Abstract
The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (CtxR) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFKs), and nuclear EGFR had a role in resistance to cetuximab. To better understand SFK-mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated upregulation of the SFK members Yes (v-Yes-1 yamaguchi sarcoma viral oncogene) and Lyn (v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog) in all CtxR clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in CtxR clones, but not in cetuximab-sensitive (CtxS) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR-expressing CtxS parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all CtxR clones exhibited upregulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101), and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101, which influences EGFR nuclear translocation in this model of cetuximab resistance.
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Abbreviations
- BCRP:
-
breast cancer-resistance protein
- CRC:
-
colorectal cancer
- CtxR:
-
cetuximab-resistant
- CtxS:
-
cetuximab-sensitive
- DMSO:
-
dimethyl sulfoxide
- EGF:
-
epidermal growth factor
- EGFR:
-
epidermal growth factor receptor
- ER:
-
endoplasmic reticulum
- FBS:
-
fetal bovine serum
- FGFR:
-
fibroblast growth factor receptor
- HNSCC:
-
head and neck squamous cell carcinoma
- iNOS:
-
inducible nitric oxide synthase
- Lyn:
-
v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog
- mAb:
-
monoclonal antibody
- NSCLC:
-
non-small cell lung cancer
- PGFR:
-
platelet-derived growth factor
- PI3K:
-
phosphatidylinositol 3-kinase
- PKC:
-
protein kinase C
- PLCγ:
-
phospholipase C-gamma
- qPCR:
-
quantitative PCR
- RTK:
-
receptor tyrosine kinase
- SFK:
-
Src-family kinases
- STAT:
-
signal transducers and activators of transcription
- Yes:
-
v-Yes-1 yamaguchi sarcoma viral oncogene
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Acknowledgements
This project was supported, in part, by grant P30CA014520 from the National Cancer Institute, grant 1UL1RR025011 from the Clinical and Translational Science Award program of the National Center for Research Resources and the National Institutes of Health (DLW) by grant RSG-10-193-01-TBG from the American Cancer Society (DLW), and by NIH grant T32 GM08.1061-01A2 from Graduate Training in Cellular and Molecular Pathogenesis of Human Diseases (TMB).
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Iida, M., Brand, T., Campbell, D. et al. Yes and Lyn play a role in nuclear translocation of the epidermal growth factor receptor. Oncogene 32, 759–767 (2013). https://doi.org/10.1038/onc.2012.90
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DOI: https://doi.org/10.1038/onc.2012.90
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