Search

PDF file - 4MB, Loss of ErbB3 impairs hyperplasia of MMTV-Neu mammary glands.

Supplementary Figures 1-2 from Tie2 Signaling Regulates Osteoclastogenesis and Osteolytic Bone Invasion of Breast Cancer

Increasing evidence suggests that HER2-amplified breast cancer cells use HER3/ErbB3 to drive therapeutic resistance to HER2 inhibitors. However, the role of ErbB3 in the earliest events of breast epithelial transformation remains unknown. Using mouse mammary specific models of Cre-mediated ErbB3 ablation, we show that ErbB3 loss prevents the progressive transformation of HER2-overexpressing mammary epithelium. Decreased proliferation and increased apoptosis were seen in MMTV-HER2 and MMTV-Neu mammary glands lacking ErbB3, thus inhibiting premalignant HER2-induced hyperplasia. Using a transgenic model in which HER2 and Cre are expressed from a single polycistronic transcript, we showed that palpable tumor penetrance decreased from 93.3% to 6.7% upon ErbB3 ablation. Penetrance of ductal carcinomas in situ was also decreased. In addition, loss of ErbB3 impaired Akt and p44/42 phosphorylation in preneoplastic HER2-overexpressing mammary glands and in tumors, decreased growth of preexisting HER2-overexpressing tumors, and improved tumor response to the HER2 tyrosine kinase inhibitor lapatinib. These events were rescued by reexpression of ErbB3, but were only partially rescued by ErbB36F, an ErbB3 mutant harboring six tyrosine-to-phenylalanine mutations that block its interaction with phosphatidyl inositol 3-kinase. Taken together, our findings suggest that ErbB3 promotes HER2-induced changes in the breast epithelium before, during, and after tumor formation. These results may have important translational implications for the treatment and prevention of HER2-amplified breast tumors through ErbB3 inhibition. Cancer Res; 72(10); 2672–82. ©2012 AACR.

PDF file - 122K, Loss of ErbB3-PI3K interactions increases lapatinib sensitivity in NiB3 cells.

PDF file - 3.7MB, Partial Cre-mediated loss of ErbB3 expression in ErbB3MMTV-KO mammary glands.

EphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. In this study, the potential role of EphA2 in this clinically relevant phenomenon is investigated as metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients. It was found that the EphA2 function in breast cancer cells promotes osteoclast activation and the development of osteolytic bone disease. Blocking EphA2 function molecularly and pharmacologically in breast tumors reduced the number and size of bone lesions and the degree of osteolytic disease in intratibial and intracardiac mouse models, which correlated with a significant decrease in the number of osteoclasts at the tumor–bone interface. EphA2 loss of function in tumor cells impaired osteoclast progenitor differentiation in coculture, which is mediated, at least in part, by reduced expression of IL‐6. EPHA2 transcript levels are enriched in human breast cancer bone metastatic lesions relative to visceral metastatic sites; EphA2 protein expression was detected in breast tumor cells in bone metastases in patient samples, supporting the clinical relevance of the study's findings. These data provide a strong rationale for the development and application of molecularly targeted therapies against EphA2 for the treatment of breast cancer bone metastatic disease. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

During pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland. We assessed mRNA expression levels by expression microarray of mouse mammary glands harvested throughout pregnancy and lactation. To study the role of ErbB3 in mammary gland lactogenesis, we used transgenic mice expressing WAP-driven Cre recombinase to generate a mouse model in which conditional ErbB3 ablation occurred specifically in alveolar mammary epithelial cells (aMECs). Profiling of RNA from mouse MECs isolated throughout pregnancy revealed robust Erbb3 induction during mid-to-late pregnancy, a time point when aMECs proliferate rapidly and undergo differentiation to support milk production. Litters nursed by ErbB3 KO dams weighed significantly less when compared to litters nursed by ErbB3 WT dams. Further analysis revealed substantially reduced epithelial content, decreased aMEC proliferation, and increased aMEC cell death during late pregnancy. Consistent with the potent ability of ErbB3 to activate cell survival through the PI3K/Akt pathway, we found impaired Akt phosphorylation in ErbB3 KO samples, as well as impaired expression of STAT5A, a master regulator of lactogenesis. Constitutively active Akt rescued cell survival in ErbB3-depleted aMECs, but failed to restore STAT5A expression or activity. Interestingly, defects in growth and survival of ErbB3 KO aMECs as well as Akt phosphorylation, STAT5A activity, and expression of milk-encoding genes observed in ErbB3 KO MECs progressively improved between late pregnancy and lactation day 5. We found a compensatory upregulation of ErbB4 activity in ErbB3 KO mammary glands. Enforced ErbB4 expression alleviated the consequences of ErbB3 ablation in aMECs, while combined ablation of both ErbB3 and ErbB4 exaggerated the phenotype. These studies demonstrate that ErbB3, like ErbB4, enhances lactogenic expansion and differentiation of the mammary gland during pregnancy, through activation of Akt and STAT5A, two targets crucial for lactation.

Breast cancers that occur in women 2–5 years postpartum are more frequently diagnosed at metastatic stages and correlate with poorer outcomes compared with breast cancers diagnosed in young, premenopausal women. The molecular mechanisms underlying the malignant severity associated with postpartum breast cancers (ppBCs) are unclear but relate to stromal wound-healing events during postpartum involution, a dynamic process characterized by widespread cell death in milk-producing mammary epithelial cells (MECs). Using both spontaneous and allografted mammary tumors in fully immune–competent mice, we discovered that postpartum involution increases mammary tumor metastasis. Cell death was widespread, not only occurring in MECs but also in tumor epithelium. Dying tumor cells were cleared through receptor tyrosine kinase MerTK–dependent efferocytosis, which robustly induced the transcription of genes encoding wound-healing cytokines, including IL-4, IL-10, IL-13, and TGF-β. Animals lacking MerTK and animals treated with a MerTK inhibitor exhibited impaired efferocytosis in postpartum tumors, a reduction of M2-like macrophages but no change in total macrophage levels, decreased TGF-β expression, and a reduction of postpartum tumor metastasis that was similar to the metastasis frequencies observed in nulliparous mice. Moreover, TGF-β blockade reduced postpartum tumor metastasis. These data suggest that widespread cell death during postpartum involution triggers efferocytosis-induced wound-healing cytokines in the tumor microenvironment that promote metastatic tumor progression.

ErbB3, a member of the ErbB family of receptor tyrosine kinases, is a potent activator of phosphatidyl inositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling, driving tumor cell survival and therapeutic resistance in breast cancers. In luminal breast cancers, ErbB3 upregulation following treatment with the antiestrogen fulvestrant enhances PI3K/mTOR-mediated cell survival. However, the mechanism by which ErbB3 is upregulated in fulvestrant-treated cells is unknown. We found that ErbB3 protein levels and cell surface presentation were increased following fulvestrant treatment, focusing our attention on proteins that regulate ErbB3 at the cell surface, including Nrdp1, NEDD4 and LRIG1. Among these, only LRIG1 correlated positively with ERα, but inversely with ErbB3 in clinical breast cancer data sets. LRIG1, an estrogen-inducible ErbB downregulator, was decreased in a panel of fulvestrant-treated luminal breast cancer cells. Ectopic LRIG1 expression from an estrogen-independent promoter uncoupled LRIG1 from estrogen regulation, thus sustaining LRIG1 and maintaining low ErbB3 levels in fulvestrant-treated cells. An LRIG1 mutant lacking the ErbB3 interaction motif was insufficient to downregulate ErbB3. Importantly, LRIG1 overexpression improved fulvestrant-mediated growth inhibition, whereas cells expressing the LRIG1 mutant were poorly sensitive to fulvestrant, despite effective ERα downregulation. Consistent with these results, LRIG1 expression correlated positively with increased disease-free survival in antiestrogen-treated breast cancer patients. These data suggest that ERα-dependent expression of LRIG1 dampens ErbB3 signaling in luminal breast cancer cells, and by blocking ERα activity with fulvestrant, LRIG1 is decreased thus permitting ErbB3 accumulation, enhanced ErbB3 signaling to cell survival pathways and blunting therapeutic response to fulvestrant.

Increasing evidence suggests that HER2-amplified breast cancer cells use HER3/ErbB3 to drive therapeutic resistance to HER2 inhibitors. However, the role of ErbB3 in the earliest events of breast epithelial transformation remains unknown. Using mouse mammary specific models of Cre-mediated ErbB3 ablation, we show that ErbB3 loss prevents the progressive transformation of HER2-overexpressing mammary epithelium. Decreased proliferation and increased apoptosis were seen in MMTV-HER2 and MMTV-Neu mammary glands lacking ErbB3, thus inhibiting premalignant HER2-induced hyperplasia. Using a transgenic model in which HER2 and Cre are expressed from a single polycistronic transcript, we showed that palpable tumor penetrance decreased from 93.3% to 6.7% upon ErbB3 ablation. Penetrance of ductal carcinomas in situ was also decreased. In addition, loss of ErbB3 impaired Akt and p44/42 phosphorylation in preneoplastic HER2-overexpressing mammary glands and in tumors, decreased growth of preexisting HER2-overexpressing tumors, and improved tumor response to the HER2 tyrosine kinase inhibitor lapatinib. These events were rescued by reexpression of ErbB3, but were only partially rescued by ErbB36F, an ErbB3 mutant harboring six tyrosine-to-phenylalanine mutations that block its interaction with phosphatidyl inositol 3-kinase. Taken together, our findings suggest that ErbB3 promotes HER2-induced changes in the breast epithelium before, during, and after tumor formation. These results may have important translational implications for the treatment and prevention of HER2-amplified breast tumors through ErbB3 inhibition. Cancer Res; 72(10); 2672–82. ©2012 AACR.

One arising challenge in the treatment of breast cancer is the development of therapeutic resistance to trastuzumab, an antibody targeting the human epidermal growth factor receptor-2 (HER2), which is frequently amplified in breast cancers. In this study, we provide evidence that elevated level of the receptor tyrosine kinase Eph receptor A2 (EphA2) is an important contributor to trastuzumab resistance. In a screen of a large cohort of human breast cancers, we found that EphA2 overexpression correlated with a decrease in disease-free and overall survival of HER2-overexpressing patients. Trastuzumab-resistant cell lines overexpressed EphA2, whereas inhibiting EphA2 restored sensitivity to trastuzumab treatment in vivo. Notably, trastuzumab treatment could promote EphA2 phosphorylation by activating Src kinase, leading in turn to an amplification of phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signaling in resistant cells. Our findings offer mechanistic insights into the basis for trastuzumab resistance and rationalize strategies to target EphA2 as a tactic to reverse trastuzumab resistance. Cancer Res; 70(1); 299–308.

Vav guanine nucleotide exchange factors modulate changes in cytoskeletal organization through activation of Rho, Rac, and Cdc42 small GTPases. Although Vav1 expression is restricted to the immune system, Vav2 and Vav3 are expressed in several tissues, including highly vascularized organs. Here, we provide the first evidence that Vav2 and Vav3 function within the tumor microenvironment to promote tumor growth, survival, and neovascularization. Host Vav2/3 deficiency reduced microvascular density, as well as tumor growth and/or survival, in transplanted B16 melanoma and Lewis lung carcinoma models in vivo. These defects were due in part to Vav2/3 deficiency in endothelial cells. Vav2/3-deficient endothelial cells displayed reduced migration in response to tumor cells in coculture migration assays, and failed to incorporate into tumor vessels and enhance tumor volume in tumor-endothelial cotransplantation experiments. These data suggest that Vav2/3 guanine nucleotide exchange factors play a critical role in host-mediated tumor progression and angiogenesis, particularly in tumor endothelium.(Mol Cancer Res 2009;7(5):615–23)

Cell death in response to physiological cues is a feature of many normal tissues, and often occurs due to tissue damage or disease. Regardless of the reason, dying cells require their rapid removal from the tissue in order to maintain homeostasis in the tissue, prevent potential cytotoxic immunity against self-antigens, and to promote wound healing. Phagocytic removal of dying cells is referred to as efferocytosis, a process necessary for sterile wound healing. Interestingly, malignant tumor progression has long been compared to wound healing, and recent evidence demonstrates a molecular link between efferocytosis and tumor a ‘wound healing’ phenotype. We discuss the impact of efferocytosis in the normal breast and in breast cancers.

Programmed cell death, or apoptosis, occurs in nearly all tissues of all multi-cellular organisms. In order to avoid leakage of intracellular contents, which could generate tissue damaging inflammation, apoptotic cells are cleared from tissues by phagocytes, which then dispatch the engulfed dying cell through the lysosomal pathway. Phagocytic clearance of apoptotic cells is referred to as efferocytosis. One key feature of efferocytosis is the production and release of wound healing cytokines by the phagocyte, which acts to resolve inflammation, and promote tissue repair. Phagocytic engulfment of apoptotic cells coupled with cytokine modulation aimed at immune suppression ensures that physiological programmed cell death does not induce inflammation and tissue damage. However, cytokines involved in wound healing and immune suppression are notorious for their role in the tumor microenvironment, increasing tumor cell motility and promoting evasion of anti-tumor immunity. Therefore, current and future studies aimed at targeting important players of efferocytosis should reveal new and efficacious therapeutic approaches for limiting cancer progression and relapse.

The breast endures vast changes during reproductive phases of a woman's life (puberty, pregnancy, lactation, post-partum involution, post-menopausal involution). Each phase uniquely shapes cancer susceptibility, formation, and progression. Although pregnancy at a young age decreases lifetime breast cancer risk, the first five years following pregnancy at any age are associated with increased breast cancer risk regardless of the woman's age, and with even greater risk with increasing age at the woman's first pregnancy [1]. Increasingly, women are postponing child-birth, which may increase the incidence of post-partum breast cancer (ppBC), defined as those breast cancers diagnosed 2–5 years after pregnancy. These ppBCs are distinguishable from those breast cancers that are diagnosed and treated during pregnancy, and which never are exposed to post-partum/post-lactational involution, and which correlate with a favorable prognosis. Currently, ppBC accounts for nearly 25% of all breast cancers in young (pre-menopausal) women. In contrast, ppBCs are highly aggressive, metastatic, and life-threatening, even when corrected for molecular breast cancer subtype and for the age of the woman at diagnosis [1]. Mouse models of ppBC that specifically compare mammary tumors from nulliparous (virgin) mice to those from age-matched parous (single pregnancy) mice confirm that post-partum involution increases metastasis by up to 10-fold [2, 3]. The molecular mechanisms underlying the exaggerated lethality of post-partum breast cancers are related to an exaggerated abundance of M2-like tumor associated macrophages, which produce immune suppressive and wound healing cytokines and proteases that modify the post-partum mammary (and tumor) microenvironment [4], although the mechanisms that trigger this shift in macrophage behavior in the post-partum mammary gland remained obscure. It was recently demonstrated that widespread cell death, a hallmark of the mammary gland during post-partum involution when milk production ceases, triggers macrophage-mediated efferocytosis, M2 macrophage polarization and Th2 cytokine production in normal mammary glands during post-partum involution [5]. Remarkably, widespread cell death efferocytosis, macrophage M2 polarization, and Th2 cytokine-mediated wound healing in malignant post-partum breast cancers was similarly observed [3]. Under physiological conditions, dying cells are rapidly removed from the breast to prevent secondary necrosis of the dying cell, wherein intracellular antigens released from the necrotic cell might trigger inflammation, tissue damage, or autoimmunity [6]. To ensure suppression of inflammation or autoimmunity, efferocytosis is coupled with production of cytokines that dampen tissue-damaging immune responses, such as interleukin (IL)-10, IL-4, and Transforming Growth Factor (TGF)-β [7]. Macrophages use multiple cell surface protein to recognize and engulf dying cells. Among these, the receptor tyrosine kinase (RTK) MerTK is essential for post-partum efferocytosis and for subsequent induction of immunosuppressive and wound healing cytokines [6]. Genetically engineered mouse models lacking MerTK activity display impaired efferocytosis and limited expression of wound healing cytokines during post-partum involution, resulting in severe immune-mediated damage and scarring to the post-partum mammary gland that interferes with the success of lactogenesis upon future pregnancies [5]. We recently found that efferocytosis was a key driver of malignant progression in ppBCs, responsible for exaggerated M2-like polarization of tumor-infiltrating macrophages and production of IL-4, TGFβ, and IL-10 [3]. Genetic MerTK ablation inhibited efferocytosis in ppBCs, blocked macrophage M2-like polarization, impaired expression of efferocytosis-induced cytokines, and repressed formation of lung metastases. Pharmacologic inhibition of MerTK for the first 7 days of post-partum involution similarly blocked efferocytosis, and significantly decreased metastatic burden. Thus, a causal relationship exists between the tissue remodeling during physiological postpartum involution and the increased metastasis of postpartum mammary tumors. Both scenarios are characterized by transient and widespread programmed cell death, efferocytosis, and the abundant M2-like macrophages and wound-healing cytokines that associate with reduced breast cancer survival. These observations highlight tumor cell death as a double-edged sword in the tumor microenvironment: although the chemotherapies, targeted therapies and radiation provide the benefit of widespread tumor cell death and tumor shrinkage, widespread efferocytosis in response to tumor cell death may enhance tumor wound healing, thus limiting the effectiveness of the targeted agent. In some cases, efferocytosis may even promote tumor metastasis. These issues require careful consideration and experimental testing, as the role of efferocytosis in modulating the stromal response to therapeutically-induced tumor cell death is not fully understood. These recent findings support future endeavors to examine efferocytosis/MerTK targeting in combination with current treatment strategies to block unhealthy ‘tumor healing’ and improve tumor response to treatment.

Studies have shown that ErbB3 and ErbB4 (the neuregulin receptors) are required for growth and survival of the two major luminal mammary epithelial cell types, the ductal luminal mammary epithelium, and the milk-producing alveolar mammary epithelium. However, as both ErbB3 and ErbB4 are capable of binding to neuregulins and heterodimerizing with other ErbB family members, there is evidence that ErbB4 may compensate for loss of ErbB3, and vice versa making it likely that the neuregulin receptors drive cell growth and survival of breast cancers derived from the luminal breast epithelium. Our early studies demonstrate loss of ErbB3 expansion of the alveolar epithelium during pregnancy, decreased expression of genes encoding milk proteins, and decreased milk delivery to nursing pups. This is similar to what is seen in ErbB4-deficient mammary glands. We are currently studying the ErbB3-dependnet molecular mechanisms required for expansion of the alveolar epithelium during pregnancy, and have generated mice that lack ErbB3 and ErbB4 in the alveolar mammary epithelium for specific analysis of neuregulin loss in mammary morphogenesis, lactational differentiation, and breast cancer development.

Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ~30% of patients with breast cancer. However, many patients have residual cancer after chemotherapy, which correlates with a higher risk of metastatic recurrence and poorer outcome than those who achieve a pCR. We hypothesized that molecular profiling of tumors after NAC would identify genes associated with drug resistance. Digital transcript counting was used to profile surgically resected breast cancers after NAC. Low concentrations of dual specificity protein phosphatase 4 (DUSP4), an ERK phosphatase, correlated with high post-NAC tumor cell proliferation and with basal-like breast cancer (BLBC) status. BLBC had higher DUSP4 promoter methylation and gene expression patterns of Ras-ERK pathway activation relative to other breast cancer subtypes. DUSP4 overexpression increased chemotherapy-induced apoptosis, whereas DUSP4 depletion dampened the response to chemotherapy. Reduced DUSP4 expression in primary tumors after NAC was associated with treatment-refractory high Ki-67 scores and shorter recurrence-free survival. Finally, inhibition of mitogen-activated protein kinase kinase (MEK) synergized with docetaxel treatment in BLBC xenografts. Thus, DUSP4 downregulation activates the Ras-ERK pathway in BLBC, resulting in an attenuated response to anti-cancer chemotherapy.

Disease or malformation of heart valves is one of the leading causes of morbidity and mortality in both children and adults. These congenital anomalies can remain undetected until cardiac function is compromised, making it important to understand the underlying nature of these disorders. Here we show that ephrin-A1, a ligand for class A Eph receptor tyrosine kinases, regulates cardiac valve formation. Exogenous ephrin-A1-Fc or overexpression of ephrin-A1 in the heart inhibits epithelial-to-mesenchymal transformation (EMT) in chick atrioventricular cushion explants. In contrast, overexpression of wild-type EphA3 receptor promotes EMT via a kinase-dependent mechanism. To analyze ephrin-A1 in vivo, we generated an ephrin-A1 knockout mouse through gene targeting. Ephrin-A1 null animals are viable but exhibit impaired cardiac function. Loss of ephrin-A1 results in thickened aortic and mitral valves in newborn and adult ephrin-A1-deficient mice. Analysis of early embryonic hearts revealed increased cellularity in outflow tract endocardial cushions and elevated mesenchymal marker expression, suggesting that excessive numbers of cells undergo EMT. Taken together, these data indicate that ephrin-A1 regulates cardiac valve development, making ephrin-A1-deficient mice a novel model for congenital heart defects.

Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The metastases are often incurable and are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared with normal and benign breast tumors and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem cell differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As CD11b myeloid cells have the potential to become osteoclasts and Tie2 is present in a certain population of these cells, we isolated Tie2+ and Tie2− myeloid cells. We observed a significant reduction of osteoclastogenesis in Tie2− compared with Tie2+ CD11b cells. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclasts and tumor angiogenesis. Collectively, these data reveal a direct and novel role of Tie2 signaling in osteoclast differentiation. These findings identify Tie2 as a therapeutic target for controlling not only tumor angiogenesis but also osteolytic bone metastasis in breast cancer. Cancer Res; 70(7); 2819–28

Eph receptor tyrosine kinases, including EphA2, are expressed in the mammary gland. However, their role in mammary gland development remains poorly understood. Using EphA2-deficient animals, we demonstrate for the first time that EphA2 receptor function is required for mammary epithelial growth and branching morphogenesis. Loss of EphA2 decreased penetration of mammary epithelium into fat pad, reduced epithelial proliferation, and inhibited epithelial branching. These defects appear to be intrinsic to loss of EphA2 in epithelium, as transplantation of EphA2-deficient mammary tissue into wild-type recipient stroma recapitulated these defects. In addition, HGF-induced mammary epithelial branching morphogenesis was significantly reduced in EphA2-deficient cells relative to wild-type cells, which correlated with elevated basal RhoA activity. Moreover, inhibition of ROCK kinase activity in EphA2-deficient mammary epithelium rescued branching defects in primary three-dimensional cultures. These results suggest that EphA2 receptor acts as a positive regulator in mammary gland development, functioning downstream of HGF to regulate branching through inhibition of RhoA. Together, these data demonstrate a positive role for EphA2 during normal mammary epithelial proliferation and branching morphogenesis.

Eph receptor tyrosine kinase signaling regulates cancer initiation and metastatic progression through multiple mechanisms. Studies of tumor-cell-autonomous effects of Eph receptors demonstrate their dual roles in tumor suppression and tumor promotion. In addition, Eph molecules function in the tumor microenvironment, such as in vascular endothelial cells, influencing the ability of these molecules to promote carcinoma progression and metastasis. The complex nature of Eph receptor signaling and crosstalk with other receptor tyrosine kinases presents a unique challenge and an opportunity to develop therapeutic intervention strategies for targeting breast cancer.

ErbB3 and ErbB4 are members of the ErbB family of receptor tyrosine kinases (RTK) that can be activated by neuregulins (NRGs) and as a result are often referred to as the neuregulin receptors. The neuregulin receptors are required for growth and survival of the ductal and alveolar luminal epithelium. We have recently shown that ErbB3 is required for survival and growth of the ductal luminal epithelium and to maintain the balance of differentiated epithelial cell types in the mammary gland, which is consistent with the fact the ErbB3 expression is highest in the ductal luminal epithelium and in luminal progenitor populations of the mouse and human mammary glands. ErbB4, is also required for growth in the luminal mammary epithelium, but not within the ductal luminal population, rather in the alveolar luminal population. In addition to neuregulin receptor signaling in development, ErbB3 and ErbB4 expression has been found to be at the highest levels in luminal breast cancers compared to other molecular breast cancer subtypes. Furthermore, human luminal breast cancer cell lines also express higher levels of ErbB3 and ErbB4 than cell lines derived from other molecular breast cancer subtypes. Since both ErbB3 and ErbB4 are capable of binding to neuregulin ligands and heterodimerizing with other ErbB family members, there is evidence that ErbB4 may compensate for loss of ErbB3, and vice versa. Thus, our goal is to understand how combined loss of ErbB3 and ErbB4 impact the normal luminal cell populations and the transformed cell populations of the breast epithelium. We hypothesize that loss of ErbB3 and ErbB4 will decrease cellular growth and survival during development and in our mouse models of breast cancer. Early studies demonstrate conditional loss of ErbB3fl/fl and ErbB4fl/fl independently and in combination decrease pup weights compared to control mice and demonstrate reduced epithelium/alveolar cell expansion in whole mount and histology analysis. In addition, early analysis of ErbB3fl/fl, ErbB4fl/fl ErbB3fl/fl/ErbB4fl/fl crossed to the p110 inducible mouse model of breast cancer are trending to a delay in tumor onset. Molecular analysis demonstrates a reduction in Pi3K/Akt signaling and Jak/Stat signaling in ErbB3fl/fl and ErbB4fl/fl respectively, with ongoing studies currently underway to analyze signaling in ErbB3fl/fl/ErbB4fl/fl mice in development. Citation Format: David B. Vaught, Donna Hicks, Violeta Sanchez, Rebecca Cook. Neuregulin signaling in development and transformation of the luminal breast epithelium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4196. doi:10.1158/1538-7445.AM2015-4196

The goal of these studies is to determine how combined loss of ErbB3 and ErbB4 impact the normal and transformed luminal population of the breast epithelium. Although little is known regarding the role of ErbB3 in luminal breast cancers, and even less about ErbB4, we have shown that ErbB3 and ErbB4 (the neuregulin receptors) are required for growth and survival of the two major luminal mammary epithelial cell types, the ductal luminal mammary epithelium, and the milk-producing alveolar mammary epithelium. However, as both ErbB3 and ErbB4 are capable of binding to neuregulins and heterodimerizing with other ErbB family members, there is evidence that ErbB4 may compensate for loss of ErbB3, and vice versa. Further, given that ErbB3 and ErbB4 are uniquely required within the luminal breast epithelium during phases of extreme physiological proliferation (i.e., puberty and pregnancy, respectively) it is likely that the neuregulin receptors drive cell growth and survival of breast cancers derived from the luminal breast epithelium. We hypothesize that elimination of NRG signaling by combined loss of ErbB3 and ErbB4 would prevent expansion of the mammary epithelium in response to physiological cues (i.e. pregnancy) and pathological cues (oncogene expression). Our early studies demonstrate loss of ErbB3 expansion of the alveolar epithelium during pregnancy, decreased expression of genes encoding milk proteins, and decreased milk delivery to nursing pups. This is similar to what is seen in ErbB4-deficient mammary glands. We are currently studying the ErbB3-dependnet molecular mechanisms required for expansion of the alveolar epithelium during pregnancy, and are generating mice that lack ErbB3 and ErbB4 in the alveolar mammary epithelium. We anticipate that combined gene targeting of ErbB3 and ErbB4 will cause a more pronounced lactational deficit. Citation Format: David B. Vaught, Donna Hicks, Rebecca Cook. Neuregulin signaling in development and transformation of the luminal breast epithelium. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B013.

HER3/ErbB3 is required in luminal mammary epithelial cells (MECs). Given that breast cancer phenotypes may reflect biological traits of the MECs from which they originate, we tested the hypothesis that HER3/ErbB3 drives luminal breast cancer growth. We found higher ERBB3 expression and frequent ERBB3 gene copy gains in clinical Luminal A/B breast cancers over other molecular subtypes. ErbB3 increased growth of luminal breast cancer cells, while ErbB3 targeting using U3-1287, a fully-human monoclonal anti-ErbB3 antibody, decreased three-dimensional colony growth, increased apoptosis, and decreased tumor growth in vivo. In response to the anti-endocrine fulvestrant, ErbB3 expression increased in clinical breast tumors, as did phosphatidylinositol 3-kinase (PI3K)/mTOR signaling. However, ErbB3 targeting with U3-1287 blocked compensatory PI3K/mTOR signaling in fulvestrant-treated tumor cells, transcriptional responses to fulvestrant, tumor cell survival and tumor growth. U3-1287 produced these effects in tumor cells with and without genetic PI3K pathway aberrations (PIK3CAH1047R, PIK3CAE545K, PTEN loss). Although fulvestrant increased phosphorylation of all ErbB family RTKs and insulin-like growth factor-1 receptor, phospho-RTK upregulation was not seen in tumors treated with U3-1287 and fulvestrant. Therefore, luminal breast cancer cells utilize ErbB3 for growth, survival, and to limit response to fulvestrant, thus defining a novel role for ErbB3 in breast cancer. Citation Format: Meghan M. Morrison, Jamie Stanford, Christian Young, David Vaught, Andrew Williams, michelle williams, Chuck M. Perou, Carlos L. Arteaga, Rebecca S. Cook. ErbB3 downregulation enhances luminal breast tumor response to antiestrogens. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A053.