Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells : Correlation with ErbB2 and p66 Shc expression

Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r = 0.91, p < 0.0002) and PY-ErbB2 levels (r = 0.89, p = 0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r = -0.86, p = 0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.