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1.  Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor 
Oncogene  2012;32(6):759-767.
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 (SFK), and nuclear EGFR played 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 up-regulation of the SFK members Yes and Lyn 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 up-regulation 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.
doi:10.1038/onc.2012.90
PMCID: PMC3381861  PMID: 22430206
nuclear EGFR; SFK; Yes; Lyn
2.  Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab 
Oncogene  2010;30(5):561-574.
KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux®) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, sprycel®) a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src Family Kinases. We analyzed 16 CRC lines for: 1) KRAS mutation status, 2) dependence on mutant KRAS signaling, 3) expression level of EGFR and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo, and HCT116) cell lines, and two KRAS wild type cell lines (SW48 and CaCo2). In vitro, using Poly-D-Lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab whereas parental controls showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater anti-proliferative effect on KRAS mutant line as compared to either agent alone both in vitro and in vivo. To investigate potential mechanisms for this anti-proliferative response in the combinatorial therapy we performed Human Phospho-kinase Antibody Array analysis measuring the relative phosphorylation levels of phosphorylation of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the classical MAPK pathway, AKT/mTOR pathway and the family of STAT transcription factors when compared to the untreated control or monotherapy treatments. Next we analyzed tumor growth with cetuximab, dasatinib or the combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an anti-tumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation as measured by Ki67 and higher rates of apoptosis as measured by TUNEL. The data presented herein indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key-signaling pathways. Further, these results suggest that signaling via the EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. This data strengthen the rationale for clinical trials in this genetic setting combining cetuximab and dasatinib.
doi:10.1038/onc.2010.430
PMCID: PMC3025039  PMID: 20956938
Cetuximab; resistance; KRAS mutation; dasatinib; EGFR; SRC; colorectal cancer

Results 1-2 (2)