Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2

Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients.Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR.Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours.Akt/protein kinase B (PKB) is a serine/threonine kinase that is involved in mediating various biological responses, such as inhibition of apoptosis and stimulation of cell proliferation (for review [1,2]). Three mammalian isoforms are currently known [1]: Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ. Akt1 was first discovered as a cellular homologue of the viral oncogene v-Akt, which causes leukaemia in mice [3] and is the predominant isoform in most tissues. High expression of Akt2 has been observed in insulin-responsive tissues, whereas Akt3 has been shown to be predominantly expressed in brain and testis [2].Phosphoinositol-3-phosphate (PIP3) is a product of phosphoinositol 3-kinase enzymatic activity and has been shown to be a prerequisite lipid modulator of Akt activity [4]. PIP3 has been described as a downstream component of a wide range of receptors, including the c-Met receptor [5], the epidermal growth factor receptor family [6], fibroblast growth factor receptor [7], insulin growth factor receptor [8] and platelet-derived