Loss of the cyclin-dependent kinase (CDK) inhibitor p16 is one of the most common genetic aberrations in human cancers. p16 is thought to function as a tumor-suppressor by inducing senescence and negatively regulating the cell cycle through binding CDK4/6 and inhibiting retinoblastoma (RB) protein phosphorylation (canonical pathway). However, many germline p16 mutations that predispose families to melanoma are reported not to compromise CDK-binding or cell cycle regulatory activity of p16, and inherited mutations have not been found in other downstream proteins (CDK6, RB) ? suggesting that additional p16-mediated pathways are involved in melanoma predisposition and may suppress melanoma development. We have discovered a novel role for p16 in regulating intracellular oxidative stress, that results in elevated reactive oxygen species (ROS) and oxidative DNA damage in p16-deficient cells and can be normalized by expression of wild-type (WT) p16. Thus genetic compromise of p16 may promote melanoma in several ways: first, loss of cell cycle control enhances proliferation and may not allow sufficient time for repair of spontaneous and UV-induced DNA damage; and second, dysregulation of ROS promotes cell migration and leads to aberrant signaling and accumulation of oxidative DNA mutations if not repaired. We have reported several findings suggesting that p16 regulates oxidative stress through an alternate, non-canonical pathway: 1) ROS are increased under conditions of partial p16 knockdown in melanocytes while cell cycle distribution is unperturbed, 2) ROS are increased in p16-deficient fibroblasts synchronized for cell cycle phase, 3) oxidative stress associated with p16 knockdown is not recapitulated by RB knockdown, and importantly 4) several melanoma-predisposing p16 mutations compromise oxidative regulatory function but not cell cycle control, while other mutations impair cell cycle but not oxidative regulatory function. We hypothesize that these two independent regulatory activities are both important for p16-mediated suppression of melanoma development, however the mechanism(s) underlying p16 regulation of ROS are unknown. We recently made two observations: first, p16-deficiency results in enhanced cell migration, and second, p16-deficient cells display increased mitochondrial biogenesis. Moreover, we found that this hyper-motility associated with p16-deficiency is dependent upon ROS and elevated mitochondrial membrane potential. The purpose of this R21 application is to define the mechanisms underlying p16 regulation of oxidative stress.
Two aims are proposed:1) Identify p16 residues required for oxidative/mitochondrial regulation and cellular migration, and 2) Define the mechanisms underlying p16 control of mitochondrial biogenesis. Together, these studies will establish a mechanistic basis for p16 control of ROS and cellular motility, and enable future studies using cell-based and animal models to investigate the role of p16-mediated regulation of oxidative stress on cellular transformation, melanoma tumor formation, growth and metastasis.
In addition to the hundreds of thousands of individuals in the U.S. with prior history of melanoma, approximately 70,000 new patients are diagnosed each year. The proposed studies will increase our understanding of a key gene that predisposes people to melanoma, and may lead to discovery of new therapeutic targets for prevention of melanoma. This project is particularly timely given the increasing incidence of melanoma, and lack of available effective therapies for patients with advanced disease.
