David B. Vaught

Profiling of residual breast cancers after neoadjuvant chemotherapy identifies DUSP4 deficiency as a mechanism of drug resistance

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.

Elevation of Receptor Tyrosine Kinase EphA2 Mediates Resistance to Trastuzumab Therapy

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.

HER3 Is Required for HER2-Induced Preneoplastic Changes to the Breast Epithelium and Tumor Formation

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.

Eph receptors in breast cancer: roles in tumor promotion and tumor suppression

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.

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution

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.

Host Deficiency in Vav2/3 Guanine Nucleotide Exchange Factors Impairs Tumor Growth, Survival, and Angiogenesis In vivo

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)

Regulation of heart valve morphogenesis by Eph receptor ligand, ephrin-A1.

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 animals. 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. Developmental Dynamics 239:3226–3234, 2010.

Tie2 Signaling Regulates Osteoclastogenesis and Osteolytic Bone Invasion of Breast Cancer

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.

Decreased LRIG1 in fulvestrant-treated luminal breast cancer cells permits ErbB3 upregulation and increased growth

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.

Clearance of dying cells accelerates malignancy.

The breast endures vast changes during reproductive phases of a womans 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 womans age, and with even greater risk with increasing age at the womans 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.