Cell-Surface Interactions in Pathogenesis

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications, trials & patents

Abstract

Our goal in this project is to characterize new roles for cellular and cell-extracellular interactions in disease pathogenesis with a goal of identifying novel molecular regulators and mediators. One area of focus is cancer progression and invasion. We have conducted a four-year collaboration with the laboratory of Prof. Luis Alberto Baena at the Oxford University Sir William Dunn School of Pathology to explore new roles of the caspase family of proteases. Caspases have been previously implicated in cancer, where their dysregulation can protect tumor cells against apoptosis to facilitate cancer progression. However, we have explored tumor-suppressing activities of caspases that are independent of apoptosis. We used a Drosophila model system that mimics human cancers with activated EGF receptors and elevated JAK/STAT signaling. We found surprisingly widespread, extensive non-apoptotic activation of initiator caspases in these tumors. Those activities suppress the usual over-activation of the JNK class of MAP kinases in this type of cancer to help retain more-normal cell fate commitment in tissues and to help suppress excessive cell proliferation and other features of malignancy that overlap phenomenologically with those of chronic wounds. This role of the caspase-2/9 system in modulating JNK signaling and the tumor microenvironment adds insight into the surprisingly wide roles of caspases, which in this case is to serve a tumor suppressor function as opposed to the classical role of caspases functioning to promote cancer. In other work still in progress, we used our 3D in vitro cancer invasion model consisting of cancer spheroids encapsulated by a basement membrane and embedded in 3D collagen gels to visualize the early events of cancer invasion using confocal microscopy and live imaging. Cancer cells generated large numbers of basement membrane perforations of varying sizes that expanded over time during cell invasion. Cells showing this phenomenon were human breast cancer cells and oral cancer cells. We used a wide variety of small molecule inhibitors to probe the mechanisms of basement membrane perforation and hole expansion. After inhibiting MMPs, the average size of the perforations decreased by 63% and invasion was blocked. The effects of other inhibitors are being compared. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we have an ongoing collaboration with the laboratory of Dr. Ashok Kulkarni in the NIDCR. We have been refining methodologies to directly visualize craniofacial pain-associated signaling by live-animal real-time intravital microscopy. The approach has been to expose the trigeminal ganglion of anesthetized mice for visualization while performing either noxious or non-noxious stimulation. The activation of calcium signaling can be visualized by epifluorescence microscopy to detect flashes of green fluorescence from the genetically encoded sensor GCaMP6. For example, gently brushing the cheek of mice can stimulate a form of calcium signaling in a sensory response normally perceived as touch and not associated with pain. Elevated temperature or exposure to capsaicin, the active ingredient of hot chili peppers that stimulates heat receptors, can induce pain signaling. Induction of local facial inflammatory pain was found to modify the pattern of pain signaling such that light brushing of the cheek induced exaggerated pain signaling in a process analogous to that of allodynia in human patients, i.e., the misperception of light touch as pain. Using transgenic and knockout mouse models of the cyclin-dependent kinase 5 (Cdk5) known to be involved in pain signaling, we confirmed the role of Cdk5 in facial pain signaling, but more importantly were able to show that the local or systemic application of a peptide inhibitor of Cdk5 was able to suppress elevated calcium signaling in response to both noxious stimuli and inflammation-induced allodynia by inhibiting the number and type of trigeminal peripheral sensory neurons that were activated. This study suggests that inhibitors of Cdk5 might potentially provide novel inhibitors of pain at the peripheral rather than at the central nervous system level, providing a possible alternative to opioids for suppressing orofacial pain. We have completed a long-time collaboration with Subhash Dhawan at the FDA exploring the role of the cell-protective enzyme HO-1 (heme oxygenase-1). We had previously found that HO-1 activation by the natural molecule hemin could suppress a wide range of types of viral infections by activation of innate immunity. Hemin is approved by the FDA for treatment of porphyria. In the current study, we found that hemin treatment of cells infected by Mycoplasma produced a dramatic >90% inhibition of DNA replication, which was confirmed by transmission electron microscopy and proteomic analysis to show the complete absence of both intact Mycoplasma organisms from cells and its characteristic proteins. The role of HO-1 was confirmed using siRNA suppression of its expression, which resulted in elevated levels of Mycoplasma as predicted. This study represents to our knowledge the first data suggesting the therapeutic potential for the use of HO-1 activation of cellular innate responses to suppress Mycoplasma infection. Our various primary and collaborative studies are providing new insights into the ways in which normal physiological cellular interactions are altered or subverted in disease pathogenesis.