Analyses of Akt and PDGF receptor expression and activation in human tumors

University dissertation from Stockholm : Karolinska Institutet, Department of Oncology-Pathology

Abstract: These studies aimed at developing methodology for detection of proteins involved in cancer and the evaluation of their prognostic significance in human tumors. Signal transduction profiling of tumors are becoming increasingly interesting based on the accumulating evidence of its prognostic and drug response-predicative value. Realization of the full potential of this information requires novel assay formats. We have explored the possibility to adapt the micro-fluidic Gyros platform for determination of Akt phosphorylation and the in situ PLA to investigate phosphorylation of PDGFR. The Gyros analyses clearly demonstrated that the drug-induced reduction in phosphorylation of Akt could be detected in the Gyrolab bioaffy device. A correlation between high pAkt/Akt expression and high Gleason score was seen when prostate tumors were analyzed. These preliminary findings encourage to further evaluation of the Gyrolab bioaffy device as a novel platform for analyses of tumor signal transduction. By in situ PLA we detected phosphorylated PDGFRbeta in cells stably over-expressing the human PDGFRbeta but not in cells transfected with the PDGF alpha-receptor and in immortalized human fibroblasts endogenously expressing the PDGFRbeta. We also demonstrated detection of tyrosine phosphorylated PDGFRbeta in tissue sections from fresh-frozen human scar tissue undergoing wound healing. PDGFR signaling has been shown in brain malignancies like glioma and medulloblastoma. We determined PDGF receptor expression in human choroid plexus tumors by immunohistochemistry and found that the majority expressed PDGFR. PDGFRbeta was more frequently expressed. Gene amplification was also more frequent for the PDGFRB. Next we also looked at grade I choroid plexus papillomas and grade II atypical choroid plexus papillomas and the activation status of both PDGF alpha and beta receptors by in situ PLA in formalin fixed paraffin embedded tumor tissue. Both the alpha- and beta-receptor was less expressed in the carcinomas compared to the two papillomas. In contrast the activated beta- receptor was found more frequently in the carcinomas whereas the activated alpha-receptor did not differ between the tumor types. In glioblastoma samples from patients of a randomized trial that compared hydroxyurea monotherapy with combination of hydroxyurea and imatinib, PDGFRalpha was expressed in 32% of the tumors. It was associated with male sex, young age at presentation, and loss of PTEN expression. PDGFRalpha expression status was independently associated with short survival in the entire series and associated with poor survival in the subset of patients treated with hydroxyurea monotherapy but not among those treated with the combination. PDGF receptor expression is common in the tumor stroma of common solid tumors. We found that PDGF alpha- and beta-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. In breast cancer, high stromal PDGFRbeta expression was significantly associated with high histopathological grade, ER negativity and high HER2 expression. High stromal PDGFRbeta expression was correlated with significantly shorter recurrence-free and breast cancer specific survival. The prognostic significance of stromal PDGF beta-receptor expression was particularly prominent in tumors from pre-menopausal women. These findings highlight the prognostic significance of stromal markers, and should be considered in ongoing clinical development of PDGF receptor inhibitors. In summary, we have developed generic methods for analyses of tumor relevant proteins; Gyrolab bioaffy and in situ PLA. Furthermore, analyses of human tumors have revealed clinically relevant previously unrecognized associations between PDGFR and survival in GBM and breast cancer.

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