Establishment and characterization of cetuximab resistant head and neck squamous cell carcinoma cell lines: focus on the contribution of the AP-1 transcription factor.
BACKGROUND
After an initial response to EGFR targeted therapy, secondary resistance almost invariably ensues, thereby limiting the clinical benefit of the drug. Hence, it has been recognized that the successful implementation of targeted therapy in the treatment of HNSCC cancer is very much dependent on predictive biomarkers for patient selection.


METHODS
We generated an in vitro model of acquired cetuximab resistance by chronically exposing three HNSCC cell lines to increasing cetuximab doses. Gene expression profiles of sensitive parental cells and resistant daughter cells were compared using microarray analysis. Growth inhibitory experiments were performed with an HB-EGF antibody and the MMP inhibitor, both in combination with cetuximab. Characteristics of EMT were analyzed using migration and invasion assays, immunofluorescent vimentin staining and qRT-PCR for several genes involved in this process. The function of the transcription factor AP-1 was investigated using qRT-PCR for several genes upregulated or downregulated in cetuximab resistant cells. Furthermore, anchorage-independent growth was investigated using the soft agar assay.


RESULTS
Gene expression profiling shows that cetuximab resistant cells upregulate several genes, including interleukin 8, the EGFR ligand HB-EGF and the metalloproteinase ADAM19. Cytotoxicity experiments with neutralizing HB-EGF antibody could not induce any growth inhibition, whereas an MMP inhibitor inhibited cell growth in cetuximab resistant cells. However, no synergetic effects combined with cetuximab could be observed. Cetuximab resistant cells showed traits of EMT, as witnessed by increased migratory potential, increased invasive potential, increased vimentine expression and increased expression of several genes involved in EMT. Furthermore, expression of upregulated genes could be repressed by the treatment with apigenin. The cetuximab resistant LICR-HN2 R10.3 cells tend to behave differently in cell culture, forming spheres. Therefore, soft agar assay was performed and showed more and larger colonies when challenged with cetuximab compared to PBS challenged cells.


CONCLUSIONS
In summary, our results indicate that increased expression of the ligand HB-EGF could contribute to resistance towards cetuximab in our cetuximab resistant HNSCC cells. Furthermore, several genes upregulated or downregulated in cetuximab resistant cells are under control of the AP-1 transcription factor. However, more studies are warranted to further unravel the role of AP-1 in cetuximab resistance.
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium.
Carolien
C
Boeckx
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium.
Lina
L
Blockx
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium ; Center for Medical Genetics, Department of Biomedical Sciences, University of Antwerp Belgium.
Ken Op
KO
de Beeck
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium.
Ridha
R
Limame
Center for Medical Genetics, Department of Biomedical Sciences, University of Antwerp Belgium.
Guy Van
GV
Camp
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium ; Department of Medical Oncology, Antwerp University Hospital Belgium.
Marc
M
Peeters
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium ; Department of Medical Oncology, Antwerp University Hospital Belgium.
Jan B
JB
Vermorken
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium ; Department of Medical Oncology, Antwerp University Hospital Belgium.
Pol
P
Specenier
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium.
An
A
Wouters
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium.
Marc
M
Baay
Center for Oncological Research (CORE) Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp Belgium.
Filip
F
Lardon
ADAM19; HB-EGF; Head and neck squamous cell carcinoma (HNSCC); cetuximab; interleukin 8; resistance; transcription factor AP-1