DESCRIPTION (provided by applicant): Chromate (Cr (VI)) compounds are toxic and carcinogenic on humans. Our preliminary studies show that exposure of human lung bronchial epithelial (BEAS-2B) cells to Cr (VI) generated reactive oxygen species (ROS). Through ROS reactions, Cr(VI) caused cell transformation, leading to tumorigenesis. However, once cells were transformed, the capability of those cells to generate ROS was decreased. Expressions of antioxidant regulative nuclear factor Nrf2, its positive regulator, p62, and several major antioxidant enzymes were increased. Apoptosis eliminates DNA-damaged or mutated cells. When cells acquire apoptosis resistance, they continue to proliferate, leading to carcinogenesis. Cr(VI)-transformed cells developed apoptosis resistance as indicated by reductions of cleaved poly(ADP-ribose) polymerase (C-PARP) and cleaved caspase 9 (C-caspase 9) and by elevation of anti- apoptotic protein Bcl-2. Binding of Nrf2 to antioxidant response element (ARE) of Bcl-2 gene was increased, indicating the possibility of Nrf2 in up-regulation of Bcl-2. In Cr (VI)-transformed cells, inhibition of p62 by its shRNA reduced Nrf2 expression, leading to induction of apoptosis. These results provide a linkage among p62, Nrf2, Bcl- 2, and apoptosis resistance. The central hypothesis of this application is that due to up-regulations of p62 and Nrf2 and decreased generation of ROS, Cr (VI)-transformed cells develop apoptosis resistance and increase cell survival, invasion, and migration, contributing to overall mechanism of Cr (VI)-induced carcinogenesis. Aim 1 will investigate the mechanism of decreased ROS generation of Cr(VI)-transformed cells. We will carry out comparative studies using non-transformed and Cr(VI)- transformed cells to study each key step of major Cr(VI)- induced ROS generation pathway and identify the specific step responsible for decreased ROS generation in Cr(VI)-transformed cells. We will also investigate the contribution of elevated antioxidant level by focusing on Nrf2 and Nrf2 targeting antioxidants. Aim 2 will investigate apoptosis resistance and its role in enhanced proliferation, invasion, and migration of Cr(VI)-transformed cells. We will alter ROS production by up-regulation of key proteins involved in ROS generation pathway and by down-regulation of key antioxidant enzymes to study the role of ROS in apoptosis resistance. We will investigate whether Nrf2-regulated Bcl-2 induction is a key event. We will alter apoptosis resistance by modifying Bcl-2, Bcl-xL, Mcl-1, or Bax expression to investigate the role of apoptosis resistance in Cr(VI)-enhanced cell proliferation, invasion, and migration. Aim 3 will investigate the role of apoptosis resistance in Cr(VI)-induced tumorigenesis and metastasis using animal models. We will investigate the role of ROS by altering ROS generation including modification of antioxidant enzymes and Nfr2. The role of p62 will be investigated by inhibiting its expression. The role of apoptosis resistance will be investigated by alternation of apoptosis regulative proteins, Bcl-2, Bcl-xL, Mcl-1, or Bax. The same approaches will be used to investigate the role of ROS, p62, and apoptosis resistance in metastasis of Cr(VI)-transformed cells using animal models.

PROJECT NARRATIVE Cr(VI)-containing compounds are human carcinogens. The mechanism of their carcinogenesis remains to be investigated. This application will study the roles of p62, Nrf2, reactive oxygen species, and apoptosis resistance in Cr(VI)-induced carcinogenesis.