Joyce A. Schroeder

Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors

Heart septation and valve malformations constitute the most common anatomical birth defects. These structures arise from the endocardial cushions within the atrioventricular canal (AVC) through dynamic interactions between cushion cells and the extracellular matrix (termed cardiac jelly). Transformation of endothelial cells to mesenchymal cells is essential for the proper development of the AVC and subsequent septation and valve formation. Atrioventricular septal defects can result from incomplete endocardial cushion morphogenesis. We show that hyaluronan-deficient AVC explants from Has2−/− embryos, which normally lack mesenchyme formation, are rescued by heregulin treatment, which restores phosphorylation of ErbB2 and ErbB3. These events were blocked using a soluble ErbB3 molecule, as well as with an inhibitor of ErbB2, herstatin. We show further that ErbB3 is activated during hyaluronan treatment of Has2−/− explants. These data provide a link between extracellular matrix-hyaluronan and ErbB receptor activation during development of early heart-valve and septal mesenchyme.

Form and function of developing heart valves: coordination by extracellular matrix and growth factor signaling

It is becoming clear that converging pathways coordinate early heart valve development and remodeling into functional valve leaflets. The integration of these pathways begins with macro and molecular interactions outside the cell in the extracellular matrix separating the myocardial and endocardial tissue components of the rudimentary heart. Such interactions regulate events at the cell surface through receptors, proteases, and other membrane molecules which in turn transduce signals into the cell. These signals trigger intracellular cascades that transduce cellular responses through both transcription factor and cofactor activation mediating gene induction or suppression. Chamber septation and valve formation occur from these coordinated molecular events within the endocardial cushions to sustain unidirectional blood flow and embryo viability. This review discusses the emerging connection between extracellular matrix and growth factor receptor signaling during endocardial cushion morphogenesis by highlighting the extracellular component, hyaluronan, and erbB receptor functions during early valve development.

MUC1 alters β-catenin-dependent tumor formation and promotes cellular invasion

MUC1 is aberrantly expressed in greater than 90% of all breast carcinomas, yet its function as a tumor antigen is not fully understood. Recently, studies have shown that MUC1 interacts with β-catenin, erbB receptors, src, GSK-3β and protein kinase Cδ, possibly in a complex that promotes the disassembly of adherens junctions and the invasion of cells. Here we show that the deletion of Muc1 expression from MMTV-Wnt-1 transgenic mice results in a significant increase in the time to mammary gland tumor onset. Analysis of MMTV-Wnt-1 tumors on a wild-type Muc1 background shows a tumor-specific complex formation between Muc1 and β-catenin that can be observed in both the membrane and the cytoplasm of transformed epithelium. Analysis of primary human adenocarcinomas revealed that this MUC1/β–catenin interaction occurs in both primary and metastatic tumors, but is dramatically increased in metastatic lesions. Addition of MUC1-cytoplasmic domain peptides to the invasive MDA-MB-468 and MDA-MB-231 cell lines increases their invasive capability, and these peptides colocalize with both β-catenin and the focal adhesion protein vinculin, primarily at sites of membrane invasion into a collagen matrix. These data indicate a potential mechanism for MUC1 promotion of invasive tumorigenesis in the breast through the modulation of β-catenin localization and subsequent cytoskeletal dynamics.

CD44 Attenuates Metastatic Invasion during Breast Cancer Progression

Metastatic invasion is the primary cause of breast cancer mortality, and adhesion receptors, such as CD44, are believed to be critical in this process. Historically, primary breast tumor epithelium has been investigated in isolation from other tissue components, leading to the common interpretation that CD44 and its primary ligand, hyaluronan, promote invasion. Here, we provide in vivo evidence showing CD44 antagonism to breast cancer metastasis. In a mouse model of spontaneously metastasizing breast cancer (MMTV-PyV mT), we found that loss of CD44 promotes metastasis to the lung. Localization studies, in combination with a novel hyaluronan synthase-GFP transgenic mouse, show a restricted pattern of expression for CD44 and hyaluronan. Whereas CD44 is expressed in tumor epithelium, hyaluronan synthase expression is restricted to stromal-associated cells. This distinct CD44 and hyaluronan pattern of distribution suggests a role for epithelial-stromal interaction in CD44 function. To define the relevance of this spatial regulation, we developed an in vitro invasion assay to emulate invasion into the extracellular matrix. Invasion of CD44-positive tumor cells was inhibited in hyaluronan-containing matrices, whereas blocking CD44-hyaluronan association increased invasion. Collectively, these data show that during breast cancer progression, hyaluronan-CD44 dynamics occurring through epithelial-stromal interactions are protective against metastasis.

Understanding the Dual Nature of CD44 in Breast Cancer Progression

CD44 has been the subject of extensive research for more than 3 decades because of its role in breast cancer, in addition to many physiological processes, but interestingly, conflicting data implicate CD44 in both tumor suppression and tumor promotion. CD44 has been shown to promote protumorigenic signaling and advance the metastatic cascade. On the other hand, CD44 has been shown to suppress growth and metastasis. Histopathological studies of human breast cancer have correlated CD44 expression with both favorable and unfavorable clinical outcomes. In recent years, CD44 has garnered significant attention because of its utility as a stem cell marker and has surfaced as a potential therapeutic target, necessitating a greater understanding of CD44 in breast cancer. In this review, we attempt to unify the literature implicating CD44 in both tumor promotion and suppression, and explain its dualistic nature. Mol Cancer Res; 9(12); 1573–86. ©2011 AACR.

MUC1 overexpression results in mammary gland tumorigenesis and prolonged alveolar differentiation

MUC1 is a transmembrane mucin that was initially cloned from malignant mammary epithelial cells as a tumor antigen. More than 90% of human breast carcinomas overexpress MUC1. Numerous studies have demonstrated an interaction between MUC1 and other oncogenic proteins such as β-catenin, erbB receptors and c-Src, but a functional role for MUC1 in transformation has not been identified. We previously reported the development of transgenic mice that overexpress human MUC1 in the mouse mammary gland (MMTV-MUC1). Analysis of these transgenic mice at an early age demonstrated the ability of MUC1 to potentiate EGF-dependent activation of MAP kinase signaling pathways in the lactating mammary gland. We now report that multiparous MMTV-MUC1 transgenic mice stochastically develop unifocal mammary gland carcinomas late in life. Molecular analysis of these tumors shows a tumor-specific coimmunoprecipitation between MUC1 and β-catenin. Examination of the contralateral glands in MMTV-MUC1 transgenics demonstrates that the development of frank carcinomas is accompanied by a failure of multiparous glands to undergo postlactational involution. Furthermore, uniparous MMTV-MUC1 transgenic mice display decreased postlactational apoptosis, elevated whey acidic protein expression and aberrant pErk2 activation. These findings are the first to determine that MUC1 overexpression promotes in vivo transformation of the mammary gland.

MUC1 is a novel regulator of ErbB1 receptor trafficking

ErbB receptors are key regulators of cell survival and growth in normal and transformed tissues. The oncogenic glycoprotein MUC1 is a binding partner and substrate for erbB1 and MUC1 expression can potentiate erbB-dependent signal transduction. After receptor activation, erbB1 is typically downregulated via an endocytic pathway that results in receptor degradation or recycling. We report here that MUC1 expression inhibits the degradation of ligand-activated erbB1. Through the use of both RNAi-mediated knock down and overexpression constructs of MUC1, we show that MUC1 expression inhibits erbB1 degradation after ligand treatment in breast epithelial cells. This MUC1-mediated protection against erbB1 degradation can increase total cellular pools of erbB1 over time. Biotinylation of surface proteins demonstrates that cell-surface associated erbB1 receptor is protected by MUC1 against ligand-induced degradation, although this is accompanied by an increase in erbB1 internalization. The MUC1-mediated protection against degradation occurs with a decrease in EGF-stimulated ubiquitination of erbB1, and an increase in erbB1 recycling. These data indicate that MUC1 expression is a potent regulator of erbB1 receptor stability upon activation and may promote transformation through the inhibition of erbB1 degradation.

MUC1 regulates nuclear localization and function of the epidermal growth factor receptor

Alteration of protein trafficking and localization is associated with several diseases, including cystic fibrosis, breast cancer, colorectal cancer, leukemia and diabetes. Specifically, aberrant nuclear localization of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is a poor prognostic indicator in several epithelial carcinomas. It is now appreciated that in addition to signaling from the plasma membrane, EGFR also trafficks to the nucleus, and can directly bind the promoter regions of genes encoding cyclin D1 (CCND1) and B-Myb (MYBL2). We have previously established that loss of MUC1 in an EGFR-dependent transgenic mouse model of breast cancer correlates with the loss of cyclin D1 expression. Here, we provide evidence for a novel regulatory function of MUC1 in the trafficking and nuclear activity of EGFR. We found that MUC1 and EGFR interact in the nucleus of breast cancer cells, which promotes the accumulation of chromatin-bound EGFR. Additionally, the presence of MUC1 results in significant colocalization of EGFR and phosphorylated RNA polymerase II, indicating that MUC1 influences the association of EGFR with transcriptionally active promoter regions. Importantly, we found that the loss of MUC1 expression resulted in a decrease in the interaction between EGFR and the CCND1 promoter, which translated to a significant decrease in cyclin D1 protein expression. This data offers insights into a novel regulatory mechanism of EGFR nuclear function and could have important implications for evaluating nuclear localization in cancer.

MUC1 and metastatic cancer: Expression, function and therapeutic targeting

MUC1 is a transmembrane mucin that is often overexpressed in metastatic cancers and often used as a diagnostic marker for metastatic progression. The extracellular domain of MUC1 can serve as a ligand for stromal and endothelial cell adhesion receptors, and the cytoplasmic domain engages in several interactions that can result in increased migration and invasion, as well as survival. In this review, we address the role of MUC1 in metastatic progression by assessing clinical studies reporting MUC1 levels at various disease stages, reviewing mouse models utilized to study the role of MUC1 in metastatic progression, discuss mechanisms of MUC1 upregulation, and detail MUC1 protein interactions and signaling events. We review interactions between MUC1 and the extracellular environment, with proteins colocalized in the plasma membrane and/or cytoplasmic proteins, and summarize the role of MUC1 in the nucleus as a transcriptional cofactor. Finally, we review recent publications describing current therapies targeting MUC1 in patients with advanced disease and the stage of these therapies in preclinical development or clinical trials.

Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel

Attachment, deformation and detachment of N-cadherin expressing prostate and breast cancer cell lines in a functionalized microchannel under hydrodynamic loading have been studied. N-cadherin antibodies are immobilized on the microchannel surface to capture the target cancer cells, PC3N and MDA-MB-231-N, from a homogeneous cell suspension. Although difficult, a significant fraction of moving cells can be captured under a low flow rate. More than 90% of the target cells are captured after a certain incubation time under no flow condition. The mechanical response of a captured cancer cell to hydrodynamic flow field is investigated and, in particular, the effect of flow acceleration is examined. The observed cell deformation is dramatic under low acceleration, but is negligible under high acceleration. Consequently, the detachment of captured cells depends on both flow rate and flow acceleration. The flow rate required for cell detachment is a random variable that can be described by a log-normal distribution. Two flow acceleration limits have been identified for proper scaling of the flow rate required to detach captured cells. A time constant for the mechanical response of a captured cell, on the order of 1 min, has been identified for scaling the flow acceleration. Based on these acceleration limits and time constant, an exponential-like empirical model is proposed to predict the flow rate required for cell detachment as a function of flow acceleration.