Patricia J. Keely, Ph.D. Associate Professor Department of Pharmacology pjkeely@wisc.edu

Appropriate cellular interactions with the extracellular matrix (ECM) help to establish normal cellular architecture and differentiation. During oncogenic transformation, these normal interactions with the ECM are profoundly altered, resulting in cells that lose their polarization and differentiation, lose anchorage dependent growth control, and acquire a migratory, invasive phenotype. Our lab is interested in understanding, at a molecular level, how cellular interactions with the ECM determine differentiation and epithelial polarization, and how these interactions are altered during carcinogenesis to result in invasive, metastatic carcinoma.

Understanding molecular mechanisms underlying breast cancer risk due to breast density

Patients with “dense” breast tissue have a four to six-fold increased risk of developing breast carcinomas.  In fact, 1/3 of all breast cancer cases are attributed to breast density, making it one of the greatest risk factors for carcinoma.  Increased breast density is associated with a significant increase in the deposition of connective tissue, or extracellular matrix (ECM) components, most notably the protein, collagen.  We have been developing model systems to understand why increased breast density results in an increased risk for developing breast carcinoma. We find in a simple in vitro model that increasing the density of collagen in the matrix is sufficient to disrupt breast epithelial differentiation, suggesting that matrix density is itself an important regulator of cellular behavior.  Additionally, we are employing a mouse strain engineered to have more collagen in its connective tissue.  Mouse tumor studies are underway to directly test whether increased collagen density will enhance tumor formation or tumor metastasis.

Mammary epithelial cells cultured in 3D collagen gels differentiate into tubule structures.  This differentiation is disrupted when cells are cultured in dense 3D collagen gels, providing a model system for understanding the molecular underpinnings for cellularresponses to matrix density

State of the art imaging approaches are being used to characterize the collagen structure in normal glands and around tumors so that we can better understand the physical relationship between cells and the collagen fibers found in breast tissue.  We find evidence for a collagen “signature” that is present in even before a tumor is palpable, predicting where a tumor will soon arise.  We are investigating whether this signature can be developed as a tool to aid in diagnosing human breast carcinoma at an earlier stage. Using biochemical and DNA microarray approaches, we are characterizing several biochemical and genetic changes that occur in cells that encounter dense matrices.  Dense collagen environments activate signal pathways within cells that result in a more tumor-like behavior: increased cell proliferation or growth, decreased cell death, and increased ability to invade into nearby tissues.  We expect that our studies to characterize the molecular response of cells to dense collagen matrices will allow us to better understand tumor progression.

Mouse mammary carcinoma cells (blue) invading into the surrounding collagen stroma (green/yellow) in fresh, unstained, unfixed tissue, and visualized by Fluorescent Lifetime Imaging Microscopy

Molecular signaling events related to cell interactions with the ECM

Cells interact with the ECM through a variety of cell surface receptors, the best understood of which are members of the integrin family. Much remains to be determined regarding the specific molecular players and signaling pathways downstream of integrins, and how these pathways are involved in the progression of various diseases. Therefore, part of the focus of the lab is to investigate signaling events through the integrin family of receptors. A second aspect of this work is to investigate how small GTPases of the Ras superfamily, some of which are known or suspected oncogenes, affect the response of cells to the ECM. Specifically, we have focused on R-Ras and Rho, which we find alter the way breast epithelial cells respond to the ECM, promoting cellular migration and invasion. We are particularly interested in studying signaling events using state of the art imaging approaches to understand how small GTPases function in a spatial and temporal manner during cell migration.

Selected Publications: Articles on PubMed

• Keely PJ, Conklin MW, Gehler S, Ponik SM, and Provenzano PP. (2007). Investigating integrin regulation and signaling events in three-dimensional systems. Methods Enzymol. 426:27-45. PMID 17697878
  
  
• Skala MC, Riching KM, Bird DK, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, Keely PJ, and Ramanujam N. (2007). In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J Biomed Opt. 12:024014. PMID 17477729
  
  
• Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG, and Keely PJ. (2007). Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med. 4:38. PMID 17190588
  
  
• Modzelewska K, Newman LP, Desai R, Keely PJ. (2007). Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas. J Biol Chem. 281:37527-37535. PMID 17038317
  

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