Abstract
Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔGbind) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics.
Highlights
Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages
We investigated the roles of Nitric oxide (NO) in complex formation and stem properties of lung cancer cells, and unveiled a novel mechanism of CSC regulation that may lead to a better understanding of cancer cell biology under nitrosative oxidative stress conditions, which may be exploited for CSC-targeted therapy
Apoptosis and necrosis studies by Hoechst 33342/propidium iodide (PI) assays showed that the lowdose (Ͻ40 M) DPTA NONOate had no significant effect on DNA condensation/fragmentation or nuclear PI fluorescence (Fig. 1, B and C), indicating the lack of apoptotic and necrotic cell death under the test conditions
Summary
To study the potential regulation of CSCs by NO, we first characterized the optimal dose response to NO in human lung cancer cells. The results showed that treatment of the cells with DPTA NONOate significantly increased the expression of Oct over time as compared with the untreated control (Fig. 4B), suggesting the stabilizing effect of NO on Oct. To test whether Cav-1 could down-regulate Oct (Fig. 5, C and D) in a post-translational modification manner, we performed a CHX protein synthesis inhibition study in which Cav-1 knockdown and overexpressing cells were treated with 50 g/ml CHX, a protein synthesis inhibitor, over time and analyzed for Oct expression by Western blotting. To investigate whether Cav-1 diminishes the activity of NO on Oct, Cav-1 knockdown (shRNA–Cav-1 ) or overexpressing (FLAG–Cav-1) cells were treated with DPTA NONOate, and the expression level of Oct in these cells was determined by Western blotting. The binding free energy and its components (kcal/mol) calculated from MM/PBSA method
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