Quantitative Proteomics Reveals Fundamental Regulatory Differences in Oncogenic HRAS and Isocitrate Dehydrogenase (IDH1) Driven Astrocytoma.
Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our proteomic datasets are available for download and provide a comprehensive catalogue of alterations in protein abundance, phosphorylation, and histone modifications in oncogenic HRAS and IDH1 driven astrocytoma cells beyond the transcriptomic level.
From the ‡Department of Pharmaceutical Chemistry, University of California, San Francisco, 94158-2517 California.
Sophia
S
Doll
From the ‡Department of Pharmaceutical Chemistry, University of California, San Francisco, 94158-2517 California.
Anatoly
A
Urisman
From the ‡Department of Pharmaceutical Chemistry, University of California, San Francisco, 94158-2517 California.
Juan A
JA
Oses-Prieto
§Department of Protein Chemistry, Genentech Inc, South San Francisco, 94158-2517 California.
David
D
Arnott
From the ‡Department of Pharmaceutical Chemistry, University of California, San Francisco, 94158-2517 California; alb@cgl.ucsf.edu.
Alma L
AL
Burlingame
D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D042421:Histone Code; D006657:Histones / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis*; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
D006657:Histones; D007521:Isocitrate Dehydrogenase; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D016283:Proto-Oncogene Proteins p21(ras); D058570:TOR Serine-Threonine Kinases; C543588:IDH1 protein, human; C546842:MTOR protein, human; C501009:HRAS protein, human