Phosphorylated EGFR and PI3K/Akt Signaling Kinases Are Expressed in Circulating Tumor Cells of Breast Cancer Patients

Introduction: The phosphoinositide-3 kinase (PI3K)/Akt pathway, operating downstream of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER)2, is implicated in cell migration and survival. EGFR and HER2 are expressed in circulating tumor cells, but the activation status of downstream signaling molecules has not yet been reported.

Methods: To investigate expression levels of EGFR, HER2, PI3K, and Akt in circulating tumor cells, we used peripheral blood mononuclear cells from 32 cytokeratin-19 mRNA-positive patients with early (n = 16) and metastatic (n = 16) breast cancer.Peripheral blood mononuclear cell cytospins were double stained with cytokeratin antibody along with one of the following: EGFR, phospho-EGFR, HER2, phospho-PI3K, or phospho-Akt antibodies.

Results: EGFR and HER2 were expressed in circulating tumor cells of 38% and 50% patients with early and 44% and 63% patients with metastatic disease, respectively. Interestingly, phospho-PI3K and phospho-Akt expression levels were similar at 88% (14 out of 16) and 81% (13 out of 16), respectively, in circulating tumor cells of patients with early and metastatic disease. Phospho-EGFR was observed in circulating tumor cells of two (33%) early and six (86%) metastatic EGFR-positive patients. Immunomagnetic separation of peripheral blood mononuclear cells, using EpCAM antibody, and subsequent double-staining experiments of circulating tumor cells showed that EGFR was co-expressed with HER2, phospho-Akt and phospho-PI3K kinases, indicating activation of the corresponding survival signaling pathway.

Conclusions: Our findings demonstrate that circulating tumor cells express receptors and activated signaling kinases of the EGFR/HER2/PI3K/Akt pathway, which could be used as targets for their effective elimination.