Protein phosphatase-2A associates with the cytoskeleton to maintain cell spreading and reduced motility of nonmetastatic Lewis lung carcinoma cells: the loss of this regulatory control in metastatic cells.

Abstract

Metastatic Lewis lung carcinoma (LLC-LN7) variants have previously been shown to have reduced levels of protein phosphatase-2A (PP-2A) activity as compared to the nonmetastatic LLC-C8 cells. The present study showed that inhibition of PP-2A in the nonmetastatic LLC-C8 cells caused a rapid change from a spread to a rounded morphology and increased their in vitro invasiveness through laminin. In contrast, the metastatic LLC-LN7 cells were rounded and invasive, which was not affected by inhibition of PP-2A. To determine whether these differences could be attributed to alterations in PP-2A association with the cytoskeleton, the extent of PP-2A colocalization with microtubules was tested. Immunostaining for tubulin showed prominent filamentous fibers in nonmetastatic LLC-C8 cells and small foci of PP-2A immunostaining along these microtubules. In contrast, the tubulin staining was diffuse throughout the metastatic LLC-LN7 cells and there was little evidence of association with PP-2A. Western blot analyses showed that this reduced level of PP-2A association with microtubules in metastatic LLC-LN7 cells was not due to differences in levels of the PP-2A subunits. Instead, it may be due to the reduced association of the subunits into the heterotrimeric form of the PP-2A holoenzyme. These studies show the importance of PP-2A in maintaining a spread morphology and in restricting invasiveness, and a loss of this regulatory control in metastatic cells. This loss of PP-2A regulatory control in metastatic cells may be due to a reduction in the trimeric form of the PP-2A holoenzyme.