The tumor suppressor protein p27kip1 is deregulated in many aggressive human cancers.  A novel role for this protein was identified in previous work from our lab, which showed that cells lacking p27 can switch to amino acids as a carbon source when glucose levels are low. The goal of this project was to investigate the mechanism of the switch using the kinase inhibitor N-[2-(bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide, or H-89. This drug inhibits several protein kinases, one of which is Protein kinase A, which is known to play an important role in cellular metabolism. Cells with and without p27 were distributed in a 96 well plate and allowed to attach. They were then treated with different concentrations of H-89 for various times. Effects on ATP production were monitored using a luciferase-based assay. Effects of H-89 on nutrient usage in cells with and without p27 were determined by monitoring glucose levels in the media to measure how much glucose was consumed. H-89 appears to force cells lacking p27 to use glucose, possibly by inhibiting glutaminolysis. Consistent with this result, the p27 -/- cells were more sensitive to the glycolysis inhibitor 2 deoxy-glucose (2DG) after adding H-89. The ATP levels were also decreased, which was measured using CellTiterGlo<sup>TM</sup>, a luciferase-based assay that correlates the amount of light produced to ATP levels in the cell. Taken together these results suggest that protein kinase A activity, or that of another kinase targeted by H-89, is required for the metabolic switch in p27-/- cells. These observations will contribute to ongoing efforts to develop methodologies for specifically targeting cells lacking the p27 tumor suppressor, which may prove therapeutically valuable for treating aggressive cancers.

Protein kinase inhibitor H-89 blocks a metabolic switch in cells lacking the tumor suppressor p27kip1

255th National Meeting (2018)

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