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The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr 992 -EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors., (© 2020 Hartman et al.)

Amplification and overexpression of erbB2/neu proto-oncogene is observed in 20-30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 because of constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2ΔEx16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16-derived mammary tumors exhibit several unique features that distinguish this model from the conventional ErbB2 ones expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2-derived tumors that express luminal keratins, ErbB2ΔEx16-derived tumors exhibit high degree of intratumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibit distinct signaling and gene expression profiles that correlate with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal., Competing Interests: There are no conflicts of interest to declare.

One of the causes of breast cancer is overexpression of the human epidermal growth factor receptor 2 (HER2). Enhanced receptor autophosphorylation and resistance to activation-induced downregulation have been suggested as mechanisms for HER2-induced sustained signaling and cell transformation. However, the molecular mechanisms underlying these possibilities remain incompletely understood. In the current report, we present evidence that show that HER2 overexpression does not lead to receptor hyper-autophosphorylation, but alters patterns in a manner that favors receptor stability and sustained signaling. Specifically, HER2 overexpression blocks epidermal growth factor receptor (EGFR) tyrosine phosphorylation on Y1045 and Y1068, the known docking sites of c-Cbl and Grb2, respectively, whereas promoting phosphorylation on Y1173, the known docking site of the Gab adaptor proteins and phospholipase C gamma. Under these conditions, HER2 itself is phosphorylated on Y1221/1222, with no known role, and on Y1248 that corresponds to Y1173 of EGFR. Interestingly, suppressed EGFR autophosphorylation on the Grb2 and c-Cbl-binding sites correlated with receptor stability and sustained signaling, suggesting that HER2 accomplishes these tasks by altering autophosphorylation patterns. In conformity with these findings, mutation of the Grb2-binding site on EGFR (Y1068F-EGFR) conferred resistance to ligand-induced degradation, which in turn induced sustained signaling, and increased cell proliferation and transformation. These findings suggest that the Grb2-binding site on EGFR is redundant for signaling, but critical for receptor regulation. On the other hand, mutation of the putative Grb2-binding site in HER2 (Y1139) did not affect stability, signaling or transformation, suggesting that Y1139 in HER2 may not serve as a Grb2-binding site. In agreement with the role of EGFR in HER2 signaling, inhibition of EGFR expression reduced HER2-induced anchorage-independent growth and tumorigenesis. These results imply that complementing HER2-targeted therapies with anti-EGFR drugs may be beneficial in HER2-positive breast cancer.

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.