Cancer Stem Cell Renewal Research Articles

Abstract 5067: Preadipocyte exosomes promote early stage breast cancer formation by enhancing cancer stem cell renewal signaling

Abstract The tumor microenvironment plays a critical role in regulating breast cancer progression. Adipocytes differentiate from preadipocytes, which possess mesenchymal stem cell properties. Preadipocytes and adipocytes are essential components of the tumor microenvironment, and signaling between preadipocytes and breast cancer cells has been found to promote breast tumor formation and metastasis. Exosomes secreted from preadipocytes are also important components of the cancer stem cell niche. The primary goal of our studies is to examine how preadipocyte-derived exosomes can regulate early-stage breast cancer formation via regulating stem cell renewal. We use human and mouse preadipocytes and early-stage breast cancer cells (ductal carcinoma in situ, DCIS), as well as xenografts in immunodeficient mice to determine the role of preadipocyte secreted exosomes in DCIS stem cell behavior and tumor formation. By using protein arrays, we reveal CD81 and FLOT-1 as key exosomal protein markers and SOX2 and SOX9 as exosomal secretion of stem cell renewal regulators. We identify a key miR-140/SOX2/SOX9 axis that regulates differentiation, stemness, and migration in the tumor microenvironment. Next, we find that the natural antitumor compound shikonin can suppress preadipocyte signaling, inhibiting nearby DCIS cells. Through co-culture experiments we show that shikonin treated preadipocytes secrete exosomes containing high levels of miR-140 that can impact nearby DCIS cells through targeting SOX9 signaling. We demonstrate that preadipocyte-derived exosomes promote tumorigenesis in vivo, providing strong support for the importance of exosomal signaling in the tumor microenvironment. Finally, our data also show that targeting the tumor microenvironment may assist in blocking tumor formation. Citation Format: Benjamin Wolfson, Ramkishore Gernapudi, Nadire Duru, Qun Zhou. Preadipocyte exosomes promote early stage breast cancer formation by enhancing cancer stem cell renewal signaling. [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 5067. doi:10.1158/1538-7445.AM2015-5067

Combination therapy with epigenetic-targeted and chemotherapeutic drugs delivered by nanoparticles to enhance the chemotherapy response and overcome resistance by breast cancer stem cells

Aberrant DNA hypermethylation is critical in the regulation of renewal and maintenance of cancer stem cells (CSCs), which represent targets for carcinogenic initiation by chemical and environmental agents. The administration of decitabine (DAC), which is a DNA hypermethylation inhibitor, is an attractive approach to enhancing the chemotherapeutic response and overcoming drug resistance by CSCs. In this study, we investigated whether low-dose DAC encapsulated in nanoparticles could be used to sensitize bulk breast cancer cells and CSCs to chemotherapy. In vitro studies revealed that treatment with nanoparticles loaded with low-dose DAC (NPDAC) combined with nanoparticles loaded with doxorubicin (NPDOX) better reduced the proportion of CSCs with high aldehyde dehydrogenase activity (ALDHhi) in the mammospheres of MDA-MB-231 cells, and better overcame the drug resistance by ALDHhi cells. Subsequently, systemic delivery of NPDAC significantly down-regulated the expression of DNMT1 and DNMT3b in a MB-MDA-231 xenograft murine model and induced increased caspase-9 expression, which contributed to the increased sensitivity of the bulk cancer cells and CSCs to NPDOX treatment. Importantly, the combined treatment of NPDAC and NPDOX resulted in the lowest proportion of ALDHhi CSCs and the highest proportion of apoptotic tumor cells, and the best tumor suppressive effects in inhibiting breast cancer growth.

Abstract 212: Withaferin A in combination with cisplatin targets CD44 and Oct4 positive cancer stem cells in ovarian cancer

Abstract Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly, carboplatin coupled with paclitaxel. Although this treatment regimen is initially effective in a high percentage of cases, it is associated with severe side effects and within 6 to 24 months of initial treatment, tumor relapse occurs, which is attributed to the carcinomas having become platinum-resistant. Due to the poor survival of women with platinum-resistant carcinomas, and the severe side effects an alternative treatment strategy is warranted. We developed a novel strategy that combines cisplatin with withaferin A (WFA). WFA, a bioactive compound isolated from the plant Withania somnifera, is used in the US as an over-the-counter dietary supplement. More recently, it has been shown to prevent tumor growth, angiogenesis, and metastasis. In addition, WFA has been reported to inhibit Notch 1 signaling an important signaling pathway for the self-renewal and maintenance of cancer stem cell population. Based on this information, we hypothesize that a novel combination of withaferin A and cisplatin can target ovarian cancer cells and also ovarian cancer stem cells, which can reduce the cisplatin side effects and recurrence of ovarian cancer. Our hypothesis is supported by our preliminary studies in which epithelial ovarian cancer cell lines (A2780 and CaOV3 and A2780/CP70) when co-treated with low doses of the WFA (1.5 µM) and cisplatin (20 µM), resulted in inhibiting 80 to 90% cell proliferation within 48 h of treatment. In contrast, same concentration of each drug when used alone exhibited only 15% to 20% inhibition, respectively. In addition, treatment of nude mice bearing orthotopic ovarian tumors with WFA/cisplatin combination resulted in 80 to 90% reduction in tumor growth and complete inhibition of metastasis to other organs. In addition, treatment of mice bearing ovarian tumors with WFA/cisplatin combination showed a significant reduction in CD44 positive cancer stem cells analyzed by immuno-staining and western blot analysis of the CD44 protein. Tumors collected from mice treated with cisplatin alone revealed enrichment of CD44 positive cancer stem cells and expression of CD44 protein. Analysis of Notch1 signaling gene and its downstream signaling gene Hes1 showed a significant down regulation of expression. Notch1 plays an important role in renewal and maintenance of cancer stem cells which are reported to be chemo-resistance and play critical role in recurrence of cancer. Based on our results, we conclude that WFA/cisplatin combination therapy not only suppresses ovarian tumor growth but minimizes/eliminates cancer stem cell population, hence reduces the drug resistance and recurrence of cancer. This work was support by funds from James Graham Brown Cancer Center. Presenting author is a post-doctoral fellow. Citation Format: Sanjay K. Singh, J C. States, Sham S. Kakar. Withaferin A in combination with cisplatin targets CD44 and Oct4 positive cancer stem cells in ovarian 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 212. doi:10.1158/1538-7445.AM2014-212

Priming cancer cells for drug resistance: role of the fibroblast niche.

Conventional and targeted chemotherapies remain integral strategies to treat solid tumors. Despite the large number of anti-cancer drugs available, chemotherapy does not completely eradicate disease. Disease recurrence and the growth of drug resistant tumors remain significant problems in anti-cancer treatment. To develop more effective treatment strategies, it is important to understand the underlying cellular and molecular mechanisms of drug resistance. It is generally accepted that cancer cells do not function alone, but evolve through interactions with the surrounding tumor microenvironment. As key cellular components of the tumor microenvironment, fibroblasts regulate the growth and progression of many solid tumors. Emerging studies demonstrate that fibroblasts secrete a multitude of factors that enable cancer cells to become drug resistant. This review will explore how fibroblast secretion of soluble factors act on cancer cells to enhance cancer cell survival and cancer stem cell renewal, contributing to the development of drug resistant cancer.

Editorial (Thematic Issue: Advances with microRNAs in Tumorigenesis and Cancer Therapy)

Recent studies have highlighted the pivotal role of microRNAs (miRNAs) in tumorigenesis. A growing body of data implicates that miRNAs are critically involved in regulation of cell growth, cell cycle, apoptosis, migration, invasion, angiogenesis, metastasis, epithelial to mesenchymal transition (EMT), cancer stem cell renewal, and drug resistance. Therefore, our special issue reviewed the important role of miRNAs in the development and progression of human cancers. It has been accepted that miRNAs play an essential role in human malignancies. Dr. Markel et al. reviewed the diverse roles of miRNAs in regulation of cell cycle, proliferation, migration, invasion and drug resistance in melanoma cell biology [1]. Moreover, Dr. Zhou et al. discussed the oncogenic miRNAs in the genesis of leukemia and lymphoma, suggesting that miRNAs could be novel targets for anti-leukemia and anti-lymphoma drug discovery [2]. Dr. Sarkar et al. summarized the function of miRNAs in breast cancer tumorigenesis and metastasis, and provided the current knowledge of miRNAs as molecular targets for diagnosis, prognosis and therapy in breast cancer [3]. Emerging evidence has suggested that miRNAs exert its biological functions through governing key signaling pathways. To support this concept, Dr. Azmi et al. summarized that a number of miRNAs control Kras-PAK4 (p21 activated kinase 4) axis in cancer [4]. Dr. Xie et al. discussed that miRNAs are key players in pancreatic cancer initiation and progression through regulating FOXM1 signaling pathway [5]. Furthermore, Dr. Batra and colleagues summarized miRNAs as biomarkers for prognosis and diagnosis of gastrointestinal cancers and gastrointestinal tract associated organs including esophagus, gastric, liver, pancreas, and colon [6]. Intriguingly, Dr. Lu et al. discussed the developments on the regulatory role of miRNAs in circulating tumor cells and thrombosis, suggesting that targeting miRNAs could be useful for the treatment and prevention of metastasis and thrombosis in cancer [7]. It is noteworthy that miRNAs have been demonstrated to be involved in EMT progress. Dr. Yang et al. described that multiple miRNAs regulated EMT-inducing transcription factors such as Notch and TGF-β, leading to regulation of EMT in cancer [8]. Since EMT and miRNAs are associated with cancer stem cells (CSCs), Dr. Watabe et al. discussed how miRNAs regulate CSCs through different self-renewal pathways in various types of human cancers [9]. More importantly, Dr. Croce et al. discussed approaches towards therapeutic miRNA-based intervention including viral or non-viral approaches of miRNA replacement therapy [10]. Remarkably, Dr. Fan et al. summarized the implication of miRNAs in clinical use and challenge for miRNA based therapy such as miRNA delivery [11]. Notably, Dr. Wang et al. demonstrated that natural agent genistein down-regulated oncomiR-27a in pancreatic cancer cells, indicating that genistein could be a miR-27a inhibitor [12]. Dr. Chen et al. observed that arsenic trioxide exerts its anti-tumor activity partly through regulation of miR-125b in glioma cells [13]. Taken together, targeting miRNAs could be a novel strategy for treatment of cancer. Lastly, as the editor, I would like to thank the authors for their contributions. I also would like to appreciate the referees for their timely reviews. We hope that this special issue will help identify novel therapeutic targets to treat human cancers.

Abstract B13: Predictive biomarkers for FAK inhibitor sensitivity in ovarian cancer

Abstract Ovarian cancer is the fifth leading cause of cancer related death among women in the US and has the highest mortality rate among all gynecologic malignancies. Approximately 80% of patients diagnosed with ovarian epithelial cancer will relapse after first-line chemotherapy. Although targeted therapies tailored to specific genetic lesions have been developed for lung cancer, melanoma and CML, the molecular heterogeneity of the ovarian cancer genome has made it challenging to couple patient stratification markers with targeted agents. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that plays important roles in proliferation, cell survival, migration, invasion and cancer stem cell (CSC) renewal. FAK amplification and overexpression in ovarian cancer have been correlated with poor clinical outcome. In addition, high levels of phosphorylated FAK (pFAK) have been linked to poor prognosis and high ovarian tumor grade. Anti-tumor activity of the FAK inhibitors VS-6063 and VS-4718 has been previously demonstrated in preclinical models including ovarian cancer xenograft models (1,2). We have aimed to identify molecular markers predictive of ovarian cancer sensitivity to FAK inhibitors. Human ovarian cancer cell lines were analyzed for FAK inhibitor sensitivity in 3D Matrigel culture that mimics the ovarian tumor microenvironment. We then performed gene expression analysis of ovarian cancer cell lines most sensitive or least sensitive to FAK inhibitor treatment and identified a 64 gene signature that unambiguously clustered cell lines into sensitive and resistant subgroups (fold change >2, AUC >0.85). The gene signature associated with increased sensitivity to VS-4718 contained 27 genes with elevated expression, such as SLIT2, and 37 genes with decreased expression, such as LPNH2. Gene expression data were validated using qPCR as well as Western blot analysis to assess corresponding protein expression levels. As these genes may be important predictive biomarkers for patient selection or stratification, the variability of gene expression across human ovarian tumors was investigated using RNA microarray data of 733 patient tumor specimens derived from 5 study groups. As desired, SLIT2, LPNH2, and other genes in the signature exhibited a broad range of expression in ovarian tumor samples. Seven genes encoded for secreted proteins which could enable development of detection assays in blood. To assess regional expression of some of the above biomarkers within the tumor, we used immunohistochemistry analysis of a human ovarian Tissue MicroArray (TMA). Potential biomarkers were then evaluated in an independent test set of ovarian cancer samples. We first analyzed expression of the 64-gene signature in RNA microarray datasets from 6 ovarian patient-derived xenograft (PDX) models to predict their response to FAK inhibitor treatment. Then, an ex vivo clonogenic 3D assay was used to assess cell viability of ovarian PDX samples treated with FAK inhibitor. Interestingly, expression of several markers correlated with sensitivity to FAK inhibitor. In a similar fashion, we analyzed cell viability of ovarian cancer surgical samples from patients following ex vivo treatment with the FAK inhibitor. Tumors that expressed high levels of a number of genes from the 64 gene signature were especially sensitive to FAK inhibitor ex vivo. Our data identify biomarkers predictive of response to FAK inhibition in ovarian cancer models and provide a rationale for validation of these markers in a clinical trial of a FAK inhibitor in patients with ovarian cancer.

HIF expression and the role of hypoxic microenvironments within primary tumours as protective sites driving cancer stem cell renewal and metastatic progression

Hypoxic microenvironments frequently exist in many solid tumours with oxygen levels fluctuating temporally and spatially from normoxia to hypoxia. The response to hypoxia in human cells is mainly regulated by hypoxia-inducible factors (HIFs), a family of transcription factors which orchestrate signalling events leading to angiogenesis and tumorigenesis. Several events conspire together to lead to the stabilization of HIF-α, commonly expressed in many cancer cell types. These events can result from low oxygen tensions occurring within the expanding tumour mass to produce hypoxic microenvironments or from mutations whereby the HIFs cause changes in expression of genes involved in several cellular functions. Hypoxia-mediated HIF-α regulation has gained significant prominence in tumour biology over recent years, and the hypoxic microenvironments have been shown to facilitate and trigger major molecular and immunological processes necessary to drive the progression of tumours to malignancy. More recently, it has been realized that the hypoxic microenvironments also play significant roles in shielding tumour cells from immune attack by promoting immune suppression. In addition, the hypoxic microenvironment promotes many other oncogenic events, such as the metabolic reconfiguration of tumour cells, neovascularization, epithelial to mesenchymal transition (EMT), and cancer stem cell renewal and accumulation. This article reviews the molecular mechanisms underlying tumour hypoxia and their pro-tumour contributions, such as immune suppression, development of nascent and more permeable tumour vasculature, selective cancer stem cell renewal, accumulation, mobilization and promotion of EMT leading to tumour cell metastasis.

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

Abstract 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

The role of intratumoral and systemic IL-6 in breast cancer

Chronic low-grade inflammation plays an important role in the pathogenesis of several cancer forms including breast cancer. The pleiotropic cytokine IL-6 is a key player in systemic inflammation, regulating both the inflammatory response and tissue metabolism during acute stimulations. Here, we review the associations between IL-6 and breast cancer ranging from in vitro cell culture studies to clinical studies, covering the role of IL-6 in controlling breast cancer cell growth, regulation of cancer stem cell renewal, as well as breast cancer cell migration. Moreover, associations between circulating IL-6 and risk of breast cancer, prognosis for patients with prevalent disease, adverse effects and interventions to control systemic IL-6 levels in patients are discussed. In summary, direct application of IL-6 on breast cancer cells inhibits proliferation in estrogen receptor positive cells, while high circulating IL-6 levels are correlated with a poor prognosis in breast cancer patients. This discrepancy reflects distinct roles of IL-6, with elevated systemic levels being a biomarker for tumor burden, physical inactivity, and impaired metabolism, while local intratumoral IL-6 signaling is important for controlling breast cancer cell growth, metastasis, and self renewal of cancer stem cells.

Abstract P6-11-09: FAK Inhibitor VS-4718 Attenuates Breast Cancer Stem Cell Function In Vitro and In Vivo

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates signal transduction by integrins as well as growth factor receptors. FAK has been implicated in multiple steps of carcinogenesis including tumor initiation, growth and metastasis. Amplification and overexpression of FAK have been observed in aggressive human cancers including breast cancer. We have now observed that VS-4718, a selective FAK kinase inhibitor, exhibits preferential inhibitory effects on breast cancer stem cells. VS-4718 is a potent and selective FAK kinase inhibitor that blocks fibronectin-stimulated FAK autophosphorylation at Tyr397 at low nanomolar concentrations. To determine if FAK plays a role in the biology of breast cancer stem cells, VS-4718 was evaluated in a multitude of cancer stem cell assays both in vitro and in vivo. In parallel, treatment of SUM159 triple negative breast cancer cells in vitro with FAK shRNA inhibits tumorsphere formation. These data taken together indicate a role of FAK in breast cancer stem cell renewal. Similarly, pre-treatment of SUM159 cells with VS-4718 in matrigel attenuated secondary tumorsphere formation. Furthermore, VS-4718 reduced the side population (SP) and the percentage of ALDEFLUOR+ cancer stem cells in SUM159 breast cancer cells in vitro. In direct contrast, standard-of-care agent paclitaxel increased the percentage of ALDEFLUOR+ cancer stem cells in all three assays. The in vivo effect of VS-4718 on cancer stem cells was evaluated in SUM159 and MDA-MB-231 human triple negative breast cancer xenograft models. Following systemic administration, VS-4718 induced significant reduction of cancer stem cells in tumors as assessed by a decrease in ALDEFLUOR+ cells and tumorsphere-forming efficiency relative to vehicle-treated tumors. In summary, our results indicate the importance of FAK in the self-renewal of breast cancer stem cells in vitro and in vivo, and support the clinical development of the selective FAK inhibitor VS-4718 to target cancer stem cells for the treatment of triple negative breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-09.

CD44+/CD24− breast cancer cells isolated from MCF-7 cultures exhibit enhanced angiogenic properties

Recent studies suggest that the relationship between cancer stem cells (CSCs) and the vascular niche may be bidirectional; the niche can support the growth and renewal of CSCs, and CSCs may contribute to the maintenance of the niche. There is little knowledge concerning the role of breast cancer stem cells in promoting tumor angiogenesis. For human breast cancers, CSCs have been shown to be associated with a CD44+/CD24- phenotype. We investigated the potential activities of CD44+/CD24- breast cancer stem cells in promoting tumor angiogenesis. The expression of pro-angiogenic genes was determined by quantitative real-time RT-PCR. Endothelial cell migration assays were employed to evaluate effects of conditioned media from CD44+/CD24- on human umbilical vein endothelial cells. A chorioallantoic membrane (CAM) assay was used to study the potential of CD44+/CD24- cells to promote angiogenesis. In our study, CD44+/CD24- cells expressed elevated levels of pro-angiogenic factors compared with CD44+/CD24+ cells. CD44+/CD24- cell-conditioned media significantly increased endothelial cell migration. Breast cancer cell lines enriched with CD44+/CD24- cells were more pro-angiogenic in the CAM assay than those lacking a CD44+/CD24- subpopulation. CD44+/CD24- cells sorted from MCF-7 cell lines were more pro-angiogenic in a CAM assay than CD44+/CD24+ cells. Furthermore, the VEGF concentration was significantly higher in CD44+/CD24- cell-conditioned media than in CD44+/CD24+ cell-conditioned media. The pro-angiogenic effect of CD44+/CD24- cells on endothelial cells was abolished by bevacizumab. Our findings demonstrate that CD44+/CD24- breast cancer stem cells have substantial pro-angiogenic potential and activity. This provides new insights to explore in the development of targeted therapies.

Abstract 5419: Targeting colon cancer stem cells using sulforaphane

Abstract Colon cancer is a leading cause of cancer death in the United States, accounting for approximately 51,000 deaths each year. For patients with advanced colon cancer, the 5-year survival rate is less than 10%. 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 signalling pathways involved in cancer stem cells will help in designing novel agents to target this refractory cell population in colon cancers. We are currently investigating the mechanism by which sulforaphane, an isothiocyanate present in cruciferous vegetables such as broccoli, Brussels sprouts and cabbage inhibits growth of colon cancer cells. Our studies demonstrate that the compound had significant cytotoxicity against HCT116 colon cancer cells in both a dose- and time-dependent manner. Colon CSCs can be allowed to divide and grow in ultra-low binding tissue culture dishes to form multicellular spheroids called colospheres. Treatment with sulforaphane decreased the number and size of the primary and secondary colospheres in a dose dependent manner, suggesting that sulforaphane targets CSCs. To understand the mechanism of inhibition of colospheres, the signaling pathways affected by sulforaphane 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 sulforaphane inhibited the expression of both Shh and smoothened in the colon CSCs. Furthermore, it inhibited the expression of patched-1 and Gli-1, downstream targets of the hedgehog signalling pathway. Taken together, these data suggest that sulforaphane effectively inhibits colon cancer stem cells by down regulating the sonic hedgehog pathway, thereby inhibiting tumorigenesis (supported in-part by the US PHS grants CA 109269 and CA 135569 to SA and CA 151727 to AD). 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 5419. doi:1538-7445.AM2012-5419

Abstract 5044: Radiation modulates expression of microRNAs in breast cancer cell lines grown under adherent and stem cell promoting culture conditions

Abstract Although mounting evidence demonstrates that cancer stem/progenitor cells are radioresistant, explicit mechanisms of resistance have not been fully explored. MicroRNAs (miRNAs) are small noncoding RNAs that are involved in regulating cancer stem cell self-renewal and play an important role in response to cellular stress. Few studies have been conducted to investigate the role of specific miRNAs in cellular radiation response. Knowledge of how radiation therapy modulates miRNA expression in cancer stem cells vs. non-stem cells is critical to developing cancer stem cell sensitizers. We isolated total RNA from adherent (2D) and non-adherent mammosphere (3D) cultures of six breast cancer cell lines (MCF12F, MCF7, SUM190, SUM149, MDA-IBC-3, and SUM159) subjected to 0Gy or 4Gy radiation and examined differentially expressed miRNAs using Exiqon's miRCURY microarray. Analysis of the data showed 62 differentially expressed miRNAs between 2D and 3D independent of radiation treatment (fold change of at least 1.5, FDR <0.1; P<0.05). While no radiation based signature was observed when comparing all irradiated samples to unirradiated samples, assessment of each cell line indicated that the levels of specific microRNAs were altered in response to radiation treatment in 2D and 3D cells. Further studies to validate the functions of top candidate miRNAs using inhibitors or mimics to alter the self renewal and radiosensitivity of cancer stem cells are underway. 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 5044. doi:1538-7445.AM2012-5044

Abstract LB-192: The FAK inhibitors VS-4718 and VS-5095 attenuate breast cancer stem cell function in vitro and tumor growth in vivo

Abstract As a key mediator of integrin signaling, focal adhesion kinase (FAK) regulates cellular responses to extracellular matrix interactions. Amplification and overexpression of FAK have been observed in aggressive human cancers including breast cancer. FAK has been implicated in multiple steps in carcinogenesis including tumor growth, metastasis and angiogenesis. We now demonstrate the importance of FAK in breast cancer stem cell function, and the reduction of cancer stem cell function by the selective FAK inhibitors VS-4718 and VS-5095. VS-4718 and VS-5095 are potent and selective FAK inhibitors which were optimized following high throughput screening. Both VS-4718 and VS-5095 block fibronectin-stimulated FAK autophosphorylation of Tyr397 with low nanomolar cellular potency and are highly selective for FAK among a panel of protein kinases. Consistent with their mechanism of action, VS-4718 and VS-5095 showed greater inhibitory potency on the growth of multiple cancer cell lines in 3D matrigel culture as compared to conventional 2D culture. To determine if FAK plays a role in the biology of breast cancer stem cells in addition to its reported function in normal mammary stem cell biology, the effects of these FAK inhibitors were characterized using two different in vitro assays. It was previously demonstrated that immortalized mammary epithelial cells (HMLEs) driven to undergo epithelial to mesenchymal transition (EMT) by knockdown of E-cadherin (HMLE-shECad) exhibit many of the characteristics of cancer stem cells and can be used to identify agents that selectively target cancer stem cells. VS-4718 exhibited 25-fold greater potency against proliferation of mesenchymal HMLE-shECad cells as compared to epithelial HMLE-shGFP control cells, suggesting preferential effects on breast cancer stem cells. Furthermore, pre-treatment of SUM159 triple negative breast cancer cells with VS-5095 in matrigel attenuated secondary tumorsphere formation, suggesting that FAK is important for the self-renewal function of breast cancer stem cells. The role of FAK in breast cancer stem cell renewal was further corroborated by the observation that FAK shRNA inhibited tumorsphere formation by SUM159 cells. The in vivo efficacy of the FAK inhibitor VS-5095 was evaluated in the MDA-MB-231 human triple negative breast cancer xenograft model. By oral administration, VS-5095 induced significant dose-dependent tumor growth inhibition. In summary, these results demonstrate the importance of FAK in the self-renewal of breast cancer stem cells, and support the clinical development of the selective FAK inhibitors VS-4718 and VS-5095 to target breast cancer stem cells for the treatment of triple negative breast 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 LB-192. doi:1538-7445.AM2012-LB-192

Abstract 5297: Autocrine stimulation of VEGFR-2 by leptin is associated with Notch signaling pathway and cancer stem cell marker expression

Abstract Obesity-related cancer is an increasing health problem. Leptin, the major adipokine, has been suggested a major factor in these relationships. Leptin is a pro-inflammatory, pro-angiogenic and mitogenic factor for breast cancer cells. Recently, we described a comprehensive mechanism for leptin induction of VEGF, and unveiled a novel crosstalk between Notch, IL-1 and leptin (NILCO) in breast cancer. NILCO regulates VEGF/VEGR-2 in breast cancer cells and could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin oncogenic actions. However, the specific mechanisms for leptin-induced VEGFR2 are unknown. To answer this question, we re-cloned mouse VEGFR-2 promoter and prepared VEGFR-2 luc, then analyzed VEGFR-2 regulation and expression by using several kinase inhibitors, leptin peptide antagonist 2 (LPrA2), a VEGFR-2 kinase inhibitor (SU5416), as well as siRNA against VEGFR-2, Notch1, Notch3 in 4T1 cells. Our results demonstrated that leptin-induced JAK2/STAT3, MAPK/ERK 1/2, PI-3K/AKT1, PKC, p38 and JNK signaling pathways may correlate with upregulated VEGFR-2. Moreover, leptin-induced activation of Notch/VEGFR-2 was linked to the upregulation of cancer stem cell markers CD44 and ALDH1. These leptin effects were inhibited by siRNA-Notch1&3, leptin signaling antagonist (PEG-LPrA2), VEGFR2-kinase inhibitor (SU5416) and siRNA-VEGFR-2. Our results show for the first time how leptin's molecular signals regulate VEGFR-2 (an autocrine survival process) that was associated to Notch and cancer stem cell marker expression. These novel findings may help to design new strategies for prevention/treatment of breast cancer through the abrogation of tumor angiogenesis and breast cancer stem cell renewal in an obesity context. [This work was supported in part by NIH/NCI1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP)]. 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 5297. doi:1538-7445.AM2012-5297

MiR-181b suppresses proliferation of and reduces chemoresistance to temozolomide in U87 glioma stem cells

Abstract MicroRNAs regulate self renewal and differentiation of cancer stem cells. There, we sought to identify the expression of miR-181b in glioma stem cells and investigate the biological effect of miR-181b on glioma stem cells in this study. MiR-181b expression was measured by real-time PCR in glioma stem cells isolated from U87 cells by FACS sorting. After miR-181b was overexpressed in U87 glioma stem cells by miR-181b lentiviral expression vector and/or treatment of temozolomide, secondary neurosphere assay, soft agar colony assay and MTT assay were performed. Compared with U87 cells, the expression of miR-181b was significantly decreased in U87 glioma stem cells. Overexpression of miR-181b decreased neurosphere formation by U87 glioma stem cells in vitro and suppressed colony formation in soft agar, and the cell growth inhibition rates increased in a time-dependent manner in U87 glioma stem cells infected with miR-181b lentivirus. Furthermore, miR-181b had a synergistic effect on temozolomide-induced inhibition of secondary neurosphere and soft agar colony, and on cell growth inhibition rates. MiR-181b functions as a tumor suppressor that suppresses proliferation and reduces chemoresistance to te-mozolomide in glioma stem cells.

The pan-HDAC Inhibitor Suberoylanilide Hydroxamic Acid Targets Self Renewal of Breast Cancer Stem Cells.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC often lack estrogen receptor and present with skin metastasis characterized by the presence of tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly over-express E-cadherin and have characteristics of stem cells. Based on our initial observations that IBC cells express multiple forms of histone deacetylase (HDAC) enzymes involved in epigenetic modulation of multiple genes associated with critical functions of both embryonic stem cells and cancer cells, the present studies evaluated the effects of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on self-renewal, expression of transcription factors associated with self renewal, homotypic aggregation, and E-cadherin expression by mammospheres of SUM149 and SUM190 established IBC cell lines and of tumor cells isolated from pleural effusion fluid of patients with IBC and LABC.Methods: Low adherence culture conditions that promote mammosphere formation and clonogenic assays were used to assess effects of SAHA on self renewal. Real time PCR arrays and protein arrays were used to evaluate the effects of SAHA on transcription factors Oct4, Nanog and Sox-2 and breast cancer associated genes including E-cadherin and ER/PR. Flow cytometry was used to document stem cell markers present on breast cancer cells grown under low adherence conditions. Confocal microscopy was used to document the effects of SAHA on E-cadherin localization and homotypic aggregation by mammospheres.Results: SAHA inhibited self-renewal of second and third passage mammospheres as assessed by clonogenic assays, which was associated with loss of Oct4, Nanog and Sox-2 transcription factors associated with stem cell self renewal an pluripotency. SAHA blocked E-cadherin mRNA and protein assessed by PCR and Western blotting and visualized by confocal imaging which demonstrated that loss of E-cadherin between cells comprising mammospheres blocked homotypic aggregation, leading to lack of integrity of the 3-dimensional spheroids. Moreover, SAHA induced ER-/low mammospheres to re-express ER mRNA and protein which was associated with responsiveness to tamoxifen and conversely, SAHA inhibited ER expression in ER+breast tumor cells. The effective inhibitory concentration50 (IC50) of SAHA to induce these effects in both established cell lines and in mammospheres derived from tumor cells isolated from pleural effusion fluid of patients with IBC and LABC was in the range of 0.37-3.31 uM, which is achievable by oral administration of SAHA. Initial in vivo studies indicate that intraperitoneal injection of SAHA effectively inhibits SUM149 breast tumor xenograft growth and formation of metastatic lesions.Conclusions: SAHA promotes differentiation of IBC stem cells and blocks E-cadherin expression, which is a hallmark of IBC skin metastasis. Taken together with the observations that SAHA can re-program ER function depending upon the ER status of the breast cancer cells, this observations suggest that SAHA may provide additional therapeutic opportunities for patients with LABC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3141.