Chemically diverse toxicants converge on Fyn and c-Cbl to disrupt precursor cell function.

Identification of common mechanistic principles that shed light on the action of the many chemically diverse toxicants to which we are exposed is of central importance in understanding how toxicants disrupt normal cellular function and in developing more effective means of protecting against such effects. Of particular importance is identifying mechanisms operative at environmentally relevant toxicant exposure levels. Chemically diverse toxicants exhibit striking convergence, at environmentally relevant exposure levels, on pathway-specific disruption of receptor tyrosine kinase (RTK) signaling required for cell division in central nervous system (CNS) progenitor cells. Relatively small toxicant-induced increases in oxidative status are associated with Fyn kinase activation, leading to secondary activation of the c-Cbl ubiquitin ligase. Fyn/c-Cbl pathway activation by these pro-oxidative changes causes specific reductions, in vitro and in vivo, in levels of the c-Cbl target platelet-derived growth factor receptor-alpha and other c-Cbl targets, but not of the TrkC RTK (which is not a c-Cbl target). Sequential Fyn and c-Cbl activation, with consequent pathway-specific suppression of RTK signaling, is induced by levels of methylmercury and lead that affect large segments of the population, as well as by paraquat, an organic herbicide. Our results identify a novel regulatory pathway of oxidant-mediated Fyn/c-Cbl activation as a shared mechanism of action of chemically diverse toxicants at environmentally relevant levels, and as a means by which increased oxidative status may disrupt mitogenic signaling. These results provide one of a small number of general mechanistic principles in toxicology, and the only such principle integrating toxicology, precursor cell biology, redox biology, and signaling pathway analysis in a predictive framework of broad potential relevance to the understanding of pro-oxidant-mediated disruption of normal development.

Redox biology in normal cells and cancer: restoring function of the redox/Fyn/c-Cbl pathway in cancer cells offers new approaches to cancer treatment.

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/ßpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries.

Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation.

Although many co-activators have been identified for various nuclear receptors, relatively fewer co-repressors have been isolated and characterized. Here we report the identification of a novel testicular orphan nuclear receptor-4 (TR4)-associated protein (TRA16) that is mainly localized in the nucleus of cells as a repressor to suppress TR4-mediated transactivation. The suppression of TR4-mediated transactivation is selective because TRA16 shows only a slight influence on the transactivation of androgen receptor, glucocorticoid receptor, and progesterone receptor. Sequence analysis shows that TRA16 is a novel gene with 139 amino acids in an open reading frame with a molecular mass of 16 kDa, which did not match any published gene sequences. Mammalian two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact strongly with TR4. The electrophoretic mobility shift assay suggests that TRA16 may suppress TR4-mediated transactivation via decreased binding between the TR4 protein and the TR4 response element on the target gene(s). Furthermore, TRA16 can also block the interaction between TR4 and TR4 ligand-binding domain through interacting with TR4-DNA-binding and ligand-binding domains. These unique suppression mechanisms suggest that TRA16 may function as a novel repressor to selectively suppress the TR4-mediated transactivation.