Volume 143, Issue 4, Pages 995-1005.e12 (October 2012) Flotillin-1 Promotes Tumor Necrosis Factor-α Receptor Signaling and Activation of NF- κB in Esophageal Squamous Cell Carcinoma Cells  Libing Song, Hui Gong, Chuyong Lin, Chanjuan Wang, Liping Liu, Jueheng Wu, Mengfeng Li, Jun Li  Gastroenterology  Volume 143, Issue 4, Pages 995-1005.e12 (October 2012) DOI: 10.1053/j.gastro.2012.06.033 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 FLOT1 is up-regulated in ESCC cell lines and primary human ESCC. (A and B) Western blotting analysis of FLOT1 expression in 2 NEECs and cultured ESCC cell lines (A) and in 8 paired primary ESCC tissues (T) and matched adjacent nontumor tissues (ANT) from the same patient (B). GAPDH was used as a loading control. (C) IHC staining indicating that FLOT1 expression is up-regulated in human ESCC (Clinical stage I–IV) compared with normal esophageal tissue. (D) Kaplan-Meier curves of ESCC patients with low vs high expression of FLOT1 (n = 432; P < .001, log-rank test). Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 FLOT1 promotes the aggressiveness of ESCC cells in vitro. (A) Overexpression of FLOT1 in Kyse30 and Kyse510 cell lines analyzed by immunoblotting. GAPDH was used as a loading control. (B) The representative pictures (left panel) and quantification (right panel) of crystal violet-stained indicated cells. (C) The representative pictures (left panel) and quantification (right panel) of colony numbers of indicated cells as determined by an anchorage-independent growth assay. Colonies larger than 0.1 mm in diameter were scored. (D) Representative images of the chicken chorioallantoic membrane blood vessels stimulated with conditioned medium from indicated cells. (E) The representative pictures (left panel) and quantification (right panel) of invaded cells were analyzed using Transwell matrix penetration assay. (F) Annexin V-FITC/PI staining of indicated cells treated with cisplatin (20 μmol/L) for 24 hours. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Silencing FLOT1 inhibits the malignant properties of ESCC cells. (A) Silencing FLOT1 in 2 specific short hairpin RNA-transduced stable ESCC cell lines. GAPDH was used as a loading control. (B) The representative pictures (left panel) and quantification (right panel) of crystal violet-stained indicated cells. (C) The representative pictures (left panel) and quantification (right panel) of colony numbers of indicated cells as determined by an anchorage-independent growth assay. Colonies larger than 0.1 mm in diameter were scored. (D) Representative images of the chicken chorioallantoic membrane blood vessels stimulated with conditioned medium from indicated cells. (E) The representative pictures (left panel) and quantification (right panel) of invaded cells were analyzed using the Transwell matrix penetration assay. (F) Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining of indicated cells treated with cisplatin (20 μmol/L) for 24 hours. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 FLOT1 contributes to ESCC progression in vivo. (A) Xenograft model in nude mice. Representative images of tumor-bearing mice (left panel) and images of the tumors from all mice in each group (right panel). (B) H&E and IHC staining demonstrated that overexpression of FLOT1 induced and suppression of FLOT1 inhibited the aggressive phenotype of ESCC cells in vivo, as indicated by the expression of Ki67 and CD31, TUNEL-positive cells, and F4/80-positive cells. (C) Electrophoretic mobility shift assay of NF-κB activity in 4 paired FLOT1-overexpressing tumors and control tumors (left panel) and FLOT1-silenced tumors and control tumors (right panel). Octamer-binding transcription factor 1(OCT-1) DNA-binding complex served as a control. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 FLOT1 activates NF-κB signaling. (A) NF-κB luciferase-reporter activities were analyzed in indicated cells. (B) Real-time PCR analysis indicating an apparent overlap between NF-κB-dependent gene expression and FLOT1-regulated gene expression. The pseudocolor represents the intensity scale of FLOT1 vs Vector, or FLOT1 short hairpin RNA vs RNAi-vector, generated by a log2 transformation. (C) Electrophoretic mobility shift assay indicating that NF-κB activity significantly increased in FLOT1-transduced ESCC cells and decreased in FLOT1-silenced cells. OCT-1 DNA-binding complex served as a control. (D) Overexpressing IκBα dominant-negative mutant (IκBα-mu) inhibited FLOT1-induced tumorigenesis as examined in a xenograft model. Representative images of tumor-bearing mice (left panel) and images of the tumors (middle panel). Tumor volumes were measured on the indicated days (right panel). Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 FLOT1 facilitates the recruitment of TNFR and sustains NF-κB activity. (A) Luciferase-reported NF-κB activities were analyzed in vector ESCC cells and FLOT1-transduced ESCC cells treated with or without methyl-β-cyclodextrin (MβCD). (B) Western blotting analysis of expression of FLOT1, TNFR, and GM1. (C) Western blotting analysis of expression of FLOT1, TRAF2, RIP, IKKβ, and NEMO in lipid rafts isolated from indicated cells. (D) Western blotting analysis of the K63-linked polyubiquitin levels of TRAF2 (left panel), RIP (middle panel), and NEMO (right panel) in indicated cells treated with TNF-α (10 ng/mL). (E) Western blotting analysis of IκBα expression in indicated cells treated with TNF-α (10 ng/mL). α-Tubulin was used as a loading control. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Model: FLOT1 overexpression facilitates the recruitment of the TNF-α receptor (TNFR) into lipid rafts and activates the NF-κB signaling pathway, and consequently leads to progression and poorer clinical outcome in human ESCC. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 FLOT1 is up-regulated in human esophageal squamous cell carcinoma. (A) Real-time polymerase chain reaction (PCR) analysis of FLOT1 messenger RNA (mRNA) in 2 primary normal human esophageal epithelial cells (NEECs) and cultured esophageal squamous cell carcinoma (ESCC) cell lines. (B) Real-time PCR analysis of FLOT1 mRNA in 8 paired primary ESCC tissues (T) and adjacent noncancerous tissues (ANT) from the same patient. Expression levels were normalized to GAPDH. (C) Western blotting analysis indicated that elevated FLOT1 mainly enriched in the lipid rafts. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 (A and B) Kaplan-Meier curves and univariate log-rank analyses of the survival of esophageal squamous cell carcinoma (ESCC) patients expressing low and high levels of FLOT1 within subgroups of (A) clinical stage I and (B) T1 classification. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 FLOT1 plays important role in lipid rafts formation. Representative images of FLOT1 and β-CT in indicated cells analyzed with immunofluorescence staining assay. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 FLOT1 promotes angiogenesis. (A and B) Representative images (left panel) and quantification (right panel) of HUVECs cultured on Matrigel-coated plates with conditioned medium from (A) vector control and FLOT1-transduced esophageal squamous cell carcinoma (ESCC) cells or (B) RNAi-vector control and FLOT1-silenced ESCC cells. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 FLOT1 contributes to esophageal squamous cell carcinoma progression in vivo. (A) Tumor volumes were measured on the indicated days. (B) Mean tumor weights. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 FLOT1 activates NF-κB pathway. Western blotting analysis of phospho-IKK-β, total IKK-β, phospho-IκBα, total IκBα expression in indicated cells. α-Tubulin was used as a loading control. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 7 Overexpression of FLOT1 promotes normal esophageal epithelial cells proliferation and activates NF-κB pathway. (A) Overexpression of FLOT1 in normal esophageal epithelial cells (NEEC) 1 and NEEC2 analyzed by immunoblotting. GAPDH was used as a loading control. (B and C) Overexpression of FLOT1 promotes growth rates of NEEC1 and NEEC2 as determined by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (B) and colony formation assay (C). (D) NF-κB luciferase-reporter activities were analyzed in indicated cells. (E) Changes of messenger RNA (mRNA) expression of NF-κB-regulated genes in indicated cells assessed by real-time PCR. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 8 Blockage of NF-κB pathway, via overexpressing IκBα-mutant, inhibits FLOT1-induced aggressiveness. (A) Changes of messenger RNA (mRNA) expression of NF-κB-regulated genes in indicated cells assessed by real-time PCR. (B) The representative pictures (left panel) and quantification (right panel) of colony number of indicated cells as determined by an anchorage-independent growth assay. Colonies larger than 0.1 mm in diameter were scored. (C) Representative images of the CAM blood vessels stimulated with conditioned medium from indicated cells. (D) Representative images (left panel) and quantification (right panel) of indicated invaded cells analyzed with transwell matrix penetration assay. (E) Annexin V-fluorescein isothiocyanate (FITC)/PI staining of indicated cells treated with cisplatin (20 μmol/L) for 24 hours. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 9 FLOT1 facilitates the recruitment of tumor necrosis factor-α receptor. (A) Western blotting analysis of TNF-α in indicated cells. (B) Western blotting analysis of tumor necrosis factor-α receptor (TNFR) and GM1 in lipid rafts isolated from indicated cells transfected with TNF-α small interfering RNA. (C) FLOT1 binds to TNFR. Kyse30 cells were stimulated with Flag-tagged TNF-α for 15 minutes, and lipid rafts were isolated and immunoprecipitated using Flag affinity agarose. Immunoprecipitates and corresponding total cell lysates were subjected to Western blotting using TNFR and FLOT1 antibodies. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 10 FLOT1 sustains NF-κB activity. (A) In vitro kinase assay measured in the indicated cells treated with TNF-α (10 ng/mL) for indicated times. IKK-β was immunoprecipitated, and kinase activity was measured by phosphorylation of a recombinant GST-IκBα substrate with a phospho-specific IκBα antibody. Equal immunoprecipitation of IKK-β was shown. (B and C) Real-time PCR of IL-6 and IL-1β in cells treated with TNF-α (10 ng/mL) for indicated times. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 11 Clinical relevance of FLOT1-triggered NF-κB activation in human esophageal squamous cell carcinoma. (A) FLOT1 levels associated with p-IKK-β (S181), Ki67, CD31, MMP-9, or VEGFC expression in 432 primary human esophageal squamous cell carcinoma (ESCC) specimens. Two representative specimens with low and high levels of FLOT1 are shown. Original magnification, ×200. (B) Percentages of specimens showing low or high FLOT1 expression relative to the level of p-IKK-β (S181), Ki67, CD31, MMP-9, or VEGFC. (C) Analysis of expression (left) and correlation (right) of FLOT1 with cyclin D1, MMP9, and VEGFC messenger RNA (mRNA) expression, as well as NF-κB DNA-binding activity in 10 freshly collected human ESCC samples. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 995-1005.e12DOI: (10.1053/j.gastro.2012.06.033) Copyright © 2012 AGA Institute Terms and Conditions