Abstract
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 (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (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 KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines 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 mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor 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 (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.
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Abbreviations
- ABL:
-
v-abl Abelson murin leukemia viral oncogene homolog 1
- BCR:
-
breakpoint cluster region
- CRC:
-
colorectal cancer
- EGFR:
-
epidermal growth factor receptor
- eNOS:
-
endothelial nitric oxide synthase
- FAK:
-
focal adhesion kinase-1
- FDA:
-
Food and Drug Administration
- GSK α/β:
-
glycogen synthase kinase α/β
- HNSCC:
-
head and neck squamous cell carcinoma
- IgG:
-
immunoglobulin G
- IHC:
-
immunohistochemistry
- MAPK:
-
mitogen-activated protein kinase
- mCRC:
-
metastatic colorectal cancer
- MEK:
-
MAPK kinase
- MSK:
-
mitogen and stress-activated protein kinase
- mTOR:
-
mammalian target of rapamycin
- NSCLC:
-
non-small cell lung cancer
- PDL:
-
poly-D-lysine
- PDGFR:
-
platelet-derived growth factor receptor
- PI3K:
-
phosphatidylinositol 3-kinase
- PKC:
-
protein kinase C
- PLCγ:
-
phospholipase C-γ
- RSK:
-
ribosomal s6 kinase
- RTK:
-
receptor tyrosine kinase
- SFK:
-
Src-family kinase
- STAT:
-
signal transducer and activator of transcription
- TUNEL:
-
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
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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, National Institutes of Health (to DLW), by grant RSG-10-193-01-TBG from the American Cancer Society (to DLW) and T32 grant CA009614-17 Physician Scientist Training in Cancer Medicine from the National Institutes of Health (to EFD).
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DLW has held a sponsored research agreement with Bristol-Myers Squibb.
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Dunn, E., Iida, M., Myers, R. et al. Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab. Oncogene 30, 561–574 (2011). https://doi.org/10.1038/onc.2010.430
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DOI: https://doi.org/10.1038/onc.2010.430
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