| | Polymorphism of FGFR4 in cancer development and sensitivity to cisplatin and radiation in head and neck cancerReceived 23 January 2008; received in revised form 12 March 2008; accepted 13 March 2008. published online 09 June 2008. Introduction  Cancer of the head and neck poses a major health problem in the world and is ranked from prevalence as number six of all cancer types. Approximately 600.000 new malignancies are diagnosed each year of which about half occur in Southern East Asia and China. The majority of all head and neck cancers are squamous cell carcinomas originating from epithelial tissues.1 Locoregionally advanced squamous cell carcinoma of the head and neck has been treated in addition to surgery with radiotherapy alone often with alternative fractionation schedules such as hyperfractionation, acceleration or concomitant boost. Moreover, in the last years concomitant chemoradiotherapy has been the treatment of choice especially in case of known risk factors such as extracapsular spread or close resection margins. Radioresistance and local recurrence are however significant problems following radiotherapy, and therefore there is a paramount need for predictive markers.2 Fibroblast growth factor receptors (FGFRs) consist of four closely related genes (FGFR1-4) with a similar protein structure all belonging to the receptor tyrosine kinase family. All four FGFRs can interact with a various number of fibroblast growth factors (FGFs) proposing that there is a high evolutionary conservation within the FGFR family. FGFRs are thought to be implicated in many human cancers e.g. cervix, bladder and breast. Furthermore, they are involved in a wide spectrum of biological processes such as cell growth, tissue development, differentiation, angiogenesis and tissue repair and can thereby contribute to tumourigenesis.3, 4, 5 Single nucleotide polymorphisms (SNPs) have for a long time been used to identify individual differences seen in clinical practice, such as drug sensitivity or immune response to malignant cells.6 A SNP in the transmembrane domain of FGFR4 (position 388 altering a glycine to an arginine) has in some studies been linked to a worse prognosis in several types of cancers e.g. in breast,7 colon,7 prostate,8 head and neck squamous cell carcinoma (HNSCC)9 and soft tissue sarcoma.10 Others, however show no correlation between the FGFR4 Gly388Arg SNP and the prognosis of malignant gliomas,11 cancer of the breast, colorectal and lung.12, 13 Bange et al. have suggested that the Arg388 allele is crucial in both breast and colon cancer progression and can be correlated to a worse prognosis.7 A study on HNSCC patients indicate that the Arg388 allele in combination with high FGFR4 expression is associated with poor clinical outcome.9 In addition, another study showed a significant reduced overall survival of HNSCC patients carrying the Arg388 allele and that the Arg388 allele might predispose for the development of distant metastasis and late recurrences.6 Summarizing these results, it seems that the Arg388 allele is associated with worse prognosis, faster progression and reduced overall survival in comparison with the Gly388 allele. However, there are no reports on the correlation between the Arg388 allele frequency and predisposition for HNSCC. The aim of the present study was to investigate the predisposition of this FGFR4 SNP for development of HNSCC and furthermore, to examine if the FGFR4 Arg388 is associated with resistance to cisplatin and radiotherapy. Materials and methods  Tissue samples and DNA isolation DNA was isolated from 110 tumour biopsies from the head and neck region collected from patients at the University Hospital in Linköping between the years 2004–2006 (Table 1; approved by the Linköping University ethical committee). Of the patients, 67 (61%) were males and 43 (39%) were females with a mean age of 66 years (range 34–91). DNA was also isolated from 39 HNSCC cell lines established from patients at the University Hospital in Turku, Finland between the years 1990–2002. These cell lines were selected for this study to represent different parts of the spectrum of in vitro radiosensitivity and cisplatin sensitivity. Furthermore, 192 individuals from the South-East region of Sweden were used as controls, of which 97 were males and 95 were females with the mean age of 45 years (range 23–78). The controls were in Hardy–Weinberg equilibrium. The DNA isolation of the tumour biopsies was performed by standard proteinase K treatment and phenol–chloroform extractions and the DNA isolation of the cell lines was performed by MaxwellTM 16 DNA Purification Kit (Promega, Madison, WI, USA). Cells and culture conditions The HNSCC cell lines were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 2 mM glutamine, 1% non-essential amino acids, 100 IU/ml penicillin-G, 50 μg/ml streptomycin, and 10% fetal bovine serum (all from GIBCO, Paisly, UK). The cells were incubated in humidified air with 5% CO2 at 37 °C and subcultured once a week using 0.25% trypsin and 0.02% EDTA. Normal oral human keratinocytes (NOK) were cultured as previously described.14 Biopsies were harvested during benign surgery in the oral cavity, mostly tonsillectomies and contained non-keratinized squamous cell epithelium (approved by the Linköping University ethical committee). Primary keratinocyte cultures were derived from trypsin-digested tissue using growth medium (Keratinocyte-SFM; GIBCO) supplemented with antibiotics solution (penicillin 100 u/ml, streptomycin 100 μg/ml) and culture flasks pre-coated with fibronectin and collagen. Medium was replaced at three day intervals, and the cultures were subcultured at about 75% confluence using 0.25% trypsin and 0.02% EDTA. Cultures in passage two and three were used for the analyses. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis The PCR was conducted on exon 9 for RFLP analysis and on the transmembrane and kinase domains (ranging from exons 9–18) for DNA sequencing. Primers were designed by our group, sequences are available upon request. Each PCR mixture had a total volume of 20 μl and they consisted of 1.0 μM of each primer (Invitrogen, Paisley, UK), 200 μM of each dNTP, 2.0 mM MgCl2, 10× Taq buffer (containing 200 mM (NH4)2SO4, 750 mM Tris (pH 9.0) and 0.1% Tween 20), 0.5 U Taq DNA polymerase (Thermowhite, Saveen Werner AB, Limhamn, Sweden) and 50 ng DNA. The amplifications were performed by annealing at 55–62 °C for 35 cycles. The samples were loaded on a 1.5% agarose gel (Invitrogen) and stained with ethidium bromide for detection of PCR products on a UV-table. The 126-bp fragment from exon 9 for RFLP analysis was digested in 1 h in 60 °C with BstN1 (New England BioLabs, Beverly, MA, USA) according to the manufacturer’s instructions. Restriction fragments were resolved on a 3% Nusieve 1% agarose gel (Invitrogen) and stained with ethidium bromide for detection of the fragments. The FGFR4 Gly/Gly388 was characterized by two different fragments of 97 and 29 base pairs (bp), the FGFR4 Gly/Arg388 was characterized by three different fragments of 97, 68 and 29 bp and the FGFR4 Arg/Arg388 by the two fragments of 68 and 29 bp. Western blot analysis Cells were washed in phosphate-buffered saline (PBS) and lysed in 63 mM Tris–HCl buffer (pH 6.8) containing 10% glycerol, 2% SDS, 5% 2-mercaptoethanol and 0.05% bromphenol blue (all from Sigma, St Louis, MO, USA). The protein concentration was determined,16 and 100 μg aliquots were separated by 4–20% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA USA). Proteins were transferred onto a nitrocellulose membrane (pore size 0.45 μm; Bio-Rad Laboratories), which was subsequently blocked for 90 min at room temperature in Tris-buffered saline (TBS; 50 mM Tris and 0.15 M NaCl, pH 7.5) containing 5% skimmed milk and 0.1% Tween-20 (Sigma). After washing in TBS, the membranes were incubated with a rabbit anti-FGFR4 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibody was diluted in TBS containing 0.1% skimmed milk and 0.1% Tween-20 and incubated at 4 °C overnight. The membrane was washed and incubated for 1 h at room temperature with a peroxidase-conjugated anti-mouse (1:1500; Dakopatts, Älvsjö, Sweden) and the bands were visualized by Western Blotting Luminol Reagent (Santa Cruz Biotechnology). Equal loading was verified by reprobing the membrane with a goat anti-actin antibody (I-19; 1:1000; Santa Cruz Biotechnology), followed by a bovine anti-goat antibody (1:1000; Santa Cruz Biotechnology). DNA sequencing with MegaBACE™ The PCR-products from the transmembrane and kinase domains were purified using the ExoSAPIT Purification Kit (Amersham Bioscience, Uppsala, Sweden). The purified PCR fragments were sequenced using the DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE™ DNA Analysis Systems (Amersham Bioscience) and the MegaBACE 1030 instrument (GE Healthcare, Uppsala, Sweden). mRNA isolation and detection mRNA was extracted from cells using the PARIS™ Kit (Protein And RNA Isolation System; Ambion, Austin, Texas, USA) according to manufacturer’s instructions and RNA concentration was measured using a UV–vis spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Wilmington, Delaware, USA). The cDNA synthesis was conducted with random primers and SuperScript II Reverse Transcriptase (Invitrogen). Primers (Invitrogen) were designed by our group, sequences available upon request. The PCR amplification and detection of cDNA was performed as described above but with an annealing temperature of 60 °C and for 40 cycles. Statistics χ2-test from Epi Info™, version 3.4.1., (Atlanta, Georgia, USA) was used to calculate Hardy–Weinberg equilibrium and also the statistical association between the frequency of the polymorphism and development of HNSCC. Overall survival was estimated with the Kaplan–Meier method and the significance of difference between survival rates for patients with different genotypes was assessed by log-rank test (SPSS 15.0 for Windows). Mann–Whitney U test was used to analyse the significance of the FGFR4 Gly/Arg polymorphism for radiation and cisplatin sensitivity (SPSS 15.0 for Windows). Results  FGFR4 genotyping and predisposition for developing HNSCC The FGFR4 Arg388 allele was found in 58% (111 out of 192) of the control individuals confirming the frequencies from previous studies,7, 8 whereas the frequencies of the Arg388 allele in the tumour biopsies from patients with HNSCC were 45% (49 out of 110). In the material from primary tumours we found that the Gly388 allele gave a statistically significant higher risk of developing cancer, OR 1.71 (p = 0.026), suggesting the Gly388 allele to be a risk allele considering predisposition (see Table 2). Furthermore, males carrying the Gly388 allele had a 2-fold risk to develop HNSCC, OR 2.00 (p = 0.031), while no significant increase of risk was seen for females. In addition, the Gly388 allele gave a statistically significant higher risk to develop cancer in the oral cavity, OR 2.49 (p = 0.002) while no significance was seen for larynx. | a HNSCC, head and neck squamous cell carcinoma. bχ2-test. |
Since this tumour material is recently collected, we have only a one-year follow-up on the patients so far. Calculations for overall survival depending on FGFR4 Arg388 genotype show no significance overall or stratified for gender or age of onset with cut-off at 65 years. Western blot analyses of the FGFR4 protein A significant reduction of overall survival in HNSCC patients was seen in a study correlating the Arg388 allele to a high FGFR4 expression.9 In the present study we investigate the hypothesis that the Arg388 allele in combination with high FGFR4 expression plays a role in treatment sensitivity. Therefore, we analysed the FGFR4 protein expression in eight cell lines with different radiation and cisplatin sensitivity (five cell lines with the Gly388 allele, three cell lines with the Arg388 allele; Table 3) and NOK. In two of the cell lines (UT-SCC-9 and UT-SCC-33) we found a low expression of FGFR4 while the cell lines UT-SCC-12 and 77 were the most similar to NOK. The remaining cell lines displayed 2–4 protein bands not represented in NOK (Fig. 3). However, these results did not show any correlation between protein expression and treatment sensitivity. | a TNM classification according to the International Union against Cancer (UICC, 1977). bRadiation sensitivity given in terms of mean inactivation dose (AUC = area under curve). Previously published by Pekkola-Heino et al.24 and Erjala et al.25 |
DNA sequencing of the FGFR4 gene and RNA expression In order to explain the Western blot pattern we sequenced the transmembrane and kinase domains of the FGFR4 gene. A novel mutation was found in six (UT-SCC-2, -9, -12A, -33, -34 and -77) of the eight cell lines analysed with Western blot. A T > C in the consensus splice site, four nucleotides into intron 13 resulted in an amino acid exchange altering a stop codon to a glutamine. This leads to a six amino acid supplement to the protein before the next stop codon and could result in a truncated protein with a loss of exons 14–18. The Western blot analysis showed only weak FGFR4 protein bands of cell lines UT-SCC-9 and -33 which both carried the novel mutation. Therefore, we analysed the mRNA expression in these cell lines and in NOK. However, mRNA was detected in both cell lines and NOK thus the solution to the weak bands in the Western blot analysis could not be explained on mRNA level. Discussion  In this study we examined whether the Arg388 allele affects the predisposition of cancer in the head and neck region. Our results were unexpected and showed that Gly388 was the risk allele. There are two previously published studies on HNSCC and FGFR4 where one Caucasian study shows no difference in frequency between the 104 cancer cases and 123 controls.9 The distribution in gender and age was similar to our study, but since our study only shows an increased risk for tumours in the oral cavity the different results may be explained by the location of the tumours. Another study on 75 Brazilian patients displayed a genotype frequency similar to our patient group, but did not include a control group.6 In this Brazilian study, the authors speculate that their mix of different ethnical groups could have the same genotype composition as Caucasians. If this is the case their data indicate the Gly388 allele as risk allele for predisposition of HNSCC. However, this is highly speculative and warrants further studies. Our results on predisposition can be summarized as healthy individuals carrying the Arg388 allele are protected against development of cancer. However, other studies have shown that if malignancy does occur they are facing a faster progression and a reduced overall survival.7, 9 This relation has been seen in for example, the matrix metalloproteinase-1 gene where individuals carrying the variant allele have an increased risk to develop colorectal cancer but patients harbouring the same variant SNP had a more favourable prognosis.18, 19 The primary mode of treatment in locally advanced HNSCC involves a combination of radiotherapy and surgery or definitive chemo-irradiation. Radio- and chemo-resistance and local carcinoma recurrences are significant problems during and after therapy. Cisplatin is one of the most frequently used drugs during chemo-irradiation in HNSCC. A different mechanism for cisplatin to induce cell death compared to radiation has been suggested.20, 21 Therefore, in this study we investigated if the Arg388 allele could have a role in treatment response of tumour cells. Our results show no impact of Arg388 allele for intrinsic radiosensitivity in HNSCC cell line but interestingly, a tendency to increase the cell lines sensitivity to cisplatin. However, these results were not statistically significant but since many other factors e.g. p5322 and Bcl-XL23 are also thought to influence the effect of cisplatin, our results are probably well in line with what can be expected from one single SNP. Another study shows that the FGFR4 Arg388 allele is associated with resistance to adjuvant therapy in primary breast cancer.17 The conflicting results could be due both to the fact that two different cancer sites are studied and the pharmacological difference between cisplatin and cyklophosphamide/methrotrexate/fluorouracil (CMF). Moreover, the primary tumours in our study come from patients that have been treated with radiotherapy alone or in combination with surgery, not chemotherapy. Therefore, our results show no correlation between overall survival and the FGFR4 Arg388 genotype in this patient group, confirming the lack of influence on radiosensitivity in the cell lines. Streit et al. have shown that high FGFR4 expression reduces the overall survival when correlating with the Arg388 allele.9 When we analysed the FGFR4 protein expression in eight HNSCC cell lines (Table 3) we found that all eight varied both in strength and in band pattern. This variation in FGFR4 protein expression in HNSCC tumours is a novel result and has not been described before. Nonetheless, no correlation between protein expression and treatment sensitivity was found. The effect of the novel mutation found in six of the eight investigated cell lines is not known. The mutation in the consensus splice site leads to a six amino acid supplement to the protein before the next stop codon and could theoretically result in a truncated protein with a loss of exons 14–18. Our results indicate a role of this protein in tumour development, however; but the impact and the frequency in the population of this novel mutation together with the impact or function of different FGFR4 expression have to be further evaluated. A major problem in clinical work is a varying response to radio- and chemotherapy. Some HNSCC even grow during ongoing therapy, and it would be of interest from both the socio-economic point of view and from that of the patient to give only the effective treatment. In order to do this, it is necessary to understand the changes that are responsible for the process of tumour progression. Theoretically, a large number of factors affecting growth control, apoptosis and other processes could be altered, which is why the recognition of a panel of multiple predictive factors is of great importance. Our long-term goal is to identify a panel of markers that predict treatment sensitivity in newly found tumours, to help clinicians predict the most effective treatment in each patient. Taken together, we have here identified FGFR4 as a risk marker to develop HNSCC and as a protein that together with other markers could be included in a panel to predict outcome of cisplatin treatment. This novel finding can in the future have impact on clinical management and lead to a more effective therapy for patients with HNSCC. Conflict of Interest Statement  None declared. Acknowledgements  We thank Jonas Ungerbäck for his skill and help with the mRNA isolation and Annette Molbaek and Åsa Schippert for technical assistance with the MegaBACE™. This study was supported by the Swedish Laryng Foundation, the County Council of Östergötland (ÖLL), the Research Council of South Eastern Sweden (FORSS), Gunnar Nilsson’s Cancer foundation, and The Swedish Cancer foundation (No. 070180), The Swedish Society of Medicine and from the Research Funds of Linköping University Hospital. References  1. 1Shah JP, Lydiatt W. Treatment of cancer of the head and neck. CA Cancer J Clin. 1995;45:352–368. MEDLINE |
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a Division of Otorhinolaryngology, University Hospital, SE-58185 Linköping, Sweden b Division of Cell Biology, Department of Biomedicine and Surgery, Linköping University, Sweden c Department of Otorhinolaryngology, Head & Neck Surgery, and Medical Biochemistry, University of Turku, Finland Corresponding author. Tel.: +46 13 221534.
PII: S1368-8375(08)00093-6 doi:10.1016/j.oraloncology.2008.03.007 © 2008 Elsevier Ltd. All rights reserved. | |
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