PO-115 HER2-amplified tumours overcome the requirement for HER3

Abstract

ABSTRACT Introduction Amplification and overexpression of HER2 underlies the pathogenesis of a large subset of cancers including more than 20% of breast cancers. A considerable body of experimental evidence strongly suggests that HER2 is the oncogenic driver of these cancers, important in the genesis and progression of these tumours. Current evidence suggests that HER2 driven tumorigenesis requires HER3, the preferred heterodimerization partner of HER2. This is felt to be due to the unique ability of HER3 to activate PI3K/Akt pathway signalling, not directly accessible to HER2. We have been interested in developing deeper insights into the role of HER3 in HER2-amplified cancers. In particular, we have been interested in understanding why HER3 is required, whether the requirement is for the activation of PI3K, and whether its requirement is absolute or perhaps conditional or transient. Material and methods Using CRISPR-Cas9 technology, we knocked-out HER3 (HER3KO) in HER2-amplified HCC1569 cells and assessed the transforming and tumorigenic properties of HER3KO cells in vitro and in vivo. Co-immunoprecipitation assays in HER3-KO cells and stable doxycycline-inducible HER3 shRNA cell lines were performed to detect the binding of HER2 to the regulatory subunit of PI3K through direct and indirect mechanisms. Site-directed mutagenesis in the C-terminal of HER2 was used to identify the tyrosine involved in this interaction. Results and discussions By genetic elimination of HER3 or shRNA knockdown of HER3 in HER2-amplified cancer cells we find residual HER2-driven activation of PI3K/Akt pathway signalling that is driven by HER2 through direct and indirect mechanisms. Indirect mechanisms involve second messenger pathways including Ras or Grb2. Direct PI3K binding occurs through p-Tyr1139 which has a weak affinity for PI3K that becomes significant at very high expression and phosphorylation. Mutation of Y1139 impairs the tumorigenic competency of HER2. The total elimination of HER3 expression in HER2-amplified cancer cells significantly impairs tumorigenicity, but the impairment is transient and overcome with further increases in HER2 expression and phosphorylation with binding and activation of PI3K. Effective treatment of these cancers must focus on much more potent inhibitors of HER2. Conclusion This study shows for first time the intrinsic ability of HER2 to activate PI3K that becomes more relevant with increased HER2 expression, and can supplant the requirement for HER3 in the growth of HER2-amplified cancers.