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Epidermal growth factor is a biomarker for poor cetuximab response in tongue cancer cells
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science.
2016 (English)In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 45, no 1, 9-16 p.Article in journal (Refereed) Published
Abstract [en]

Background: Epidermal growth factor receptor (EGFR) is a target for treatment in tongue cancer. Here, EGFR ligands were evaluated for their potential uses as predictive biomarkers of cetuximab treatment response.

Methods: In three tongue cancer cell lines the influences of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumour cell proliferation and cetuximab response were evaluated by the addition of recombinant human (rh) proteins or the siRNA-mediated downregulation of endogenous ligand production.

Results: EGF or AR downregulation suppressed the proliferation of all investigated cell lines. Furthermore, all cell lines displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing resulted in an improved cetuximab response in one cell line.

Conclusions: Our data suggest that EGF and AR are critical components of the EGFR signalling network required for full proliferative potential. Moreover, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2016. Vol. 45, no 1, 9-16 p.
Keyword [en]
Head and neck cancer; Tongue cancer; Erbitux; EGFR ligands; treatment response
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:liu:diva-100675DOI: 10.1111/jop.12310ISI: 000369990100003OAI: oai:DiVA.org:liu-100675DiVA: diva2:663300
Note

Funding agencies: Foundation of Ake Wiberg; Swedish Cancer Society [2008/552, 2010/545]; County Council of Ostergotland; Linkoping University Hospital; Foundation of Magnus Bergvall; Cancer Foundation of Ostergotland

Vid tiden för disputation förelåg publikationen endast som manuskript

Available from: 2013-11-11 Created: 2013-11-11 Last updated: 2017-05-03
In thesis
1. Identification of Tumor Cell- and Stroma Derived Biomarkers of Treatment Response in Head and Neck Cancer
Open this publication in new window or tab >>Identification of Tumor Cell- and Stroma Derived Biomarkers of Treatment Response in Head and Neck Cancer
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck squamous cell carcinoma (HNSCC) poses a major health problem in the world with approximately 600 000 new cases yearly. Treatment resistance is a major problem within this patient group and despite advances in treatment strategies the overall survival rate has unfortunately not increased.

One of the major components of the tumor microenvironment is the cancer associated fibroblasts (CAFs) which can modulate the treatment sensitivity, tumor growth, and the invasive potential of tumor cells.

The aim of this thesis was to identify predictive markers for treatment response in HNSCC and to study the crosstalk between tumor cells and CAFs that may underlie treatment resistance.

In paper I, we identified gene expression differences between one cisplatin sensitive cell line and two cisplatin resistant cell lines, by microarray analysis, and found that a high expression of matrix metalloproteinase (MMP) -7 was associated with resistance to cisplatin. In paper II, the epidermal growth factor (EGF) receptor ligands EGF, amphiregulin, and epiregulin were evaluated regarding their potential use as predictive biomarkers for cetuximab treatment response in tongue cancer cell lines and it was shown that EGF may serve as a marker for poor cetuximab response. In paper III and IV, we investigated the influence of CAFs on the proliferation, migration, gene expression, and cetuximab response of tumor cells. It was found that CAFs induced resistance to cetuximab in a MMP-dependent manner. In addition, a microarray analysis, comparing tumor cells co-cultured with CAFs and tumor cells cultured alone, revealed that CAFs induced multiple gene expression changes in tumor cells some of which are related to epithelial to mesenchymal transition. Some of these changes were found to be dependent on cell-cell contact.

Taken together, we here suggest MMP-7 and EGF to be predictive markers of cisplatin and cetuximab response, respectively. We also show that CAFs protect HNSCC cells from cetuximab treatment; however, the factor responsible for the protective effect is yet to be discovered.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. 89 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1382
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-100734 (URN)10.3384/diss.diva-100734 (DOI)978-91-7519-492-9 (ISBN)
Public defence
2013-11-29, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2013-11-11 Created: 2013-11-11 Last updated: 2013-11-11Bibliographically approved
2. Understanding the Role of EGFR in the Treatment of Head and Neck Squamous Cell Carcinoma
Open this publication in new window or tab >>Understanding the Role of EGFR in the Treatment of Head and Neck Squamous Cell Carcinoma
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck squamous cell carcinoma (HNSCC) originates from the epithelial lining of the upper aerodigestive tract. It accounts for over 90 % of the malignancies found in the head and neck region. 600,000 new cases of HNSCC occur each year worldwide. Apart from causing painful lesions, HNSCC directly impacts the patient’s fundamental functions such as breathing and eating and also can disrupt the patient’s senses such as smell, taste, speech and even vision. Most cases of HNSCC require a combination of different treatments such as surgery, chemotherapy (primarily cisplatin based), and radiotherapy. Treatment decisions are largely based on the size of the tumor, the involvement of local lymph nodes, and distant spread. Treatment resistance and local recurrence are significant problems and to date no form of clinical treatment sensitivity  prediction is available.

A majority of HNSCC tumors overexpresses the epidermal growth factor receptor (EGFR). This receptor is involved in proliferation and DNA repair and is the target of a monoclonal antibody named cetuximab that selectively binds and inhibits EGFR. It is the only targeted therapy available to HNSCC patients and reserved for late stage patients in  Sweden.

Numerous investigators have searched for predictive markers and we hypothesized that since HNSCC is a very heterogeneous disease a single factor would not be able to predict the treatment outcome. In paper I we explore a panel of predictive factors using a point system, called the number of negative points (NNP), in which we could combine both proteins and genetic variations in an attempt to find a set of markers that could predict the intrinsic cisplatin sensitivity (ICS). The expression level of EGFR, Hsp70, Bax, Bcl-XL, survivin, and COX-2 was determined in 39 HNSCC cell lines. Moreover, the p53, MDM2, FGFR4, XPC, XPD, XRCC1, and XRCC3 genes were analyzed for the presence of specific single nucleotide polymorphisms (SNPs). Pearson’s correlation tests showed that EGFR was the only protein that alone correlated to ICS (r=0.388, P=0.015). The strongest correlation to ICS was found when combining SNPs in XRCC3 and XPD with the expression of EGFR, Hsp70, Bax, and Bcl-2 using the NNP system (r=0.614 P≤0.001).

In paper II we assess the intrinsic radiosensitivity (IR), the ICS, and the intrinsic cetuximab sensitivity (ICmabS) as well as their combinations in 25 HNSCC cell lines established from HNSCC biopsies taken at the Department of Otorhinolaryngology and Head and Neck Surgery at Linköping University Hospital. Furthermore we investigate the EGFR status (consisting of EGFR gene copy number, EGFR mRNA, EGFR protein, pEGFR), pAkt and mRNA levels of the seven known EGFR ligands. No correlation was found between the different treatment sensitivities. Cetuximab treatment response was significantly correlated to epiregulin (EREG) mRNA expression (r=-0.408, P=0.043). Cetuximab resistant cell lines tended to have low levels of pEGFR (P=0.13) while resistant cell lines had a significantly lower expression of EGFR protein (P=0.04) and tended to have decreased levels of pAkt (P=0.13) and amphiregulin (AREG) mRNA (p=0.18).

In paper III the functional importance of EGFR ligands in relation to proliferation and cetuximab sensitivity was investigated. Here we tried to diminish the tumor heterogeneity by selecting three cell lines that are derived from the same anatomical location but display different ICmabS. Signaling through the EGFR was stimulated with recombinant EGF, AREG or EREG or reduced by siRNA-mediated silencing of the aforementioned EGFR ligands. EGF downregulation suppressed the proliferation of all investigated tumor cell lines whereas the response to an increased level of EGF differed between EGFR overexpressing and EGFR non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of EGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AREG and EREG were inconclusive.

In paper IV we wanted to validate in vitro drug sensitivity testing of HNSCC cell lines in an in vivo xenograft model, and to identify treatment-induced changes in the EGFR signaling pathway that could be used as markers for cetuximab treatment response. In vitro ICmabS for the HNSCC cell lines UT-SCC-14 and UT-SCC-45 was established using a crystal violet assay. In order to determine the corresponding in vivo sensitivity, UT-SCC-14 and UT-SCC-45 xenografts were generated in female BALB/c (nu/nu) nude mice. Mice were given three injections of intraperitoneal cetuximab or PBS and the tumor volume was recorded continuously. The expression of EGFR, pEGFR, pSrc, and Ki67 in the tumor tissue was investigated by immunohistochemistry. The in vitro sensitivity was reproduced in the in vivo model. Furthermore a clear reduction of EGFR, pEGFR, and pSrc after cetuximab treatment was noted in UT-SCC-14, the cetuximab sensitive cell line while the cetuximab resistant UT-SCC-45 showed a slight increase in EGFR, pEGFR and pSrc.

In conclusion, the EGFR ligand EGF is a potential predictive marker of poor cetuximab response and a possible treatment target. Moreover, treatment-induced downregulation of EGFR and pEGFR is associated with a good cetuximab response.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 74 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1432
National Category
Other Health Sciences
Identifiers
urn:nbn:se:liu:diva-113744 (URN)10.3384/diss.diva-113744 (DOI)978-91-7519-176-8 (ISBN)
Public defence
2015-03-06, Eken, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2015-01-29Bibliographically approved

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Ansell, AnnaJedlinski, AdamJohansson, Ann-CharlotteRoberg, Karin

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Division of Neuro and Inflammation ScienceFaculty of Medicine and Health SciencesDepartment of Otorhinolaryngology in LinköpingDepartment of Otorhinolaryngology in Linköping
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