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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.
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.
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.
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.
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.
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,” J Cell Biol. 2014 Mar 3; 204(5):839-56.
Huebner RJ, Lechler T, Ewald AJ, “Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells,” Development. 2014 Mar; 141(5):1085-94.
Ellison D, Mugler A, Brennan M, Lee SH, Huebner RJ, Shamir ER, Woo LA, Kim J, Amar P, Nemenman I*, Ewald AJ*, Levchenko A*, “Cell-cell communication enhances the capacity of cell ensembles to sense shallow gradients during morphogenesis,” Proc Natl Acad Sci U S A 2016 Feb 9; 113(6):E679-88
Cheung KJ, Padmanaban V, Silvestri V, Schipper K, Cohen JD, Fairchild AN, Gorin MA, Verdone JE, Pienta KJ, Bader JS, Ewald AJ, “Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters,” Proc Natl Acad Sci U S A. 2016 Feb 16 ;113(7):E854-63.
Huebner RJ, Neumann NM, Ewald AJ, “Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration.,” Development. 2016 Mar 15; 143(6):983-93.
Neumann NM, Perrone MC, Vedlhuis JH, Zhan H, Devreotes PN, Brodland GW, Ewald AJ, "Coordination of receptor tyrosine kinase signaling and interfacial tension dynamics drive radial intercalation and tube elongation," Dev Cell. 2018 Apr 9;45(1):67-82.
Sirka OK, Shamir ER, Ewald AJ, "Myoepithelial cells are a dynamic barrier to epithelial dissemination," Journal of Cell Biology, 2018 Oct 1;217(10):3368-3381.
Padmanaban V, Krol I, Suhail Y, Szczerba BM, Aceto N, Bader JS, Ewald AJ, "E-cadherin is required for metastasis in multiple models of breast cancer," Nature. 2019 Sep;573(7774):439-444.
Padmanaban V, Cheung KJ, Ewald AJ*, Bader JS*, Between-tumor and within-tumor heterogeneity in invasive potential," PLoS Computational Biology, 16 (1), e1007464 2020 Jan 21 eCollection Jan 2020.
Georgess D, Padmanaban V, Sirka OK, Choi A, Frid G, Neumann NM, Ewald AJ, "Twist1-induced epithelial dissemination requires Prkd1 signaling," Cancer Research, 80 (2), 204-218 2020 Jan 15.
Jiao Z, Cai H, Long Y, Sirka OK, Padmanaban V, Ewald AJ, Devreotes PN, "Statin-induced GGPP depletion blocks macropinocytosis and starves cells with oncogenic defects," PNAS, 117 (8), 4158-4168, 2020 Feb 25.
Huang W, Xia L, Jeong YJ, Chianchiano P, Serer BN, Trujilo MA, Lionheart G, Luchini G, Veronese N, Nguyen-Ngoc KV, Neumann NM, Groot VP, Singhi AD, Gaida MM, Wolfgang CL, He J, Thompson ED, Roberts NJ, Ewald AJ, Wood LD, "Pattern of invasion in human pancreatic cancer organoids is associated with Smad4 loss and clinical outcome," In Press, Cancer Research, April 21, 2020.
Chan IS, Knútsdóttir H, Ramakrishnan G, Padmanaban V, Warrier M, Ramirez JC, Zhang H, Jaffee EM, Bader JS, Ewald AJ, " Cancer cells educate natural killer cells to a metastasis promoting cell state, In Press, Journal of Cell Biology, May 4, 2020.