Elevated expression of flotillin-1 is associated with lymph node metastasis and poor prognosis in early-stage cervical cancer

Abstract

Accumulating evidence has revealed that the expression of the lipid raft protein flotillin-1 is elevated in various human cancers, but the role flotillin-1 plays in the carcinogenesis of cervical cancer remains unclear. The expression profile of flotillin-1 was assayed using real-time PCR, western blotting, and immunohistochemical (IHC) staining in cervical cancer cell lines and cancer tissues with paired adjacent noncancerous cervical tissues. The expression of flotillin-1 protein was detected by IHC staining in a large cohort of 308 paraffin-embedded cervical cancer tissues. Ectopic expression and the short hairpin RNA interference approach were employed to determine the role of flotillin-1 in cervical cancer cell metastasis and the possible mechanism involved. Flotillin-1 expression protein and mRNA were significantly upregulated in cervical cancer cell lines and cancer tissues; elevated expression of flotillin-1 protein in early-stage cervical cancer was significantly associated with pelvic lymph node metastasis (P < 0.001), and was an independent predictive factor of poor overall survival. Moreover, flotillin-1 up- and downregulation remarkably affected cervical cancer cell motility and invasion, respectively, through epithelial-mesenchymal transition (EMT) regulated by the Wnt/β-catenin and nuclear factor-κB (NF-κB) pathways. Our results suggest that flotillin-1 facilitates cervical cancer cell metastasis through Wnt/β-catenin and NF-κB pathway-regulated EMT and that the flotillin-1 expression profile serves not only as novel predictor of pelvic lymph node metastasis, but also as neoteric risk factor for patients with early-stage cervical cancer.

Keywords: EMT; Flotillin-1; Wnt/β-catenin and NF-κB pathways; cervical cancer; lymph node metastasis; prognosis.

Figure 1

Expression of flotillin-1 protein and mRNA in cells and tissues. A. Expression of flotillin-1 protein (left) and mRNA (right) in hKC cells and HeLa, Ca Ski, C33A, and SiHa cervical cancer cells. B. Expression of flotillin-1 protein (left) and mRNA (right) in five paired cervical cancer (T) and adjacent noncancerous cervical tissues (ANT). Expression levels were normalized by GAPDH. Error bars represent SD calculated from three parallel experiments. C. IHC assay of flotillin-1 protein expression in five paired cervical cancer (T) and adjacent noncancerous cervical tissues (ANT).

Figure 2

IHC assay of flotillin-1 protein expression in 308 paraffin-embedded cervical cancer tissues. A, B. Flotillin-1 expression was not detectable in cervical cancer tissues (this patient also did not present lymph node metastasis). C, D. Representative images of weak flotillin-1 staining in cervical cancer tissues. E, F. Representative images of moderate flotillin-1 staining in cervical cancer tissues. G, H. Representative images of strong flotillin-1 staining in cervical cancer tissues. I, J. Representative images of moderate flotillin-1 staining in cervical cancer tissues without lymph node metastasis. K, L. Representative images of strong flotillin-1 staining in cervical cancer tissues with lymph node metastasis. M. Average IRS of flotillin-1 in patients with and without lymph node metastasis. Error bars represent SD; p-value was calculated by Spearman correlation analysis.

Figure 3

Kaplan-Meier curves with univariate analyses (log-rank) in cervical cancer patients with low versus high flotillin-1 expression. A. OS curves of all patients. B. OS curves of patients with squamous cell carcinoma; C. OS curves of patients without lymph node metastasis. D. OS curves of patients without recurrence.

Figure 4

Flotillin-1 promotes cervical cancer cell motility and invasion through EMT. A. Invasion ability induced by FBS as analyzed by Transwell migration assay (× 200, *P < 0.001). B. Motility as measured by testing the wound closure rate at 0 and 36 h (× 200, *P < 0.001)). C. Western blotting analysis of the expression of flotillin-1 and EMT markers. Vector: Cells transfected with vector; OV: Cells transfected with flotillin-1 cDNA; RNAi-vector: cells transfected with scramble shRNA; RNAi#1/RNAi#2: cells transfected with two human shRNA sequences to repress flotillin-1.

Figure 5

Flotillin-1 activates the NF-κB and Wnt/β-catenin pathways in cervical cancer cells. A. Luciferase reporter activities analyzed in cells according to the TOP-flash versus FOP-flash intensity scale. B. Real-time PCR indicates an apparent overlap between Wnt/β-catenin-dependent gene expression and flotillin-1-regulated gene expression. Pseudo-color represents the intensity scale of flotillin-1 shRNA versus RNAi vector or flotillin-1 cDNA vs. Vector, generated by log₂ transformation. C. NF-κB luciferase reporter activity. D. Real-time PCR indicates an apparent overlap between NF-κB-dependent gene expression and flotillin-1-regulated gene expression. Pseudo-color represents the intensity scale of flotillin-1 cDNA vs. Vector or flotillin-1 shRNA versus RNAi-vector, generated by log₂ transformation. Vector: cells transfected with vector; OV: cells transfected with flotillin-1 cDNA; RNAi-vector/Scramble: cells transfected with scramble shRNA; RNAi#1/RNAi#2/Ri: cells transfected with two human shRNA sequences to repress flotillin-1.