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Published on: March 26, 2014
Insulin-like growth factor 1 receptor stabilizes the ETV6-NTRK3 chimeric oncoprotein by blocking its
Cristina E Tognon1, Bo Rafn1, Naniye Malli Cetinbas1
1From the Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.
Abstract:
Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.
Insights
Blocking insulin-like growth factor 1 receptor (IGF1R) triggers rapid degradation of the EN oncoprotein. This occurs via ubiquitylation and proteasomal pathways, offering a new therapeutic strategy for cancers driven by chimeric tyrosine kinases.
Area of Science:
- Molecular Oncology
- Cancer Cell Signaling
Background:
- Many oncogenes, including chimeric oncoproteins like ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN), rely on the insulin-like growth factor 1 receptor (IGF1R) for cell transformation.
- The precise mechanisms by which IGF1R mediates EN activation and transformation remain incompletely understood.
Purpose of the Study:
- To elucidate the mechanistic details of IGF1R-mediated EN activation and identify potential therapeutic vulnerabilities.
- To investigate the role of IGF1R in regulating EN stability and degradation.
Main Methods:
- Utilized in vitro and in vivo models, including fibroblast, breast epithelial, and human leukemia cell lines (MO91).
- Employed IGF1R/insulin receptor (INSR) inhibitors and assessed EN tyrosine phosphorylation and degradation.
- Utilized Stable Isotope Labeling with Amino acids in Cell Culture (SILAC)-based MS to identify interacting proteins, including E3 ligase RING-finger protein 123 (Rnf123/KPC1).
- Performed in vitro ubiquitylation assays and knockdown/overexpression studies of KPC1/Rnf123.
Main Results:
- IGF1R stimulation enhances EN tyrosine phosphorylation; blocking IGF1R or reducing insulin receptor substrate 1/2 (IRS1/2) leads to rapid EN degradation.
- KPC1/Rnf123 was identified as an EN interactor upon IGF1R/INSR inhibitor treatment and mediates EN ubiquitylation and degradation.
- KPC1/Rnf123 overexpression decreased EN levels, while its knockdown conferred resistance to IGF1R inhibitor-induced degradation.
- IGF1R protects EN from KPC1/Rnf123-mediated proteasomal degradation.
Conclusions:
- IGF1R plays a critical role in protecting the EN oncoprotein from KPC1/Rnf123-mediated proteasomal degradation.
- Inhibition of IGF1R induces rapid, reversible, proteasome-dependent ubiquitylation and degradation of the EN oncoprotein.
- Targeting the IGF1R axis represents a potential therapeutic strategy for cancers harboring oncogenic chimeric tyrosine kinases like EN.
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