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.