Mammosphere Formation Efficiency Research Articles

The role of effective energy restriction on metastatic tumor growth

Obesity is a modifiable lifestyle factor known to exacerbate breast cancer risk, progression, and mortality. Conversely, energy restriction and weight loss are associated with a decreased breast cancer risk in women, and inhibit mammary tumor growth and metastasis in multiple tumor models. Although continuous energy restriction (CER) is the standard approach to weight loss, it is associated with poor patient adherence. Intermittent energy restriction (IER) is a dietary regimen whereby periods of energy restriction are interspersed with periods of normal food intake. IER is as effective or even superior to CER in achieving weight loss, and enjoys a higher level of patient compliance, rendering IER a promising lifestyle intervention. The epithelial‐mesenchymal transition (EMT) plays important roles in breast cancer invasion and metastasis. The induction of EMT elicits the conversion of sessile epithelial carcinoma cells into intrinsically motile mesenchymal counterparts, and bestows cancer stem cell (CSC) traits. EMT is accompanied by extensive metabolic reprogramming including enhanced aerobic glycolysis and increased expression of glycolytic pathway components. Recent evidence suggests that the EMT‐inducing transcription factors TWIST and SLUG promote both EMT and aerobic glycolysis. Here, we hypothesize that EMT may be regulated by energy balance, and that energy restriction may be leveraged to inhibit EMT and curtail breast cancer progression. We aim to investigate the effects of IER on cell differentiation, EMT, and metastasis in triple negative breast cancers (TNBCs), which are enriched for EMT features. MDA‐MB‐231‐IER and SUM159‐IER TNBC cells, cultured under an IER regimen, exhibited an epithelial‐like phenotype, and decreased invasiveness and migratory potential, compared with controls. Immunoblotting and immunofluorescence showed decreased protein levels of TWIST and SLUG in IER‐treated cells, compared with controls, while mRNA levels were unchanged. IER‐treated cells also exhibited decreased expression of the CSC‐associated cell surface marker, CD44, and a markedly reduced mammosphere‐forming efficiency, indicative of diminished self‐renewal capacity. Accordingly, tumor formation was compromised in mice injected with MDA‐231‐IER cells relative to controls. IER‐treated cells displayed intense punctate staining for the autophagosome marker LC3‐II suggesting that IER may stimulate autophagy‐mediated TWIST and SLUG degradation. We are currently evaluating the impact of the IER regimen on the metastatic potential of TNBC cells and their response to standard‐of‐care drugs, and investigating the molecular mechanisms underpinning the inhibition of EMT by IER. Understanding how to leverage IER to curtail cancer progression, and identifying the molecular mechanisms underlying IER‐dependent cancer preventive effects may lead to novel drugs and intervention strategies for the prevention and treatment of metastatic breast cancers, including TNBCs.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Doxycycline targets aldehyde dehydrogenase‑positive breast cancer stem cells.

Targeting cancer stem cells (CSCs) is a key strategy to prevent cancers from developing drug resistance and metastasis. Mitochondria have been reported to be a vulnerability of CSCs by multiple studies. Here, we report that doxycycline, functioning as an inhibitor of mitochondrial biogenesis, can effectively target breast cancer stem cells (BCSCs). Our results revealed that doxycycline significantly decreased the frequency of aldehyde dehydrogenase‑positive (ALDH+) BCSCs as well as mammosphere formation efficiency in HER2+ and triple‑negative breast cancer (TNBC) subtypes. Doxycycline also ameliorated paclitaxel‑induced enrichment of ALDH+ BCSCs in TNBC. Mechanistically, we showed that doxycycline decreased the level of reactive oxygen species and their downstream p38MAPK pathway. In agreement with the key role for p38 in maintaining BCSCs, a specific inhibitor targeting this MAPK pathway significantly decreased the number of ALDH+ cells. Doxycycline is a FDA‑approved drug with minor and limited side‑effects. Given doxycycline's low toxicity and strong effect on BCSC inhibition, we report that doxycycline should be safe to be used concomitantly with chemotherapy drugs to eradicate both CSCs and bulk tumor cells.

WITHDRAWN: Tannic acid, a secondary metabolite of higher plants, inhibits mammosphere formation of breast cancer cells via modulation of NF-κB pathway

// Dal-Ah Kim 1, 2 , Hack Sun Choi 1, 2 , Eun-Sun Ryu 1, 2 , Jiyeon Ko 1, 2 , Heesung Chung 3 , Eun-Sung Jun 4 , Eok-Soo Oh 3 and Duk-Hee Kang 1, 2 1 The Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul 07985, Republic of Korea 2 Ewha Medical Research Center, Ewha Womans University School of Medicine, Seoul 07985, Republic of Korea 3 Department of Life Science, The Research Center Homeostasis, Ewha Womans University, Seoul 03760, Republic of Korea 4 Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, South Korea Correspondence to: Duk-Hee Kang, email: dhkang@ewha.ac.kr Keywords: cancer stem cells; tannic acid; mammosphere; NF-kB signaling; p65 Received: October 16, 2017      Accepted: January 03, 2018      Epub: January 02, 2018 ABSTRACT Cancer stem cells (CSCs) are known to mediate metastasis and recurrence, and are therefore a promising therapeutic target. Likewise, aberrant NF-κB signaling has been identified in many cancers, and shown to promote CSC formation via several mechanisms, including induction of epithelial-to-mesenchymal transition (EMT). Tannic acid (C76H52O46, TA) is reported to inhibit the proliferation of cancer cells, however there is no data regarding the effect of TA on CSCs. The present study investigated the effects of TA on CSC formation, NF-κB signaling, and EMT in breast cancer cells. TA inhibited the formation and growth of mammosphere in MCF7 cells expressed as a decrease in mammosphere formation efficiency (MFE) and ALDH1 activity. An activation of NF-κB pathway was observed in MCF7-derived mammosphere indicated by an up-regulation of p65, a degradation of IκBα and an increased IL-6. The inhibition of NF-κB pathway via gene silencing of p65 (sip65), NF-κB inhibitor (PDTC) and IKK inhibitor (Bay11-7082) alleviated MFE. Other markers of CSCs such as an increase in ALDH1 and the number of CD44 high /CD24 low cells were ameliorated by interfering p65. TA alleviated the markers of NF-κB activation in MCF7-derived mammosphere. TGFβ-induced EMT, increase in MFE and NF-κB activation was alleviated by TA. In in-vivo murine xenograft model, tumor volume was decreased by TA with a decrease in CD44 and IKK phosphorylation. Taken together, these results strongly suggest TA as a promising potential therapeutic agent capable of inhibiting NF-κB signaling in breast CSCs, and thereby preventing cancer cells from undergoing EMT and subsequent metastasis.

Abstract 4769: Targeting HER2 enriches Jagged1 high cancer stem cells in breast cancer

Abstract The human epidermal growth factor receptor 2 (HER2) positive subtype of breast cancer is characterized by gene amplification and/or protein overexpression of HER2. It is driven by a subpopulation of cells possessing stem cell properties of self-renewal and differentiation, known as Cancer Stem cells (CSCs). CSCs are implicated in tumor growth as well as radiotherapy and chemotherapy associated resistance. Notch receptors promote breast CSCs survival and self-renewal, and overexpression of a Notch ligand Jagged1 mRNA predicts poor prognosis in women with breast cancer. Our lab has published that Jagged1 or Notch1 is a critical target in trastuzumab/lapatinib (LAP) resistant HER2+ breast cancer. The study aimed to determine whether anti-HER2 therapy selects for Jagged-1/Notch-dependent CSCs that are responsible for tumor initiation. Surface expression of Notch1 and Jagged1 upon HER2 blockade using LAP was measured in HER2+ breast cancer cell lines (MDA-MB-453 and HCC1954) using flow cytometry. LAP treatment increased the Jagged1-positive subpopulation compared to vehicle. Cells were sorted based on Jagged1 cell surface expression and assessed for CSC-like properties (i.e. mammosphere forming efficiency, Aldefluor activity, expression of CD44high /CD24low, Notch target transcripts expression, and limiting dilution tumor initiating potential in athymic, nude mice). In addition, immunohistochemistry was performed on 145 HER2+ breast tumor microarray to detect cytoplasmic, membrane, or nuclear Jagged1 protein expression. Kaplan-Meier analysis was performed to determine overall survival. The results showed that upon HER2 inhibition, Jagged1 cell surface expression increased and Notch1 cell surface expression was unchanged. The Jagged1high subpopulation of cells showed elevated levels of Aldehyde dehydrogenase activity, Notch target gene transcripts, and mammosphere formation efficiency compared to vehicle treated cells. The MRK-003 γ-secretase inhibitor (GSI) prevented mammosphere formation in the Jagged1high cells indicating that Notch activation drives Jagged1high CSC survival. Also, we confirmed that Jagged1 expression is required for the enrichment of CSCs using a Jagged1 siRNA. Combined knockdown of Notch1 and Notch3 receptors was necessary to reduce LAP-enriched mammospheres suggesting that targeting HER2 enriches for a Jagged1-Notch1+Notch3 driven CSC phenotype. Importantly, higher membrane expression of Jagged1 protein in 145 HER2+ breast tumor specimens correlated with significantly lower overall cumulative survival. These results reveal that HER2 blockade in breast cancer cells enriches for a Jagged1high subpopulation that has higher CSC potential and is resistant to HER2 inhibitors. The implications of this work are that dual blockade of Jagged-1/Notch1/Notch3 and HER2 could be more effective than either therapy alone to eliminate both HER2 and Jagged-1-dependent cancer cells. Citation Format: Deep S. Shah, Debra Wyatt, Andrew Baker, Andrew Green, Aleksandra Filipovic, Lucio Miele, Clodia Osipo. Targeting HER2 enriches Jagged1 high cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4769. doi:10.1158/1538-7445.AM2017-4769

Abstract 4915: Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells

Abstract Background and Objective: The epidemiological and experimental studies suggest that alcohol consumption is associated with increased risk for breast cancer development and metastasis. However, the mechanisms of alcohol-induced breast cancer progression and metastasis remain unknown. In this study, we examined the roles of CCN5, a tumor suppressor gene in alcohol induced breast cancer cell migration/invasion, epithelial to mesenchymal transition (EMT) and stemness. Methods: Estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 and immortalized normal human epithelial breast cell line MCF-12A were treated with ethanol at various concentrations in the presence and absence of human recombinant human CCN5 protein (hrCCN5) . Invasive/migratory ability of treated and untreated cells was measured by Boyden chamber assays. The mRNA and protein expression level of CCN5 was determined by real-time qRT-PCR and Western blot. EMT and stemness markers were evaluated by Western blot. Single-cell suspensions from pre-treated cells were re-suspended at a density of 500 cells/ml mammocult media in ultralow attachment dishes. Number as well as the size of the mammosphere in specified experimental set-up was monitored and recorded alternate day for 8-10 days. Results: The studies demonstrated that alcohol promotes significantly the invasive/migratory ability and EMT phenotypes of breast cancer cells through the suppression of CCN5. Interestingly, hrCCN5 protein treatment suppresses the effect of alcohol and reprograms MET (mesenchymal to epithelial transition). In addition, alcohol-induced mammosphere formation efficiency is also suppressed by hrCCN5 protein-treatment. Conclusions: Our findings suggest that the CCN5 signaling plays a preventive role in alcohol-induced reprogramming of tumor plasticity in breast cancer cells. Citation Format: Inamul Haque, Arvind Subramanian, Vijayalaxmi Gupta, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4915. doi:10.1158/1538-7445.AM2017-4915

Abstract 3922: NR2F1 limits dissemination and stemness of early breast cancer cells

Abstract Tumor cells derived from MMTV-Neu mouse models or human ErBb2+ primary tumors are known to be able to complete all steps of the metastatic cascade. However, new evidence show that Neu+ early cancer cells derived from early stages of tumor progression (before detection of palpable tumors), can disseminate and, as disseminated cancer cells (early DCCs), enter a non-proliferative state for prolonged periods of time at secondary organs. We recently showed that early DCCs contributed to metastasis formation together with late-arriving DCCs (those coming from overt tumors). However, the mechanisms that would allow these early cancer cells, considered sessile and with few genetic alterations, to complete all steps of metastasis are unknown. Here we report that downregulation of the orphan nuclear receptor NR2F1 in Neu+ early cancer mammary epithelial cells (MECs) further activated a motile phenotype and this was coincident with previously described detection of circulating cancer cells (CCCs) and lung and bone marrow DCCs in MMTV-Neu mouse model. Knock down of basal NR2F1 levels in Neu+ early cancer cells induced cell motility, loss of laminin-V deposition, β-catenin delocalization from the membrane, dramatic loss of E-cadherin junctions and increased percentage of leader cells positive for CK14 and PRRX1 staining. Interestingly, TWIST and PRRX1 levels became upregulated upon NR2F1 depletion in 3D structures. These results suggest that NR2F1 expression maintains epithelial identity and suppresses epithelial-mesenchymal transition (EMT). Knock down of NR2F1 in Neu+ early cancer cells enhanced mammosphere formation efficiency and this was accompanied by upregulation of the pluripotency transcription factor NANOG and EMT master regulator TWIST. Interestingly, early dormant DCCs (negative for proliferation markers) showed re-expression of NR2F1 levels while micrometastases (cycling cells) had reduced levels of NR2F1. This result suggests that downregulation of NR2F1 in early DCCs may contribute to the dormancy escape of early DCCs and consequently metastatic relapse. Our findings provide for the first time evidence that NR2F1 functions as a suppressor of stemness and dissemination during early stages of tumor progression. We propose that therapies that restore expression of NR2F1 might limit early dissemination and the progression of early and late-arriving DCCs toward metastatic outgrowth. Citation Format: Maria Soledad Sosa. NR2F1 limits dissemination and stemness of early breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3922. doi:10.1158/1538-7445.AM2017-3922

Abstract PD2-02: SFX-01 targets Wnt signalling to inhibit stem-like cells in breast cancer patient-derived xenograft tumours

Abstract Background: SFX-01 is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within a-cyclodextrin. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are likely drivers of breast cancer metastasis and treatment resistance. We recently established that CSC activity in ER+ BC, represent a source of therapeutic resistance (Simões et al, Cell Reports, 2015). Material and methods: We investigated SFX-01 effects on breast CSC activity using mammosphere formation efficiency (MFE) and aldehyde dehydrogenase (ALDH) activity using the ALDEFLUOR assay in patient samples and patient-derived xenograft (PDX) tumours. Cells from primary (n=12) and metastatic (n=15) samples were treated with SFX-01 (5 μM) or vehicle control.Using a 2 or 8 week in vivo treatment, early (HBCx34) and metastatic (BB3RC31) ER+ PDX tumours were treated with SFX-01 (300mg/Kg/day) alone or in combination with tamoxifen (TAM, 10 mg/kg/day) or fulvestrant (FULV, 200 mg/kg/week). Tumours were dissociated and MFE and ALDH activity assessed. Results: SFX-01 in vitro reduced MFE of both primary (0.19%±0.02 vs control 0.52%±0.06: p<0.001) and metastatic patient samples (0.43±0.04 vs control 0.93%±0.07: p<0.001). SFX-01 treatment in vivo for 2 weeks reduced MFE of HBCx34 (0.35%±0.03 vs control 0.64%±0.09; p<0.01) and BB3RC31 (0.78%±0.04 vs control 0.89%±0.06: p<0.05) and also ALDH activity of HBCx34 (3%±0.6 vs control 6.3%±0.4: p<0.01) and BB3RC31 (1%±0.2 vs control 3%±0.6: p<0.05). TAM and FULV increased MFE and ALDH activity after 2 weeks of treatment in vivo, which was abrogated by combination with SFX-01; for example HBCx34 MFE with TAM alone: 0.81%±0.07 vs TAM+SFX-01: 0.34%±0.02 (p<0.01) and ALDH+ with TAM alone 10%±0.4 vs TAM+SFX 4.2%±0.4 (p<0.01). TAM+SFX-01 suppressed tumour growth at 8 weeks vs TAM alone in HBCx34 but not BB3RC31. FULV treatment maintained tumour growth suppression at 8 weeks and no additive effect was seen with SFX-01, although MFE and ALDH activity were suppressed. Mechanistically, SFX-01 potently inhibited the canonical Wnt pathway in MCF-7 cells and their endocrine-resistant derivatives and we are currently exploring SFX-01 activity on other CSC regulatory pathways. SFX-01 has been shown to be well tolerated in SAD and MAD studies in normal volunteers and clinical studies designed to test tolerability and efficacy in combination with the three major classes of endocrine therapy (AI, TAM and FULV) in advanced BC will begin in Q4 2016. Conclusions: Our data demonstrate the potential of SFX-01 for clinically meaningful improvements to endocrine therapy in ER+ breast cancer by reversing CSC mediated resistance. Citation Format: Howell SJ, Simoes BM, Alferez D, Eyre R, Spence K, Santiago-Gomez A, Sarmiento-Castro A, Tanaka I, Howat D, Clarke RB. SFX-01 targets Wnt signalling to inhibit stem-like cells in breast cancer patient-derived xenograft tumours [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-02.

Tissue Factor promotes breast cancer stem cell activity in vitro.

Cancer stem cells (CSCs) are a subpopulation of cells that can self-renew and initiate tumours. The clotting-initiating protein Tissue Factor (TF) promotes metastasis and may be overexpressed in cancer cells with increased CSC activity. We sought to determine whether TF promotes breast CSC activity in vitro using human breast cancer cell lines. TF expression was compared in anoikis-resistant (CSC-enriched) and unselected cells. In cells sorted into of TF-expressing and TF-negative (FACS), and in cells transfected to knockdown TF (siRNA) and overexpress TF (cDNA), CSC activity was compared by (i) mammosphere forming efficiency (MFE) (ii) holoclone colony formation (Hc) and (iii) ALDH1 activity. TF expression was increased in anoikis-resistant and high ALDH1-activity T47D cells compared to unselected cells. FACS sorted TF-expressing T47Ds and TF-overexpressing MCF7s had increased CSC activity compared to TF-low cells. TF siRNA cells (MDAMB231, T47D) had reduced CSC activity compared to control cells. FVIIa increased MFE and ALDH1 in a dose-dependent manner (MDAMB231, T47D). The effects of FVIIa on MFE were abrogated by TF siRNA (T47D). Breast CSCs (in vitro) demonstrate increased activity when selected for high TF expression, when induced to overexpress TF, and when stimulated (with FVIIa). Targeting the TF pathway in vivo may abrogate CSC activity.

Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial–mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

Abstract 2124: Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer

Abstract ErbB-2 (HER2) is over expressed in 15 - 25% of breast cancer. First line therapy for HER2+ breast cancer is a humanized, monoclonal antibody, trastuzumab. Resistance is a major clinical problem. Previous work has suggested that Notch-1 is critical for resistance. PTEN, a breast tumor suppressor and potent inhibitor of the PI3-K/AKT is decreased in HER2+ breast cancer and loss of PTEN predicts for worst outcome. Notch-1 downregulates PTEN expression and we sought to determine the mechanism by which Notch-1 attenuates PTEN and whether this decrease is necessary for trastuzumab resistance. Our hypothesis is that Notch-1 decreases PTEN to maintain trastuzumab resistance. Human HER2+ breast cancer cell lines: sensitive (BT474HS) or resistant (BT474HR and HCC1954) to trastuzumab were used to measure Notch-1 modulation of PTEN expression when treated with trastuzumab. Protein and transcript expression of PTEN were measured by Western blot and RT-PCR, respectively. ChIP assays were preformed to identify whether Notch-1 regulated PTEN expression was mediated by direct recruitment of Notch-1 to the PTEN promoter. Growth and mammosphere forming assays were performed to determine if trastuzumab resistance could be rescued in trastuzumab treated cells with simultaneous Notch-1 and PTEN knockdown, or increased downstream PI3K pathway activity using a constitutive active form of AKT1 (Myr-AKT1). Western blot and RT-PCR results showed that Notch-1 protein levels are increased while PTEN mRNA and protein levels are decreased in trastuzumab resistant cells (BT-474HR) compared to sensitive (BT-474HS) cells. Results revealed that Notch-1 knockdown increased PTEN transcript and protein expression. Notch-1 ChIP assays demonstrated that Notch-1 was enriched on the PTEN promoter and a Notch target gene promoter, HEY1 in resistant cells. Notch1 knockdown significantly decreased proliferation and mammosphere forming efficiency (MFE) of resistant cells regardless of trastuzumab treatment. PTEN knockdown rescued the decrease in proliferation and MFE mediated by Notch-1 knockdown. As PTEN is an attenuator of PI3-K/AKT signaling, we asked whether constitutive activation of AKT1 was sufficient to rescue proliferation upon Notch-1 knockdown. The results showed that myr-AKT1 increased AKT1 signaling and rescued the decrease in proliferation mediated by Notch-1 knockdown. These results demonstrate, to our knowledge, for the first time that Notch-1 is a direct transcriptional repressor of PTEN in HER2+ breast cancer cells. Increased activation of the PI3K pathway through Notch-1 mediated inhibition of PTEN is necessary to maintain proliferation and possibly survival of the cancer stem cell population of trastuzumab resistant cells. Inhibiting the expression and/or activity of Notch-1 in HER2+ breast cancer could prove to be an effective therapy to prevent expansion of cancer stem cells and/or re-sensitize HER2+ breast cancer to ant-HER2 therapy. Citation Format: Andrew T. Baker, Kinnari Pandya, Clodia Osipo. Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ 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 2124.

Abstract 3076: Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer

Abstract The expression and activities of the Rho GTPases Rac and Cdc42, and their downstream effector P21-activated kinase (PAK), have been correlated with metastatic cancer. Our published studies with the Rac/PAK inhibitor EHop-016 demonstrate the validity of using Rac inhibitors as anti metastatic cancer therapeutics. However, the relatively high effective concentrations (Rac activity IC50 1μM, tumor inhibition at 25 mg/kg body weight (BW)), and the moderate bioavailability (∼30%, t1/2 4.5h) of EHop-016 need improvement. Therefore, we developed EHop-016 derivatives and identified EHop-167 as a Rac inhibitor at nM concentrations. Unlike EHop-016, which does not substantially change breast cancer cell shape, but only reduces cell surface actin based invadopodia, EHop-167 induced a marked decrease in breast cancer cell polarity, cell surface extensions, and cell-extracellular matrix (ECM) attachments (focal adhesions). This phenotype of cell rounding and detachment in response to EHop-167 was demonstrated only by breast cancer cell lines that have undergone epithelial to mesenchymal transition, but not by epithelial breast cancer cells, or MCF-10A mammary epithelial cells. As assessed by pulldown assays and western blotting, Rac and PAK activities were reduced by 80-90% in response to 250 nM EHop-167, in the detached cells. As demonstrated by caspase assays, the cell rounding and detachment from the ECM ultimately resulted in anoikis (cell death due to loss of focal adhesions). Accordingly, Transwell assays of mesenchymal breast cancer cells following 250 nM Ehop-167 showed a ∼90% reduction in cell migration in the detached breast cancer cells, and a ∼60% inhibition in the attached cells. EHop-167 also reduced the mammosphere formation efficiency of metastatic cancer cells by 50%, indicating an inhibitory effect on cancer stem cells. To determine the in vivo efficacy of EHop-167, athymic nude mice, bearing mammary fatpad tumors of MDA-MB-435 metastatic cancer cells, were treated 3X a week with 0, 1, or 10 mg/kg BW EHop-167 for 50 days. Treatment with 1.0 mg/kg BW EHop-167 resulted in a 50% reduction in tumor growth, while 10.0 mg/kg BW EHop-167 induced an ∼95% reduction in tumor growth, compared to controls. Additionally, these mice did not show gross signs of toxicity or significant weight loss. Since the parental compound EHop-016 has no anticancer effects at similar concentrations, we conclude that EHop-167 is an improved Rac/PAK inhibitor that holds promise as an anti metastatic breast cancer therapeutic. Citation Format: Linette Castillo-Pichardo, Tessa Humphries-Bickley, Ingrid Forrestier-Roman, Luis Borrero-Garcia, Fabiola Pagan Melendez, Eliud Hernandez, Cornelis Vlaar, Luis A. Cubano, Surangani Dharmawardhane. Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in 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 3076.

Abstract LB-121: DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ breast cancer

Abstract Breast cancer is a heterogeneous disease that is best treated based on the expressed biomarker profile. Currently, the major clinical challenges are drug resistance and metastatic spread. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and disease progression despite therapy. We previously identified novel biomarkers that could potentially be used with novel therapeutic strategies that target CSCs. We demonstrated in a presurgical window biomarker study of 20 breast cancer patients with ER-positive disease that signaling through Notch receptors mediates expression of 18 genes. Twenty women with ER-positive tumors were treated with 14 days of ET (tamoxifen or letrozole), with the addition of the oral GSI MK-0752 on day 15 (3 days on, 4 days off, 3 days on). Definitive surgery was on day 25. Analysis of biomarkers using microarray and RT-PCR during ET combined with MK-0752 GSI revealed statistically significant modulation of 18 genes: pro-apoptotic DAXX and NOXA; a tumor suppressor LFNG; Notch signaling such as NOTCH1, NOTCH4, HEYL, HES1, and HEY2; as well as proliferation-associated transcripts MIK67, CCND1, CCNA2; stem cell markers RUNX1 and ALDH1; and novel genes such as RICTOR, RPTOR, MMP7, ADAM19, and PgR (Albain et al. Proc SABCS 2014). The goal of the current study was to identify whether Notch1 directly regulated the 18 identified genes. We measured binding of Notch1 to CBF-1 (CSL/RPBJκ) binding sites of the 18 genes using a Chromatin Immunoprecipitation assay (ChIP) to determine whether Notch1 directly regulates the 18 genes. We scanned and designed primers 5 kb upstream and downstream of transcription start sites of the 18 genes and demonstrated that Notch1 was recruited to CBF-1 sites of 16 out of 18 genes. Different distribution of Notch1 binding was observed across all of the genes. Classical Notch targets, HEY1 and HES1 were among the most responsive genes and used as positive controls. Notch1 was found to be highly enriched on CBF-1 regulatory elements for the DAXX and NOXA genes. Binding of Notch1 to DAXX promoter elements increased in response to estrogen deprivation and this increase was attenuated upon GSI treatment. The biological activity of most genes was measured using the mammosphere-forming assay as a surrogate for CSC survival. Estrogen deprivation increased mammosphere-forming efficiency of ER+ breast cancer cells more than 2 fold compared to estrogen treatment. GSI blocked mammosphere formation by 95%-98% in response to anti-estrogen treatment. DAXX expression, was found to be necessary for GSI-mediated blockade of mammosphere formation. ChIP and mammosphere forming data calculated by ANOVA were found to be statistically significant. These results demonstrated that Notch1 is a direct transcriptional regulator of 16 genes identified by the clinical trial and in particular, DAXX, a pro-apoptotic gene that could serve as a cancer stem cell biomarker for anti-Notch therapy in ER+ breast cancer. Supported by a grant from the Breast Cancer Research Foundation. Citation Format: Andrei Zlobin, Debra Wyatt, Jeffrey C. Bloodworth, Susan Hilsenbeck, Suzanne Fuqua, Lucio Miele, Kathy S. Albain, Clodia Osipo. DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ 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 LB-121.

Histone deacetylase inhibitor-induced cancer stem cells exhibit high pentose phosphate pathway metabolism

PurposeWe recently demonstrated that histone deacetylase (HDAC) inhibitors can “reprogram” differentiated triple-negative breast cancer cells to become quiescent stem-like cancer cells. We hypothesized that the metabolic state of such cells differs from that of their differentiated progeny.ResultsIn untreated cells, glucose uptake was higher in ALDH+ cells than in ALDH− cells (p = 0.01) but lactate production was not different; treating ALDH− or ALDH+ cells with VA or SAHA similarly increased glucose uptake without changing lactate production but upregulated G6PD, a rate-limiting enzyme in pentose phosphate pathway metabolism. NADPH production was higher in HDAC inhibitor-treated stem-like cells than in vehicle-treated cells (p < 0.05). Two G6PD inhibitors, 6-aminonicotinamide and dehydroepiandrosterone, decreased mammosphere formation efficiency and ALDH activity and 6-aminonicotinamide reduced the VA-induced increase in ALDH+ cells. Finally, patients expressing high G6PD mRNA had significantly worse overall survival (p < 0.001), and patients with high G6PD protein showed a similar trend towards worse disease-specific survival (p = 0.06).MethodsGlucose consumption, lactate and NADPH production, and reactive oxygen species generation were compared in aldehyde dehydrogenase (ALDH)-positive and –negative cells in the presence or absence of the HDAC inhibitors valproic acid (VA) or suberoylanilide hydroxamic acid (SAHA). Glucose-6-phosphate dehydrogenase (G6PD) expression was evaluated in a tissue microarray from 94 patients with node-positive invasive breast carcinoma and in two publically available databases and correlated with overall survival.ConclusionsEnergy metabolism in HDAC inhibitor-induced stem-like cancer cells differed sharply from that of differentiated cell types. HDAC inhibitor-induced dedifferentiation promoted metabolic reprogramming into the pentose phosphate pathway, which is targeted effectively by G6PD inhibition. These findings highlight a potential dual-therapy approach to targeting bulk differentiated cells with HDAC inhibitors and CSCs with G6PD inhibitors.

PO-11 - Thrombin and cancer stem-like cells: in vitro support for breast cancer anticoagulation

Cancer stem-like cells (CSCs) are tumour initiating, resistant to chemotherapy and play a role in metastasis. We have previously shown thrombin pathway signalling regulates CSC activity: thrombin pathway activation with FVIIa or through TF overexpression increases CSC activity whilst TF siRNA knockdown decreases it. We hypothesise that NOACs, by targeting specific factors in the thrombin pathway, may inhibit CSC activity. In breast cancer cell lines, to determine the effect of i) thrombin and ii) dabigatran (Boehringer Ingelheim), a direct thrombin inhibitor on cancer stem cell activity. MDA-MB-231, MCF-7, SKBR3 and MDA-MB-157 cell lines representing the spectrum of breast cancer subtypes were cultured with: i) 0.1 NIH Units/ml human thrombin (Sigma) ii) 0.5μM Dabigatran (clinically relevant plasma concentration). Mammosphere culture is an established in vitro assay to measure CSC activity. Mammosphere forming efficiency (MFE) was calculated from the proportion of plated cells forming mammospheres in non-adherent culture, (each experiment, n=≥4, two-tailed independent t-test). Thrombin increased MFE in the high PAR-1 expressing MDA-MB-231 and MDA-MB-157 cell lines, but not the low PAR-1 expressing MCF-7/SKBR3 cell lines as compared to untreated controls (MDA-MB-231 mean (range): thrombin treated: 0.91 (0.70-1.11) vs control: 0.73 (0.51-0.93)%, p=<0.04; MDA-MB-157: thrombin treated: 0.58 (0.45-0.69) vs control: 0.37 (0.31-0.47)%, p=<0.001). Dabigatran abrogated the stimulatory effect of thrombin on MFE in thrombin-treated MDA-MB-231 and MDA-MB-157 cells (MDA-MB-231 mean (range) thrombin+Dabigatran: 1.10 (0.83-1.41) vs thrombin only: 1.45 (1.32-1.66)%, p=<0.01); MDA-MB-157 thrombin+Dabigatran: 0.48 (0.39-0.56) vs thrombin only: 0.58 (0.45-0.69)%, p=<0.05). The stimulation of mammosphere formation by thrombin and reduction by thrombin inhibitor treatment indicates a functional relationship between cancer stem-like cells and coagulation. This suggests possible clinical utility of novel oral anticoagulants in targeting this critical cancer cell subpopulation.

Abstract B35: NR2F1 limits early dissemination and phenotypic plasticity of early-progressed ErbB2+ tumor cells by maintaining luminal differentiation

Abstract Tumor cells derived from MMTV-Neu primary tumors (tumor-derived cells, TDC) are known to be able to complete all steps of the metastatic cascade. However, new evidence shows that cells derived from early stages of tumor progression (before detection of palpable tumors, ErbB2+ early-progressed tumor cells, EPTC) can disseminate and, as disseminated tumor cells (early DTCs), enter a non-proliferative state for prolonged periods at secondary organs. When EPTC are co-cultured with TDC, the tumor sphere formation capacity of the later is increased suggesting a synergistic contribution of early-progressed tumor cells to the growth of TDC. Thus, early DTCs are proposed to contribute to metastasis, but the mechanisms that would allow these cells, considered sessile and with few genetic alterations, to complete all steps of metastasis are unknown. Here we report that the orphan nuclear receptor NR2F1 is downregulated in human pre-malignant and malignant lesions and mouse ErbB2+ EPTC and TDC when compared with normal mammary tissue. In ErBb2+ EPTC further downregulation of NR2F1 activates a motile phenotype and this is coincident with the detection of circulating tumor cells (CTCs) and DTCs in lungs and bone marrow. Knock down of basal NR2F1 levels in ErBb2+ EPTC induced cell motility, loss of laminin-V deposition, β-catenin delocalization from the membrane and dramatic loss of E-cadherin junctions. Interestingly, TWIST, SMAD2, CK14 and ROR1 levels became upregulated upon NR2F1 depletion. This anti-EMT NR2F1-regulated program is ErbB2 oncogen-dependent. These results suggest that NR2F1 expression maintains epithelial identity and suppresses epithelial-mesenchymal transition (EMT), possibly by blocking WNT signaling. Knock down of NR2F1 in ErBb2+ EPTC also enhanced mammosphere formation efficiency and this was accompanied by upregulation of the pluripotency transcription factor NANOG and TWIST. These findings suggest that NR2F1 limits the spreading of ErBb2+ EPTC cells and it promotes a luminal differentiation-associated program by limiting the activation of a stem cell-like program. Our findings provide for the first time evidence that NR2F1 functions in mammary epithelial cells as a suppressor of pluripotency and dissemination during early stages of tumor progression. We propose that therapies that might restore expression of NR2F1 might limit early dissemination and the progression to metastasis of already disseminated tumor cells. Citation Format: Maria Soledad Sosa, Julie Cheung, Julio A. Aguirre-Ghiso. NR2F1 limits early dissemination and phenotypic plasticity of early-progressed ErbB2+ tumor cells by maintaining luminal differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B35.

Abstract P3-06-15: Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer

Abstract Background: Breast cancer is the second leading cause of cancer-associated death in women. Triple negative breast cancer (TNBC) accounts for 10-15% of breast cancer, lacks expression of ER, PR, and HER2 gene amplification. Due to lack of targeted therapy, TNBC has the worst prognosis. TNBC is a heterogeneous disease with six distinct subtypes. Current treatment includes combination of surgery, radiation therapy and chemotherapy. Notch signaling is increased in TNBC and predicts for worst overall outcome. The goal of the study is to identify novel Notch-biomarkers that predict sensitivity of subtypes of TNBC to Notch inhibition in both bulk and cancer stem cells. Methods: Two subtypes of TNBC, Mesenchymal stem-like MDA-MB-231 and basal like MDA-MB-468 cells were used. Notch signaling was evaluated using both RNA-sequencing and quantitative real time PCR (q-RTPCR). Cancer stem cell markers were evaluated using Aldefluor assay, CD44 high/ CD24 low expression, and mammosphere forming assay. To study the effect of chemotherapy and Notch, MDA-MB-231 and MDA-MB-468 cells were treated with carboplatin or a pan Notch inhibitor- gamma secretase inhibitor (GSI) or combination of both and cell viability was measured using the trypan blue exclusion test. Results: The RNA-seq and RT-PCR results showed that Notch3 is differentially expressed in the two cell lines. Notch3 was knocked down using siRNA and its effect on cell viability was assessed after GSI or carboplatin or combination treatment. MDA-MB-468 cells had higher Notch receptors and activity, most notably Notch3. MDA-MB-468 cells had high levels of Aldefluor whereas MDA-MB-231 cells showed higher levels of CD44 High/CD24 Low expression. MDA-MB-468 cells had higher mammosphere forming efficiency (MFE) compared to MDA-MB-231. Notch inhibition decreased MFE of MDA-MB-468 whereas it increased MFE of MDA-MB-231 cells. MDA-MB-468 cells have lower IC50 for carboplatin or GSI compared to MDA-MB-231 cells, as measured by cell viability assay. However, combination treatment lowered IC50 for GSI in MDA-MB-231 cells as compared to MDA-MB-468 cells. Since Notch3 levels were strikingly different between the two cell lines, we hypothesized that Notch3 levels are necessary for GSI sensitivity. Carboplatin treatment increased Notch3 in MDA-MB-231 cells and the increase in Notch3 could be the reason for lower IC50 of GSI during combination treatment as increased Notch3 would provide the substrate for gamma-secretase. In MDA-MB-468 cells, Notch3 knockdown significantly reduced cell viability and was similar to GSI, suggesting that GSI acts through Notch3. Furthermore, Notch3 knockdown and carboplatin treatment reduced viability comparable to carboplatin and GSI treatment, reinforcing that the GSI acts through Notch3 in TNBC. Using the Kaplan-Meier Plotter, high Notch3 predicted poor recurrence free survival post chemotherapy in patients with ER-, HER2-, basal breast cancer. Conclusions: GSI acts through Notch3 in two TNBC subtypes and combination of chemotherapy with Notch inhibition results in a better outcome as compared to either drug alone. Future experiments would elucidate the role of Notch3 inhibition in targeting cancer stem cells post chemotherapy treatment in different subtypes of TNBC. Citation Format: Shah D, Osipo C. Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-15.

Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells.

BackgroundDeveloping novel strategies against treatment-resistant triple negative breast cancer (TNBC) cells remains a significant challenge. The ErbB family, including epidermal growth factor receptor (EGFR), plays key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSC) which are believed to be responsible for tumor initiation and maintenance. Ixabepilone is a new generation microtubule-stabilizing agent, which has been expected to be more efficacious than conventional taxanes. Here we aim to investigate whether the EGFR monoclonal antibody Cetuximab, in combination with Ixabepilone, is more effective in eliminating CSC populations compared to chemotherapy alone in TNBC.MethodsRepresentative TNBC cell lines (MDA-MB-231 and SUM159) were used to evaluate breast CSC populations. We used fluorescence-activated cell sorter analysis (CD44+ and CD24-/low, or Aldefluor+) and a self-renewal assay called mammosphere formation efficiency (MSFE) to measure CSC population size after treatment with Cetuximab, or Cetuximab plus Ixabepilone in vitro.ResultsAlthough there was no significant decrease in cell viability, Cetuximab reduced MSFE and the CSC population in breast cancer cells in vitro and in vivo through inhibition of autophagy. Also, SUM159 and MDA-MB-231 orthotopic tumors demonstrated partial response to Centuximab or Ixabepilone monotherapy; however, the effect of the combination treatment was significant only in SUM159 tumors (p <0.0001), when compared to Ixabepilone alone.ConclusionsOverall, our findings demonstrate that EGFR-targeted therapy by Cetuximab effectively reduces the CSC population in TNBC tumors. However, combination therapy with Ixabepilone may be effective only in a small subset of TNBCs, warranting further investigation of alternative approaches to target multiple pathways for TNBC treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0662-4) contains supplementary material, which is available to authorized users.

MEK Inhibitor Selumetinib (AZD6244; ARRY-142886) Prevents Lung Metastasis in a Triple-Negative Breast Cancer Xenograft Model.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis because TNBC often metastasizes, leading to death. Among patients with TNBC, those with extracellular signal-regulated kinase 2 (ERK2)-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors (hazard ratio, 2.76; 95% confidence interval, 1.19-6.41). The MAPK pathway has been shown to be a marker of breast cancer metastasis, but has not been explored as a potential therapeutic target for preventing TNBC metastasis. Interestingly, when we treated TNBC cells with the allosteric MEK inhibitor selumetinib, cell viability was not reduced in two-dimensional culture. However, in three-dimensional culture, selumetinib changed the mesenchymal phenotype of TNBC cells to an epithelial phenotype. Cells that undergo epithelial-mesenchymal transition (EMT) are thought to contribute to the metastatic process. EMT leads to generation of mesenchymal-like breast cancer cells with stem cell-like characteristics and a CD44(+)CD24(-/low) expression pattern. We tested the hypothesis that targeted inhibition of the MAPK pathway by selumetinib inhibits acquisition of the breast cancer stem cell phenotype and prevents lung metastasis of TNBC. TNBC cells treated with selumetinib showed inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity (P < 0.005), and decreases in the CD44(+)CD24(-/low) fraction, ALDH1 activity, and mammosphere-forming efficiency. Mice treated with selumetinib formed significantly fewer lung metastases than control mice injected with vehicle (P < 0.05). Our data demonstrate that MEK inhibitors can inhibit breast cancer stem cells and may have clinical potential for the prevention of metastasis in certain cases in which tumors are MAPK dependent.

LncRNA-Hh Strengthen Cancer Stem Cells Generation in Twist-Positive Breast Cancer via Activation of Hedgehog Signaling Pathway.

SummaryCancer stem cells (CSCs) are a subpopulation of neoplastic cells with self‐renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial‐mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non‐coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT‐associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist‐positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs‐associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh‐GLI1 pathway associated lncRNA‐Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere‐formation efficiency (MFE) and the self‐renewal capacity in vitro, and oncogenicity in vivo in Twist‐positive breast cancer cells are elevated. lncRNA‐Hh silence in Twist‐positive breast cells attenuates the activated Shh‐GLI1 signaling and decreases the CSC‐associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist‐induced EMT cells to gain the CSC‐like stemness properties. Stem Cells 2016;34:55–66

Combinatorial TGF-β attenuation with paclitaxel inhibits the epithelial-to-mesenchymal transition and breast cancer stem-like cells.

Distant relapse after chemotherapy is an important clinical issue for treating breast cancer patients and results from the development of cancer stem-like cells (CSCs) during chemotherapy. Here we report that blocking epithelial-to-mesenchymal transition (EMT) suppresses paclitaxel-induced CSCs properties by using a MDA-MB-231-xenografted mice model (in vivo), and breast cancer cell lines (in vitro). Paclitaxel, one of the cytotoxic taxane-drugs such as docetaxel, increases mesenchymal markers (Vimentin and Fibronectin) and decreases an epithelial marker (Zo-1). Blocking TGF-β signaling with the TGF-β type I receptor kinase (ALK5) inhibitor, EW-7197, suppresses paclitaxel-induced EMT and CSC properties such as mammosphere-forming efficiency (MSFE), aldehyde dehydrogenase (ALDH) activity, CD44+/CD24- ratio, and pluripotency regulators (Oct4, Nanog, Klf4, Myc, and Sox2). The combinatorial treatment of EW-7197 improves the therapeutic effect of paclitaxel by decreasing the lung metastasis and increasing the survival time in vivo. We confirmed that Snail is increased by paclitaxel-induced intracellular reactive oxygen species (ROS) and EW-7197 suppresses the paclitaxel-induced Snail and EMT by attenuating paclitaxel-induced intracellular ROS. Knock-down of SNAI1 suppresses paclitaxel-induced EMT and CSC properties. These data together suggest that blocking the Snail-induced EMT with the ALK5 inhibitor attenuates metastasis after paclitaxel-therapy and that this combinatorial approach could prove useful in treating breast cancer.