Although reactive oxygen species (ROS) are considered important in Cr(VI)-induced malignant cell transformation, the underlying mechanism remains to be investigated. Recent studies show that ROS are important in the induction of autophagy and subsequently upregulation of p62. Our preliminary results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) upregulated p62 and that chronic exposure of animals to Cr(VI) particles upregulated this protein in the lung. Forced expression of p62 in BEAS- 2B cells caused malignant transformation of these cells and generated tumor in xenograft model. Thus, it is likely that in BEAS-2B cells ROS upregulate p62 and its downstream, leading to cell transformation. Our preliminary studies show that in Cr(VI)-transformed cells p62 was upregulated. Among six main domains/regions of p62, we will study (a) the TRAF6-binding domain, which binds to TRAF6 and causes its phosphorylation, leading to activation of NF-κB, and (b) the Keap-interacting region, which binds to Keap1 (Nrf2 inhibitor) and causes constitutive activation of Nrf2. The central hypothesis is that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and then upregulates p62, leading to malignant cell transformation and that in Cr(VI)-transformed cells p62 activates NF-κB through TRAF6-binding domain and causes constitutive Nrf2 activation through Keap-interacting region and subsequently upregulates its downstream anti- inflammatory, antioxidant, and anti-apoptotic proteins, resulting in increased survival and tumorigenesis of these transformed cells. Aim 1 will test the hypothesis that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and upregulates p62, leading to malignant cell transformation. We will carry out the following studies. (a) ROS generated by Cr(VI) upregulate p62. We will inhibit ROS to show the decrease of p62. (b) We will inhibit ROS or p62 to demonstrate the inhibition of Cr(VI)-induced malignant cell transformation. (c) We will use whole transcriptome (RNA Seq) analysis to identify p62 downstream genes responsible for Cr(VI)-induced malignantly transformation. Aim 2 will test the hypothesis that p62 upregulates NF-κB through its TRAF6- binding domain and that p62 causes constitutive Nrf2 activation through its Keap-interacting region, resulting in the generation of microenvironment favorable for survival of Cr(VI)-transformed cells and subsequent tumorigenesis. We will show (a) the role of TRAF6-binding domain in NF-κB activation; (b) the role of Keap- interacting region in constitutive Nrf2 activation and upregulation of its downstream inflammatory, antioxidant, and anti-apoptotic proteins; and (c) the role of p62 and its downstream NF-κB and Nrf2 pathways in tumorigenesis of Cr(VI)-transformed cells. Aim 3 will investigate the role of p62 in the mechanism of Cr(VI) carcinogenesis using p62 wildtype and knockout mice. We will measure p62 and its downstream proteins in lung issues of animals chronically exposed to Cr(VI) and of workers occupationally exposed to Cr(VI) to strengthen the cellular and animal Cr(VI) exposure studies.

NARRATIVE This application investigates the role of p62 in the mechanisms of malignant cell transformation and of tumorigenesis induced by Cr(VI) at cellular, animal, and human tissue levels. If aims are achieved, the study will demonstrate that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and upregulates p62, leading to malignant cell transformation and that in Cr(VI)-transformed cells p62 activates NF-κB through TRAF6-binding domain and causes constitutive Nrf2 activation through Keap-interacting region and subsequently upregulates its downstream anti-inflammatory, antioxidant, and anti-apoptotic proteins, resulting in increased survival of Cr(VI)-transformed cells and tumorigenesis.