Lung cancer causes more mortality in the United States than any other form of cancer. Non-small lung cancer (NSCLC) is the most common form of the disease, constituting over 85% of all newly diagnosed lung cancer. The poor clinical prognosis for NSCLC is due to late stage diagnosis and high frequency of metastatic disease. NF-?B is an immediate-early transcription factor that is commonly up regulated in NSCLC tumors. NSCLC tumors display elevated NF-?B activity, which correlates with invasive de-differentiated morphology, tumor staging, and poor clinical outcomes. Many laboratories, including our own, have shown that NF-?B provides cellular resistance to many forms of apoptosis. To understand better how NSCLC cells resist apoptotic cues, we have characterized the molecular pathways used by NF-?B to block apoptosis. Preliminary data presented in this proposal indicate that the p65 component of NF-?B provides a novel avenue of cell survival by blocking detachment from the extracellular matrix. Pro inflammatory cytokines inhibit this form of apoptosis in a process referred to as anoikis. Both IKK and p65 activities are required to block anoikis by inhibiting Bim, a pro-apoptotic protein important for induction of anoikis in NSCLC cells. IKK and p65 are required to stimulate Bim protein turnover in response to pro-inflammatory cytokines. IKK? directly phosphorylates Bim, regulating its half-life through a process involving polyubiquitination and proteasome-dependent degradation. Knockdown of the adaptor molecule COMMD1, a member of ECSSOCS1 E3 ligase complex that regulates p65 degradation, results in a significant up regulation of endogenous Bim protein expression. Using an in vitro model for invasion and metastasis that induces epithelial-mesenchymal transition (EMT), we demonstrate that pro-inflammatory cytokines facilitate TGF?-induced EMT. The EMT phenotype is associated with an elevation in IKK and p65 activity, loss of Bim expression, and resistance to anoikis. Since EMT has been proposed to link de-differentiation with the self-renewing properties observed in cancer stem cells, we hypothesize that loss of Bim protein expression is one of the rate-limiting steps in NSCLC metastasis. The goal of this proposal is to understand how p65 regulates Bim protein turnover and to determine whether this pro-inflammatory responsive mechanism governs NSCLC metastasis. To address our hypothesis three Specific Aims will be addressed.
Aim 1 will determine whether stimulation or inactivation of IKK and p65 activities alters the sensitivity of NSCLC cells to anoikis. Experiments will identify the minimal interacting domain required to stimulate Bim degradation and will determine if localization of p65 to the microtubule complex or to the mitochondria alters Bim induced apoptosis.
Aim 2 will determine the importance of the COMMD1-containing E3 ligase for ubiquitination of Bim and will determine the role of p65 and IKK in this process.
Aim 3 will use in vitro and in vivo models to determine whether regulation of Bim protein stability governs EMT and metastasis in lung cancer cells. Understanding the mechanisms used by inflammatory mediators to stimulate Bim degradation will potentially result in the identification of novel approaches to preventing NSCLC metastasis.
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