Abstract A29: Different PIK3CA activation mutations can lead to distinct signaling mechanisms and EGFR inhibitor sensitivities in colorectal cancer cells

Abstract

Abstract Introduction: The PI3K/AKT pathway is activated in a wide variety of human cancers, and it has previously been shown that its dysregulation can lead to EGFR inhibitor resistance. In colorectal cancer, activating mutations of the PIK3CA gene have been identified in 13–17% of patients, and typically occur in either the helical domain (E542K or E545K) or the kinase domain (H1047R). Here, we used the colorectal cancer cell SW48, which has an EGFR G719S activating mutation, as well as the isogenic lines with knock-in of either PIK3CA E545K or H1047R mutation to study the differential effects of these two mutations on cell signaling, and the impact that each mutation has on the activity of the EGFR inhibitors erlotinib (Tarceva®), and GDC-0068, an AKT inhibitor. Results: The effect on cell growth of the erlotinib was measured in parental, PIK3CA E545K, and PIK3CA H1047R SW48 cells. Compared to the parental SW48 cells, knock-in of E545K led to erlotinib resistance, while knock-in of H1047R significantly sensitized cells to erlotinib. The GI50 of cells exposed to erlotinib was 2,400, 21,000, and 20 nM, respectively. The EGFR/ErB2 inhibitor lapatinib (Tykerb®) had GI50 values of >30,000, >30,000, and 18 nM, respectively. To explore the mechanism of resistance and sensitivity rendered by the two PIK3CA mutations, we measured the phosphorylation levels of 80 proteins that represent several key signaling pathways in these isogenic cells. Compared to parental SW48 cells, H1047R cells were found to have increased activation of EGFR and ErbB2, as well as PDGFRβ and Ron, receptor tyrosine kinases that have been shown to heterodimerize with members of the ErbB family. Furthermore, these cells showed increased activity of the MEK/ERK pathway. Treatment with erlotinib led to attenuation of EGFR, ErbB2, PDGFRβ, Ron, and MEK/ERK pathway signaling, providing a mechanistic basis for the increased effect of erlotinib in H1047R cells. While cells with both the E545K and H1047R mutations had increased activity of the PI3K/AKT pathway, a greater increase was seen in E545K cells. To determine the impact of each mutation on the cell's dependence on the PI3K/AKT pathway, we measured the effect of GDC-0068 on cell proliferation. SW48 parental, E545K, and H1047R cells had GI50 values of 2,500, 520, and 1,800 nM, respectively, highlighting the differential dependence on the PI3K/AKT pathway in cells with the E545K. While GDC-0068 inhibited downstream targets of PI3K/AKT in all three lines, increased suppression was seen in the E545K mutant. In addition, more substantial inhibition of targets that converge downstream of both PI3K/AKT and MEK/ERK was seen in E545K cells. Conclusion: Previous studies have shown that activating mutations of the PIK3CA gene lead to independence from receptor tyrosine kinase signaling, and insensitivity to EGFR inhibition. However, we have demonstrated that two distinct types of activating mutation of PIK3CA lead to differential sensitivity to EGFR inhibitors in CRC cells. PI3K with the H1047R mutation led to increased receptor tyrosine kinase activities and increased sensitivity to EGFR inhibitors, whereas PI3K with the E545K mutation caused hyperactivity of the PI3K/AKT pathway, which led to resistance to EGFR inhibitors and increased sensitivity to an Akt inhibitor. Our results, which added to potential explanations for the variability of clinical outcomes of EGFR inhibitors in patients with colorectal cancer, demonstrate the importance of informed patient selection.