Human Cancer Biology Downregulation of miR-138 Sustains NF-kB Activation and Promotes LipidRaft Formation in Esophageal SquamousCell Carcinoma

Purpose: Constitutive activation of NF-kB signaling plays vital roles in esophageal squamous cell carcinoma (ESCC) progression. The aim of this study was to evaluate the effect of miR-138 on NF-kB activation and ESCC progression. Experimental Design: Expression of miR-138 in ESCC cell lines, ESCC tissues, and 205 archived ESSC specimens was determined using real-time PCR analysis. Anchorage-independent growth, chicken chorioallantoic membrane, Transwell matrix invasion and Annexin V–binding assays, and a xenograft tumor model were used to determine the role of miR-138 in ESCC progression. The effect of miR-138 on NF-kB activation was investigated using IKK in vitro kinase, electrophoretic mobility shift, lipid raft isolation, and luciferase reporter assays. Results: miR-138 was downregulated and inversely correlated with tumor progression and patient survival in ESCCs. Downregulation of miR-138 enhanced, whereas upregulation of miR-138 reduced, the aggressive phenotype of ESCC cells both in vitro and in vivo. Silencing miR-138 promoted K63-linked polyubiquitination of the NF-kB signaling intermediaries TRAF2 and RIP1 and sustained NF-kB activation. Furthermore, downregulation of miR-138 induced lipid raft formation via upregulating multiple components of lipid rafts, including FLOT1, FLOT2, and caveolin-1. Importantly, the in vitro analysiswas consistent with a significant inverse correlation betweenmiR-138 expression andNF-kB hyperactivation in a cohort of human ESCC specimens. Conclusion: Our results show that miR-138 functions as a tumor-suppressive miRNA and that downregulation of miR-138 contributes to constitutive NF-kB activation and ESCC progression. Clin Cancer Res; 19(5); 1083–93. 2013 AACR.