| Description |
Introduction:The overexpression of HER-2 (c-erbB2/Neu) in breast cancer is associated with poor prognosis, tumor recurrence and shortened survival. The administration of the trastuzumabu, humanized monoclonal antibody against HER-2 receptor, significantly improves patient prognosis. However, in spite of these successful results, trastuzumab is effective only about 40% of cases. Several mechanisms contributing to resistance are proposed and strategies to overcome it are in developing, as well as searching for feasible biomarkers to predict the response of trastuzumab-based targeted therapy. PI3K/Akt/mTOR/S6K and Ras/MAPK signaling pathways are activated through HER-2 receptor and both play important role in tumor behaviour. Increasingly it becomes obvious that not only the activity but also the subcellular compartmentalization of activated protein kinase could play an important role. Methods: Our study included 76 women with verified HER-2 positive (IHC or/and FISH) metastatic breast cancer (MBC) who were treated with trastuzumab based palliative chemotherapy. Immunohistochemistry was performed on formalin fixed, paraffin embedded tissue sections with antibodies against Akt1, Akt2, phospho(p)Akt Ser473 and pAkt Thr308, PTEN, S6K, pS6K Ser235/236, MAPK, pMAPK Thr202/Tyr204. Except PTEN, the cytoplasmic and nuclear fractions were assessed separately. The expression was considered weak positive if 5%- 80% of tumor cells were stained with the antibody and strong positive if more than 80% of cells were positive. The sensitivity of seven breast cancer cell lines to paclitaxel, adriamycin and trastuzumab was evaluated using an MTT cell viability test and we used the western blotting analysis to confirm the subcellular compartmentalization of Akt2 and pAkt-S473 kinase in the cell lines. P-value was based on Gehan-Wilcoxons test or Chi-square test and P-values below 0,05 were considered significant. The Kaplan-Meier method was used to estimate time to progression (TTP). Results: Patients whose tumors showed high Akt2 expression (above 80% positive cells) accompanied with nuclear and cytoplasmic (n+c) positivity of: a) pAkt-S473, b) pAkt-T308, c) both pAkts (pAkt-S473/T308) exhibited improved TTP compared to those with any Akt2 expression, but negative for nuclear staining of any pAkt: a) TTP 13,1 vs 7,6 months; P=0,018, Hazard Ratio 2,09, CI95% 1,13-3,5; b) TTP 17,1 vs 7,6 months; P=0,007, Hazard Ratio 2,41, CI95% 1,25-4,0; c) TTP 13,1 vs 7,6 months; P=0,028, Hazard Ratio 1,9, CI95% 1,12-3,49. Of the remaing results, only S6K kinase had significant impact on TTP. Activation of S6K was associated with shorter TTP (see table). Significant differencies were found between the expression and subcellular localization of total Akt-2 and p-Akt-S473 and tumor cell line sensitivity to the drug tested (data not presented). Conclusions: We have confirmed that prediction of the response to trastuzumab treatment depends on the Akt kinase isoform, activity and compartmentalization. Patients, whose tumors had high level of total Akt2 and concurrent nuclear and cytoplasmic presence of the activated forms of Akt kinase (pAkt), have greater benefit from trastuzumab based therapy compared to those with any expression of total Akt2, but negative for nuclear staining of any pAkt. In contrast, activated S6K was associated with worse response to therapy.
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