MMTV-neu Mice Research Articles

Abstract IA28: Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer

Abstract Integrin-mediated adhesion and signaling via focal adhesion kinase is required for mammary tumorigenesis in the PyMT mouse model. Moreover, PTK2 encoding FAK is amplified in a substantial fraction of human breast cancers. We have investigated the mechanism through which integrin signaling controls mammary tumorigenesis and found that integrin-mediated mechano-transduction controls invasive growth in stiff 3D matrices through activation of YAP/TAZ. Mechanistic studies revealed that integrin signaling induces—via activation of FAK and Src family kinases (SFKs) —tyrosine phosphorylation and inactivation of LATS. The ensuing activation of YAP/TAZ is necessary for invasive growth of HER2+ breast cancer cells. Engagement of HER2/HER3 with neuregulin impinges on SFKs to amplify activation of YAP/TAZ in HER2+ breast cancer cells, suggesting that integrin-mediated mechano-transduction functions as a rheostat to regulate HER2 oncogenic signaling. Finally, administration of dasatinib significantly increases the efficacy of HER2 inhibition in the MMTV-Neu mouse model of HER2+ breast cancer. These findings reveal a major mechanism through which the Hippo pathway is disabled in breast cancer and suggest that targeting HER2 and SFKs simultaneously may be a rational strategy in selected cases of breast cancer. Citation Format: Shimin Wang, Young-Mi Kim, Ralph Garippa, Larry Norton, Filippo G. Giancotti. Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA28.

Increased Arginase1 expression in tumor microenvironment promotes mammary carcinogenesis via multiple mechanisms.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/-;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/-;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

CD36-Mediated Metabolic Rewiring of Breast Cancer Cells Promotes Resistance to HER2-Targeted Therapies

SUMMARYAlthough it is established that fatty acid (FA) synthesis supports anabolic growth in cancer, the role of exogenous FA uptake remains elusive. Here we show that, during acquisition of resistance to HER2 inhibition, metabolic rewiring of breast cancer cells favors reliance on exogenous FA uptake over de novo FA synthesis. Through cDNA microarray analysis, we identify the FA transporter CD36 as a critical gene upregulated in cells with acquired resistance to the HER2 inhibitor lapatinib. Accordingly, resistant cells exhibit increased exogenous FA uptake and metabolic plasticity. Genetic or pharmacological inhibition of CD36 suppresses the growth of lapatinib-resistant but not lapatinib-sensitive cells in vitro and in vivo. Deletion of Cd36 in mammary tissues of MMTV-neu mice significantly attenuates tumorigenesis. In breast cancer patients, CD36 expression increases following anti-HER2 therapy, which correlates with a poor prognosis. Our results define CD36-mediated metabolic rewiring as an essential survival mechanism in HER2-positive breast cancer.

Retinoid X receptor agonist LG100268 modulates the immune microenvironment in preclinical breast cancer models

Despite numerous therapeutic advances in the past decade, breast cancer is expected to cause over 42,000 deaths in the United States in 2019. Breast cancer had been considered an immunologically silent tumor; however recent findings suggest that immune cells play important roles in tumor growth even in the breast. Retinoid X receptors (RXRs) are a subclass of nuclear receptors that act as ligand-dependent transcription factors that regulate a variety of cellular processes including proliferation and differentiation; in addition, they are essential for macrophage biology. Rexinoids are synthetic molecules that bind and activate RXRs. Bexarotene is the only rexinoid approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Other more-potent rexinoids have been synthesized, such as LG100268 (LG268). Here, we report that treatment with LG 268, but not bexarotene, decreased infiltration of myeloid-derived suppressor cells and CD206-expressing macrophages, increased the expression of PD-L1 by 50%, and increased the ratio of CD8/CD4, CD25 T cells, which correlates with increased cytotoxic activity of CD8 T cells in tumors of MMTV-Neu mice (a model of HER2-positive breast cancer). In the MMTV-PyMT murine model of triple negative breast cancer, LG268 treatment of established tumors prolonged survival, and in combination with anti-PD-L1 antibodies, significantly (p = 0.05) increased the infiltration of cytotoxic CD8 T cells and apoptosis. Collectively, these data suggest that the use of LG268, a RXR agonist, can improve response to immune checkpoint blockade in HER2+ or triple-negative breast cancer.

Abstract LB-186: The rexinoid LG100268 decreases immunosuppressive immune cells and activates T cells in two preclinical models of breast cancer

Abstract Breast cancer (BC) was considered an immunologically silent tumor, however recent findings suggest that immune cells present in the tumor microenvironment play important roles in tumor growth. Retinoid X receptors (RXRs) are a subclass of nuclear receptors that act as ligand-dependent transcription factors that regulate a variety of cellular processes including proliferation and differentiation. Rexinoids are synthetic molecules that bind and activate RXRs. Bexarotene (Targretin®) is the only rexinoid approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Other more potent rexinoids have been synthesized, such as LG100268 (LG268) and LG101506. Because recent studies have suggested that RXRs are involved in immune regulation, we studied the effects of LG268 as an immune modulator in a preclinical model of breast cancer. MMTV-Neu mice with tumors approximately 32 mm3 in volume were treated with control diet or LG268 (100 mg/kg diet) for 5 days. Rexinoid treatment significantly (p=0.0047) reduced tumor weight from 3.2% of body weight in the control group to 1.1% in the treated group. Bexarotene (100 mg/kg diet), however, did not reduce tumor weight even after 10 days of treatment. Analysis of the tumor by flow cytometry showed a significant (p=0.0013) reduction in the percentage of myeloid derived suppressor cells (MDSCs) in the treated mice (3.4% vs 1.6%). MDSCs are associated with a lack of immune surveillance against tumor cells, and in BC, are correlated with higher metastatic burden and poor survival. The reduction in MDSCs was accompanied by a 80% reduction in p-STAT1 (p=0.02). The percentage of activated CD4 T cells (CD45, CD3, CD4, CD25) also decreased when mice were treated with LG268 (27.5% vs 13%; p=0.012). Moreover, the ratio of CD8/CD4, CD25 cells was increased in the tumors of treated mice (1.58 vs 3.07; p=0.04), suggesting increased activation of cytotoxic T cells. Higher CD8/CD4, CD25 ratios correlate with better outcomes in patients with several solid tumors. To further elucidate the effects of LG268 on immunosuppressive T cells, CD4 cells were isolated from spleen and stimulated with CD28, IL-2 and TGFβ, skewing the cells towards regulatory T cells. CD4 Treg FOXP3 expressing cells contribute to the immunosuppressive milieu in tumors. Treatment with LG268 at 100 nM reduced the mRNA expression of FOXP3 by 40% (p=0.03) when compared with untreated CD4 T cells. In the MMTV-PyMT model of triple negative BC, LG268 (100 mg/kg diet) significantly (p=0.018) extended survival by an average of 29 days when compared with the controls. Histopathological analysis revealed that tumors treated with LG268 had higher infiltration of CD8 cytotoxic T cells, which correlated with increased cleaved caspase 3. These results show that RXR agonists can be used to modulate immune cell populations present in the tumor microenvironment, and reduce immunosuppressive MDSC cells and CD4 T regulatory cell populations in breast cancer models. We thank the Breast Cancer Research Foundation for support. Citation Format: Ana Sofia Leal, Sarah Carapellucci, Kayla ZydecK, Michael B. Sporn, Karen T. Liby. The rexinoid LG100268 decreases immunosuppressive immune cells and activates T cells in two preclinical models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-186.

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.

Treatment-Induced Tumor Cell Apoptosis and Secondary Necrosis Drive Tumor Progression in the Residual Tumor Microenvironment through MerTK and IDO1.

Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor 'homeostasis' and progression. SIGNIFICANCE: These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.

Abstract LB-344: The rexinoid LG100268 modulates immune cell populations in mammary gland tumors of MMTV-neu mice

Abstract Despite numerous therapeutic advances in the past decade, breast cancer is expected to cause around 41,000 deaths in 2018. Breast cancer was considered an immunologically silent tumor, however recent findings suggest that immune cells present in the tumor microenvironment play important roles in tumor growth. Retinoid X receptors (RXRs) are a subclass of nuclear receptors that act as ligand-dependent transcription factors that regulate a variety of cellular processes including proliferation and differentiation. Rexinoids are synthetic molecules that bind and activate RXRs. Bexarotene (Targretin®) is the only rexinoid approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Other more potent rexinoids have been synthesized, such as LG100268 and LG101506. The rexinoid LG100268 delays the development of mammary tumors in MMTV-Neu mice. Because recent findings suggested an involvement of RXRs in immune regulation, we studied the effects of LG100268 as an immune modulator in preclinical breast cancer. MMTV-Neu mice with tumors approximately 32 mm3 in volume were treated with control diet or LG100268 (100 mg/kg diet) for 5 days. Rexinoid treatment significantly (p<0.05) reduced tumor weight from 3.2% of body weight in the control group to 1.1% in the treated group. Analysis of the tumor by flow cytometry showed a significant (p<0.05) reduction in the percentage of myeloid derived suppressor cells (MDSCs) in the treated mice (3.4% vs 1.6%). MDSCs associate with a lack of immune surveillance against tumor cells, and in breast cancer, are associated with higher metastatic burden and poor survival. The percentage of activated CD4 T cells (CD45, CD3, CD4, CD25) also decreased when mice were treated with LG100268 (27.5% vs 13%; p<0.05). Moreover, the ratio of CD8/CD4, CD25 cells was increased in the tumors of treated mice (1.58 vs 3.07), suggesting more activation of cytotoxic T cells. Higher CD8/CD4, CD25 ratios correlate with better outcomes in patients with several solid tumors. Tumors treated with LG100268 also showed decreased expression of CD31, indicating reduced vascularization. To further elucidate the effects of LG100268 on immunosuppressive T cells, CD4 cells were isolated from spleen and stimulated with CD28, IL-2 and TGFβ, skewing the cells towards regulatory T cells. CD4 Treg FOXP3 expressing cells contribute to the immunosuppressive milieu in tumors. Treatment with LG100268 at 100 nM reduced the mRNA expression of FOXP3 by 45% when compared with untreated CD4 T cells. These results show that RXR agonists can be used to modulate the immune populations present in the tumor microenvironment in breast cancer, and reduce immunosuppressive MDSC cells and CD4 T regulatory cell populations. We thank the Breast Cancer Research Foundation for supporting this research. Citation Format: Ana Sofia Leal, Sarah Carapellucci, Kayla Zydeck, Nupur Raychaudhuri, Michael B. Sporn, Karen T. Liby. The rexinoid LG100268 modulates immune cell populations in mammary gland tumors of MMTV-neu mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-344.

Abstract 4386: Regulation of FIP200 by Her2 and its role in Her2-driven mammary tumors

Abstract Autophagy has been shown to play both tumor promoting and suppressive roles in various cancers, although the underlying mechanisms are not well understood. Compared to other subtypes of breast cancer (BCa), Her2+ BCa displays a lower autophagy gene signature, suggesting a potentially tumor suppressive function of autophagy in this subtype of BCa. FIP200 is a component of the ULK1/ATG13/FIP200/Atg101 complex essential for autophagy induction in mammalian cells, and our previous studies showed that Fip200 deletion suppressed BCa in MMTV-PyMT mouse model. Indeed, analysis of TCGA database showed that higher FIP200 expression correlated with decreased survival of BCa patients including all subtypes. In contrast, in Her2+ BCa population specifically, higher expression of FIP200 correlated with better patient survival, suggesting a differential function of FIP200. To uncover the role of FIP200 and autophagy in Her2+ BCa, we studied Fip200 in MMTV-Neu mouse tumor cells that model Her2+ BCa. We found that inhibiting autophagy by deletion of Fip200 or Atg13 using Crispr-Cas9 led to increased migration and partial EMT for MMTV-Neu cells. Further, deletion of Fip200 or Atg13 as well as autophagy inhibition by Spautin1 significantly enhanced β-catenin/LEF1/TCF reporter activity, which may promote cell migration and EMT. These changes in Fip200-null tumor cells could be rescued by re-expression of ectopic FIP200, excluding any potential off-target effects of gene knockout. We also found that Her2 knockdown or lapatinib treatment of MMTV-Neu cells increased Fip200 expression and autophagy. Together, these results raised the interesting possibility that Her2 may promote tumor development and/or progression at least in part by reducing FIP200 expression to inhibit autophagy, which might play a tumor suppressive role in Her2-driven BCa. Citation Format: Mingang Hao, Jun-Lin Guan. Regulation of FIP200 by Her2 and its role in Her2-driven mammary tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4386.

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.

Sepiapterin Enhances Tumor Radio- and Chemosensitivities by Promoting Vascular Normalization.

Previously, we demonstrated that nitric oxide (NO) synthase (NOS) is uncoupled in a wide range of solid tumors and that restoring NOS coupling with the tetrahydrobiopterin precursor sepiapterin (SP) inhibits tumor progression. Endothelial dysfunction characterizes the poorly functional vasculature of solid tumors, and since NO is critical for regulation of endothelial function we asked whether SP, by recoupling NOS, improves tumor vasculature structure and function-enhancing chemotherapeutic delivery and response to radiotherapy. MMTV-neu mice with spontaneous breast tumors were treated with SP by oral gavage and evaluated by multispectral optoacoustic tomographic analysis of tumor HbO2 and by tissue staining for markers of hypoxia, blood perfusion, and markers of endothelial and smooth muscle proteins. Recoupling tumor NOS activity results in vascular normalization observed as reduced tumor hypoxia, improved tumor percentage of HbO2 and perfusion, as well as increased pericyte coverage of tumor blood vessels. The normalized vasculature and improved tumor oxygenation led to a greater than 2-fold increase in radiation-induced apoptosis compared with radiation or SP alone. High-performance liquid chromatography analysis of tumor doxorubicin levels showed a greater than 50% increase in doxorubicin uptake and a synergistic effect on tumor cell apoptosis. This study highlights for the first time the importance of NOS uncoupling and endothelial dysfunction in the development of tumor vasculature and presents a new approach for improving the tumoricidal efficacies of chemotherapy and radiotherapy.

Abstract P4-04-15: Hormonal prevention of HER2 positive mammary cancers

Abstract Introduction: Approximately 25-30% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2). HER2-positive breast cancers are more likely to be diagnosed in younger women and are highly aggressive. Currently, there are few treatments options available specifically for HER2-positive breast cancer which includes Herceptin, Kadcyla, Perjeta and Tykerb. There is a higher risk of recurrence of HER2-positive breast cancers compared to HER2-negative breast cancers. Hence it is important to devise effective strategies to prevent/treat HER2-positive breast cancer. We had earlier shown that short-term treatment with pregnancy levels of estrogen plus or minus progesterone was highly effective in inhibiting mammary tumor growth in MMTV. Here we attempt to understand the mechanism behind this protection. Materials and Methods: MMTV-neu transgenic mice were treated for 3 weeks, starting at 7 weeks of age, with 100 μg of estradiol in Silastic capsules. This dose of estradiol used in the Silastic capsules results in pregnancy levels of estradiol in the circulation. The control animals received empty Silastic capsules for the same duration. Mice were palpated weekly for 9 months to monitor for mammary cancer development. A set of mice were sacrificed at 7, 10, 16, 22, 28 and 36 weeks of age. Mammary gland was surgically isolated. Inguinal mammary glands were prepared as wholemounts. A piece of the mammary gland was fixed in formalin for histopathology and the rest was snap frozen in liquid nitrogen and stored at -80oC for further molecular analysis. Pathway focused microarrays (Cancer Pathway Finder, and PI3K-AKT Signaling) were performed and the differential regulated genes were validated using real-time RTPCR and immunoblotting. All data obtained were statistically analyzed using Graphpad Prism version 5.03. The repeated measures analysis of variance was used to evaluate the dose and time response to hormone treatment. Multiple comparisons between groups with significant differences were analyzed using Dunnett post–hoc test. Paired t–test was done to analyze intergroup differences. The values of p <0.05 were considered as statistically significant. Results: Short-term treatment with pregnancy levels of estradiol significantly reduced the mammary cancer incidence and multiplicity, while it significantly increased mammary cancer latency. Pathway focused microarray analysis of mammary glands at different time points demonstrated that mice treated with short-term estradiol has persistent changes in gene expression involved in key signaling pathways involved in cancer. Pro-angiogenic genes (Angpt1, Fgf2, Vegfc), anti-apoptotic genes (Bcl2, Birc3), pro-proliferative/survival genes (Akt, Ccnd1, Igf1r, Mtor, Pi3k) were significantly down-regulated in the mammary gland of short-term estradiol treated mice. On contrary, pro-apoptotic genes (Bad,Casp, Cflar) and anti-proliferative/survival genes (Pten, p53) were up-regulated. Conclusion: Our data demonstrates that short-term treatment with pregnancy levels of estradiol is highly effective in inhibiting mammary carcinogenesis in MMTV-neu mice. This protection against HER2 positive mammary cancers is achieved by altering key signaling pathways involved in cancer growth and progression. Citation Format: Subramani R, Lakshmanaswamy R. Hormonal prevention of HER2 positive mammary cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-15.

Frequent overexpression of AMAP1, an Arf6 effector in cell invasion, is characteristic of the MMTV-PyMT rather than the MMTV-Neu human breast cancer model

BackgroundThe small GTPase Arf6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) constitute a signaling pathway promoting cell invasion, in which AMAP1 interacts with several different proteins, including PRKD2, EPB41L5, paxillin, and cortactin. Components of this pathway are often overexpressed in human breast cancer cells, to be correlated with poor prognosis of the patients, whereas overexpression of the Arf6 pathway did not correlate with the four main molecular classes of human breast tumors. In this pathway, receptor tyrosine kinases, including EGFR and Her2, activate Arf6 via GEP100. MMTV-PyMT mice and MMTV-Neu mice are well-established models of human breast cancer, and exhibit the early dissemination and the lung metastasis, by utilizing protein tyrosine phosphorylation for oncogenesis. PyMT-tumors and Neu-tumors are known to have overlapping gene expression profiles, which primarily correspond to the luminal B-type of human mammary tumors, although they differ in the time necessary for tumor onset and metastasis. Given the common usage of protein tyrosine phosphorylation, as well as the frequent use of these animal models for studying breast cancer at the molecular level, we here investigated whether mammary tumors in these mouse models utilize the Arf6-based pathway for invasion.MethodsExpression levels of Arf6, AMAP1, and GEP100 were analyzed in PyMT-tumors and Neu-tumors by western blotting. Expression of Arf6 and AMAP1 was also analyzed by immunohistochemistry. The involvement of AMAP1 in invasion, and the possible correlation of its high expression levels with cancer mesenchymal properties were also investigated.ResultsWe found that PyMT-tumors, but not Neu-tumors, frequently overexpress AMAP1 and use it for invasion, whereas both types of tumors expressed Arf6 and GEP100 at different levels. High levels of the AMAP1 expression among PyMT-tumor cells were frequently correlated with loss of the epithelial marker CK8 and also with expression of the mesenchymal marker vimentin both at the primary sites and at sites of the lung metastases.ConclusionsPyMT-tumors appear to frequently utilize the Arf6-based invasive machinery, whereas Neu-tumors do not. Our results suggest that MMTV-PyMT mice, rather than MMTV-Neu mice, are useful to study the Arf6-based mammary tumor malignancies, as a representative model of human breast cancer.

In Vivo Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior.

Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers.Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models.Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography.Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 23(24); 7531-42. ©2017 AACR.

Targeting Aberrant p70S6K Activation for Estrogen Receptor-Negative Breast Cancer Prevention.

The prevention of estrogen receptor-negative (ER-) breast cancer remains a major challenge in the cancer prevention field, although antiestrogen and aromatase inhibitors have shown adequate efficacy in preventing estrogen receptor-positive (ER+) breast cancer. Lack of commonly expressed, druggable targets is a major obstacle for meeting this challenge. Previously, we detected the activation of Akt signaling pathway in atypical hyperplasic early-stage lesions of patients. In the current study, we found that Akt and the downstream 70 kDa ribosomal protein S6 kinase (p70S6K) signaling pathway was highly activated in ER- premalignant breast lesions and ER- breast cancer. In addition, p70S6K activation induced transformation of ER- human mammary epithelial cells (hMEC). Therefore, we explored the potential of targeting Akt/p70S6K in the p70S6K activated, ER- hMEC models and mouse mammary tumor models for the prevention of ER- breast cancer. We found that a clinically applicable Akt/p70S6K dual inhibitor, LY2780301, drastically decreased proliferation of hMECs with ErbB2-induced p70S6K activation via Cyclin B1 inhibition and cell-cycle blockade at G0-G1 phase, while it did not significantly reverse the abnormal acinar morphology of these hMECs. In addition, a brief treatment of LY2780301 in MMTV-neu mice that developed atypical hyperplasia (ADH) and mammary intraepithelial neoplasia (MIN) lesions with activated p70S6K was sufficient to suppress S6 phosphorylation and decrease cell proliferation in hyperplasic MECs. In summary, targeting the aberrant Akt/p70S6K activation in ER- hMEC models in vitro and in the MMTV-neu transgenic mouse model in vivo effectively inhibited Akt/S6K signaling and reduced proliferation of hMECs in vitro and ADH/MIN lesions in vivo, indicating its potential in prevention of p70S6K activated ER- breast cancer. Cancer Prev Res; 10(11); 641-50. ©2017 AACR.

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

Abstract 1839: TGFBR1*6A mouse model mimics human breast cancer development and progression

Abstract Transforming Growth Factor-ß (TGF-β) is one of the most commonly altered cellular signaling pathways in human cancer. Our group was the first to discover TGFBR1*6A (*6A), a common hypomorphic variant of the type I TGF-ß receptor (TGFBR1), which has been associated with increased risk for breast cancer in multiple epidemiological studies. Functional analysis also suggests that *6A is associated with decreased tumor suppressive TGF-β signaling in early tumor development and is involved with promoting tumorigenesis in advanced carcinomas. To better understand the functional impact of *6A and constitutively reduced TGF-ß signaling on breast cancer development and progression, we have recently developed a series of transgenic knock-in mouse model variants by replacing exon 1 of the mouse Tgfbr1 sequence with human exon 1 sequences harboring both *6A and wild-type (*9A) TGFBR1 variants and crossed them with MMTVneu mice. We generated a total of 5 FVB double and/or triple transgenic mouse strains: MMTVneu;9A/9A, MMTVneu;6A/6A, MMTVneu;9A/6A, MMTVneu;*9A/-, and MMTVneu;6A/-. Following tumor development, measurements were performed three times per week to determine tumor volume. All mice were allowed to live up to 18 months (548 days) or until they died of natural causes. Specimens were collected for later analysis of blood, tumors, normal mammary tissue, and were evaluated for lung metastases. Statistical comparisons performed using Student’s t-test. Only 34.4% of MMTVneu;9A/9A mice (11/32) developed mammary tumors, while 80% of MMTVneu;6A/6A mice (24/30; p=0.0002) and 62.5% of MMTVneu;6A/- mice (19/32, p=0.046) developed tumors. The percentage of MMTVneu;9A/6A and MMTVneu;*9A/- mice that developed tumors was higher than MMTVneu;9A/9A mice (48.3% and 50%, respectively) but was not statistically significant. Additionally, 66.7% of MMTVneu;6A/6A mice (20/30) developed 2 or more tumors, while only 25% of MMTVneu;9A/9A mice (8/32) developed multiple tumors (p=0.0007). Our data suggests that we have developed transgenic mouse strains that accurately mimic human breast cancer susceptibility associated with *6A and constitutively decreased TGFBR1 expression in humans, as evidenced by other studies. These mouse models may be a tool to provide invaluable insight to better understand the role of TGFBR1 variants in the development and progression of breast cancer. Citation Format: Michael James Pennison, Minghui Wang, Hugo Jimenez, Boris Pasche. TGFBR1*6A mouse model mimics human breast cancer development and progression [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 1839. doi:10.1158/1538-7445.AM2017-1839

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

Genetic causes of resistance to entinostat in luminal breast cancer model systems.

1065 Background: Based on promising phase II data, the histone deacetylase inhibitor Entinostat (ENT) is in phase III trials for patients with metastatic ER-positive breast cancer. Predictors of sensitivity and resistance, however, remain unknown. Methods: Luminal cell lines SKBR3 (ER-/HER2+), BT474 (ER+/HER2+) and MCF7 (ER+/HER2-) were treated with or without ENT at their IC50 doses and their gene expression profiles determined. In addition, a total of 27 MMTV-Neu mouse tumors (luminal) were untreated (N = 8), or treated with ENT at 12 mg/kg for 3 weeks (N = 5), 6 weeks (N = 6), or until progression after complete response (N = 8). We investigated their gene expression profiles by microarray and copy number (CN) by arrayCGH, and utilized the Dawnrank analysis, a network-based bioinformatics tool that integrates DNA and RNA data to identify driver genes, to find predictors of resistance to ENT. Results: Supervised analysis of gene expression data coming from the 3 treated cell lines showed significant upregulation of multiple MYC gene signatures. Therefore, we constitutively overexpressed MYC using lentiviral MYC shRNA in SKBR3 and MCF7, and MYC overexpression made cell lines more resistant to ENT. In MMTV-Neu mice, both MYC gene mRNA and gene signatures were downregulated while cells responded to ENT, and became upregulated when the tumors progressed. aCGH CN analysis revealed that a large portion of mouse Chromosome 4 had DNA CN loss and low gene expression in tumors that progressed while on ENT. Within this region, JUN was computationally identified to be a top driver gene associated with resistance. JUN was next knocked down using lentiviral JUN shRNA in BT474 and T47D, and JUN knock-down repeatedly made cell lines more resistant to ENT. MYC gene-expression was also upregulated in JUN-knockdown BT474 and T47D. Finally, JUN CN loss was found in 22% (132/588) of luminal tumors in The Genome Cancer Atlas breast cancer, and all the MYC signature scores were significantly higher in JUN-deleted TCGA samples. Conclusions: ENT was an effective drug for all of our luminal models, both in vitro and in vivo. Using these models, we selected for resistant variants and identified MYC signature expression, and JUN CN deletion as being associated with resistance.

Abstract P6-10-02: MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates

Abstract Background: Lymphocyte-activation gene 3 (LAG-3) is a T-cell checkpoint regulator and a current target in immunotherapy trials. LAG-3's main ligand is MHC class II (MHC-II), to which it binds with higher affinity than CD4. Binding of LAG3 to MHC-II antigen-presenting cells negatively regulates cellular proliferation, activation, and homeostasis of T cells, similarly to CTLA-4 and PD-1, suggesting that antibodies targeting LAG-3 may demonstrate similar anti-tumor immune effects. Hypothesis: We recently reported an association of MHC-II on tumor cells and its involvement in mediating sensitivity to PD-1/PD-L1 monoclonal antibodies. MHC-II demonstrates a strong bimodal expression pattern on tumor cells from a variety of tissues, including those of the breast. In breast cancer patients, tumor-specific MHC-II expression on TNBCs is correlated with a 'hot' immune environment. We hypothesized that 1) MHC-II expression may drive potent anti-tumor immune responses and 2) MHC-II-positive tumors that generate immunotolerance may develop a specific immune checkpoint dependency on LAG-3, since LAG-3 is the inhibitory receptor for MHC-II-mediated antigen presentation. Methods: To determine the functionality of MHC-II in driving anti-tumor immune responses, we constitutively expressed the MHC-II master regulator CIITA in MMTV-neu mouse tumor cells and determined their ability to form tumors in immunocompetent syngeneic hosts. To evaluate the association of MHC-II+ tumors with LAG-3 expression, we evaluated LAG-3-positivity by immunohistochemistry (IHC) in lymphocytic infiltrates in a series of 111 post-NAC TNBC specimens from patients with residual disease remaining after presurgical chemotherapy. Tumor-infiltrating lymphocytes (TILs) were scored by H&E, PD-L1 and MHC-II (HLA-DR) were scored in the stroma and tumor compartments using automated quantitative immunofluorescence (AQUA). Results: Enforced expression of MHC-II via constitutive expression of CIITA caused rejection in 60% of mice, while only 11% of mice rejected MMTV-neu tumors expressing the vector control (Fisher's exact p=0.04). All rejecting mice were immune to rechallenge with parental (non-CIITA-expressing) MMTV-neu cells, suggesting a memory effector response. Clinically, 11/102 patients (10.8%) had LAG-3+ immune cells in their tumor. LAG-3+ tumors were strongly correlated with MHC-II positivity in tumor cells (p<0.0001). Presence of LAG-3+ cells also correlated strongly with overall TILs (p<0.0001), and PD-L1 expression on TILs (p<0.02). Since the likelihood of identifying LAG3+ lymphocytes is confounded by the inclusion of poorly-infiltrated tumors, we performed a subset analysis on only those tumors with substantial TILs (>20%). When this subset was analyzed, LAG-3 positivity retained its association with tumor MHC-II expression (p=0.0001), while the association of LAG-3 with stromal PD-L1 was reduced below the level of significance (p=0.052). Conclusions: MHC-II expression causes increased immune activation in breast cancers, consistent with our previous findings. MHC-II positivity in breast tumors may identify a population with preferential dependence on the LAG-3 checkpoint, which may be important for future immunotherapy trials. Citation Format: Balko JM, Loi S, Giltnane JM, Combs S, Estrada MV, Sanchez V, Rimm D, Sanders ME, Salgado R, Gomez H, Johnson DB. MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates [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 P6-10-02.