed bladdosphere formation, suggesting that stem cells are affected. Notch signaling pathway plays a significant role in stem cell behavior, and previous studies have suggested that the pathway may be a therapeutic target for bladder cancer. Notch receptor activation, which occurs following ligand binding, involves specific intracellular cleavage by the γ-secretase complex. First, RT-PCR array analyses suggested that CPX inhibits the Notch signaling pathway. Furthermore, CPX significantly inhibited the expression of the γ-secretase complex proteins Presenilin1, Nicastrin, APH-1 and PEN-2. In addition, there was a reduction in the expression of downstream targets of the pathway including Hes1 and Cyclin-D1. These data suggest that CPX significantly inhibits Notch intracellular signaling pathway proteins. Ectopic expression of the activated/cleaved Notch protein reversed CPX mediated inhibition of cell proliferation. Conclusion: CPX is a novel chemotherapeutic and preventive agent for bladder cancers, and this occurs by suppressing the stem cells. We are confident that there will be no failure of the compound as a result of unacceptable toxicity because drug safety profile of CPX has been previously characterized in animals and humans. These studies provide insight into understanding the role of CPX as a potent chemotherapeutic and preventive agent. Citation Format: Parthasarathy Rangarajan, Satish Ramalingam, Dharmalingam Subramaniam, Michael J. Baltezor, Robyn Wood, Shrikant Anant, Scott Weir. Ciclopirox prodrug for the prevention and therapy of non-muscle invasive bladder cancer. [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 1895. doi:10.1158/1538-7445.AM2015-1895

Abstract 5163: JMJD1a: A potential target for prevention and therapy in pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Pancreatic cancer is the fourth leading cause of cancer related death in the United States. Therapeutic options for patients with advanced disease are time limited and non-curative. A growing body of evidence suggests that cancer stem cells (CSCs) within a solid tumors including in pancreatic cancer initiate and sustain tumor growth. Our preliminary data implicates Jumanji demethylases1a (JMJD1a) as a prime regulator of stem cell renewability for cancer progression. Hypoxia most prominently controls malignant properties of cancer cells by stimulating hypoxia inducible factor 1 (HIF1). HIF1 binds to its response elements (HREs) in the JMJD1a promoter and subsequently up-regulates JMJD1a. This could explain why hypoxia exacerbates malignancy. Therefore, we hypothesize that hypoxia induces JMJD1a that regulate self-renewability of CSCs, thereby promoting tumor initiation. Recently, two small molecules analogues of crocetinic acid, a carotenoid molecule isolated from saffron, have revealed to have potent antimitotic effects in pancreatic cancer models. Experimental Procedure: We have studied pancreatic CSCs during hypoxic and normoxic conditions allow to form multicellular spheroids called pancospheres and monitored expression of JMJD1a using western blots and immunohistochemistry on those CSCs. We also used shRNA to silence JMJD1a using lentiviral vectors in different pancreatic cancer cell lines. Results: JMJD1a was significantly overexpressed in human pancreatic cancer patient samples compared to their adjacent normal tissues. JMJD1a is overexpressed in pancreatic cancer cells and it is significantly reduced when it is knocked down through shRNA in those cell lines. The colony formation and migration assays were significantly reduced after knock-down of JMJD1a. Hypoxia stimulated pancospheres formation associated with concomitant overexpression of JMJD1a. Knock-down of JMJD1a using shRNA demonstrated significant reduction of pancospheres forming ability in both normoxic and hypoxic conditions. The demethylation of specific substrate of JMJD1a, H3-K9me2, was significantly increased by knock-down of JMJD1a in pancreatic cancer cells. Hypoxia significantly increases overexpression of cancer stem-cell and angiogenic markers in pancreatic cancer cells in vitro. Knock-down of JMJD1a significantly affected stem-cell specific markers in both hypoxia and normoxia. Treatment with gemcitabine did not affect in JMJD1a expression in those cell lines particularly during hypoxic conditions. Crocetinic acid and its analogues inhibited JMJD1a expression in both in vivo and in vitro pancreatic cancer models. Conclusions: JMJD1a is overexpressed in pancreatic cancer and stimulates the self-renewability of pancospheres. In conclusion, JMJD1a is one of important factor for pancreatic cancer progression and could be a novel preventive and therapeutic target for pancreatic cancer. Citation Format: Kanagaraj Palaniyandi, Santanu Paul, Parthasarathy Rangarajan, Deep Kwatra, Satish Ramalingam, Dharmalingam Subramaniam, Tomoo Iwakuma, Subhash Padhye, Shrikant Anant, Animesh Dhar. JMJD1a: A potential target for prevention and therapy in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5163. doi:10.1158/1538-7445.AM2014-5163

Abstract 3734: Cancer stem cells: novel chemopreventive targets for pancreatic cancer.

Pancreatic cancer is the fourth leading cause of mortality in the United States and no significant treatment is currently available. Although an increasing number of therapeutic options exist for patients with advanced disease, their efficacy is time limited and non-curative. Presently approximately close to 60% of cancer patients in the United States are believed to utilize therapies derived from plants, herbs, flowers, or nutrients either exclusively or concurrently with traditional chemotherapy or radiation therapy. Recent evidence suggests the existence of a small population of tumorigenic stem cells responsible for tumor initiation, metastasis and resistance to chemotherapy and radiation. Identification of the regulatory mechanisms and signaling pathways involved in cancer stem cells (CSCs) will help in designing novel agents to target this refractory cell population in pancreatic cancers. We are currently investigating the mechanisms by which purified crocetinic acid, a carotenoid molecule isolated from saffron, inhibits growth of pancreatic cancer in vitro and in vivo models. Our studies demonstrate that the compound had significant cytotoxicity against pancreatic cancer cells in both a dose- and time-dependent manner. Pancreatic CSCs can be allowed to divide and grow in ultra-low binding tissue culture dishes to form multicellular spheroids called pancospheres. Treatment with purified crocetinic acid decreased the number and size of the primary and secondary pancospheres in a dose dependent manner, suggesting that crocetinic acid targets CSCs. To understand the mechanism of inhibition of pancospheres, the signaling pathways affected by crocetinic acid were dissected. Aberrant activation of Sonic Hedgehog signaling pathway has been associated with renewal of cancer stem cells, and in the development of several solid cancers. Sonic hedgehog (Shh) upon binding to its cognate receptor patched, allows smoothened to accumulate and activate Gli transcription factor. Treatment with crocetinic acid inhibited the expression of both Shh and smoothened in the pancreatic CSCs with concomitant reduction of the expression of a novel pancreatic CSC marker, DCLK-1 (Doublecortin Calcium/Calmodulin-Dependent Kinase-1). Furthermore, it inhibited the expression of patched-1 and Gli-1, downstream targets of the hedgehog signaling pathway. Crocetinic acid also inhibited tumor formation in pancreatic cancer in vivo xenograft models. Taken together, these data suggest that crocetinic acid effectively inhibits pancreatic CSCs by down regulating the sonic hedgehog pathway, thereby inhibiting tumorigenesis. Citation Format: Parthasarathy Rangarajan, Dharmalingam Subramaniam, Deep Kwatra, Shamima Islam, Kanagaraj Palaniyandi, Shrikant Anant, Animesh Dhar. Cancer stem cells: novel chemopreventive targets for pancreatic cancer. [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 3734. doi:10.1158/1538-7445.AM2013-3734

Abstract 2584: Tandutinib inhibits the PI3 Kinase/Akt/mTOR signaling pathway to inhibit colon cancer growth.

Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. The c-Kit receptor can activate distinct signaling pathways including phosphatidylinositol-3- kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Aberrant c-Kit activation protects cells from apoptosis and enhances invasion of colon carcinoma cells. Tandutinib is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases including c-Kit and PDGFR. The current study is designed to determine the effect of tandutinib on colon cancer growth and identified a mechanism of action. Method: Colon cancer cell lines HCT116, HT-29 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Cell cycle analysis was measured by Flow cytometry. Signaling proteins were quantified by western blot analysis. For in vivo effects, HCT116 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for CD31 and Akt/mTOR signaling proteins. Results: Tandutinib treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in all three cell lines but did not affect normal colonic epithelial cells. Treatment also induced colon cancer cells to undergo apoptosis and G0/G1 arrest. Apoptosis was further confirmed by increased levels of activated caspase 3 and Bax/Bcl2 ratio, coupled with a reduction in cyclin D1. There was also a downregulation of cyclooxygenase 2 (COX-2), vascular endothelial growth factor (VEGF) and interleukin-8 expression suggesting effects on cancer promoting genes. Moreover, tandutinib inhibited phosphorylation of c-Kit, Akt, mTOR, p70S6 Kinase and 4EBP1. Overexpression of constitutively active Akt1 overcame this inhibitory effect suggesting a critical role for this pathway. To determine the effect of tandutinib on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Tandutinib treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following tandutinib treatment when compared to controls suggesting inhibition of angiogenesis. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression cancer promoting genes COX-2 and VEGF, and suppressed the activation of Akt/mTOR signaling proteins in the tandutinib-treated xenograft tissues. Conclusion: Together, these data suggest that tandutinib is a novel potent therapeutic agent that can target the Akt/mTOR/p70S6K signaling pathway to inhibit tumor growth and angiogenesis. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, David Standing, Parthasarathy Rangarajan. Tandutinib inhibits the PI3 Kinase/Akt/mTOR signaling pathway to inhibit colon cancer growth. [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 2584. doi:10.1158/1538-7445.AM2013-2584

Abstract C40: Histone demethylase Jmjd1a: A novel preventive target in pancreatic cancer

Purpose: Pancreatic cancer is the fourth leading cause of mortality in the United States with no significant treatment currently available. Therapeutic options for patients with advanced disease are time limited and non-curative. A growing body of evidence suggests that cancer stem cells/tumor initiating cells (CSC/TIC) within a solid tumor including in pancreatic cancer initiate and sustain tumor growth. Our preliminary data implicates Jumanji demethylases1a (Jmjd1a) as a prime regulator of stem cell renewability for cancer progression. Hypoxia most prominently controls malignant properties of cancer cells by stimulating hypoxia inducible factor 1 (HIF1). The fact that HIF1 binds to its responsible elements (HREs) in the Jmjd1 promoters and subsequently up-regulates Jmjd1 could explain why hypoxia exacerbates malignancy. In this context, the availability of small molecules will allow us to determine mechanistic aspects of the interconnection among hypoxia, Jmjd1a and CSC/TIC self-renewal. Indeed, the inhibition of the members of the Jmjd family, 2-oxoglutarate (2OG)-dependent histone lysine demethylases (KDMs), suppresses tumor growth in many types of cancer. Therefore, we hypothesize that hypoxia induces Jmjd1a that regulate self-renewability of CSC/TIC, thereby promoting tumor initiation. Recently, two small molecules analogues of crocetinic acid, a carotenoid molecule isolated from saffron, have revealed to have potent antimitotic effects in pancreatic cancer models. Experimental Procedure: In this study, we have developed pancreatic CSC/TIC during hypoxic and normoxic conditions using ultra-low binding tissue culture dishes to form multicellular spheroids called pancospheres and monitored expression of Jmjda1a using western blots and immunohistochemistry on those CSC/TIC. We also used RNA technology to silenced Jmjd1a using lenntiviral vectors different pancreatic cancer cell lines. We have also determined Jmjd1a expression in pancreatic cancer patients9 samples. Results: Jmjd1a is highly expressed in pancreatic cancer cells and it is significatly reduced when it is knocked down through shRNA in those cell lines. The proliferation of those knockdown cell lines was significantly reduced using colony formation and wound healing assays. Hypoxia stimulated pancospheres formation associated with concomitant overexpression of Jmjd1a. Knockdown of Jmjd1a using shRNA demonstrated significant reduction of pancreatic pancospheres formation ability in both normoxic and hypoxic conditions. The demethylation of specific substrate of Jmjd1a, H3-K9me2, was significantly affected by knockdown of Jmjd1a in pancreatic cancer cells. Treatment with gemcitabine, drug of choice for pancreatic cancer, did not affect in Jmjd1a expression in those cell lines particularly during hypoxic conditions. Crocetinic acid and its analogues inhibited Jmjd1a expression in both in vivo and in vitro pancreatic cancer models. We also observed that Jmjd1a was significantly overexpressed in human pancreatic cancer patient samples than the adjacent normal tissues. Conclusions: Jmjd1a is overexpressed in pancreatic cancer and stimulates the self-renewability of pancospheres. Taken together, Jmjd1a is one of important factors for pancreatic cancer progression and could be a novel preventive target for pancreatic cancer. Citation Format: Kanagaraj Palaniyandi, Shamima Islam, Parthasarathy Rangarajan, Deep Kwatra, Satish Ramalingam, Dharmalingam Subramaniam, Subhash Padhye, Shrikant Anant, Animesh Dhar. Histone demethylase Jmjd1a: A novel preventive target in pancreatic cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C40.

Abstract PR05: Effects of Hsp90 inhibitors on triple-negative breast cancer: Notch as a therapeutic target for stem cells

Background: Breast cancer is the second leading cause of death for woman. Within breast cancer subtypes, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Tumors contain heterogeneous cell populations and it has numerically rare cancer stem cells with indefinite proliferative potential that is responsible for tumor invasiveness, heterogeneity, and therapy resistance. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. The aim of this study was to elucidate whether the two natural inhibitors of Hsp90, celastrol and triptolide inhibit triple negative breast cancer growth. Both these compounds are terpenoids and were obtained from the Chinese herb “Thunder God of Vine” ( Tripterygium wilfordii ). Methods: BT20, BT549, MDA-MB-231 and MDA-MB-157 cells (all TNBC cells) were obtained and grown in DMEM containing 10% FBS as per ATCC recommendations. Cell proliferation was assessed by hexoseaminidase activity, and IC50 values calculated using GraphPad Prism5. For clonogenicity, 500 cells were treated with IC50 concentration of each compound for 24h, and then allowed to grow and form colonies. Mammosphere assay was performed using 5000 cells/ml in ultra low attachment plates. Images were captured after 5 days. For in vivo, BT20 cells were injected into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively. Results: Celastrol and triptolide treatment suppressed the proliferation and colony formation ability of all four TNBC cell lines BT20, BT549, MDA-MB-231 and MDA-MB-157. Interestingly, the mammosphere assay (an assay used to evaluate the self-renewal capacity of the cancer stem cells) revealed that celastrol or triptolide significantly reduces the size and number of spheroids. Furthermore, expression of breast cancer stem cell markers ALDH1 and CD133 were significantly reduced in BT20 cells upon the treatment. Recently, Notch signaling has been shown to be critical for self-renewal of cancer stem cells. Activation of the Notch receptor, a membrane spanning receptor involves the interaction with a ligand resulting in a series of proteolytic cleavage events culminating in the release of the Notch intracellular domain (NICD). This NICD translocates to the nucleus, and together with its interacting partner CSL/RBPJ binds to cognate element and activates the expression of downstream target genes such as Hes-1. In cells treated with either celastrol or triptolide, there was a significant reduction in NICD, and its downstream target Hes-1. Furthermore, there was a reduction in ALDH+ cells. However, in cells where we ectopically overexpressed NICD, neither compound was as potent as control vector transfected cells in reducing proliferation, colony formation or mammosphere formation, suggesting the direct role for inhibiting Notch activation as a mechanism of action for the two compounds. We confirmed these finding in vivo using BT20 tumor xenografts grown in athymic nude mice. There was a reduction in the size of tumors in mice treated with celastrol or triptolide. In addition, western blot and immunohistochemistry analyses demonstrated a reduction in the number of ALDH+ and CD133+ cells. Conclusion: Taken together these data suggest that both celastrol and triptolide affect cancer stem cells in TNBC, in part through inhibition of Notch signaling. This abstract is also presented as Poster B31. Citation Format: Prabhu Ramamoorthy, Sydney Byrne, Satish Ramalingam, Parthasarathy Rangarajan, Dharmalingam Subramaniam, Scott Weir, Shrikant Anant, Roy Jensen. Effects of Hsp90 inhibitors on triple-negative breast cancer: Notch as a therapeutic target for stem cells. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR05.