Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of Head and Neck cancers

Antisense (AS) induced down-regulation of uPAR in ACCS adenoid-cyctic carcinoma cells decreased the cellular adhesion and invasion on various extracellular matrices. Additionally, ACCS-AS cells showed an increased EGFR expression and other behavioral similarities to NA-SCC, a typical highly proliferative but less invasive squamous cell carcinoma (SCC) cell line of the head and neck. ACCS, ACCS-AS and NA-SCC cells were used to elucidate the relationships between uPAR down-regulation and EGFR inhibition. Results Tyrosine kinase inhibitor Gefitinib (IRESSA, ZD 1839) significantly reduced the chemotactic cell migration and adhesion. This was associated with reduced EGFR and ERK activation. In addition, anti-proliferative effect of gefitinib in uPAR down-regulated ACCS-AS was significantly higher than parental ACCS, to levels comparable to gefitinib-sensitive NA-SCC cells. This was evidenced by both reduced dosage and duration of treatment. Furthermore, time-lapse videography showed that treatment with gefitinib was also associated with cell rounding and loss of pseudopodia, mostly in ACCS-AS rather than parental ACCS cells. There were also evidences of formation and exocytosis of vacuole-like structures in ACCS-AS, as well as NA-SCC, but not in parental ACCS cells. Interestingly, immunocytochemistry showed that the exocytotic vacuoles actually contained de-activated EGFR. Conclusion Our results suggested that down-regulation of uPAR affected the fate of EGFR in high EGFR expressing cells. Furthermore, combining the uPAR down-regulation with EGFR inhibition showed a synergistic anti-tumor effect and might provide an alternative method to increase anti-proliferative effect of tyrosine kinase inhibitors with lower doses and duration to reduce their side effects during cancer control.