Heavy metal-stimulated signal transduction: new metal-regulatory and -responsive mechanisms

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PROJECT SUMMARY/ABSTRACT From a human physiological perspective, heavy metals (HM) are divided into two groups: essential (eHM) and toxic (tHM). eHM and tHM overexposure are both linked to numerous pathophysiological conditions; however, the signal transduction pathways and genomic activities stimulated by eHM dyshomeostasis and tHM exposure have yet to be adequately established. For example, the mammalian Cu-sensor protein responsible for relaying changes in extracellular Cu levels into signaling responses has not been identified. The goals of this application are to define (i) eHM- and tHM-stimulated and -regulatory signaling networks (including resultant genomic activities), and (ii) the underlying molecular biological, biochemical, and biophysical mechanisms. Through the work in this application, I have characterized the signaling and genomic responses induced by Cu exposure and identified a mechanism of Cu-stimulated epidermal growth factor receptor (EGFR, a cell surface receptor tyrosine kinase (RTK)) signal transduction along the MAPK/ERK pathway, thereby identifying EGFR as a candidate Cu- sensor protein. Biochemical experiments are underway investigating the EGFR-Cu binding site and geometry. Relatedly, through application of newly-developed next generation sequencing (NGS), molecular biological, and transcriptomic (in vitro and in vivo) techniques, I discovered a novel mechanism by which intracellular Cu levels are regulated. Elevated extracellular Cu levels stimulate EGFR/MAPK/ERK activation, which induces expression of the transcription factor (TF) EGR1 and EGR1-corepressor NAB1/2; the EGR1-NAB1/2 complex subsequently represses transcription of the mammalian Cu-importer, CTR1. EGR1 ChIP-seq experiments are underway for further corroboration. This work will pair with experiments assessing synergism of an inhibitor of Cu-chaperones and MAPK/ERK signaling (to increase CTR1 levels) with a platinum anticancer drug (cisplatin, imported via CTR1), as well as genomic insights into the TF activities of EGR1 in EGFR-driven lung cancer (a role that is currently unknown). My research establishing the Cu-regulatory network lays the foundation for my planned independent career (i) evaluating eHM- and tHM-regulatory and -responsive mechanisms and (ii) characterizing disruption of native signaling by tHM. In my independent career, I will apply phosphorylation arrays and NGS techniques to cells stimulated with non-Cu eHM and high-priority environmental pollutant tHM, with emphasis on the effects of tHM exposure in disrupting the eHM-regulatory and -responsive signal transduction networks. In summary, my identification of EGFR as a conduit for relaying elevated extracellular Cu levels into genomic activities via the MAPK/ERK pathway has linked a major signal transduction network (implicated in cancer pathophysiology, for example) to eHM-regulation, and thereby opens new avenues of understanding HM- regulatory networks in general. More broadly, the comprehensive research program established in this proposal stands to identify biomarkers of harm preceding development and progression of cancer and other human diseases, establish novel HM-regulatory mechanisms, and enrich tHM-toxicological mechanistic understanding.