P3-01-04: Differential Impact of Gefitinib and PLX4720 on Proliferation of MCF10A and Isogenic Lines as Measured with a Metastasis Expression Score.

Abstract

Abstract Background: We previously reported that a published “Metastasis Score” (MS) could be used to evaluate the effects of a PI3K inhibitor (GDC-0941) on mutated human isogenic breast cancer cell lines. MS, based on the expression of14 genes, has been shown to predict distant metastasis in ER(+), node (−), breast cancer. We were interested to determine the impact of gefitinib (EGFR inhibitor) and PLX-4720 (selective inhibitor of BRAF V600E) on MS to assess the applicability of this score to a broader class of targeted agents. In addition, given the cross-talk between metabolism and proliferation, we also profiled genes involved in glycolysis, fatty acid metabolism and oxidative phosphorylation. Methods: Parental MCF10A (WT for all genes) and isogenic lines of MCF10A harboring PI3K (H1047R), p53 null or KRAS(G12V) mutations were cultured overnight in DMEM:F12 media under identical conditions. Cells were then treated with gefitinib, PLX4720, or DMSO (vehicle control) and further incubated for 24hr. Expression analysis was performed by RT-PCR. Results: The MS of the PI3K (H1047R) and p53 null lines was higher than the parental line and lower for the KRAS (G12V) line. Treatment of parental, PI3K and KRAS cell lines with gefitinib resulted in dose-dependent decreases in MS, as reported for GDC-0941, with a higher dose required to inhibit growth of PI3K (H1047R) cells. Treatment of p53 null cells with gefitinib, however, had only a modest effect on MS. In contrast to gefitinib, MS increased with PLX-4720 treatment in all 4 lines; the greatest increase was observed in KRAS (G12V) cells. The expression of metabolic genes differed significantly depending on the oncogenic mutation harbored by the cell line. ACTA2, ACLY, RPIA, KHK, GLS2 were most highly expressed in p53 null but lowest in KRAS (G12V), while GLUT5, PFKFB4, ENO3, SLC27A1, PGK2, GLUT1, TKT were expressed most highly in KRAS (G12V) but lowest in PI3K(H1047R). Upon treatment with gefitinib or PLX-4720, genes that were downregulated in the mutated lines generally showed a dose-dependent increase; those that were upregulated relative to the parental, showed a dose-dependent decrease with treatment. Discussion: The decrease in MS of the parental, PI3K (H1047R) and KRAS (G12V) cell lines treated with gefitinib supports the impact of this EGFR inhibitor on cell proliferation. The modest effect on MS in p53 null cells supports the findings that sensitivity to gefitinib requires active p53 in order to induce apoptosis through a p53-dependent pathway. Increases in MS were observed in all 4 cell lines treated with PLX-4720, with the largest increase observed in KRAS (G12V). The enhanced proliferation of a BRAF WT cell line with a KRAS mutation is consistent with the Ras-dependent nature of this pathway. The metabolic genes showed diverse expression patterns that differed with different oncogenic mutations and likely reflect the multiple mechanisms controlling metabolism in cancer. An improved understanding of the expression of metabolic genes relative to proliferation in cell lines with various oncogenic mutations may provide additional insights into the dysregulation of these cellular processes and the possible role of anti-metabolite intervention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-04.