Regulation of ezrin tension by S-nitrosylation mediates non-small cell lung cancer invasion and metastasis

Cancer invasion and metastasis depend on accurate and rapid modulation of both chemical and mechanical activities. The S-nitrosylation (SNO) of membrane cytoskeletal cross-linker protein ezrin may regulate the malignant process in a tension-dependent manner. Methods: The level of nitrosylated ezrin in non-small cell lung Cancer (NSCLC) tissues and A549 cell line were evaluated by biotin-switch assay. A few cysteine mutated plasmids of ezrin were used to identify active site for SNO. Newly designed ezrin or mutated-ezrin tension probes based on Förster resonance energy transfer (FRET) theory were applied to visually observe real-time tension changes. Cytoskeleton depolymerizing and motor molecular inhibiting experiments were performed to reveal the alternation of the mechanical property of ezrin after SNO. Transwell assays and xenograft mouse model were used to assess aggressiveness of A549 cells in different groups. Fluorescent staining was also applied to examine cellular location and structures. Results: High inducible nitric oxide synthase (iNOS) levels were observed to induce ezrin-SNO, and then promote malignant behaviors of NSCLC cells both in vitro and in vivo. Cys¹¹⁷ was identified as the only active site for ezrin-SNO. Meanwhile, an increased level of ezrin tension was observed after iNOS-induced SNO. Enhanced ezrin tension was positively correlated with aggressiveness of NSCLC. Moreover, Microfilament (MF) forces instead of microtubule (MT) forces played dominant roles in modulating ezrin tension, especially after ezrin nitrosylation. Conclusion: This study revealed a SNO-associated mechanism underlying the mechanical tension of ezrin. Ezrin-SNO promotes NSCLC cells invasion and metastasis through facilitating mechanical transduction from the Cytoskeleton to the membrane. These studies implicate the therapeutic potential by targeting ezrin in the inhibition NSCLC invasion and metastasis.