Constitutively Active Akt Reduces Lapatinib Sensitivity, while Kinase Dead Sample Clauses

Constitutively Active Akt Reduces Lapatinib Sensitivity, while Kinase Dead. Akt Improves Lapatinib Sensitivity Based on this initial data (Figure 3-2) showing that lapatinib sensitivity correlates with reduced p-Akt in BT474 and HCC1419 cells in contrast to JIMT-1 and HCC1954 cells, we hypothesized that Akt inhibition is important for achieving response to lapatinib. To test this hypothesis, HCC1419 cells, which are relatively sensitive to lapatinib, were transfected with constitutively active Akt double mutant T308D/S473D. Alternatively, JIMT-1 cells, which showed relatively low response to single-agent lapatinib, were transfected with dominant-negative kinase-dead Akt mutant K179A. Transfection was confirmed by blotting for p-Akt and total Akt (Figure 3-3A). In comparison to control transfectants, HCC1419 cells transfected with constitutively active Akt showed significantly reduced sensitivity to lapatinib (p=0.006) (Figure 3-3B). Sensitivity to the mTOR inhibitor rapamycin was also reduced in the presence of constitutively active Akt, although this effect did not reach statistical significance (p=0.08). In the presence of hyperactive Akt, combination mTOR inhibition plus lapatinib resulted in a significant reduction in cell viability versus either drug alone (p=0.01). These results suggest that combination lapatinib plus rapamycin may be an effective therapeutic strategy in tumors that show elevated PI3K signaling and low response to single-agent lapatinib. In a background of kinase dead Akt, JIMT-1 cells showed a statistically significant increase in lapatinib sensitivity (p=0.03) (Figure 3-3C). In contrast, sensitivity to rapamycin was not significantly affected. The combination of lapatinib plus rapamycin showed a trend towards reduced cell viability versus single-agent lapatinib, although this did not reach statistical significance (p=0.06). However, JIMT-1 cells did retain sensitivity to this drug combination in the presence of kinase dead Akt. Collectively, these results suggest that Akt activation status affects lapatinib sensitivity. Hyperactive Akt signaling significantly reduced response to lapatinib in HCC1419 cells, whereas kinase dead Akt significantly improved response to lapatinib in JIMT-1 cells. In addition, pharmacologic inhibition of mTOR significantly increased response to lapatinib in HCC1419 cells transfected with constitutively active Akt. Thus, this combination may be effective in HER2-overexpressing breast cancers that show poor response to lapatinib and high baseline Akt activity.