EphA2 Is a Clinically Relevant Target for Breast Cancer Bone Metastatic Disease.

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.

ErbB3 drives mammary epithelial survival and differentiation during pregnancy and lactation.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Efferocytosis creates a tumor microenvironment supportive of tumor survival and metastasis.

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.

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-ß. 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-ß expression, and a reduction of postpartum tumor metastasis that was similar to the metastasis frequencies observed in nulliparous mice. Moreover, TGF-ß 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.

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.

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.

Abner Doubleday, Marc Bloch, and the cultural significance of baseball in rural America.

In 1907 baseball's promoters decreed that Civil War hero Abner Doubleday created the game in the village of Cooperstown, New York, in 1839. Baseball thus acquired a distinctly rural American origin and a romantic pastoral appeal. Skeptics have since presented irrefutable evidence that America's pastime was neither born in the United States nor was a product of rural life. But in their zeal to debunk the myth of baseball's rural beginnings, historians have fallen prey to what Annales School founder Marc Bloch famously called the "idol of origins," and all but neglected the very real phenomenon of rural baseball itself. The claim that baseball has always been "a city game for city men" does not stand up to empirical scrutiny anymore than the Doubleday myth itself, as this address demonstrates with three case studies -- Cooperstown in the 1830s, Davisville, California, in the 1880s, and Milroy, Minnesota, in the 1950s. Baseball may have been a source of rural nostalgia for city people, but it was the sport of choice for farmers and a powerful cultural agent.

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.

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.

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.

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.

Regulation of mammary gland branching morphogenesis by EphA2 receptor tyrosine kinase.

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 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.