Growth Of Breast Cancer Stem Cells Research Articles

Brain infiltration of breast cancer stem cells is facilitated by paracrine signaling by inhibitor of differentiation 3 to nuclear respiratory factor 1.

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood-brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.

Discovery of Novel Tetrahydro-β-carboline Containing Aminopeptidase N Inhibitors as Cancer Chemosensitizers.

Aminopeptidase N (APN, CD13) is closely associated with the development and progression of cancer. Previous studies suggested APN as a biomarker for cancer stem cells. APN inhibitors have been intensively evaluated as chemosensitizers for cancer treatments. In the present study, tetrahydro-β-carboline scaffold was introduced to the structure of APN inhibitors. The synthesized compounds showed potent enzyme inhibitory activities compared with Bestatin, an approved APN inhibitor, in cell-based enzymatic assay. In combination with chemotherapeutic drugs, representative APN inhibitor molecules D12, D14 and D16 significantly improved the antiproliferative potency of anticancer drugs in the in vitro tests. Further mechanistic studies revealed that the anticancer effects of these drug combinations are correlated with decreased APN expression, increased ROS level, and induction of cell apoptosis. The spheroid-formation assay and colony-formation assay results showed effectiveness of Paclitaxel-APN inhibitor combination against breast cancer stem cell growth. The combined drug treatment led to reduced mRNA expression of OCT-4, SOX-2 and Nanog in the cancer stem cells tested, suggesting the reduced stemness of the cells. In the in vivo study, the selected APN inhibitors, especially D12, exhibited improved anticancer activity in combination with Paclitaxel compared with Bestatin. Collectively, potent APN inhibitors were discovered, which could be used as lead compounds for tumor chemo-sensitization and cancer stem cell-based therapies.

Increased lipogenesis is critical for self-renewal and growth of breast cancer stem cells: Impact of omega-3 fatty acids.

Aberrant lipid metabolism has recently been recognized as a new hallmark of malignancy, but the characteristics of fatty acid metabolism in breast cancer stem cells (BCSC) and potential interventions targeting this pathway remain to be addressed. Here, by using the in vitro BCSC models, mammosphere‐derived MCF‐7 cells and HMLE‐Twist‐ER cells, we found that the cells with stem cell‐like properties exhibited a very distinct profile of fatty acid metabolism compared with that of their parental cancer cells, characterized by increased lipogenesis, especially the activity of stearoyl‐CoA desaturase 1 (SCD1) responsible for the production of monounsaturated fatty acids, and augmented synthesis and utilization of the omega‐6 arachidonic acid (AA). Suppression of SCD1 activity by either enzyme inhibitors or small interfering RNA (siRNA) knockdown strikingly limited self‐renewal and growth of the BCSC, suggesting a key role for SCD1 in BCSC proliferation. Furthermore, elevated levels of SCD1 and other lipogenic enzymes were observed in human breast cancer tissues relative to the noncancer tissues from the same patients and correlated with the pathological grades. Interestingly, treatment of BCSC with omega‐3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, effectively downregulated the expression of the lipogenic enzymes and markedly suppressed BCSC self‐renewal and growth. Dietary supplementation of nude mice bearing BCSC‐derived tumors with omega‐3 fatty acids also significantly reduced their tumor load. These findings have demonstrated that increased lipogenesis is critical for self‐renewal and growth of BCSC, and that omega‐3 fatty acids are effective in targeting this pathway to exert their anticancer effect.

Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell-like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor-suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3-dimensional (3D), semisolid culture system was established to culture MCF-7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF-7 parental cells, BCSC-enriched MCF-7 spheroid cells, and CD44+/CD24- MCF-7 cells were evaluated by miRNA microarray, and the high expression of miR-210 in MCF-7 spheroid cells and CD44+/CD24- MCF-7 cells was verified by quantitative RT-PCR. MCF-7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR-210 expression and the stemness of the cells. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT), transwell, and sphere-formation assays were performed to detect the proliferation, migration, and self-renewal ability of miR-210-overexpressed MCF-7 cells and MCF-7 spheroid cells with miR-210 knocked down. The target of miR-210 was validated with a dual-luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR-210 targeting E-cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF-7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR-210 was up-regulated in those cells compared with MCF-7 parental cells. High miR-210 expression was also detected in CD44+/CD24- MCF-7 cells and human CD44+/CD24- breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF-7 spheroids or solid tumors. Ectopic expression of miR-210 in MCF-7 cells promoted their migration, invasion, proliferation, and self-renewal in both in vitro and in vivo studies. We further reported that miR-210 suppressed E-cadherin expression by targeting the open reading frame region of E-cadherin mRNA and by up-regulation of E-cadherin transcription repressor, Snail. Accordingly, E-cadherin overexpression compromises the migration, invasion, proliferation, and self-renewal ability of miR-210-overexpressed MCF-7 both in vitro and in vivo. These findings reveal a novel regulatory pathway centered on hypoxia-mediated miR-210 targeting of E-cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.-Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Esculentoside A suppresses breast cancer stem cell growth through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway.

Breast cancer stem cells (CSCs) survive in inflammatory microenvironment, their survival are regulated by inflammatory cytokines and signaling pathways. Esculentoside A (EsA), a triterpene saponin derived from the root of Phytolacca esculenta, possesses antiinflammation effects; but whether it has anticancer activity is unknown. The purpose of this study is to test the inhibitory effect of EsA on the growth of breast CSCs and to elucidate its probable mechanisms of action. The proliferation inhibitory effect of EsA on breast CSCs in vitro were determined by cytotoxicity, mammosphere formation inhibition, apoptotic cell detection assays, and in vivo tumor growth inhibition experiment. The possible molecular mechanisms elucidating the inhibitory effect of EsA on breast CSC growth were examined with western blotting. EsA caused proliferation and mammosphere formation inhibition of breast CSCs; induced breast CSCs apoptotic death; suppressed the growth of tumors generated from breast CSCs significantly; the expressions of stemness proteins including ALDH1A1, Sox2, and Oct4 were downregulated; proapoptotic proteins, Bax and cleaved caspase-3 were upregulated, whereas the antiapoptotic protein Bcl-2 was reduced; IL-6/STAT3 pathway proteins including IL-6, phosphorylated STAT3 (Tyr705), and STAT3 (Ser727) were downregulated significantly in EsA-treated breast CSCs and tumor tissues. EsA inhibited breast CSC growth in vitro and in vivo through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway; it might serve as a novel candidate agent for human breast cancer treatment and/or prevention.

A Virus-Like-Particle immunotherapy targeting Epitope-Specific anti-xCT expressed on cancer stem cell inhibits the progression of metastatic cancer in vivo

ABSTRACTAggressive forms of breast cancer, such as Her2+ and triple negative breast cancer (TNBC), are enriched in breast cancer stem cells (BCSC) and have limited therapeutic options. BCSC represent a key cellular reservoir for relapse, metastatic progression and therapeutic resistance. Their ability to resist common cytotoxic therapies relies on different mechanisms, including improved detoxification. The cystine-glutamate antiporter protein xCT (SLC7A11) regulates cystine intake, conversion to cysteine and subsequent glutathione synthesis, protecting cells against oxidative and chemical insults. Our previous work showed that xCT is highly expressed in tumorspheres derived from breast cancer cell lines and downregulation of xCT altered BCSC function in vitro and inhibited pulmonary metastases in vivo. We further strengthened these observations by developing a virus-like-particle (VLP; AX09-0M6) immunotherapy targeting the xCT protein. AX09-0M6 elicited a strong antibody response against xCT including high levels of IgG2a antibody. IgG isolated from AX09-0M6 treated mice bound to tumorspheres, inhibited xCT function as assessed by reactive oxygen species generation and decreased BCSC growth and self-renewal. To assess if AX09-0M6 impacts BCSC in vivo seeding, Her2+ TUBO-derived tumorspheres were injected into the tail vein of AX09-0M6 or control treated female BALB/c mice. AX09-0M6 significantly inhibited formation of pulmonary nodules. To evaluate its ability to impact metastases, AX09-0M6 was administered to mice with established subcutaneous 4T1 tumors. AX09-0M6 administration significantly hampered tumor growth and development of pulmonary metastases. These data show that a VLP-based immunization approach inhibits xCT activity, impacts BCSC biology and significantly reduces metastatic progression in preclinical models.

A recombinant protein TmSm(T34A) can inhibit proliferation and proapoptosis to breast cancer stem cells(BCSCs) by down-regulating the expression of Cyclin D1.

Cancer stem cells (CSCs), a small fraction of cancer cells lines proved with stem cell characteristics, were regarded as "bad seeds" related to recurrence, metastasis and chemotherapy resistance of breast carcinoma in recent years. So inhibiting the growth or inducing the differentiation and apoptosis of CSCs were considered as one of the effective pathways to fight against breast cancer. Based on the recombinant protein TmSm(T34A) that was designed and prepared in our previous experiments for targeting survivin, an inhibitor of apoptosis protein(IAP), in this study, we explored the effects of TmSm(T34A) on BCSCs obtained by enriching in serum-free suspension, sorting and characterizing of MCF-7/ADM. The results showed that TmSm(T34A) could not only inhibit the proliferation and growth of BCSCs by decreasing CD44+CD24- proportion and down-regulating the expression of Cyclin D1 significantly, but also induce BCSCs apoptosis evidently. Furthermore, in BCSCs xenograft nude mice administrated TmSm(T34A), the tumor growth was slower than that of the control obviously. Thus it can be seen TmSm(T34A) would be a promising potential protein for treatment of breast cancer by effecting on BCSCs.

Abstract 3317: Tumor microenvironment of metastasis (TMEM) in breast cancer patients represents a stem cell niche; likely mediated by the Wnt pathway

Abstract Cell surface biomarkers CD44, CD24, CD133 and intracellular biomarker ALDH1 have been used to identify breast cancer stem cells (BCSC). BCSC are clinically significant because they are resistant to chemotherapy and in very small numbers can initiate tumor growth and metastasis. Recent studies suggest that juxtacrine signaling from monocytes and macrophages support the BCSC niche. Macrophages represent one of the components of the micro-anatomical sites of hematogenous dissemination of breast cancer cells called Tumor MicroEnvironment of Metastasis (TMEM) which are predictors of metastatic outcome in patients. We hypothesize that TMEM rich tumor microenvironments support BCSCs which are a prerequisite for systemic metastatic outgrowths. We used flow cytometry to quantify CD44high/CD24low cells, as well as mRNA fluorescent in situ hybridization (FISH) and qRT-PCR to quantify CD133 and ALDH1 expression in breast cancer cells from 50 invasive ductal carcinomas obtained from patients’ cancer excisions by fine needle aspiration (FNA) before formalin fixation. Formalin-fixed paraffin embedded tissue from each sample was also analyzed for TMEM score using triple immunohistochemistry and the stem cell marker expression was compared to TMEM scores for each corresponding cancer excision. We observed very strong correlation between the percentage of CD44high/CD24low cells and TMEM scores (r = 0.91), as well as the percentage of CD133 and ALDH1 expressing cells with TMEM scores (r = 0.88 and 0.86 respectively). FISH results were validated using qRT-PCR and showed very strong correlation with TMEM scores (r = 0.76 and 0.73 for CD133 and ALDH1 respectively). Wnt/β-catenin signaling pathway is involved in stem cell generation and pathogenesis of various types of cancer. Aberrant activation of Wnt-signaling pathway in normal stem cells can promote their transformation into cancer stem cells. We hypothesize that interactions between cancer cells and macrophages, and/ or cancer cells and endothelial cells (i.e. both interactions among the cellular components of TMEM) induce cancer stemness via Wnt/-β catenin pathway. Indeed, co-culture of mouse macrophages (BAC) with MCF-7 cells, as well as human endothelial cells (HUVEC) with MCF-7, significantly increased the percentage of cells expressing stem cells markers, while salinomycin, selective inhibitor of breast cancer stem cell growth and progression, significantly reduced BAC or HUVEC induced increase in the percentage of cancer stem cells. Our findings indicate that TMEM-rich microenvironments support breast cancer stem cells likely via Wnt signaling involving juxtacrine cancer cell-macrophage and cancer cell-endothelial cell interactions. Citation Format: Gargi Bandyopadhyaya, Eli Grunblatt, Sweta Roy, Nathan Agi, Esther Adler, Joan Jones, John S. Condeelis, Maja H. Oktay, Sumanta Goswami. Tumor microenvironment of metastasis (TMEM) in breast cancer patients represents a stem cell niche; likely mediated by the Wnt pathway. [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 3317.

3D-QSAR and Docking Simulation Studies of Some Benzopyrone Derivatives as Inhibitors for Breast Cancer Stem Cell Growth via PGlycoprotein Mediated Efflux

Benzopyrone derivatives (Coumarins) are well known inhibitors of P-glycoprotein (P-gp) mediated efflux. The high expression level of these efflux proteins promotes the growth of breast cancer stem cells (CSCs). The activity of breast CSCs is directly affected by the inhibition of efflux proteins by benzopyrone derivatives. Ligand based pharmacophoric study and structure based docking studies have been exploited for assessing this inhibitory activity. Based on QSAR results, a three point pharmacophore comprising of one hydrogen bond acceptor (A) and two condensed aromatic groups (R) has been designed. The atom based QSAR study was conducted to predict partial least square (PLS) statistical factors for test and training data sets. Some specific amino acids have been demonstrated to be actively involved in the ligand protein interaction. These structural features of ligands and active site residues of target protein provide new pathways to develop therapeutically important drugs for the inhibition of breast cancer stem cells. Keywords: Breast cancer stem cells, P-glycoprotein (P-gp), Efflux, Quantitative structure activity relationship (QSAR), Benzopyrones.

Inhibition of P-Glycoprotein Mediated Efflux of Paclitaxel by Coumarin Derivatives in Cancer Stem Cells: An In Silico Approach.

P-glycoprotein (P-gp) is well known to cause multidrug resistance (MDR) in cancer cells. This MDR leads to cancer recurrence which is a major obstacle in cancer treatment. High P-gp expression has been observed in the population of cancer stem cells (CSCs) having self-renewal potential. Early detection and inhibition of these CSCs is directly beneficial to cancer treatment. In this study coumarin derivatives are used to inhibit efflux process and thereby enhance bioavailability of various drugs like paclitaxel (PTX). This drug is most commonly used for the treatment of cancers of breast, ovary, head and neck. Coumarin derivatives can be used to reduce the growth of breast cancer stem cells through P-gp mediated efflux inhibition and paclitaxel bioavailability enhancement. With the use of computational approaches including molecular docking simulation and pharmacophore study, few coumarin derivatives have been found to be more potential inhibitors of P-gp mediated efflux. Based on high affinity inhibitors, new coumarin derivatives have been designed and docked at active site cavity of P-gps. Some newly designed coumarin derivatives were found to be more potent due to their higher binding affinity towards target protein. The finding that newly designed coumarins can be exploited for inhibition of P-gp mediated efflux in order to enhance paclitaxel bioavailability and can inhibit breast cancer stem cell growth is significant for designing potent anticancer drugs.

Designing and Development of Novel Curcumin Analogues/Congeners as Inhibitors of Breast Cancer Stem Cells Growth

Curcumin, a polyphenolic yellow pigment of the spice turmeric is a unique, multi-targeted molecule having potential for cancer therapy. The inhibition of cancer stem cell growth is an essential step for breast cancer treatment. The main objective of the present work is to identify a potential target of curcumin against breast cancer stem cells and also to predict potency of designed curcumin analogues/congeners on predicted target. The high expression level of P-glycoprotein (P-gp) in the population of breast cancer stem cells promotes the cancer stem cells growth. The anticancer activity of curcumin is directly associated with the inhibition of P-gp mediated efflux process. This efflux mechanism is considered as a major reason for the failure of multidrug resistance in the cancer treatment. Using computational tools, the strong potency of curcumin against P-gp for the inhibition of breast cancer stem cells growth has been determined. Few analogues of curcumin were found to be more effective than curcumin towards cancer stem cells inhibition. In this way, the present study provides the pathway to design and improve the efficacy of novel and therapeutically important herbal drugs for the inhibition of breast cancer stem cells via inhibiting the P-gp mediated efflux mechanism.

Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.

BackgroundBreast cancer stem cells (BCSCs) have been reported as the origin of breast cancer and the radical cause of drug resistance, relapse and metastasis in breast cancer. BCSCs could be derived from mutated mammary epithelial stem cells (MaSCs). Therefore, comparing the molecular differences between BCSCs and MaSCs may clarify the mechanism underlying breast carcinogenesis and the targets for gene therapy. Specifically, the distinct miRNome data of BCSCs and MaSCs need to be analyzed to find out the key miRNAs and reveal their roles in regulating the stemness of BCSCs.MethodsMUC1−ESA+ cells were isolated from normal mammary epithelial cell line MCF-10A by fluorescence-activated cell sorting (FACS) and tested for stemness by clonogenic assay and multi-potential differentiation experiments. The miRNA profiles of MaSCs, BCSCs and breast cancer MCF-7 cells were compared to obtain the candidate miRNAs that may regulate breast tumorigenesis. An miRNA consecutively upregulated from MaSCs to BCSCs to MCF-7 cells, miR-200c, was chosen to determine its role in regulating the stemness of BCSCs and MaSCs in vitro and in vivo. Based on bioinformatics, the targets of miR-200c were validated by dual-luciferase report system, western blot and rescue experiments.ResultsIn a 2-D clonogenic assay, MUC1−ESA+ cells gave rise to multiple morphological colonies, including luminal colonies, myoepithelial colonies and mixed colonies. The clonogenic potential of MUC1−ESA+ (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05). In a 3-D matrigel culture, MUC1−ESA+ cells grew into mammospheres with duct-like structures. A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs. In gain- and lost-of-function assays, miR-200c was sufficient to inhibit the self-renewal of BCSCs and MaSCs in vitro and the growth of BCSCs in vivo. Furthermore, miR-200c negatively regulated programmed cell death 10 (PDCD10) in BCSCs and MaSCs. PDCD10 could rescue the tumorigenesis inhibited by miR-200c in BCSCs.DiscussionAccumulating evidence shows that there is a milignant transformation from MaSCs into BCSCs. The underlying mechanism remains unclear. In present study, miRNA profiles between MaSCs and BCSCs were obtained. Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells. These findings reveal a novel insights of breast tumorigenesis.ConclusionsPDCD10 is a target gene of miR-200c and also a possible mechanism by which miR-200c plays a role in regulating the stemness of BCSCs and MaSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1655-5) contains supplementary material, which is available to authorized users.

Abstract 1528: GD2+ breast cancer stem cell growth is dependent on NFκB signaling and suppressed by the IKK inhibitor BMS345541 in vitro and in vivo

Abstract Recurrence of breast cancer after treatment could be caused by breast cancer stem-like cells (BCSCs) or breast cancer-initiating cells. Therefore, targeting BCSCs is an important strategy that could complement standard chemotherapy. We recently reported that the ganglioside GD2 is exclusively expressed on BCSCs and that targeting its metabolic enzyme (GD3 synthase, GD3S), reduced breast tumor growth and metastasis in immuno-deficient mice (Battula et al., JCI, 2012). To identify signaling pathways that are activated in GD2+ BCSCs, we performed proteomic analysis in GD2+/- cells from breast cancer cell lines MDA-MB-231 and SUM159 cells using Kinexus® antibody arrays. Data analysis employing Ingenuity® pathway analysis revealed activation of NFκB signaling in GD2+ cells in both cell lines. To validate the array data, we tested activation of NFκB (phospho p65) by CyTOF mass cytometry using metal tagged antibodies and found that NFκB was indeed activated in GD2+ but not in GD2- cells, suggesting activation of canonical NFκB signaling in GD2+ BCSCs. Systemic phospho- and total-protein analyses by immuno blotting revealed that proteins involved in non-canonical signaling, including RelB, NFκB2, and Traf2, were also up-regulated (2-3 fold) in GD2+ compared to GD2- cells. These data suggest that both canonical and non-canonical NFκB signaling are active in GD2+ cells and that it may require inhibition of both pathways to block GD2+ BCSC growth. We found that BMS345541 (IC50 for IKKβ = 0.4μM (canonical) and IC50 for IKKα = 4μM (canonical and non-canonical) reduced GD2+ cells and inhibited GD3S expression (determined by qRT-PCR) in a dose- and time-dependent fashion. Treatment with BMS345541 at 5μM for 72hrs reduced GD2+ cell frequency from 18.5±2.5% to 1.7±0.4% (P &amp;lt;0.001) which is &amp;gt;90% reduction of GD2+ cells in a dose-dependent manner. In contrast, treatment with doxorubicin increased the percentage of GD2+ cells, from 13.5±2.5% to 21±2.6% in MDA-MB-231 cells, suggesting that GD2+ cells are resistant to doxorubicin. BCSCs generate mammospheres under low adhesion conditions. Treatment with the BMS compound inhibited the ability of breast cancer cells to form mammospheres app.12 fold in vitro. To test if BMS345541 inhibits in vivo tumor growth, NOD/SCID mice were transplanted with MDA-MB-231 cells (0.5×106) injected into the mammary fat pad. Following engraftment (2 weeks after transplantation), mice were treated with 50mg/kg of BMS345541 or with DMSO daily for only 5 days (IP, n = 5). The BMS345541-treated group showed a significant decrease (P &amp;lt;0.01) in tumor volume, and prolonged survival compared to the control group: median survival was 78 days for the BMS345541-treated group vs. 58 days for controls (P&amp;lt;0.002). Our data suggest that NFκB inhibition by BMS345541 reduces tumor growth in vivo and prolongs survival of tumor-bearing mice by inhibiting NFκB-mediated GD2+ BCSC growth. Citation Format: Venkata Lokesh Battula, Jeffrey Sun, Gabriel N. Hortobagyi, Michael Andreeff. GD2+ breast cancer stem cell growth is dependent on NFκB signaling and suppressed by the IKK inhibitor BMS345541 in vitro and in vivo. [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 1528. doi:10.1158/1538-7445.AM2015-1528

The role of microRNAs in breast cancer stem cells

Cancer stem cells are a kind of cancer cell group with the ability of self-renewal and various diferentiation potentials.Recent studies have implicated that they play a significant role in tumor formation,metastasis,resistance to anticancer therapies and cancer recurrenee.Many studies show that microRNAs are involved in the maintenance,growth and behavior of breast cancer stem cells （BCSCs）,paving a new way for diagnosis,prognosis and therapy of breast cancer. Key words: MicroRNAs; Neoplastic stem cells; Breast neoplasms

Breast cancer stem cells, EMT and therapeutic targets

A small heterogeneous population of breast cancer cells acts as seeds to induce new tumor growth. These seeds or breast cancer stem cells (BCSCs) exhibit great phenotypical plasticity which allows them to undergo “epithelial to mesenchymal transition” (EMT) at the site of primary tumor and a future reverse transition. Apart from metastasis they are also responsible for maintaining the tumor and conferring it with drug and radiation resistance and a tendency for post-treatment relapse. Many of the signaling pathways involved in induction of EMT are involved in CSC generation and regulation. Here we are briefly reviewing the mechanism of TGF-β, Wnt, Notch, TNF-α, NF-κB, RTK signalling pathways which are involved in EMT as well as BCSCs maintenance. Therapeutic targeting or inhibition of the key/accessory players of these pathways could control growth of BCSCs and hence malignant cancer. Additionally several miRNAs are dysregulated in cancer stem cells indicating their roles as oncogenes or tumor suppressors. This review also lists the miRNA interactions identified in BCSCs and discusses on some newly identified targets in the BCSC regulatory pathways like SHIP2, nicastrin, Pin 1, IGF-1R, pro-inflammatory cytokines and syndecan which can be targeted for therapeutic achievements.

Benzyl Isothiocyanate: Double Trouble for Breast Cancer Cells

An inverse association between dietary intake of cruciferous vegetables and cancer risk has been established for different types of malignancies, including breast cancer. The anticarcinogenic effect of cruciferous vegetables has been attributed to chemicals with an isothiocyanate (ITC) functional moiety. Research over the past three decades has provided extensive preclinical evidence for the efficacy of various ITCs against cancer in preclinical models. Benzyl isothiocyanate (BITC) is one such compound with the ability to inhibit chemically induced cancer, oncogenic-driven tumor formation, and human tumor xenografts in rodent cancer models. Prior work also has established that BITC has the ability to influence carcinogen metabolism and signaling pathways relevant to tumor progression and invasion. In this issue, Kim and colleagues show that BITC inhibits breast cancer stem cell growth, both in vitro and in vivo, in association with suppression of the full-length receptor tyrosine kinase RON as well as its activated truncated form (sfRon), both of which seem to drive stemness in breast cancer cells. Overexpression of RON or sfRon prevented the BITC effect. These data complement prior work from this group showing elimination of mammary tumor cells via tumor cell apoptosis by BITC administration. The inhibition of breast cancer stem cells is observed at pharmacologic concentrations of BITC. This perspective briefly reviews epidemiologic evidence, preclinical efficacy data, and molecular and cellular mechanistic attributes of BITC. Critical issues relevant to clinical development of BITC are discussed briefly.

Tanshinone IIA inhibits breast cancer stem cells growth in vitro and in vivo through attenuation of IL‐6/STAT3/NF‐kB signaling pathways

Cancer stem cells (CSCs) are maintained by inflammatory cytokines and signaling pathways. Tanshinone IIA (Tan-IIA) possesses anti-cancer and anti-inflammatory activities. The purpose of this study is to confirm the growth inhibition effect of Tan-IIA on human breast CSCs growth in vitro and in vivo and to explore the possible mechanism of its activity. Human breast CSCs were enriched and expanded under serum-free mammosphere culture condition, and identified through mammosphere formation, toluidine blue staining, immunofluorescence staining, and flow cytometry analysis of stemness markers of CD44/CD24 and ALDH, and tumorigenecity in vivo; the growth inhibition effect of Tan-IIA on human breast CSCs in vitro were tested by cell proliferation and mammosphere formation assays; inflammatory signaling pathway related protein expression in response to Tan-IIA, IL-6, STAT3, phospho-STAT3 (Tyr705), NF-κBp65 in cytoplasm and nucleus and cyclin D1 were evaluated with Western blotting; the growth inhibition effect of Tan-IIA on human breast CSCs growth were tested in vivo. A useful model of human breast CSCs for researching and developing the agents targeting CSCs was established. After Tan-IIA treatment, cell proliferation and mammosphere formation of CSCs were decreased significantly; the expression levels of IL-6, STAT3, phospho-STAT3 (Tyr705), NF-κBp65 in nucleus and cyclin D1 proteins were decreased significantly; the tumor growth and mean tumor weight were reduced significantly. Tan-IIA has the potential to target and kill CSCs, and can inhibit human breast CSCs growth both in vitro and in vivo through attenuation of IL-6/STAT3/NF-kB signaling pathways.

All-trans retinoic acid effectively inhibits breast cancer stem cells growth in vitro

To detect the inhibitory effect of all-trans retinoic acid(ATRA) on breast cancer stem cells (CSCs). The inhibitory effect of ATRA on MCF-7 and SK-BR-3 cell lines was analyzed using a Cell Counting Kit-8 (CCK-8). The proportion of CD44(+)CD24(-) tumor cells of the two cell lines were measured before and after the ATRA treatment, and the role of ATRA in the regulation of CSC self-renewing ability was evaluated with a tumor sphere assay. The tumor spheres were grown in an adherent culture to evaluate the ATRA-induced differentiation of breast cancer stem cells. ATRA effectively inhibited the unsorted cells and stem cells, but the CSCs were more sensitive to ATRA. At a concentration of 10(-6) mol/L, the inhibitory rate of MCF-7 unsorted cells and stem cells were (8.66 ± 1.06)% and (21.09 ± 3.25)%, respectively (P = 0.004). For SK-BR-3 cells, the rates were (39.19 ± 1.47)% and (51.22 ± 2.80)%, respectively (P = 0.005). The self-renewing ability of the CSCs was impaired by ATRA at a concentration of 10(-6) mol/L. The rate of MCF-7 and SK-BR-3 stem cells to form tumor sphere was 5.2% (5/96) and 13.5% (13/96), respectively. For the control group, it was 86.5% (83/96) and 93.8% (90/96), respectively (P < 0.001). ATRA also promoted the CD44(+)CD24(-) subpopulation to differentiate. SK-BR-3 stem cells were grown in an adherent culture. After using ATRA, the proportion of CD44(+)CD24(-) cells was (48.1 ± 2.5)% and that of the control group was (86.6 ± 2.5)% (P < 0.001). ATRA effectively inhibits breast NCSCs and CSCs, but CSCs are more sensitive to ATRA. ATRA impairs the self-renewing ability of CSCs and promotes CSCs to differentiate.

Abstract P4-04-06: Histone H2A Inhibits Breast Cancer Stem Cell Growth

Abstract Introduction: Recent studies have identified epigenetic abnormalities as the underlying cause of many breast cancers. In particular, the aberrant acetylation of core histones may be a pivotal source of dysregulated expression of both oncogenes and tumour suppressor genes. Histone deacetylase inhibitors are known to restore histone acetylation and to induce cellular differentiation and growth arrest in breast cancer cells and are the object of extensive research for novel anticancer treatments. We hypothesize that acetylated proteins, in particular histone H2A, could also serve as an alternative substrate for histone deacetylase, thereby promoting cellular histone acetylation and arrest of cancer cell growth. Indeed, our previous results indicate that exogenous calf thymus histone H2A penetrates cells and their nuclei, and induces differentiation and senescence in human breast cancer cells. In contrast, human recombinant histone H2A and H2AX, which lack post-translational modifications, fail to yield these results in spite of the fact that human and bovine histone H2A are virtually identical in their amino acid sequences. However, these recombinant molecules were obtained from bacteria, which lack the intracellular machinery that is necessary to generate post-translational modifications. Thus, post-translational histone modifications are pivotal to the inhibition of breast cancer cell proliferation. Objective: We have extended our study to establish if exogenous calf thymus histone H2A could also induce growth arrest of breast cancer stem cells, and subsequently to determine which portion (s) of the histone H2A molecule is responsible for this action, using overlapping synthetic peptides. Materials and Methods: Breast cancer stem cells were incubated with exogenous calf thymus histone H2A and seven synthetic peptides corresponding to overlapping portions of the entire protein and include all of its post-translational modifications. Cellular morphology was observed and viable cells were counted using trypan blue exclusion. Control experiments were performed with normal breast cells, cells from fibrocystic breast tissues, and breast cancer cells that were not significantly enriched in cancer stem cells. Results and Discussion: Histone H2A inhibits breast cancer stem cell growth and increases their adhesion. Stem cells have recently been identified in many malignant tumours, including breast cancer. Since cancer stem cells are thought to be an important source of therapeutic resistance, they provide a novel research focus for alternative treatment strategies. Our results are very encouraging and may lead to the development of new pharmacological molecules targeting epigenetic abnormalities in breast cancer stem cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-06.

Obesity, hyperinsulinemia and breast cancer: novel targets and a novel role for metformin

The relationship between obesity, metabolic syndrome, diabetes and cancer has been recognized for many years. Multiple studies conducted in the last 20 years have identified molecular mechanisms responsible for this phenomenon. Elucidation of the important role of insulin, IGF receptor, mTOR and AMP-activated protein kinase in breast cancer biology has led to the development and subsequent clinical evaluation of novel targeted therapies, including IGF-1 receptor-specific antibodies or tyrosine kinase inhibitors and inhibitors of mTOR. There is also a growing interest in the use of metformin, which has been shown to possess antitumor activity resulting from activation of AMP-activated protein kinase and subsequent inhibiton of mTOR, as well as from decreased circulating insulin levels. Metformin has been shown to inhibit proliferation, invasion and angiogenesis of neoplastic cells and to overcome resistance of breast cancer to chemotherapy, hormonal therapy and HER2 inhibition. Recently, metformin has been demonstrated to inhibit breast cancer stem cell growth and to synergize with chemotherapy in suppression of tumor growth and prolongation of survival of breast tumor-bearing animals. Several currently ongoing Phase II and III clinical studies are evaluating the therapeutic efficacy of metformin in the treatment of early and advanced breast cancer patients.