Identification of breast cancer risk biomarkers in clinical cohorts.
The most commonly hypothesized model of breast cancer development posits an evolution through incremental steps of progressively increasing cellular abnormalities from normal epithelium through proliferative disease without atypia (PDWA), atypical hyperplasia, ductal carcinoma in situ, and then invasive breast cancer.
This model is supported by epidemiologic studies that show a step-wise increase in relative risk (RR) of subsequent development of invasive breast cancer from PDWA (RR=2) to atypical hyperplasia (RR=4) to DCIS (RR=10). To define the critical factors that influence whether a premalignant lesion will develop into invasive cancer, we are investigating an extensive cohort of patients with benign breast disease (BBD) that was assembled at the Mayo Clinic and for which subsequent cancer progression status is known.
We are using transcriptional profiling and pathological observations to identify specific features that are most associated with progression to invasive cancer. We are using this information, in combination with our animal and cell culture models, to develop novel approaches to target cancer-associated processes towards the ultimate goal of prevention of breast cancer formation.
MMP-induced EMT and genomic instability in breast cancer.
MMPs are essential for many physiological processes, but inappropriate expression of MMPs can facilitate the development and progression of tumors. Recognition of the relationship between MMPs and malignancy led to clinical trials of broad-spectrum MMP inhibitors as cancer therapeutics, but the results were disappointing.
The failure of the clinical trials was due in large part to the propensity of the MMP inhibitors to inhibit essential physiological processes. Therapeutic strategies that target tumor-specific MMP-dependent effects may prove more promising. Previous studies and our preliminary data show that exposure of mammary epithelial cells to selected MMPs causes cleavage of a cell surface molecule that stimulates cellular production of reactive oxygen species (ROS). MMP-dependent production of ROS causes cells to undergo epithelial-mesenchymal transition (EMT), a fundamental phenotypic alteration associated with progression to metastasis, and compromises the cellular genomic stability. We seek to identify the specific steps associated with the MMP-induced malignant transformation so as to determine potential points for therapeutic intervention.
Currently, we are working to identify the proteolytic target of MMPs that stimulates the development of ROS by testing the specific role of cleavage of E-cadherin by MMPs supplemented by a proteolytic screen for additional/alternative targets, to determine the MMP-induced factors responsible for the increased expression of Snail and Twist, and to dissect the relative role of these transcription factors on the MMP-induced EMT, and to define how MMP/ROS induce cellular aneuploidy and mitotic abnormalities through stimulation of centrosome amplification.
Microenvironmental control of myofibroblast activation in the breast.
Malignant transformation of the breast and other organs is associated with dramatic changes in the microenvironment surrounding neoplastic cells, including a reactive fibrotic stroma characterized by increased production of inflammatory cytokines, excessive accumulation of extracellular matrix (ECM), and an increase in tissue stiffness.
Contractile myofibroblasts are key mediators of the biochemical and biophysical properties of the fibrotic tumor microenvironment. Additionally, the transdifferentiation of myofibroblasts from tissue cells and their subsequent activation is controlled by a combination of soluble factors and contractile tension. The increased tissue stiffness associated with fibrosis may thereby generate a positive feedback loop to facilitate tumor progression and metastatic invasion; delineating the microenvironmental effects and effectors will require sophisticated, tractable model systems.
We are developing an experimental model to define how alterations of the biochemical and biophysical cellular microenvironment can stimulate myofibroblast development and activation, and how formation and activation of myofibroblasts in tissue structures affects progression to malignancy.
We are using cell culture and animal studies to determine the biochemical and biomechanical requirements of the substratum microenvironment for the transdifferentiation process, and we are making use of a novel three-dimensional microlithography-based organotypic culture system of the mammary epithelial ductal network to determine how myofibroblast transdifferentiation affects the microenvironment of the duct at the biochemical, mechanical, and cell population levels.
Given that the presence of fibrotic foci in breast tumors correlates with metastasis and negative prognosis, and might hinder the efficacy of tumor therapies, the new physiologically relevant models developed in this work will have significant impact for discovery and evaluation of novel therapeutic targets to combat fibrosis genesis and tumor progression.
MMP/Rac1b activation of lung fibrosis and cancer.
MMPs also play a critical role in the development of lung fibrosis and lung cancer. We have previously determined that exposure of mammary epithelial cells to MMPs induces expression of Rac1b, a splice isoform of Rac1 that is found in many different tumor types. We have also determined that MMP-induced Rac1b stimulates a specialized form of epithelial-mesenchymal transition (EMT) in which the cells acquire myofibroblast-like characteristics. Our preliminary examination of human lung tumor tissue samples revealed that MMPs and Rac1b show increased expression in lung tumors and that expression of these genes is associated with a poor prognosis. We have developed transgenic mice which express MMP-3 or Rac1b in lung epithelial cells and have found that these mice exhibit ECM deposition and tissue disruption characteristic of fibrosis as well as accelerated and spontaneous tumor development.
We are currently defining the molecular mechanisms involved in MMP-induced EMT using sophisticated 3D cultures of lung tumor cell lines and primary lung epithelial cells, determining the participation of MMP-induced EMT in fibrosis using a transgenic model in which epithelial cells are permanently tagged and fibrosis is induced by MMP-3, and defining the stage at which MMP expression stimulates tumor progression.