Regulation Of ID1 Following Chemotherapy In GBM Is Dependent On Src/TGF-_ Signaling Pathway

Megan WU, Hospital for Sick Children, Canada
SACHDEVA R. 1, 2 , CELEBRE A. 1, , DAS S. 1, 2

1 Brain Tumor Research Center, Sick Kids Hospital, Toronto, Canada
2 Department of Neurosurgery, St. Michaelís Hospital, Toronto, Canada

The inhibitor of DNA binding (ID) family of proteins are present at high levels in stem and progenitor cells. They are known to control the timing of cell fate determination and differentiation in stem and progenitor cells during normal development and adult fate specification. ID genes are frequently disregulated in many types of cancers. ID1 has been implicated as a key regulator of cell phenotype in Glioblastoma multiforme (GBM).
In this study, we aimed to investigate the role of ID1 as a mediator of GBM chemo-resistance. Immunoprecipitation (IP) of ID1 protein complex showed ID1 protein is stabilized by de-ubiquitination as a result of TMZ treatment. We further analyzed ID1 protein stability by using the Cycloheximide chase assay to determine its half-life. Thereafter we developed a glioblastoma stem cell line expressing ID1 with a Flag-tag. IP of ID1-Flag protein complex followed by mass spectrometry demonstrated a decrease in the E3 ubiquitin-protein ligase TRIM21 to undetectable levels as a result of TMZ treatment. Furthermore, we identified the proto-oncogene tyrosine-protein kinase Src as a TMZ-induced binding partner of ID1. Western blot analysis along with IP of Src protein complex resulted in correlation of ID1/Src with pSmad2/3 (TGF-β signaling pathway marker) and Smad4 protiens. Using the TGF-β inhibitor LY-36497 and BMP inhibitor Noggin, we determined that the interaction of ID1 with Src, pSmad2/3 and pSmad 1/5, is dependent on  activation of the TGF-β signaling pathway.
Together, our results suggest that impaired ubiquitination and stabilization induces ID1 to partner with Src and elements of the TGF-β signaling pathway upon TMZ treatment. This interaction promotes tumor cell proliferation and cell self-renewal, and drives tumor recurrence. We propose that promoting ID1 degradation will provide therapeutic potential for GBM patients.