Flotillin-1 promotes tumor necrosis factor-α receptor signaling and activation of NF-κB in esophageal squamous cell carcinoma cells.
OBJECTIVE
The flotillin family of proteins, including flotillin-1 (FLOT1 or Reggie-2), are lipid raft proteins that initiate receptor kinase signaling and are up-regulated in several tumor types. We investigated the role of FLOT1 signaling and activation of the transcription factor nuclear factor (NF)-κB in esophageal squamous cell carcinoma (ESCC) cells.


METHODS
We used immunoblot and immunochemical analyses to determine levels of the lipid raft-associated protein FLOT1 in ESCC cell lines and 432 ESSC samples from patients; primary normal esophageal epithelial cells and matched adjacent nontumor tissues were used as controls. We determined the ability of FLOT1 to activate NF-κB using kinase, electrophoretic mobility shift, and luciferase reporter assays. We measured the effects of FLOT1 overexpression and knockdown with short hairpin RNAs in ESCC cell lines using colony formation, anchorage-independent growth, chicken chorioallantoic membrane, transwell matrix penetration, and Annexin V-binding assays. We analyzed growth of ESCC xenograft tumors in nude mice.


RESULTS
Levels of FLOT1 were increased in ESCC cell lines and samples from patients, compared with controls; protein levels correlated with disease stage and survival time. Overexpression of FLOT1 in Kyse30 and Kyse510 ESCC cell lines increased proliferation, anchorage-independent growth, and invasive activity and protected them from apoptosis. FLOT1-transduced ESCC cells formed larger tumors in nude mice than control cells (transduced with only the vector). FLOT1 facilitated recruitment of the tumor necrosis factor-α receptor to lipid rafts; promoted K63-linked polyubiquitination of the signaling intermediaries tumor necrosis factor receptor associated factor 2, receptor interacting protein, and NEMO; and sustained the activation of NF-κB. Levels of FLOT1 correlated with activation of NF-κB in ESCC samples from patients.


CONCLUSIONS
The lipid raft protein FLOT1 is up-regulated in ESCC cell lines and samples from patients and promotes ESCC cell proliferation and tumor growth in mice. FLOT1 activates tumor necrosis factor-α receptor signaling and sustains activation of NF-κB in ESCC cells.
State Key Laboratory of Oncology in Southern China, Department of Experimental Research, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong, China.
Libing
L
Song
Hui
H
Gong
Chuyong
C
Lin
Chanjuan
C
Wang
Liping
L
Liu
Jueheng
J
Wu
Mengfeng
M
Li
Jun
J
Li
D000818:Animals; D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016009:Chi-Square Distribution; D018450:Disease Progression; D004727:Endothelium / Q000378:metabolism; D004938:Esophageal Neoplasms / Q000378:metabolism* / Q000473:pathology; D004947:Esophagus / Q000378:metabolism; D005260:Female; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D015999:Multivariate Analysis; D016328:NF-kappa B / Q000378:metabolism*; D011379:Prognosis; D016016:Proportional Hazards Models; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015398:Signal Transduction; D014183:Transplantation, Heterologous; D025801:Ubiquitin / Q000378:metabolism; D015854:Up-Regulation
D008565:Membrane Proteins; D016328:NF-kappa B; D018124:Receptors, Tumor Necrosis Factor; D025801:Ubiquitin; C106354:flotillins; D051550:I-kappa B Kinase