Nrf2, autophagy, and arsenic carcinogenesis

? DESCRIPTION (provided by applicant): Chronic exposure to inorganic arsenic is a worldwide public health problem that has been associated to increased risks of developing cancers of the lung, skin, and bladder. Among these malignancies, arsenic- induced lung cancer presents with the highest mortality rate. The precise carcinogenic mechanism of arsenic has not yet been fully elucidated despite many years of research and the severity of the health effects associated to its exposure. We have previously reported that environmentally relevant, low doses of arsenic block autophagy, which resulted in prolonged activation of Nrf2, the main orchestrator of the adaptive antioxidant and pro-survival response. Specifically, arsenic-induced Nrf2 activation does not occur through the canonical, reactive oxygen species (ROS)-sensing mechanism but through the autophagy-dependent, non- canonical mechanism. Autophagy is a bulk degradation pathway that degrades damaged organelles and protein aggregates. Autophagy blockage can result in accumulation of defective mitochondria and excessive ROS production, which cause DNA mutations. Nrf2 activation is well known for its cellular protection against ROS stress, which can enable arsenic-exposed cells to survive and sustain gene mutations that drive malignant transformation. Therefore, we hypothesize that prolonged activation of Nrf2 resulting from arsenic-induced autophagy blockage is essential for the arsenic-mediated malignant transformation. Our hypothesis is supported by several recent publications demonstrating that the tumorigenic effect of prolonged Nrf2 activation in autophagy-deficient mice was abolished by concurrent Nrf2 knockout. Moreover, earlier studies have found high constitutive levels of Nrf2 in many cancer cells, which favor their proliferation and chemo resistance, an effect known as the dark side of Nrf2. We have generated substantial amounts of data indicating that arsenic blocks autophagy by inhibiting the autophagosome-lysosome fusion step. Three SNAREs mediate the fusion: Stx17 on the outer membrane of the autophagosome interacts through SNAP29 with VAMP8 that resides on the lysosome membrane. We believe that genetic ablation of any of these proteins should prevent the fusion of the autophagosome with the lysosome, and the effects of this ablation should mimic the effects of arsenic- mediated p62-dependent Nrf2 up regulation. To better understand arsenic carcinogenicity, we propose: Aim 1: Elucidate the detailed molecular mechanism by which arsenic blocks autophagosome- lysosome fusion. Aim 2: Determine if autophagy dysregulation and prolonged Nrf2 activation are essential for malignant transformation. Aim 3: Test the tumorigenicity of cell lines and correlate it with prolonged Nrf2 activation. Impact: A detailed and thorough understanding of the molecular events leading to the prolonged Nrf2 activation in arsenic-induced carcinogenesis will prove extremely valuable in the generation of preventive and therapeutic strategies, as well as in the identification of biomarkers, for the populations at risk.