Role of MMPs in Breast Tumor Initiation and Aggressiveness

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Jimmie Fata, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6FB-0131 Award: $75,422
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2000)
The development of breast cancer (pathogenesis) is mediated by many factors that contribute to enhancing both the initiation of a tumor cell and the progression of a tumor. Targeting factors believed to increase the risk of breast cancer onset (initiating factors) is often separate from therapeutics used against controlling tumor development. This proposal centers around a family of secreted proteins that are implicated in both tumor cell initiation and tumor progression. This group of proteins, which have been labeled the matrix metalloproteinases (MMPs), are enzymes capable of degrading extracellular factors that surround a cell's environment. MMPs can directly cleave the matrix molecules that cells reside on, can process growth factors to an active form and can mediate cleavage of cell bound proteins that are exposed on the outside of the cell. Certain normal physiological processes require the action of these proteinases, however dysregulation of MMPs is often seen in many diseases, including breast cancer. In breast cancer and other cancers, MMP dysregulation enhances tumor blood supply and their activity is necessary for many steps involved in metastatic spread. Recently, we have found that a certain MMP (stromelysin-l) can induce a unusual and pathological characteristic in normal mammary epithelial cells called epithelial to mesenchymal transition (EMT). In humans EMT is associated with the most aggressive breast cancers. A particular molecule involved in cell-cell contact (E-cadherin) is known to be lost in EMT. Interestingly, we found that stromelysin-1 induces cleavage of E-cadherin a process that may be the initial step in EMT and subsequent tumor formation. Our approach is to elucidate how MMPs, either produced by the individual or by breast-pathogens such as bacteria, induce EMT. We believe by inhibiting dysregulated MMPs we will inhibit both tumor cell initiation and tumor progression. To date there exists many synthetic MMP inhibitors being evaluated in clinical trials on cancer patients. Our results will help in bettering the trial design which is a high priority with breast cancer activists and organizations, add insight into the pathogenesis of breast cancer and increase the awareness of internal environmental factors (MMPs) implicated in the genesis and progression of this disease.


Final Report (2003)
In tissue, cells are often simultaneously connected to similar cells and to a scaffolding extracellular protein matrix, called the extracellular matrix (ECM). These connections maintain tissue architecture, cell viability and cell function. The loss of either of these attachments is seen in cancer and is mediated by a class of secreted enzymes known as matrix metalloproteinase (MMPs). The aim of this proposal is to elucidate how a particular MMP, called stromelysin-1 (MMP3), induces mammary tumors when overexpressed in the mouse mammary gland. Our initial evidence suggested a mechanism in which MMP-3 cleaved a necessary cell-cell attachment protein called E-cadherin. This transformation involves acquiring increased invasive ability and developing non-epithelial characteristics (mesenchymal characteristics) a process termed epithelial to mesenchymal transition (EMT). Those cancers that exhibit EMT are considered more aggressive and are indicative of a poor prognosis. Therefore, understanding the factors and processes involved in EMT is of considerable importance.

A wounding assay was generated in which E-cadherin is lost from mammary epithelial cells at the wound edge and E-cadherin fragments could be recovered. Those cells at the wound edge detach from each other, acquire scattering characteristics and increased migration that are all processes indicative of EMT. Inhibiting MW activity with a synthetic inhibitor suppresses E-cadherin loss and subsequent cell scattering. Importantly, protease inhibitors against other classes of proteases did not inhibit scattering or sheet migration. Having generated a suitable model, the next goal was to manipulate E-cadherin in two particular ways. One was to produce an E-cadherin that is resistant to MMP cleavage and the other is to generate an E-cadherin that can be cleaved in a regulated way. To test that E-cadherin mutants function in cell-cell adhesion while also exhibiting MMP-resistance or inducible cleavage, I have expressed them in a breast cancer cell line (SKBR3) that lacks endogenous E-cadherin. I have confirmed that non-mutant E-cadherin expression in this cell line leads to cell-cell adhesion and other E-cadherin functional characteristics. I have now established stable lines and am beginning to analyze each to elucidate the signals downstream E-cadherin cleavage.

In conjunction with the above-mentioned progress, these findings will be packaged for publication. E-cadherin mutants will be continually used in various assays that involve migration, differentiation, EMT, apoptosis, tumor growth and metastasis in order to fully determine the relevance of E-cadherin cleavage. Cell-cell detachment is a necessary event during both normal and pathological processes. Yet, we are far from a full understanding on how cells achieve this non-social behavior. Elucidating the mechanism behind this process may in many ways increase our knowledge on the most deleterious step in cancer progression, that being metastasis.


Symposium Abstract (2003)
The development of breast cancer is mediated by many factors that contribute to enhancing both the initiation of a tumor cell and the progression of the tumor. Therapeutics that target factors believed to increase the risk of breast cancer onset (initiating factors) is often separate from therapeutics used against controlling tumor development. This research centers on a family of secreted proteins that are implicated in both tumor cell initiation and tumor development. These proteins, called the matrix metalloproteinases (MMPs), are enzymes capable of degrading extracellular factors that surround a cell within the tissue. For instance, MMPs can directly cleave the matrix molecules that cells reside on, can process growth factors to an active form and can mediate cleavage of cell bound proteins that are exposed on the outside of the cell. Certain normal physiological processes require the action of these proteinases. However, deregulation of MMPs is often seen in many diseases, including breast cancer. Recently, we have found that a certain MMP (stromelysin-1) can induce a pathological characteristic in normal mammary epithelial cells called epithelial to mesenchymal transition (EMT). In humans EMT is associated with the most aggressive breast cancers. Our data suggest that other MMPs also can induce EMT. A particular molecule involved in cell-cell contact (E-cadherin) is shed from the cell surface during MMP-mediated EMT. To address whether E-cadherin shedding is necessary for, or can induce EMT we have generated E-cadherin mutants. These mutants are being analyzed to follow molecular signaling associated with E-cadherin loss to better understand the role MMPs play during EMT.


Symposium Abstract (2007)
The goal of this research was to model normal development of the breast and subsequently evaluate the importance of the mitogen activated kinase (MAPK) signaling pathway, which is often deregulated in breast cancer.

Transforming growth factor-alpha (TGF-alpha) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through MAPK; however, their unique and/or combined contributions to mammary morphogenesis has not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK for 1 hour, induced by TGF-alpha, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 minutes) of MAPK, induced by FGF7, led to growth without branching. Unlike TGF-alpha, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. Simultaneous stimulation by FGF7 and TGF-alpha indicated that the FGF7-induced MAPK signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGF-alpha-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1) and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary morphogenesis are intimately dependent on MAPK activation.

Our results indicate that the MAPK pathway, which is deregulated in the majority of breast cancers, is absolutely needed in the normal development of the breast.