Overexpression of cFLIP in head and neck squamous cell carcinoma and its clinicopathologic correlations

Abstract

Purpose: The aim of this study was to determine the expression of cellular FLICE-like inhibitory protein (cFLIP) in head and neck squamous cell carcinoma (HNSCC) and revealed its possible correlation to Fas protein and tumour clinical parameters.

Methods: The expression of cFLIP was analysed in 58 HNSCC samples and 30 morphologically normal tissues adjacent to the carcinomas using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Furthermore, its possible correlation to the expression of Fas protein and tumour clinicopathologic parameters were discussed.

Results: Cellular FLICE-like inhibitory protein was demonstrated to be up regulated in most HNSCC than in normal tissues by immunohistochemistry (p<0.01). Although the mRNA levels of both isoforms of cFLIP, long form (cFLIP(L)) and short form (cFLIP(S)), in HNSCC were higher than those in normal tissues (p<0.01), only cFLIP(L) protein could be detected by western blot. Furthermore, the expression of cFLIP(L) protein was significantly associated with tumour clinical stage (p<0.01) and lymph node metastasis (p=0.01). Since all of the tumours with Fas immunostaining also express cFLIP protein, there was no significant correlation between them (p>0.05).

Conclusions: Overexpression of cFLIP(L) is a frequent event in HNSCC and HNSCC cells in vivo may need it to evade apoptosis mediated by Fas or other receptors, which might contribute to tumour development and progression.

Fig. 1

Immunohistochemical staining of cFLIP and Fas protein in normal tissue and HNSCC: a cFLIP staining in normal tissue; b Weak expression of cFLIP in HNSCC; c, d Medium and high cFLIP protein expression in HNSCC without and with lymph node metastasis, respectively; e, f Negative control for cFLIP and Fas staining, respectively; g Fas protein in normal tissue; h Fas protein in HNSCC with lymph node metastasis. Original magnification, ×400; Scale bars = 20 μm

Fig. 2

Expression of cFLIP_L and cFLIP_S mRNA by RT-PCR. a Ethidium bromide stained agarose gel showing the RT-PCR products of three representative cases; N normal tissues, T tumour samples. b Densitometry analysis of cFLIP_L and cFLIP_S mRNA expression relative to β-actin in HNSCC compared with normal tissues. Each value is the mean ± SD (n = 30, * p < 0.01 compared to normal tissues )

Fig. 3

Expression of cFLIP_L and cFLIP_S protein by western blot and the correlation between cFLIP_L mRNA and cFLIP protein. a Western analyses using anti-cFLIP_S/L antibody only detected cFLIP_L protein in normal tissues and HNSCC samples. b Significant correlation was found between cFLIP_L/β-actin ratio and cFLIP protein weighted scores in HNSCC samples (filled diamond). not in normal tissues (open triangle)