Hypoxia is known to directly or indirectly confer resistance of cancer cells to radio-and chemo-therapies leading to treatment failure. However, the molecular events that ultimately confer resistance to current therapies in hypoxic cancer cells are not well defined. Our long-range goal is to induce apoptosis of hypoxic cancer cells through targeting the molecules that confer such a resistance. SAG (Sensitive to Apoptosis Gene) is a RING component of SCF (Skpl, Cullins, F-box proteins) E3 ubiquitin ligase. We found that SAG is induced under hypoxia and when over-expressed, inhibits hypoxia-induced apoptosis both in vitro and in vivo. The objective of this application is to identify SAG-SCF E3 ubiquitin ligase substrates/targets whose degradation contributes to apoptosis resistance under hypoxia. The central hypothesis is that hypoxia induces SAG which complexes with other components of SCF E3 ubiquitin ligase to promote the ubiquitination and degradation of several pro-apoptotic proteins/substrates, thus inhibiting hypoxia-induced apoptosis as a cellular defensive/adaptive response. The rationale is to elucidate ligase associated mechanism of SAG apoptosis suppression through identification of critical cellular proteins whose degradation confers such suppression, thus validating SAG-SCF E3 ubiquitin ligase as novel hypoxic cancer targets.
Specific aims to test the hypothesis are 1) to identify and characterize substrates of SAG-SCF E3 ubiquitin ligase that are degraded during SAG protection against apoptosis induced by hypoxia; and 2) to define the role of selected substrates of SAG-SCF E3 ubiquitin ligase in regulation of cellular response to hypoxia as well as radiation. Through this proposed research, we expect to identify several SAG-SCF E3 ligase substrates whose degradation contributes to apoptosis inhibition under hypoxia, thus elucidating the molecular mechanism of SAG action and further validating SAG-associated E3 ligase as a promising cancer target. Furthermore, identified substrates can serve as biomarkers for the development of the specific inhibitors of SAG-SCF E3 ubiquitin ligase. These initial studies will set the stage for a more detailed investigation of the mechanism of SAG mediated suppression of apoptosis. ? ?
