Epidermal growth factor-induced modulation of cytokeratin expression levels influences the morphological phenotype of head and neck squamous cell carcinoma cells.

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9.

Head and neck squamous cell carcinomas (HNSCCs) are the most frequent malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis concludes that CSCs constitute the dangerous tumor cell population due to their ability of self-renewal and being associated with relapse of tumor disease, invasiveness and resistance to chemo(radio)therapy. The aim of this study was to look for CSC candidates and expression of MMP-9 that previously was implicated in HNSCC invasiveness. Immunohistochemical, immunofluorescence and Western blot analysis were performed on HNSCC tumor specimens using antibodies specific for MMP-9, CD44, ALDH1 and CK14. Gelatinolytic activity was assessed by zymography. Pearson correlation analysis was used for statistical comparison. Immunohistochemical analysis found CD44 and MMP-9 to co-localize in tumor cells at the invasive front. Western blot analysis demonstrated a significant correlation (p=0.0047) between CD44 and MMP-9 in the tested tissues. In addition gelatinolytic activity of HNSCC tissues was found to significantly correlate (p=0.0010) with MMP-9 expression. The CD44(+) invasive front of the tumor was also positive for ALDH1 and CK14, all of them being typically expressed by cells in the basal cell layer of normal stratified squamous epithelia that also harbors the epithelial stem cells. The observations point to a role of a MMP-9 positive basal-cell-like cell layer in the process of HNSCC invasiveness. This compartment likely contains CSCs since it is expressing the putative CSC markers CD44, ALDH1 and CK14. This cell layer therefore should be considered a major therapeutic target in the treatment of head and neck cancer.

The rotamase Pin1 is up-regulated, hypophosphorylated and required for cell cycle progression in head and neck squamous cell carcinomas.

The peptidyl-prolyl cis/trans isomerase Pin1 has been implicated in malignant transformation in multiple studies, however, little is known about its potential impact in head and neck cancer. This study evaluates the role of Pin1 in head and neck squamous cell carcinomas (HNSCCs). Pin1 expression and level of phosphorylation was evaluated by Western blot analysis and 2D-gel-electrophoresis. Pin1 was inhibited with juglone (5-hydroxy-1,4-naphthalenedione) or Pin1 specific siRNA and its influence on cell cycle checkpoint distribution was assessed by FACS analysis. Pin1 overexpression was found in HNSCC tissues and cell lines. 2D-gel-electrophoresis data pointed to Pin1 being hypophosphorylated in HNSCC cells which is consistent with overactivation of this rotamase. Inhibition of HNSCC cells with juglone or Pin1 siRNA induced the cell cycle inhibitor p21(WAF1/Cip1) with a concomitant reduction of cells in G2/M and an increased fraction of cells with fragmented DNA. Cell death did not correlate with significant levels of apoptosis in Pin1 depleted HNSCC cells. In summary, the data shows that Pin1 is overexpressed and hypophosphorylated in HNSCC, and that inhibition of Pin1 blocks cell cycle progression and triggers tumor cell death. Pin1 therefore could represent a new target for the development of improved HNSCC targeting drugs.