Abstract
Problem: Increased migration and invasion are characteristics of more highly invasive and metastatic tumor cells. Prior studies in a murine tumor model have shown that metastatic cells have diminished activity of the serine/threonine protein phosphatase PP-2A compared to non-metastatic cells. In nonmetastatic cells, PP-2A co-localizes with the cytoskeleton, maintains cytoskeletal integrity, and restricts cellular motility, but these functions are lost in metastatic cells. The overall hypothesis of this study is that inhibition of PP-2A activity increases cell invasiveness by reducing the phosphotyrosine content of cytoskeletal-associated proteins FAK and paxillin, shifting them to a cytosolic location and creating a malignant phenotype.
Methods: Murine keratinocytes and SCC-VII-SF cells (murine HNSCC model) were grown in culture. PP-2A in these cells was chemically inhibited with 100 nM okadaic acid. Cells were immune stained for paxillin, FAK, and phosphotyrosine and then bound by secondary fluorescent antibody. Cells were then viewed using confocal microscopy.
Results: In normal keratinocytes, paxillin and FAK exist in a tyrosine phosphorylated state at focal adhesions. However, in malignant cells, these proteins assume a non-phosphorylated cytosolic location. When PP-2A is then inhibited in the normal cells, they exhibit rounding and a morphology similar to the malignant cells. In addition, in PP-2A inhibited cells, FAK and paxillin are both found predominantly in the cytosol, as seen in the malignant cells.
Conclusion: These studies show that PP-2A is important in maintaining cytoskeletal integrity and thus restricting cellular motility. Inhibition of PP-2A activity in normal cells reduces tyrosine phosphorylation of FAK and paxillin, stimulating a malignant phenotype.
Significance: The significance of these studies is that they help define how inhibition of PP-2A stimulates metastatic behavior in normal cells. The identification of the signaling pathways that regulate HNSCC cell motility will allow the use of pharmacological or genetic means to limit invasion and metastasis of HNSCC.
Support: Research conducted in association with the laboratory of Dr Rita Young, researcher at the Hines VA Hospital Department of Pathology and the Loyola University School of Medicine. No grants or other support was personally obtained by the presenter of this research.