Signaling from the Epidermal Growth Factor Receptor (EGFR) family is commonly dysregulated in various cancer types by amplification or activating mutations. For example, HER2 amplification occurs in 15-20% of breast cancers, and is a well-defined driver of tumorigenesis. EGFR is amplified in approximately 2% of breast cancer patients with unknown significance. It is amplified more frequently in TNBC (5.7-6.7%) and ER-/HER2+ cancers (5.8-6.5%). Basal (4.3-8.1%) and HER2 enriched (3.9-45.4%) molecular subtypes show the highest amplification than other molecular subtypes. EGFR amplification leads to shorter overall survival among breast cancer patients compared to those without amplified EGFR. Approximately 71% of EGFR amplified tumors have activated PI3K signaling, indicating a possible dual-inhibition therapeutic approach targeting both EGFR and PI3K. In order to study the role of EGFR amplification and PI3K aberrations in TNBC, we used the TNBC cell lines BT20 (amplified EGFR, PIK3CA activating mutation), MDA-MB-468 (amplified EGFR, PTEN deletion), and MDA-MB-231 (no amplification of EGFR or PI3K pathway mutations). EGFR amplification in both MDA-MB-468 and BT20 cell lines was confirmed by western blotting and Fluorescent in situ Hybridization; MDA-MB-231 did not have EGFR amplification. Activating mutations in both alleles of PIK3CA in BT20 was confirmed by sequencing. Loss of PTEN in MDA-MB-468 was confirmed by immunoblotting. Inhibition of EGFR with erlotinib resulted in decreased ERK signaling in BT20 and MDA-MB-468, but not in MDA-MB-231. Inhibition of the PI3K with Ly294002 or BKM120 decreased activation of AKT and mTOR signaling in BT20 and MDA-MB-468, but not in MDA-MB-231. The combination of erlotinib and PI3K inhibitors more dramatically reduced mTOR and AKT signaling in the BT20 and MDA-MB-468 cells. Combination of erlotinib and PI3K inhibitors reduced cell viability in these 3 cell lines, but inhibition was greater in MDA-MB-468 and BT20 compared to MDA-MB-231, and only MDA-MB-468 and BT20 cells had an increased fraction of apoptotic cells, signifying that combination therapy is most effective in cells with amplified EGFR and aberrant PI3K. It has been previously shown that HER2/HER3 activation plays a role in self renewal of tumor initiating cells. We demonstrate that the combination of erlotinib and BKM120 reduced the cancer stem cell population as measured by an aldeflour assay and by mammosphere formation. In summary, these data demonstrate that EGFR amplification in TNBC drives cell viability through EGFR and PI3K signaling, and EGFR/PI3K inhibitor combination causes apoptosis as well as reduction in the cancer stem cell population. We present evidence therefore that dual inhibition of EGFR/PI3K presents as a potential targetable pathway in TNBC with EGFR amplification and aberrant PI3K signaling.
Citation Format: David John Wisniewski, Donna Voeller, Stanley Lipkowitz. Combined inhibition of EGFR and PI3Kinase signaling in EGFR amplified triple negative breast cancer cells induces apoptosis and reduces cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1977.