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Proteins and single nucleotide polymorphisms involved in apoptosis, growth control, and DNA repair predict cisplatin sensitivity in head and neck cancer cell lines
Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
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, Cell Biology. Linköping University, Faculty of Health Sciences.
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2009 (English)In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 24, no 4, 549-556 p.Article in journal (Refereed) Published
Abstract [en]

The present study was undertaken to evaluate the possibility of using a panel of proteins and single nucleotide polymorphisms (SNPs) involved in apoptosis, growth control, and DNA repair as predictive markers for cisplatin sensitivity. For this purpose the intrinsic cisplatin sensitivity (ICS) was determined in 39 cell lines derived from squamous cell carcinomas of the head and neck using a colony-forming assay. In these cell lines and in normal oral keratinocytes (NOK), the expression of epidermal growth factor receptor (EGFR), Hsp70, Bax, Bcl-2, Bcl-XL, survivin, and COX-2 was determined. Moreover, the p53, MDM2, FGFR4, XPC, XPD, XRCC1, and XRCC3 genes were analyzed for the presence of specific single nucleotide polymorphisms (SNPs). Pearsons correlation test showed that EGFR was the only protein that was significantly correlated to the ICS (r=0.388, p=0.015). The combination of EGFR, Hsp70, Bax, and Bcl-2 gave the strongest correlation (r=0.566, p andlt;= 0.001), whereas Bax alone had the second highest influence on the ICS. Furthermore, all four SNPs within genes involved in DNA repair, i.e. XPC, XPD, XRCC1, and XRCC3, tended to influence the ICS. In order to find the combination of factors, on both protein and gene levels, with the highest correlation to ICS, a multivariate statistical calculation was performed. Our results indicate that SNPs in DNA repair genes (XRCC3(241) and XPD751) influence the ICS and together with the expression of EGFR, Hsp70, Bax, and Bcl-2, they could predict the cisplatin sensitivity of head and neck cancer cell lines (r=0.614, p andlt;= 0.001).

Place, publisher, year, edition, pages
2009. Vol. 24, no 4, 549-556 p.
Keyword [en]
epidermal growth factor receptor, Bax, Bcl-2, heat shock protein 70, DNA repair genes
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-21513DOI: 10.3892/ijmm_00000264OAI: oai:DiVA.org:liu-21513DiVA: diva2:241465
Available from: 2009-10-02 Created: 2009-10-02 Last updated: 2017-12-13
In thesis
1. Predictive markers: for treatment sensitivity in head and neck squamous cell carcinoma
Open this publication in new window or tab >>Predictive markers: for treatment sensitivity in head and neck squamous cell carcinoma
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck cancer is the sixth most common cancer world wide. In Sweden approximately 850 new cases are diagnosed each year, and two thirds are men. The past decades of improved treatment strategies have unfortunately not significantly improved the five-year survival rates for this group of patients. Therefore, it is important to rapidly find combinations of new and strong predictive markers for treatment response. Different predictive markers have been investigated for decades, without succeeding in finding means to securely predict response to treatment. Models to combine markers are called for.

The aim of this thesis was to test multiple predictive markers on both gene and protein level to evaluate their predictive value for radiotherapy and cisplatin response. Furthermore, to combine, and correlate them to treatment response in order to extract the panel of markers that strongest correlated to the investigated treatment. Cell lines derived from 42 patients with head and neck squamous cell carcinoma (HNSCC) were used for protein quantification with Western blot and ELISA of the proteins survivin, Epidermal Growth Factor Receptor, Bcl-2, Bcl-XL, Bax, Bad, Bak, PUMA, Heat shock protein 70, MDM2, p53, SMAD4, Cyclooxygenase-2, and Cyclin D1. The expression of the selected proteins was related to the mean expression of normal oral keratinocytes (NOK) from healthy individuals. Furthermore, mutations in the p53 gene, along with single nucleotide polymorphisms in the genes of p53, MDM2, FGFR4, XRCC1, XRCC3, XPD, and XPC were analysed. To allow a large number of predictive markers on both protein and gene level to be combined and correlated to treatment response, the number of negative points (NNP) model was introduced. Both correlations of sensitivity to radiotherapy and to cisplatin treatment was analysed among the cell lines. In the first paper, including nine cell lines, the panel of EGFR, survivin, and splice site/missense p53 mutations correlated strongest to radioresponse. In paper II, 42 cell lines were used and the combination of survivin, Bcl-2, Bcl-XL, Bax, COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse. In paper IV, the panel correlating strongest with cisplatin sensitivity consisted of EGFR, Hsp70, Bax, and Bcl-2 in combination with SNPs in the DNA-repair genes XRCC3 and XPD.

The predisposition of the FGFR4 Gly388Arg polymorphism for the development of HNSCC was investigated in paper III. DNA was isolated from 110 tumour biopsies, and restriction fragment length polymorphism analysis showed that 58% of the individuals in the control group carried the FGFR4 Arg388 allele, whereas the frequency in the tumour group was 45%. The Gly388 allele gave a significantly higher risk of developing HNSCC, suggesting Gly388 to be the risk allele for cancer development. Furthermore, a novel mutation was found in the FGFR4 gene. The influence of this new mutation is however unknown.

In conclusion, predictive markers for treatment sensitivity need to be combined to receive an accurate prediction of treatment response.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. 84 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1205
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-61586 (URN)978-91-7393-319-3 (ISBN)
Public defence
2010-11-12, Elsa Brändströmsalen, Campus US, Linköpings universitet, Linköping, 09:00
Opponent
Supervisors
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2012-05-09Bibliographically 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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Farnebo, LovisaJedlinski, AdamAnsell, AnnaVainikka, LindaThunell, LenaJohansson, Ann-CharlotteRoberg, Karin

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Oto-Rhiono-Laryngology and Head & Neck Surgery Faculty of Health SciencesDepartment of ENT - Head and Neck Surgery UHLDepartment of Clinical and Experimental MedicineCell BiologyExperimental Pathology
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