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Oncology, Center for Cell Dynamics, Member, Breast Cancer Research Program, Sydney Kimmel Comprehensive Cancer Center
855 N. Wolfe Street
452 Rangos Building
Baltimore MD 21205
Cellular mechanisms and molecular regulation of epithelial morphogenesis during development and breast cancer
Background and Summary: Epithelial cells in a tissue live a crowded life connected to, and interacting with, other cell types, the extracellular matrix, and diverse signaling molecules. A fundamental question in biology is: how do the constituent cells of an epithelium collaborate to build and remodel its structure? This seemingly simple question also has great relevance to human disease. Cancer is a disease of disregulated proliferation, but also of disregulated tissue growth and invasion. Tumors could grow through novel cellular mechanisms, or they could grow through normal cellular mechanisms occurring in abnormal contexts or to abnormal extents. Until we understand the cellular mechanisms of normal epithelial morphogenesis, it is not possible to distinguish these possibilities. Experimental Approach: We combine advanced time-lapse microscopy techniques and 3D organotypic cultures to study the cell behavioral basis of normal and neoplastic epithelial growth and invasion. Organotypic culture allows us to contrast different epithelia under identical conditions or to contrast similar epithelia under different microenvironmental conditions. We use a common set of imaging, genetic, and molecular interference tools to study epithelial morphogenesis, whether from normal or cancerous epithelium. Summary of Recent Work: We seek to answer a simple question: how does an epithelium grow and invade? To answer this question, we observed the cell behaviors that drive mammary branching morphogenesis. We found that ductal elongation was accomplished by a multilayered epithelium, within which cells rearranged vigorously. Surprisingly, cells at the elongation front lacked forward oriented actin protrusions. We have shown that during morphogenesis, mammary epithelium transitions from a bilayered to a multilayered organization, with dramatic, reversible changes in epithelial polarity and cell motility. We are collaborating with Manfred Auer's electron microscopy group at the Lawrence Berkeley Lab to study the intercellular junctional basis of this transition. We have now dissected the process of branching morphogenesis into discrete, observable subprocesses and have identified molecular regulators of each subprocess. (For further information see Ewald et al, Developmental Cell, April 2008).
Project 1. Quantitative analysis of the cell behavioral basis of epithelial morphogenesis The foundation for a cell biological understanding of epithelial morphogenesis is to resolve the tissue level process of ductal initiation, elongation and bifurcation into discrete changes in the properties and behaviors of individual cells. We are currently using 4D confocal microscopy to dissect the relative contributions of cell movement, cell proliferation, cell shape change and extracellular matrix dynamics to mammary branching morphogenesis.
Project 2. Molecular and cellular regulation of epithelial morphogenesis Building on a our understanding of the cell behavioral basis of epithelial morphogenesis, we are taking a combined candidate and systematic approach to identify molecules regulators of these behaviors. We are currently using microarrays to identify potential regulatory molecules and we will use pharmacologic inhibitors, function blocking antibodies, and virally mediated gene inactivation to validate targets. We also want to understand the role of microenvironmental factors such as extracellular matrix (ECM) and stromal cell populations. Preliminary data suggest that the cellular mechanisms of invasion depend on the protein composition of the ECM. We predict that the genetic regulation of epithelial morphogenesis will intimately depend on the ECM and stromal cell context in the epithelial microenvironment.
Project 3. Contrasting epithelial morphogenesis in mammary development and breast cancer The varied morphologic appearance of invasive tumors could reflect differences in the fundamental mechanisms of cellular invasion. Alternately, similar underlying invasion mechanisms might generate different outcomes, and different morphologic appearances, in response to a changing tumor microenvironment. It is difficult to distinguish these possibilities in fixed sections, as the tumor and stroma are both changing dramatically as a function of stage of progression.
We are addressing this question by comparing the cellular mechanisms of growth and invasion of normal and neoplastic epithelia, cultured in identical solution, matrix, and stromal cellular conditions. We have developed protocols to culture epithelium from normal ducts, hyperplasias, adenomas, and advanced tumors, either alone or in co-culture with corresponding stromal cells. We are committed to identifying molecular strategies for limiting the growth and spread of epithelial tumors, with a specific focus on identifying extracellular inhibitors of breast tumor invasion and metastasis.
Shamir ER and Ewald AJ, “3D Organotypic Culture: Experimental Models of Mammalian Biology and Disease,”, Nature Reviews Molecular Cell Biology.
Cheung KJ and Ewald AJ, “Illuminating breast cancer invasion: diverse roles for cell-cell interactions”, Current Opinion in Cell Biology, Cell Adhesion and Migration Issue, 2014.
Huebner RJ and Ewald AJ, Seminars in Cell and Developmental Biology, Cellular Foundations of Mammary Tubulogenesis, 31 (2014) 124-31.
Nguyen-Ngoc KV, Shamir ER, Huebner RJ, Beck JN, Cheung KJ, Ewald AJ, “3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion,” Methods in Molecular Biology, 2014.
Chen Q, Zhang N, Gray RS, Li H, Ewald AJ, Zahnow CA, and Pan DJ, A temporal requirement for Hippo signaling in mammary gland differentiation, growth and tumorigenesis, Genes Dev. 2014 Mar 1;28(5):432-7.
Huebner RJ, Lechler T, Ewald AJ, “Mammary epithelial stratification occurs through symmetry breaking vertical divisions of luminal cells,” Development. 2014 Mar;141(5):1085-94.
Shamir ER, Papallardo E, Jorgens DM, Coutinho K, Tsai WT, Aziz K, Auer M, Tran PT, Bader JS, Ewald AJ, “Twist1-induced dissemination preserves epithelial identity and requires E-cadherin,” The Journal of Cell Biology. 2014 Mar 3;204(5):839-56.
Cheung KJ, Gabrielson E, Werb Z, Ewald AJ, “Collective invasion in breast cancer requires a conserved basal epithelial program,” Cell. 2013 Dec 19;155(7):1639-51.
Beck JN, Singh A, Rothenberg AR, Elisseeff JH, Ewald AJ, “The independent roles of mechanical, structural and adhesion characteristics of 3D hydrogels on the regulation of cancer invasion and dissemination,” Biomaterials, 2013 Dec;34(37):9486-95.
Nguyen-Ngoc KV and Ewald AJ, “Mammary epithelial elongation and myoepithelial migration are regulated by the composition of the extracellular matrix,” J Microsc. 2013 Sep;251(3):212-23 Journal of Microscopy
Nguyen-Ngoc KV, Cheung KJ, Brenot A, Shamir ER, Gray RS, Hines WC, Yaswen P, Werb Z, Ewald AJ, “The ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium” Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2595-604.
Ewald AJ, Huebner RJ, Palsdottir H, Lee JK, Perez MJ, Jorgens DM, Tauscher AN, Cheung KJ, Werb Z, Auer M, "Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium", J Cell Sci. 2012 Jun 1;125(Pt 11):2638-54.
Nakasone ES, Askautrud HA, Kees T, Park JH, Plaks V, Ewald AJ, Fein M, Rasch MG, Tan YX, Qiu J, Park J, Sinha P, Bissell MJ, Frengen E, Werb Z, Egeblad M, "Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance", Cancer Cell. 2012 Apr 17;21(4):488-503.
Ewald AJ, Brenot A, Duong M, Chan BC, Werb Z. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Developmental Cell, 2008 Apr; 14(4): 570-81.