Protein phosphatases limit tumor motility

Abstract

Elevators of cAMP, such as prostaglandin E₂ (PGE₂), activate protein kinase A (PKA) and induce PKA‐stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC‐LN7). Non‐metastatic LLC (LLC‐C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP‐1/2A) within non‐metastatic LLC‐C8, the effects of the PP‐1/2A inhibitor okadaic acid on the migration and invasion by non‐metastatic LLC‐C8 cells was measured. Okadaic acid stimulated motility of non‐metastatic LLC‐C8 cells to a level that was comparable to that of metastatic LLC‐LN7 cells. PGE₂ further increased the motility of the non‐metastatic LLC‐C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose‐response studies with concentrations of okadaic acid that selectively inhibited PP‐2A or both PP‐2A and PP‐1 showed a progressive increase in migration of non‐metastatic LLC‐C8 cells, suggesting that both PP‐1 and PP‐2A limit their motility. By contrast, metastatic LLC‐LN7 cells were more motile than were non‐metastatic LLC‐C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP‐1/2A enzyme activities in the LLC variants showed more activity in non‐metastatic LLC‐C8 than in metastatic LLC‐LN7 cells. The identity of the PP whose activity was increased in the non‐metastatic LLC‐C8 was assessed by using okadaic acid, which selectively inhibits PP‐2A activity at low concentrations and PP‐1 and PP‐2A at high concentrations, and calyculin A, which inhibits PP‐2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP‐1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP‐1 and of PP‐2A in non‐metastatic LLC‐C8 cells, but the presence of PP‐1 and a reduction in PP‐2A in metastatic LLC‐LN7 cells. The sum of these data suggests that PKA‐stimulated motility is restricted both by PP‐1 and by PP‐2A in non‐metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.