Immunohistochemical assessment of phosphorylated mTORC1-pathway proteins in human brain tumors

Patrick N. Harter, Lukas Jennewein, Peter Baumgarten, Elena Ilina, Michael C. Burger, Anna Luisa Thiepold, Julia Tichy, Martin Zörnig, Christian Senft, Joachim P. Steinbach, Michel Mittelbronn, Michael W. Ronellenfitsch

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10 Citations (Scopus)

Abstract

Background: Current pathological diagnostics include the analysis of (epi-)genetic alterations as well as oncogenic pathways. Deregulated mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated in a variety of cancers including malignant gliomas and is considered a promising target in cancer treatment. Monitoring of mTORC1 activity before and during inhibitor therapy is essential. The aim of our study is to provide a recommendation and report on pitfalls in the use of phospho-specific antibodies against mTORC1-targets phospho-RPS6 (Ser235/236; Ser240/244) and phospho-4EBP1 (Thr37/46) in formalin fixed, paraffin embedded material. Methods and Findings: Primary, established cell lines and brain tumor tissue from routine diagnostics were assessed by immunocyto-, immunohistochemistry, immunofluorescent stainings and immunoblotting. For validation of results, immunoblotting experiments were performed. mTORC-pathway activation was pharmacologically inhibited by torin2 and rapamycin. Torin2 treatment led to a strong reduction of signal intensity and frequency of all tested antibodies. In contrast phospho-4EBP1 did not show considerable reduction in staining intensity after rapamycin treatment, while immunocytochemistry with both phospho-RPS6-specific antibodies showed a reduced signal compared to controls. Staining intensity of both phospho-RPS6-specific antibodies did not show considerable decrease in stability in a timeline from 0-230 minutes without tissue fixation, however we observed a strong decrease of staining intensity in phospho-4EBP1 after 30 minutes. Detection of phospho-signals was strongly dependent on tissue size and fixation gradient. mTORC1-signaling was significantly induced in glioblastomas although not restricted to cancer cells but also detectable in nonneoplastic cells. Conclusion: Here we provide a recommendation for phospho-specific immunohistochemistry for patientorientated therapy decisions and monitoring treatment response.

Original languageEnglish
Article numbere0127123
JournalPLoS ONE
Volume10
Issue number5
DOIs
Publication statusPublished - 19 May 2015
Externally publishedYes

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