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EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
2013 (English)In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 42, no 1, p. 26-36Article in journal (Refereed) Published
Abstract [en]

Background: Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. Methods: Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. Results: Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). Conclusions: Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

Place, publisher, year, edition, pages
John Wiley and Sons , 2013. Vol. 42, no 1, p. 26-36
Keyword [en]
chemoradiotherapy, EGFR, epiregulin, Erbitux (R), HB-EGF, SCCHN, TGF-a, treatment response
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-87963DOI: 10.1111/j.1600-0714.2012.01177.xISI: 000312998500004OAI: oai:DiVA.org:liu-87963DiVA, id: diva2:601030
Note

Funding Agencies|Swedish Laryng Foundation||Foundation of Olle Engkvist, Building Contractor||Linkoping University Hospital||

Available from: 2013-01-28 Created: 2013-01-28 Last updated: 2017-12-06
In thesis
1. 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. p. 74
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)
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Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2015-01-29Bibliographically approved

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Jedlinski, AdamAnsell, AnnaJohansson, Ann-CharlotteRoberg, Karin

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Department of Clinical and Experimental MedicineFaculty of Health SciencesDepartment of ENT - Head and Neck Surgery UHLOto-Rhiono-Laryngology and Head & Neck SurgeryExperimental Pathology
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