Our major research goals in this project are to characterize the roles of cellular interactions in disease pathogenesis. A major current focus is on tumor cell invasion and metastasis, with the goal of identifying novel molecular regulators and mediators. A fundamental question in cancer biology is the relationship of the local matrix environment to cancer progression. For example, a common feature of advanced carcinomas is the induction of a dense collagenous matrix surrounding tumors during the process of desmoplasia. After completing studies establishing the role of dense fibrillar collagen in induction of proteolytically active invadopodia, we turned to the question of how alterations in the physical rigidity of the matrix substrate can alter the nature of the cell migration responses of a variety of human tumor cell lines. Durotaxis -- cell migration towards regions of increasing stiffness -- has been characterized for a variety of non-malignant cell types. It was not previously known, however, whether human cancer cells lose this form of regulation of cell migration, since durotaxis might potentially contribute to patterns of tumor cell invasion. For example, tumor microenvironments often become stiffer during tumor progression, but whether cancer cells can mechanosense and respond to such gradients of stiffness was not known. We established that all four of the cancer lines we tested, including metastatic breast cancer, fibrosarcoma, and glioblastoma, displayed effective durotaxis by migrating directionally in response to changes in extracellular stiffness. Their efficiency of durotaxis was relatively similar, indicating that cancer cells do not lose this feature of normal cell migration, and that durotaxis could potentially contribute to patterns of cancer cell dissemination. An oral carcinoma cell line was found to require fibronectin to invade effectively in a transfilter invasion assay using Matrigel to mimic a basement membrane barrier. This and other findings suggested the possibility of two-way interactions between cells and basement membrane. We are characterizing the interactions between a variety of cell types and 2D basement membrane-like substrates using Matrigel basement membrane extracts, as well as purified collagen IV or laminin. Tumor cell invasiveness may be promoted by epithelial-mesenchymal transition (EMT). In studies of normal tissue morphogenesis, we previously characterized the novel regulator Btbd7 as an inducer of the EMT transcription factor Snail2 and a disruptor of E-cadherin mediated cell-cell adhesion. How general this response might be is still unclear. We are currently establishing biological assay systems for Btbd7 to better understand its mechanisms of action. Tumor expansion can be influenced by multiple factors, including immune cells. A collaborative study with a joint postdoctoral fellow in the laboratory of Dr. Alberto Baena at Oxford is evaluating the interplay between Drosophila tumors and immune cells, with particular focus on a role for caspases separate from their role in apoptosis; it appears to involve specific signal transduction. Another collaborative study established that the newly characterized anti-angiogenic protein fibulin-7 interacts with human endothelial cells through their alpha5-beta1 integrin receptors, which modulate their morphology. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we are maintaining a joint research collaboration with Dr. Ashok Kulkarni's laboratory to use the GCaMP6 mouse system for direct visualization of calcium signaling. Current research is focused on the regulatory kinase Cdk5 in characterizing and comparing orofacial pain signaling and mechano-sensation in the mouse trigeminal ganglion. A methods paper has been published to provide detailed protocols for accessing the trigeminal ganglion; it included methods for real-time fluorescence imaging and quantification of neuronal signaling. Current research is quantifying trigeminal neuronal responses during the sensing of acute and inflammatory pain, as well as sensing light touch and its pathological conversion to allodynia. Effects of inhibiting Cdk5 activity on real-time trigeminal neuronal signaling are being quantified.
Showing the most recent 10 out of 16 publications
