ABSTRACT Although the mechanism of Cd(II)-induced carcinogenesis remains to be investigated, recent studies have indicated that autophagy plays a significant role. Our preliminary studies have shown that exposure of human lung bronchial epithelial BEAS-2B cells to Cd(II) generates reactive oxygen species (ROS), which are responsible for Cd(II)-induced malignant cell transformation. We have also shown that Cd(II) is able to induce autophagy in normal BEAS-2B cells. The activation of autophagy by Cd(II) is likely to be a cell self- defense mechanism against Cd(II)-induced oxidative damage. Our preliminary studies have also shown that Cd(II)-transformed BEAS-2B cells exhibit autophagy deficiency (autophagy incompetence), resulting in accumulation of autophagosomes and increased level of p62 protein. The increased p62 causes a constitutive activation of Nrf2, which in turn upregulates its target antioxidant proteins, superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2), and anti-apoptotic proteins, Bcl-2 and Bcl-xL, most likely due to increased binding of Nrf2 to antioxidant response element (ARE) sites of these target proteins. The upregulations of these antioxidants (decrease ROS) and anti-apoptotic proteins result in development of apoptosis resistance of Cd(II)-transformed cells. The constitutively elevated p62 and Nrf2 are responsible for survival advantage of Cd(II)-transformed cells. Natural compound sulforaphane increased autophagy in normal cells exposed to Cd(II) and restored autophagy competence in Cd(II)-transformed cells. The central hypothesis of this application is that autophagy is a cell defense mechanism in Cd(II)-induced malignant cell transformation and that autophagy deficiency is responsible for tumorigenesis of Cd(II)-transformed cells. Aim 1 will investigate the protective role of autophagy against Cd(II)-induced malignant cell transformation. The hypothesis of this aim is that autophagy protects against Cd(II)-induced malignant cell transformation by decreasing ROS through facilitating mitochondrial turnover in normal cells and that sulforaphane enhances autophagy and decreases the cell transformation. Aim 2 will demonstrate that autophagy deficiency in Cd(II)- transformed cells increases cell survival through constitutive activation of p62/Nrf2 signaling. The hypothesis of this aim is that Cd(II)-transformed cells are autophagy deficient, resulting in accumulation of autophagosomes, constitutive activations of p62/Nrf2, elevated levels of antioxidants, Bcl-2, and Bcl-xL, decreased levels of ROS, and acquisition of apoptosis resistance. Aim 3 will investigate roles of autophagy deficiency, elevation of p62, constitutive activation of Nrf2, and apoptosis resistance in tumorigenesis of Cd(II)-transformed cells and protection by sulforaphane in vivo. The hypothesis of this aim is that autophagy deficiency in Cd(II)-transformed cells promotes tumorigenesis through elevation of p62, constitutive activation of Nrf2, and acquisition of apoptosis resistance and that sulforaphane restores autophagy competence and inhibits tumorigenesis.

NARRATIVE This application investigates the role of autophagy in Cd(II)-induced carcinogenesis. If goals are accomplished, this study will demonstrate that autophagy protects against Cd(II)-induced malignant cell transformation by decreasing ROS through facilitating mitochondrial turnover in normal cells, that autophagy deficiency in Cd(II)-transformed cells increases cell survival through constitutive activation of p62/Nrf2 signaling, that autophagy deficiency in Cd(II)-transformed cells promotes tumorigenesis through constitutive activations of p62 and Nrf2 and acquisition of apoptosis resistance, and that sulforaphane restores autophagy competency and inhibits tumorigenesis.