Differential Signaling by ErbB Receptor (HER) Dimers: Implications for Response to Anti-HER2 Therapies in Breast Cancer.

The ErbB family of receptor tyrosine kinases comprises of EGFR, HER2, HER3 and HER4. Ligand binding induces receptor homo- and heterodimerization and activation of downstream signaling. No ligand(s) have been identified for HER2. Recruitment of HER2 to ligand-activated co-receptors potentiates signaling by HER2-containing heterodimers. To identify differential signaling induced by HER2/HER3 and HER2/EGFR heterodimers and HER2 homodimers, we developed a cell system where HER2 dimerization can be conditionally regulated. A retroviral vector encoding HA-tagged full-length human HER2 fused to ligand-binding domain of FK506 binding protein (FKBP) in the C-terminus was stably transduced into MCF10A mammary epithelial cells. Treatment of MCF10A-HER2-FKBP-HA cells with the intracellular dimerizer AP1510 resulted in homodimerization and tyrosine phosphorylation of the HER2 chimera. The EGFR ligands EGF and TGFα and the HER3 ligand heregulin induced HER2/EGFR and HER2/HER3 heterodimers. Microarray analysis with RNA isolated from vehicle, AP1510, TGFα and heregulin treated cells identified unique transcriptional activities associated with HER2/HER2, HER2/HER3 and HER2/EGFR induced signaling. Ingenuity Pathway analysis of the microarray data showed that HER2 homodimers had the most significant effect on the “G 2 /M DNA damage checkpoint regulation pathway” whereas HER2/EGFR dimers had the most pronounced effect on the “pyrimidine metabolism pathway”. Active HER2/HER3 dimers affected the NF-κB signaling pathway the most.To determine if differential signaling by ErbB dimers have any implications for the response to anti-HER2 therapies, we treated MCF10A-HER2-FKBP-HA cells with AP1510, TGFα or heregulin in the presence or absence of trastuzumab, pertuzumab or lapatinib. In 3D Matrigel, AP1510-treated cells formed invasive acini while untreated cells failed to grow. EGF and TGFα and heregulin also stimulated acini growth in 3D. The anti-HER2 antibody trastuzumab completely inhibited AP1510-stimulated but not EGF, TGFα, or heregulin-stimulated growth. Lapatinib, a dual inhibitor of the HER2 and EGFR tyrosine kinases, blocked EGF, heregulin, and AP1510-induced growth. Pertuzumab, an antibody that blocks ligand-induced HER2 heterodimerization, inhibited heregulin-induced but not AP1510-induced growth. Reverse Phase Protein Array performed with lysates from cells treated with AP1510, TGFα or heregulin indicated that AP1510 failed to activate S473 p-AKT. This is a key enzyme in the PI3K pathway, which has been associated with resistance to HER2 inhbitors. Western blot analyses showed that AP1510 induced p-ERK whereas TGFα and Heregulin activated p-AKT. Binding assays with 125 I-labeled trastuzumab indicated that treatment with AP1510 enhanced binding of trastuzumab compared to untreated cells. This result suggests that HER2 homodimers present a conformation to which trastuzumab binds better. Taken together, these data suggest that 1) because of their inability to activate Akt, cells dependent on HER2-containing homodimers are less able to bypass trastuzumab action; and 2) high levels of HER2 homodimers in breast cancers with HER2 gene amplification may represent a biomarker predictive of response to trastuzumab. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 705.