Volume 141, Issue 5, Pages 1749-1761.e1 (November 2011) Neurotensin Signaling Activates MicroRNAs-21 and -155 and Akt, Promotes Tumor Growth in Mice, and Is Increased in Human Colon Tumors  Kyriaki Bakirtzi, Maria Hatziapostolou, Iordanes Karagiannides, Christos Polytarchou, Savina Jaeger, Dimitrios Iliopoulos, Charalabos Pothoulakis  Gastroenterology  Volume 141, Issue 5, Pages 1749-1761.e1 (November 2011) DOI: 10.1053/j.gastro.2011.07.038 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 NT-regulated microRNAs in colon epithelial cells. (A) Heat map representation of differentially expressed microRNAs after NT treatment (0.5, 6 h) of NCM460-NTR1 cells. Red: up-regulated microRNAs, green: down-regulated microRNAs. Clustering of (B) NT-induced and (C) NT-suppressed microRNAs according to their response dynamics. Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 NT regulates microRNA expression through NTR1 in colon cancer cells. (A) NTR1 mRNA expression levels in nontransformed (NCM460) and cancer cell lines. (B) MicroRNA expression levels in HCT-116 and (C) DLD1 colon cancer cells treated with NT (0.5, 6 h), assessed by real-time PCR analysis for the top 7 NT up-regulated and down-regulated microRNAs in NCM460-NTR1 cells. (D) NTR1 mRNA expression levels, assessed by real-time PCR analysis, in HCT116 and DLD1 colon cancer cells after siRNA treatment (48 h) against NTR1 (siNTR1#1 or siNTR1#2, 100 nmol/L) and negative control (siRNA NC, 100 nmol/L). (E) NTR1 expression was inhibited in HCT116 and DLD1 colon cancer cells by siRNA treatments (siNTR1#1 or siNTR1#2, 100 nmol/L, 48 h); cells were treated with NT (6 h, 100 nmol/L) and expression levels of miR-21, miR-210, and miR-155 were assessed by real-time PCR analysis. Data show mean ± standard deviation of 3 independent experiments. Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 NT controls microRNA expression through NF-κB activation in colon cancer cells. (A) NF-κB phosphorylation assessed by enzyme-linked immunosorbent assay, after time-course treatment of HCT116 and DLD1 cells with NT (20, 50, 100 nmol/L). (B) NTR1 expression was suppressed by siRNA in HCT116 and DLD1 cells (48 h), cells were treated with NT (100 nmol/L, 6 h) and NF-κB phosphorylation was assessed by enzyme-linked immunosorbent assay. (C) Chromatin immunoprecipitation (ChIP) detected enrichment of NF-κB transcription factor in the promoters of miR-21, miR-210, miR-155, and HNRPA2 (negative control) after NT treatment (0.5, 1, 6 h) in HCT116 and DLD1 cells. (D) Expression levels of miR-21, miR-210, and miR-155 and (E) their primary transcripts, assessed by real-time PCR, in HCT116 and DLD cells treated with a pharmacologic inhibitor of NF-KB pathway (BAY-117082, 5 umol/L) or an siRNA negative control (siRNA NC, 100 nmol/L) or an siRNA against p65 (sip65, 100 nmol/L) for 48 hours and treated with NT (100 nmol/L, 6 h). Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 NT regulates miR-21 and miR-155 signaling pathways in colon cancer cells. (A) Luciferase activity of the PTEN and SOCS1 3′UTRs after NT treatment (100 nmol/L) for 24 hours in HCT116 and DLD1 cells (untreated, as-miR-NC–treated, as-miR-21–treated, or as-miR-155–treated). (B) PTEN and SOCS1 mRNA levels, assessed by real-time PCR, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells (untreated, as-miR-NC–treated, as-miR-21–treated, or as-miR-155–treated). (C) PTEN, SOCS1, and β-actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (D) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), and/or as-miR-155 (100 nmol/L) for 24 hours and treated with NT (100 nmol/L) for 24 hours. Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 NT activates AKT through suppression of PPP2CA by direct interaction with miR-155. (A) miR-155 binding sites in 3′UTRs of PPP2CA predicted by Lever algorithm analysis. (B) Luciferase activity of the PPP2CA 3′UTRs in HCT116 and DLD1 cells transfected with as-miR-155 (100 nmol/L) or as-miR-155 (100 nmol/L) for 24 hours. (C) Luciferase activity of the PPP2CA 3′UTRs in HCT116 and DLD1 cells treated with NT (100 nmol/L). (D) PPP2CA and β-actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (E) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with miR-155 (100 nmol/L) or si-PPP2CA (100 nmol/L) for 24 hours. (F) NF-κB/p65 activity, assessed by enzyme-linked immunosorbent assay, in HCT cells transfected with NT (100 nmol/L) and as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), as-miR-155 (100 nmol/L), or with 10 nmol/L of an AKT pharmacologic inhibitor (MK-2206). Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 NT affects the tumorigenicity and invasiveness of colon cancer cells through regulation of miR-21 and miR-155 pathways. (A) Number of colonies and (B) invading cells in NT-treated HCT116, DLD1, and SW480 cells in which miR-21 and/or miR-155 were knocked down. (C) NT effects on tumor volume in a HCT116-xenograft model described in the Materials and Methods section. The P value indicates differences between NT/as-miR-NC–treated mice vs NT/as-miR-21/as-miR-155–treated mice. (D) PTEN, SOCS1, and PPP2CA mRNA levels in NT-treated HCT-116-xenograft tumors (day 35). Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 NT-microRNA signaling pathway in human colon cancers. (A) NTR1, PTEN, SOCS1, miR-21, and miR-155 expression levels assessed by real-time PCR in 18 normal and 34 (13 stage I, 7 stage II, and 14 stage III) colon cancer tissues. (B) Correlation between NTR1, PTEN, and SOCS1 mRNA levels and miR-21 and miR-155 expression levels and colon tumor stage. (C) NT-microRNA signaling pathway in colon cancer. Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Table 1 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Table 2 Gastroenterology 2011 141, 1749-1761.e1DOI: (10.1053/j.gastro.2011.07.038) Copyright © 2011 AGA Institute Terms and Conditions