NUAK1 overexpression correlates with tumor progression, lymph node infiltrates, and reduced overall survival (OS) in human colorectal cancer. NUAK1 overexpression.

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NUAK1 overexpression correlates with tumor progression, lymph node infiltrates, and reduced overall survival (OS) in human colorectal cancer. NUAK1 overexpression correlates with tumor progression, lymph node infiltrates, and reduced overall survival (OS) in human colorectal cancer. A, Summary of NUAK1 mRNA detection in a human colorectal cancer TMA (N = 660), sorted by Dukes’ grade A–C; N, normal colon. Asterisks indicate significance (one-way ANOVA and post hoc Tukey test). B, Box and whisker plots of median-centered NUAK1 mRNA expression from the TCGA colorectal adenocarcinoma cohort accessed via Oncomine: left, early (T1, 2) versus late (T3, 4) stage colorectal cancer; right, tumors with (N1, 2) or without (N0 lymph node metastasis. C, Overall survival of human patients with colorectal cancer (N = 947) separated by high versus low NUAK1 expression. Log-rank P value, hazard ratio (HR), and 95% confidence interval (CI) shown. D, Box and whisker plots of NUAK1 mRNA levels in human colorectal cancer separated by risk group as per C. T test P value shown. C and D, Data were mined and graphs adapted from Metabase SurvExpress. E, Apoptosis induced in human colorectal cancer cell lines 48 hours after treatment with the indicated concentrations of HTH-01-015. Red bars, Annexin V/propidium iodide (AV/PI) double-positive cells; black bars, Annexin V–positive cells. Mean and SEM of 3 independent experiments shown; asterisks, significance [ANOVA and post hoc Tukey test, relative to vehicle-treated controls (−)]. F, Immunoblots of lysates from human colorectal cancer cell lines show reduction in MYPT1 Ser445 phosphorylation upon inhibition of NUAK1 (8 hours). The asterisk indicates a nonspecific band. G, Apoptosis induced in U2OS cells 48 hours after treatment with 10 μmol/L HTH-01-015 or WZ4003. Mean and SEM of 3 independent experiments shown; asterisks, significance [ANOVA and post hoc Tukey test, relative to vehicle-treated controls (vc)]. Ns, not significantly increased relative to untreated controls. Immunoblot (bottom) shows suppression of phospho-MYPT1S445 upon NUAK1 inhibition. H, Apoptosis induced in U2OS-MycER cells upon NUAK1 inhibition in the presence (+OHT) or absence (−OHT) of 4-hydroxy-Tam-dependent activation of overexpressed MycER. Mean and SEM of 3 independent experiments shown; asterisks, significance (two-way ANOVA and post hoc Tukey test). Immunoblot shows nuclear stabilization of MycER by 4-OHT. I, Rescue of colorectal cancer cells from HTH-01-015–induced apoptosis upon depletion of MYC. Mean and SEM of a representative experiment (N ≥ 2) in each cell line shown; asterisks, significance (ANOVA and post hoc Tukey test). Immunoblots show depletion of MYC in the same cell lines (bottom). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Jennifer Port et al. Cancer Discov 2018;8:632-647 ©2018 by American Association for Cancer Research