Mouse Mammary Tumor Virus-neu Research Articles

Withaferin A inhibits expression of ataxia telangiectasia and Rad3-related kinase and enhances sensitivity of human breast cancer cells to cisplatin.

Ataxia telangiectasia and Rad3-related (ATR) is a serine/threonine-specific kinase that plays an important role in the maintenance of genomic integrity. In this study, we investigated the role of ATR in cell-cycle arrest by withaferin A (WA), a cancer preventative steroidal lactone derived from Withania somnifera plant abundant in India and surrounding countries. The WA treatment decreased the viability of MCF-7, MDA-MB-231, and SUM159 cells. Exposure of breast cancer cells to WA also resulted in suppression of protein level as well as phosphorylation of ATR and its downstream effector kinase (checkpoint kinase 1; CHK1). Both transcriptional and posttranscriptional mechanisms were involved in the WA-mediated downregulation of ATR protein. Downregulation of ATR protein expression resulting from WA exposure was not attenuated by overexpression of manganese superoxide dismutase. In contrast, the overexpression of CHK1 attenuated WA-mediated G2 /M arrest and augmented S10 phosphorylation of histone H3, a marker of mitotic arrest. The protein level of ATR was lowered by about 50% in breast tumors of WA-treated mouse mammary tumor virus-neu mice when compared with vehicle-treated controls but the difference was not significant due to small sample size. WA treatment sensitized MDA-MB-231 and SUM159 cells to growth inhibition and apoptosis induction by cisplatin. Cisplatin treatment resulted in increased autophosphorylation of ATR (T1989) and CHK1 (S345) phosphorylation that was markedly suppressed in the presence of WA. These results indicate that WA is an inhibitor of ATR in human breast cancer cells.

AKT-dependent sugar addiction by benzyl isothiocyanate in breast cancer cells.

The overall promise of breast cancer chemoprevention is exemplified by clinical success of selective estrogen receptor modulators and aromatase inhibitors. Despite clinical efficacy, these interventions have limitations, including rare but serious side effects and lack of activity against estrogen receptor-negative breast cancers. We have shown previously that dietary administration of benzyl isothiocyanate (BITC), which occurs naturally as a thioglucoside conjugate in edible cruciferous vegetables, inhibits development of estrogen receptor-negative breast cancer in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice. This study demonstrates AKT-mediated sugar addiction in breast cancer chemoprevention by BITC. BITC-treated MMTV-neu mice exhibited increased 2-deoxy-2-(18 F)-fluoro-D-glucose (18 F-FDG) uptake in mammary tumors in vivo in comparison with mice fed basal diet. Cellular studies using MDA-MB-231 and SUM159 human breast cancer cell lines revealed BITC-mediated induction and punctate localization of glucose transporter GLUT-1, which was accompanied by an increase in intracellular pyruvate levels. BITC treatment resulted in increased S473 phosphorylation (activation) of AKT in cells in vitro as well as in mammary tumors of MMTV-neu mice in vivo. Increased glucose uptake, punctate pattern of GLUT-1 localization, and intracellular pyruvate levels resulting from BITC exposure were significantly attenuated in the presence of a pharmacological inhibitor of AKT (MK-2206). Inhibition of AKT augmented BITC-mediated inhibition of cell migration and colony formation. BITC-induced apoptotic cell death was also increased by pharmacological inhibition of AKT. These results indicate increased glucose uptake/metabolism by BITC treatment in breast cancer cells suggesting that breast cancer chemoprevention by BITC may be augmented by pharmacological inhibition of AKT.

Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells.

We have reported previously that withaferin A (WA) prevents breast cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice, but the mechanism is not fully understood. Unbiased proteomics of the mammary tumors from control- and WA-treated MMTV-neu mice revealed downregulation of peptidyl-prolyl cis/trans isomerase (Pin1) protein by WA administration. The present study extends these findings to elucidate the role of Pin1 in cancer chemopreventive mechanisms of WA. The mammary tumor level of Pin1 protein was lower by about 55% in WA-treated rats exposed to N-methyl-N-nitrosourea, compared to control. Exposure of MCF-7 and SK-BR-3 human breast cancer cells to WA resulted in downregulation of Pin1 protein. Ectopic expression of Pin1 attenuated G2 and/or mitotic arrest resulting from WA treatment in both MCF-7 and SK-BR-3 cells. WA-induced apoptosis was increased by Pin1 overexpression in MCF-7 cells but not in the SK-BR-3 cell line. In addition, molecular docking followed by mass spectrometry indicated covalent interaction of WA with cysteine 113 of Pin1. Overexpression of Pin1C113A mutant failed to attenuate WA-induced mitotic arrest or apoptosis in the MCF-7 cells. Furthermore, antibody array revealed upregulation of proapoptotic insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, in Pin1 overexpressing MCF-7 cells following WA treatment when compared to empty vector transfected control cells. These data support a crucial role of the Pin1 for mitotic arrest and apoptosis signaling by WA at least in the MCF-7 cells.

Abstract 5270: Disease subtype independent biomarkers of breast cancer prevention by withaferin a

Abstract Breast cancer is a rather complex and heterogeneous disease broadly grouped into four major subtypes, including luminal-type, basal-like, HER2 amplified, and normal-like, and each with a distinct molecular signature. A non-toxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study not only demonstrates chemoprevention of breast cancer in rats by the Ayurvedic medicine phytochemical withaferin A (WA) but also identifies its mechanistic biomarkers common to different subtypes of this disease. Chemopreventive efficacy of WA (4 and 8 mg per kg body weight) was determined using a rat model of breast cancer induced by N-methyl-N-nitrosourea (MNU). The mechanisms underlying breast cancer chemoprevention by WA were elucidated by western blotting, biochemical assays, immunohistochemistry, and cytokine profiling using plasma and tumors from the MNU-rat and/or mouse mammary tumor virus-neu (MMTV-neu) models. Inhibitory effect of WA on exit from mitosis and leptin-induced oncogenic signaling was determined using MCF-7 and MDA-MB-231 cells. Incidence, multiplicity, and burden of MNU-induced breast cancer in rats were decreased by WA administration. For example, the tumor weight in the 8 mg per kg group was lower by 67% compared with controls (P = 0.004). Mitotic arrest and apoptosis induction were common determinants of breast cancer chemoprevention by WA in the MNU-rat and MMTV-neu models. Cytokine profiling showed suppression of plasma leptin levels by WA in rats. WA inhibited leptin-induced oncogenic signaling in cultured MCF-7 and MDA-MB-231 cell lines. WA is a promising phytochemical with the ability to inhibit at least two different subtypes of breast cancer, including neu-driven estrogen receptor negative (ER-) breast cancer in MMTV-neu model and MNU-induced ER+ breast cancer in rats. This study was supported by the grant RO1 CA142604-07 awarded by the National Cancer Institute. Citation Format: Eun-Ryeong Hahm, Suman K. Samanta, Anuradha Sehrawat, Su-Hyeong Kim, Subrata K. Pore, Krishna B. Singh, Susan M. Christner, Yongli Shuai, Jan H. Beumer, Ruchi Roy, Nancy E. Davidson, Shivendra V. Singh. Disease subtype independent biomarkers of breast cancer prevention by withaferin a [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 5270. doi:10.1158/1538-7445.AM2017-5270

Abstract 5265: Breast cancer stem cell inhibition by benzyl isothiocyanate is hampered by induction of Kruppel-like factor 4

Abstract Breast cancer is a leading cause of cancer-related deaths among women in the United States. Over the last decade, breast cancer stem cells (bCSC) have been identified and characterized as a key player in breast cancer progression and recurrence. Benzyl isothiocyanate (BITC) is a highly promising cancer chemopreventive constituent of edible cruciferous vegetables such as garden cress. We have shown previously that BITC inhibits bCSC in cultured breast cancer cells (MCF-7 and SUM159 cells) and in spontaneous tumors of mouse mammary tumor virus-neu (MMTV-neu) mice in vivo. Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, is known as both oncogene and tumor suppressor in various types of human cancers and plays an important role in reprogramming differentiated somatic cells into induced pluripotent stem cells. The present study demonstrates that KLF4 impedes bCSC inhibition by BITC. Exposure of breast cancer cells (MCF-7, MDA-MB-231, and SUM159) to pharmacological concentrations of BITC (2.5 and 5 µM) resulted in induction of KLF4 protein, which was accompanied by increase of its transcription. In addition, BITC treatment increased nuclear level of KLF4 in breast cancer cells. KLF4 protein expression was also higher in tumors form BITC-treated MMTV-neu mice compared with the control group. Ectopic expression of KLF4 in MCF-7 cells conferred marked protection against BITC-mediated inhibition of bCSC as evidenced by aldehyde dehydrogenase 1 (ALDH1) activity. Consistent with these observations, BITC-mediated inhibition of bCSC in MCF-7, MDA-MB-231 and SUM159 cells was significantly augmented by RNA interference of KLF4 as evidenced by ALDH1 activity and frequency of mammospheres. KLF4 overexpression also increased cell migration capacity of MCF-7 cells. Furthermore knockdown of KLF4 augmented BITC-mediated inhibition of cell migration in SUM159 cells. These results suggest that anticancer activity of BITC may be enhanced by KLF4 inhibition or knockdown in breast cancer cells. This study was supported by the grant RO1 CA129347-09 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Yong Wan, Shivendra V. Singh. Breast cancer stem cell inhibition by benzyl isothiocyanate is hampered by induction of Kruppel-like factor 4 [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 5265. doi:10.1158/1538-7445.AM2017-5265

Disease Subtype-Independent Biomarkers of Breast Cancer Chemoprevention by the Ayurvedic Medicine Phytochemical Withaferin A.

A nontoxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study was undertaken to determine the efficacy of an Ayurvedic medicine phytochemical (Withaferin A, [WA]) for chemoprevention of breast cancer and to elucidate its mode of action. Chemopreventive efficacy of WA (4 and 8 mg/kg body weight) was determined using a rat model of breast cancer induced by N-methyl-N-nitrosourea (MNU; n = 14 for control group, n = 15 for 4 mg/kg group, and n = 18 for 8 mg/kg group). The mechanisms underlying breast cancer chemoprevention by WA were elucidated by immunoblotting, biochemical assays, immunohistochemistry, and cytokine profiling using plasma and tumors from the MNU-rat (n = 8-12 for control group, n = 7-11 for 4 mg/kg group, and n = 8-12 for 8 mg/kg group) and/or mouse mammary tumor virus-neu (MMTV-neu) models (n = 4-11 for control group and n = 4-21 for 4 mg/kg group). Inhibitory effect of WA on exit from mitosis and leptin-induced oncogenic signaling was determined using MCF-7 and/or MDA-MB-231 cells. All statistical tests were two-sided. Incidence, multiplicity, and burden of breast cancer in rats were decreased by WA administration. For example, the tumor weight in the 8 mg/kg group was lower by about 68% compared with controls (8 mg/kg vs control, mean = 2.76 vs 8.59, difference = -5.83, 95% confidence interval of difference = -9.89 to -1.76, P = .004). Mitotic arrest and apoptosis induction were some common determinants of breast cancer chemoprevention by WA in the MNU-rat and MMTV-neu models. Cytokine profiling showed suppression of plasma leptin levels by WA in rats. WA inhibited leptin-induced oncogenic signaling in cultured breast cancer cells. WA is a promising chemopreventative phytochemical with the ability to inhibit at least two different subtypes of breast cancer.

The breast cancer susceptibility FGFR2 provides an alternate mode of HER2 activation.

Fibroblast growth factor receptor 2 (FGFR2) has been shown reproducibly in genome-wide association studies to be associated with increased breast cancer risk. Here we show that mouse mammary tumor virus-neu mice develop breast carcinomas with FGFR2 immunoreactivity that parallels HER2 expression. FGFR2 signaling promotes HER2 shedding through the metalloprotease ADAM10 leading to intracellular accumulation of the truncated p95HER2 protein. This is accompanied by enhanced HER2 signaling and diminished sensitivity to trastuzumab. Functionally, FGFR2 facilitates HER2-mediated cell proliferation, acinar growth in three-dimensional morphogenesis assays and promotes tumor progression in mouse xenografts. These data implicate FGFR2 in a novel mechanism of ErbB activation and demonstrate an important interaction between FGFR2 and HER2 in promoting breast cancer progression.Oncogene advance online publication, 2 February 2015; doi:10.1038/onc.2014.440.

Abstract 227: Estrogen receptor-α and tumor initiating cells are novel targets of diallyl trisulfide in breast cancer cells

Abstract Epidemiological studies suggest a protective role for Allium vegetables, such as garlic, against breast and other cancers. Diallyl trisulfide (DATS) is a promising oil-soluble anticancer metabolite generated after cutting or chewing of Allium vegetables. We have shown previously that DATS inhibits viability of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developed tumor of a mouse mammary tumor virus-neu mouse by causing apoptosis induction. We also found that a non-tumorigenic normal human mammary epithelial cell line (MCF-10A) is resistant to cell death induction by DATS. Studies by others have demonstrated in vivo efficacy of DATS against MCF-7 xenografts in nude mice. The present study builds upon these observations and identifies novel targets of DATS in breast cancer cells. MCF-7 and T47D cells exposed to DATS exhibited downregulation of estrogen receptor-α (ER-α) protein, which peaked between 12 and 24 h post-treatment. Diallyl sulfide and diallyl disulfide analogs were relatively less effective in suppressing protein level of ER-α compared with DATS. The 17β-estradiol (E2)-induced expression of pS2, an ER-α target gene product, was also decreased in the presence of DATS. Downregulation of ER-α protein expression resulting from DATS treatment was accompanied by a decrease in nuclear level of ER-α, suppression of ER-α mRNA, and inhibition of ERE2e1b-luciferase reporter activity. Moreover, apoptotic cell death induced by DATS was partially but statistically significantly attenuated in the presence of E2 in MCF-7 cells as well as by overexpression of ER-α in MDA-MB-231 cells. DATS treatment inhibited breast cancer stem cell (bCSC) population as evidenced by flow cytometric analysis of CD44high/CD24low/epithelial specific antigen positive population, aldehyde dehydrogenase 1 activity, and mammosphere frequency in MCF-7 and SUM159 cells. Inhibition of bCSC by treatment with DATS was associated with downregulation of EZH2 and Kruppel-like factor 4 proteins in both MCF-7 and SUM159 cells. In conclusion, the present study identifies ER-α and bCSC as novel targets of DATS in mammary cancer cells. This study was supported by the grant RO1 CA113363-09 awarded by the National Cancer Institute. Citation Format: Eun-Ryeong Hahm, Su-Hyeong Kim, Shivendra V. Singh. Estrogen receptor-α and tumor initiating cells are novel targets of diallyl trisulfide in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 227. doi:10.1158/1538-7445.AM2014-227

Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells.

Current dogma favors elimination of therapy-resistant cancer stem cells for chemoprevention of breast cancer. We showed recently that mammary cancer development in a transgenic mouse model (mouse mammary tumor virus-neu; MMTV-neu) was inhibited significantly upon treatment with withaferin A (WA), a steroidal lactone derived from a medicinal plant. Herein, we demonstrate that the mammary cancer prevention by WA is accompanied by in vivo suppression of breast cancer stem cells (bCSC). In vitro mammosphere formation was dose-dependently inhibited by WA treatment in MCF-7 and SUM159 human breast cancer cells. Other markers of bCSC, including aldehyde dehydrogenase 1 (ALDH1) activity and CD44(high)/CD24(low)/epithelial-specific antigen-positive (ESA+) fraction, were also decreased significantly in the presence of plasma achievable doses of WA. However, WA exposure resulted in cell line-specific changes in Oct4, SOX-2, and Nanog mRNA expression. WA administration to MMTV-neu mice (0.1 mg/mouse, 3 times/week for 28 weeks) resulted in inhibition of mammosphere number and ALDH1 activity in vivo. Mechanistic studies revealed that although urokinase-type plasminogen activator receptor overexpression conferred partial protection against bCSC inhibition by WA, Notch4 was largely dispensable for this response. WA treatment also resulted in sustained (MCF-7) or transient (SUM159) downregulation of Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion region-1) protein. Ectopic expression of Bmi-1 conferred partial but significant protection against ALDH1 activity inhibition by WA. Interestingly, WA treatment caused induction of Kruppel-like factor 4 (KLF4) and its knockdown augmented bCSC inhibition by WA. In conclusion, this study shows in vivo effectiveness of WA against bCSC.

Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice

Human epidermal growth factor receptor2/Neu, which is overexpressed in about 30% of human breast cancers, transduces growth signals in large part via the Ras-Raf-MEK-ERK pathway. Nevertheless, it is a matter of controversy whether high ERK activity in breast cancer tissues correlates with better or worse prognosis, leaving the role of ERK activity in the progression of breast cancers unresolved. To address this issue, we live-imaged ERK activity in mammary tumors developed in mouse mammary tumor virus-Neu transgenic mice, which had been crossed with transgenic mice expressing a Förster resonance energy transfer biosensor for ERK. Observation of the tumor by two-photon microscopy revealed significant heterogeneity in ERK activity among the mammary tumor cells. The level of ERK activity in each cell was stable up to several hours, implying a robust mechanism that maintained the ERK activity within a limited range. By sorting the mammary tumor cells on the basis of their ERK activity, we found that ERK(high) cells less efficiently generated tumorspheres in vitro and tumors in vivo than did ERK(low) cells. In agreement with this finding, the expressions of the cancer stem cell markers CD49f, CD24 and CD61 were decreased in ERK(high) cells. These observations suggest that high ERK activity may suppress the self-renewal of mammary cancer stem cells.

Dietary Chemopreventative Benzyl Isothiocyanate Inhibits Breast Cancer Stem Cells In Vitro and In Vivo

A small subset of mammary tumor-initiating cells (also known as breast cancer stem cells; bCSC), characterized by expression of different markers [CD44(high)/CD24(low)/epithelial-specific antigen (ESA)+], aldehyde dehydrogenase-1 (ALDH1) activity, and ability to form mammospheres under ultra-low attachment culture conditions, are suspected to evade conventional therapies leading to disease recurrence. Elimination of both therapy-sensitive epithelial tumor cells and therapy-resistant bCSC is therefore necessary for prevention of breast cancer. We have shown previously that a nontoxic small-molecule constituent of edible cruciferous vegetables (benzyl isothiocyanate; BITC) inhibits mammary cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice by causing epithelial tumor cell apoptosis. The present study shows efficacy of BITC against bCSC in vitro and in vivo. Mammosphere formation frequency and CD44(high)/CD24(low)/ESA+ and/or ALDH1+ populations in cultured MCF-7 (estrogen receptor-positive) and SUM159 (triple-negative) human breast cancer cells were decreased significantly in the presence of plasma achievable concentrations of BITC. BITC administration in the diet (3 μmol BITC/g diet for 29 weeks) resulted in a marked decrease in bCSCs in the MMTV-neu mice tumors in vivo. Overexpression of full-length Ron as well as its truncated form (sfRon), but not urokinase-type plasminogen activator receptor, conferred near complete protection against BITC-mediated inhibition of bCSCs in MCF-7 cells. The BITC treatment downregulated protein levels of Ron and sfRon in cultured breast cancer cells and in tumor xenografts. Ron overexpression resulted in upregulation of bCSC-associated genes Oct-4, SOX-2, and Nanog. In conclusion, the present study indicates that BITC treatment eliminates bCSCs in vitro and in vivo.

Metabolic Alterations in Mammary Cancer Prevention by Withaferin A in a Clinically Relevant Mouse Model

Efficacy of withaferin A (WA), an Ayurvedic medicine constituent, for prevention of mammary cancer and its associated mechanisms were investigated using mouse mammary tumor virus-neu (MMTV-neu) transgenic model. Incidence and burden of mammary cancer and pulmonary metastasis were scored in female MMTV-neu mice after 28 weeks of intraperitoneal administration with 100 µg WA (three times/week) (n = 32) or vehicle (n = 29). Mechanisms underlying mammary cancer prevention by WA were investigated by determination of tumor cell proliferation, apoptosis, metabolomics, and proteomics using plasma and/or tumor tissues. Spectrophotometric assays were performed to determine activities of complex III and complex IV. All statistical tests were two-sided. WA administration resulted in a statistically significant decrease in macroscopic mammary tumor size, microscopic mammary tumor area, and the incidence of pulmonary metastasis. For example, the mean area of invasive cancer was lower by 95.14% in the WA treatment group compared with the control group (mean = 3.10 vs 63.77 mm2, respectively; difference = -60.67 mm2; 95% confidence interval = -122.50 to 1.13 mm2; P = .0536). Mammary cancer prevention by WA treatment was associated with increased apoptosis, inhibition of complex III activity, and reduced levels of glycolysis intermediates. Proteomics confirmed downregulation of many glycolysis-related proteins in the tumor of WA-treated mice compared with control, including M2-type pyruvate kinase, phospho glycerate kinase, and fructose-bisphosphate aldolase A isoform 2. This study reveals suppression of glycolysis in WA-mediated mammary cancer prevention in a clinically relevant mouse model.

Abstract 3671: The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells.

Abstract We have shown previously that breast cancer chemoprevention by benzyl isothiocyanate (BITC) in mouse mammary tumor virus-neu transgenic mice is associated with induction of E-cadherin protein expression. Loss of E-cadherin expression concomitant with induction of mesenchymal markers (eg, vimentin and fibronectin) is a biochemical hallmark of epithelial-to-mesenchymal transition (EMT). EMT is a developmental process but implicated in progression of cancer to aggressive state. We have also shown previously that BITC treatment inhibits EMT in cultured MDA-MB-231 human breast cancer cells. The present study extends these observations and investigates the mechanism by which BITC inhibits EMT. Initially, we designed experiments using another triple-negative human breast cancer cell line (SUM159) to determine generality of the BITC-mediated inhibition of EMT. Exposure of SUM159 cells to pharmacologically-relevant concentrations of BITC (2.5 and 5 μM) resulted in induction of E-cadherin protein expression, which was accompanied by suppression of vimentin protein level especially at the 5 μM dose. Protein levels of slug and snail, which are transcriptional repressors of E-cadherin, were also decreased markedly after treatment of SUM159 cells with BITC, but this effect was relatively more pronounced on slug protein level compared with snail. Similar to MDA-MB-231 cells, the BITC-mediated induction of E-cadherin protein expression in SUM159 cells was accompanied by its transcriptional upregulation as revealed by E-cadherin luciferase reporter assay. Immunofluorescence microscopy confirmed induction of E-cadherin protein and suppression of vimentin protein expression in BITC-treated SUM159 cells. BITC treatment resulted in downregulation of Forkhead Box Q1 transcription factor (FOXQ1) protein and FOXQ1 mRNA in MDA-MB-231, SUM159, and MDA-MB-468 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with suppression of FOXQ1 protein level. Ectopic expression of FOXQ1 in a mammary epithelial cell line conferred marked protection against BITC-mediated inhibition of EMT as well as cell migration. In conclusion, the present study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells. This study was supported by the grant CA129347-06 awarded by the National Cancer Institute. Citation Format: Anuradha Sehrawat, Su-Hyeong Kim, Andreas Vogt, Shivendra V. Singh. The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3671. doi:10.1158/1538-7445.AM2013-3671

Targeting the IKKβ/mTOR/VEGF Signaling Pathway as a Potential Therapeutic Strategy for Obesity-Related Breast Cancer

Clinical correlation studies have clearly shown that obesity is associated with breast cancer risk and patient survival. Although several potential mechanisms linking obesity and cancers have been proposed, the detailed molecular mechanism of obesity-mediated breast tumorigenesis has not yet been critically evaluated. In this study, we evaluated the effects of obesity on mammary tumor initiation and progression using mice with genetic and diet-induced obesity bearing mammary tumor xenografts and mouse mammary tumor virus-neu transgenic mice that were fed a high-fat diet. We show that obesity promoted mammary tumor growth and development in these animal models. Moreover, the expressions of TNFα, VEGF, IKKβ, and mTOR are upregulated in mammary tumors of obese mice, suggesting that the IKKβ/mTOR/VEGF signaling pathway is activated by TNFα in the tumors of obese mice. More importantly, inhibitors (rapamycin, bevacizumab, and aspirin) that target members of the pathway suppressed tumorigenesis and prolonged survival more effectively in obese mice than in nonobese mice. Here, we not only identified a specific signaling pathway that contributes to mammary tumorigenesis in obese mice but also a strategy for treating obesity-mediated breast cancer.

PreImplantation Factor (SPIF*) selective modulation of NK cytotoxicty and activity in vitro in IVF patients. SPIF* Proprietary

Mouse mammary tumor virus-Neu (MMTV/neu) transgenic mice on an FVB-background (FVB-neuN) have increased numbers of myeloid derived suppressor cells (MDSCs) and regulatory T-cells (T-regs) in the spleen during mammary tumor induction and progression. Using this transgenic tumor model, we assessed the therapeutic activity of sunitinib, a multi-targeted, tyrosine kinase (TK) inhibitor and its effects on immune-regulatory cells. Our preliminary results show that sunitinib at 40 mg/kg/day, p.o. (per os), delayed the time to tumor induction and reduced the incidence and growth of tumors in FVB-neuN mice. In association with its therapeutic activity, sunitinib reduced the absolute number of splenic T-reg cells (CD4+CD25+CD62L+) and MDSCs (CD11b+Gr1+) that were increased during tumor progression with less activity in mice with gross tumors. A significant decrease in the absolute number of splenic T-regs, dendritic cells (DCs), MDSCs and hematopoietic progenitors (Lin−Sca1+CD90dull) was observed following sunitinib treatment. The frequency of splenic T-regs and hematopoietic progenitors, but not MDSCs was also reduced by sunitinib treatment. Additionally immune-regulatory cytokines and enzymes were down regulated by sunitinib treatment, including TGFβ and NOS2 in the spleen cells of sunitinib treated mice as compared to untreated tumor bearing (TB) mice. We conclude that sunitinib has therapeutic activity, in association with the down regulation of MDSCs and T-regs and has a trend towards the normalization of the inflammatory cytokine levels induced by tumor progression and growth. Based on these results, we suggest that sunitinib reduction of immune suppressive cells is a critical part of its adjuvant immune therapeutic activity.

MMTV-neu mice deficient in STAT1 are susceptible to develop ovarian teratomas

Signal transducer and activator of transcription 1 (STAT1) serves in the protection of the organism against pathogens and other harmful insults. It is implicated in innate immune response, immunosurveillance, tumor-suppression, and the response to genotoxic as well as oxidative stress. We report here that 9 of 140 examined STAT1 deficient mouse mammary tumor virus-neu (MMTV-neu) mice developed differentiated ovarian teratomas, which histologically resemble benign dermatoid cysts. Conventional karyotyping revealed diploidy without structural rearrangements of the chromosomes. STAT1 proficient MMTV-neu mice with the same genetic background (FVB/N), and STAT1 deficient C57BL/6 mice failed to develop this type of tumor. This indicates that STAT1 deficiency promotes teratoma formation and this depends on MMTV-neu expression and/or the genetic background. Since ovarian teratomas are considered to develop as a consequence of alterations in the maturation of oocytes and follicular cells, we compared the ovaries from non-tumor bearing STAT1 deficient and proficient MMTV-neu mice. No detectable alterations in the number and proportion of the different follicular developmental stages were detected, implying the absence of non-redundant functions of STAT1 in normal folliculogenesis, as well as in follicular atresia. However, strong staining for STAT1 was detectable in granulosa and theca cells. These results point to a role for STAT1 in protecting from teratoma formation in a later step of tumorigenesis, e.g. by inducing apoptosis and eliminating premature or aberrantly formed follicles which have the potential to transform into teratomas.

Abstract 3333: Overexpression of EZH2 accelerates ERB2 driven tumorigenesis in the mammary gland

Abstract BACKGROUND: EZH2 protein is overexpressed mainly in ER- tumors with concomitant HER-2/neu overexpression and/or p53 mutations and is an independent predictor of metastasis and death. A role for EZH2 in stem cell maintenance was recently reported. We previously demonstrated that mammary specific EZH2 overexpression causes ductal hyperbranching and intraductal hyperplasia but is not sufficient in isolation to induce carcinomas. To address the biological impact of EZH2 overexpression in mammary tumorigenesis, we generated a novel transgenic mouse model by crossing transgenic mice carrying mouse mammary tumor virus/EZH2 (MMTV-EZH2) and MMTV-inactivated neu (MMTV-neu) to derive progeny that coexpress the transgenes in the mammary epithelium (EZH2+ neu). METHODS: MMTV-neu mice were purchased from Jackson laboratories (FVB/N-TgN (MMTVneu)202 Mul/J, or MMTV neu, stock number 002376). They are homozygous for the MMTV neu (rat) transgene, and overexpress the neu proto-oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). We used synchronized breeding methods with male EZH2 heterozygous transgenic mice and female MMTV-neu transgenic mice to obtain EZH2+; neu and EZH2 wt; neu mice. Mouse handling and experimental procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the UM. Tumors were measured with a caliper. Statistical analyses of tumor initiation time were performed. Flow cytometry and western blot were performed in mouse mammary glands and MCF10A cells. RESULTS: EZH2 overexpression accelerates the process of ErbB-2-mediated mammary tumorigenesis. EZH2+ neu mice (n=30) showed accelerated tumor initiation (median time to tumor initiation=234 days) than EZH2 wt neu control group (n=25) (median time to tumor initiation=295 days) (Log-rank p-value < 0.0001). Despite no differences in the size of the tumors or tumor histology, flow cytometry analysis revealed that EZH2+; neu bitransgenic mice had an expansion of the luminal progenitor cell population (CD24+CD29loCD61+) in the mammary glands at 8 weeks of age, preceding tumor development. This was associated with upregulation of the luminal progenitor regulator STAT5 mRNA, protein, and signaling pathway. Consistently, conditional EZH2 overepresssion in MCF10A cells showed expansion of the luminal progenitor population (CD49f+EpCAM+). CONCLUSION: Our experiments demonstrate that overexpression of EZH2 accelerates the initiation of ErbB2-induced mammary tumors and lead to the novel hypothesis that EZH2 overexpression activates STAT5 signaling pathway to expand mammary luminal progenitor cells, which is under investigation in our lab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2011-3333

Critical Roles of DMP1 in Human Epidermal Growth Factor Receptor 2/neu-Arf-p53 Signaling and Breast Cancer Development

Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis.

Therapeutic activity of sunitinib for Her2/neu induced mammary cancer in FVB mice

Mouse mammary tumor virus-Neu (MMTV/neu) transgenic mice on an FVB-background (FVB-neuN) have increased numbers of myeloid derived suppressor cells (MDSCs) and regulatory T-cells (T-regs) in the spleen during mammary tumor induction and progression. Using this transgenic tumor model, we assessed the therapeutic activity of sunitinib, a multi-targeted, tyrosine kinase (TK) inhibitor and its effects on immune-regulatory cells. Our preliminary results show that sunitinib at 40 mg/kg/day, p.o. (per os), delayed the time to tumor induction and reduced the incidence and growth of tumors in FVB-neuN mice. In association with its therapeutic activity, sunitinib reduced the absolute number of splenic T-reg cells (CD4 +CD25 +CD62L +) and MDSCs (CD11b +Gr1 +) that were increased during tumor progression with less activity in mice with gross tumors. A significant decrease in the absolute number of splenic T-regs, dendritic cells (DCs), MDSCs and hematopoietic progenitors (Lin −Sca1 +CD90 dull) was observed following sunitinib treatment. The frequency of splenic T-regs and hematopoietic progenitors, but not MDSCs was also reduced by sunitinib treatment. Additionally immune-regulatory cytokines and enzymes were down regulated by sunitinib treatment, including TGFβ and NOS2 in the spleen cells of sunitinib treated mice as compared to untreated tumor bearing (TB) mice. We conclude that sunitinib has therapeutic activity, in association with the down regulation of MDSCs and T-regs and has a trend towards the normalization of the inflammatory cytokine levels induced by tumor progression and growth. Based on these results, we suggest that sunitinib reduction of immune suppressive cells is a critical part of its adjuvant immune therapeutic activity.

A New Rexinoid, NRX194204, Prevents Carcinogenesis in Both the Lung and Mammary Gland

We evaluated the anti-inflammatory and growth-inhibitory properties of the novel rexinoid NRX194204 (4204) in vitro and then tested its ability to prevent and/or treat experimental lung and estrogen receptor (ER)-negative breast cancer in vivo. In cell culture studies, we measured the ability of 4204 to block the effects of lipopolysaccharide and induce apoptosis. For the lung cancer prevention studies, A/J mice were injected with the carcinogen vinyl carbamate and then fed 4204 (30-60 mg/kg diet) for 15 weeks, beginning 1 week after the administration of the carcinogen. For breast cancer prevention studies, mouse mammary tumor virus-neu mice were fed control diet or 4204 (20 mg/kg diet) for 50 weeks; for treatment, tumors at least 32 mm3 in size were allowed to form, and then mice were fed control diet or 4204 (60 mg/kg diet) for 4 weeks. Low nanomolar concentrations of 4204 blocked the ability of lipopolysaccharide and tumor necrosis factor-alpha to induce the release of nitric oxide and interleukin 6 and the degradation of IKBalpha in RAW264.7 macrophage-like cells. In the A/J mouse model of lung cancer, 4204 significantly (P < 0.05) reduced the number and size of tumors on the surface of the lungs and reduced the total tumor volume per slide by 64% to 81% compared with the control group. In mouse mammary tumor virus-neu mice, 4204 not only delayed the development of ER-negative mammary tumors in the prevention studies but also caused marked tumor regression (92%) or growth arrest (8%) in all of the mammary tumors when used therapeutically. The combined anti-inflammatory and anticarcinogenic actions of 4204 suggest that it is a promising new rexinoid that should be considered for future clinical trials.