Regulation of Mammary Epithelial Invasion by MMPs and FGFs

Institution: University of California, San Francisco
Investigator(s): Andrew Ewald, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0015 Award: $135,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2005)
The ductal network of the mammary gland is established during puberty by the invasion of the mammary epithelium through the gland. The mammary epithelium is a unique tissue as cell proliferation, cell migration, and cell invasion are highly regulated throughout development and reproductive life, and can be activated dramatically during each pregnancy. Breast cancer occurs when mammary epithelial cell proliferation, invasion, and migration escape these normal developmental controls. We are studying the regulation of mammary proliferation and invasion by matrix metalloproteinases (MMPs) and fibroblast growth factors (FGFs). Both gene families are implicated in the progression of human breast cancer and are key mediators of epithelial invasion during mammary gland development.

This study focuses on the cellular events and molecular regulation of primary ductal invasion through the analysis of genetic knockout mice. We have demonstrated that our cell culture assay reproduces a robust epithelial invasion program, and provides a highly controlled environment for the evaluation of the effect of growth factor signaling and MMPs on the branching process. We have developed methods to image the organotypic cultures in large numbers and over long periods, enabling us to observe and intervene in the invasion process.

Our work is centered around matrix metalloproteinase 2 (MMP2), which is required for the primary invasion of the mammary epithelium and acts as a cell survival signal. Fibroblast growth factor receptor 2 (FGFR2) is required both for the primary invasion of the mammary epithelium and for the hyperplastic transformation of the mammary epithelium in the Wnt-1 mouse tumor model.

Our objectives/aims include: 1) Determine the cellular and molecular basis for the genetic requirement for MMP2 during primary ductal invasion. Test for interactions with FGF signaling pathways. 2) Characterize the changing responsiveness of the mammary epithelium with age, and the relative effect of MMP2 on ductal invasion with age, in an organotypic assay. 3) Test the genetic requirement for FGFR2 in the primary invasion of the mammary epithelium both in vivo and in vitro. 4) Test the requirement for FGFR2 for hyperplasia formation in the Wnt-1 tumor model.

The epithelial invasion process is incompletely understood at present. Increasing our understanding of the cellular events underlying invasion, and their regulation by MMP and FGF signaling pathways will increase our knowledge of normal development of the epithelium. Cell invasion during development has many parallels with cell invasion during cancer, and we will directly apply our new insights into the development of the mammary gland to epithelial invasion in an organotypic model of breast cancer.

Final Report (2008)
The mammary gland develops in an ongoing, iterative fashion throughout the life cycle. It is first specified in the embryo, but its mature form does not develop until the end of puberty. The structure of the mammary gland is then extensively changed during each cycle of pregnancy, lactation, and involution. Our hypothesis was that the normal development strategies for tissue invasion and morphogenesis are reused, in a corrupt or incomplete form, by mammary tumors. Our strategy was to use culture mammary epithelium in 3D embedded cultures of extracellular matrix proteins.

Building on years 1 & 2 we used advanced light microscopy techniques to determine the cellular mechanisms of normal mammary tissue invasion. We described a novel, and potentially general, cellular mechanism for the growth and invasion of mammary epithelium that is preceded by dramatic reductions in cell adhesion and cell polarity and large increases in cell proliferation. We showed that the epithelial architecture of normal mammary epithelium during morphogenesis is markedly similar to that of human mammary tumors. We then used genetic and pharmacologic techniques to demonstrate the requirement for fibroblast growth factor receptor 2, rho kinase, myosin light chain kinase, rac, matrix metalloproteinses and DNA synthase during the process of invasion. We extended our previous studies in three critical new directions. The first was to use advanced electron microscopy techniques to determine the adhesion and polarity basis of normal invasion. The second was to apply advanced light microscopy techniques to the study of the interactions between mammary tumor cells and host immune cells in vivo, in living mice. The third was to extend our 3D organotypic culture models to study primary human mammary epithelium and mammary tumors.

Subsequent to the fellowship the PI has established his own laboratory at Johns Hopkins Medical School and is using techniques developed in this fellowship to study the regulation of mammary tumor invasion by stromal cells and by the proteins of the extracellular matrix. The goal is to identify extracellular regulators of tumor invasion and metastasis that can be developed into anti-cancer therapeutics for human breast cancer patients.

Symposium Abstract (2007)
The mammary gland is built during embryonic and adult development, but is then subject to repeated, hormonally driven cycles of invasion and regression during the normal hormonal cycle, pregnancy, lactation, and involution. We sought to determine the underlying cellular mechanisms driving these normal mammary epithelial invasion events as a means of understanding pathological mammary epithelial invasion during breast cancer. We determined the cellular mechanisms underlying epithelial invasion using organotypic cultures of normal mammary tissue. We found that epithelial invasion involves a transition from an “inactive”, bilayered, polarized state to an “active” partially polarized, multilayered state, which exhibits vigorous, continual cell rearrangements. This transition from a bilayer to a multilayer is reversible, involves coordinate changes in multiple cell adhesion pathways (adherens junctions, tight junctions, and desmosomes), and depends on proliferation and signaling through matrixmetalloproteinases (MMPs) and Rho kinase (ROCK).

Importantly, we observed that normal mammary epithelial invasion in vivo involves cyclical entry and exit from this same “active” epithelial state. Invading normal ducts were in the “active” epithelial state, while adjacent, non-invasive ducts were in the “inactive” state. We also saw cyclical “activation” of this epithelial state during normal estrus-associated remodeling of the mammary epithelium in mice. We then used this characteristic signature of the “activated” epithelial state to test if common mechanisms were utilized during pathologic invasion in a clinically validated mouse model of breast cancer (MMTV-PymT). Importantly, hyperplasias, adenomas, and carcinomas displayed features of the “active” epithelial state. Early aspects of breast cancer progression may therefore arise from inappropriate persistence of a fundamentally normal “active” epithelial state.

The MAPK(ERK-1,2) pathway integrates distinct and antagonistic signals from TGFalpha and FGF7 in morphogenesis of mouse mammary epithelium.
Periodical:Developmental Biology
Index Medicus: Dev Biol
Authors: Fata JE, Mori H, Ewald AJ, Zhang H, Yao E, Werb Z, Bissell MJ
Yr: 2007 Vol: 306 Nbr: 1 Abs: Pg:193-207

Matrix metalloproteinases and the regulation of tissue remodelling.
Periodical:Nature Reviews, Molecular Cell Biology
Index Medicus: Nature Rev Mol Cell Biol
Authors: Page-McCaw A, Ewald AJ, Werb Z
Yr: 2007 Vol: 8 Nbr: 3 Abs: Pg:221-33

Formation of cysts by alveolar type II cells in three-dimensional culture reveals a novel mechanism for epithelial morphogenesis.
Periodical:Molecular Biology of the Cell
Index Medicus: Mol Biol Cell
Authors: Yu W, Fang X, Ewald A, Wong K, Hunt CA, Werb Z, Matthay MA, Mostov K.
Yr: 2007 Vol: 18 Nbr: 5 Abs: Pg:1693-700

GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model.
Periodical:Cancer Cell
Index Medicus:
Authors: Kouros-Mehr H, Bechis SK, Slorach EM, Littlepage LE, Egeblad M, Ewald AJ, et al.
Yr: 2008 Vol: 13 Nbr: 2 Abs: Pg:141-52

Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis.
Periodical:Developmental Cell
Index Medicus: Dev Cell
Authors: Ewald AJ, Brenot A, Duong M, Chan BS, Werb Z
Yr: 2008 Vol: 14 Nbr: 4 Abs: Pg:570-81

Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis.
Periodical:Current Biology
Index Medicus: Curr Biol
Authors: Martín-Belmonte F, Yu W, Rodríguez-Fraticelli AE, Ewald AJ, Werb Z, Alonso MA, Mostov K
Yr: 2008 Vol: 18 Nbr: 7 Abs: Pg:507-13

Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis.
Periodical:Developmental Biology
Index Medicus: Dev Biol
Authors: Lu P, Ewald AJ, Martin GR, Werb Z
Yr: 2008 Vol: 321 Nbr: 1 Abs: Pg:77-87

Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.
Periodical:Disease Models and Mechanisms
Index Medicus: Dis Model Mech
Authors: Egeblad M, Ewald AJ, Askautrud HA, Truitt ML, Welm BE, Bainbridge E, Peeters G, et al.
Yr: 2008 Vol: 1 Nbr: 2-3 Abs: Pg:155-67