- Email: [email protected]
BRCA pathway genes in laryngeal carcinoma
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/ppotor
Summary of Doctoral Thesis/Streszczenie pracy doktorskiej
The analysis of Fanconi anemia/BRCA pathway genes in laryngeal carcinoma
Keywords: Fanconi anemia/BRCA pathway DNA repair Laryngeal carcinoma DNA methylation microRNA mRNA level FANCA gene
PhD dissertation Poznań 24.06.2014 Promoters: Prof. dr Krzysztof Szyfter Institute of Human Genetics, Polish Academy of Sciences, Poznań Prof. dr Med. Reiner Siebert Institute of Human Genetics Christian-Albrechts-University Kiel University Hospital Schleswig-Holstein Campus Kiel, Germany Reviewers: Prof. dr Wanda Baer-Dubowska Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań Prof. dr Barbara Tudek Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw Institute of Genetics and Biotechnology, University of Warsaw
The majority of head and neck cancers (HNSCCs) in Poland are located in the larynx. During the last decades, the number of diagnosed laryngeal cancer cases in Europe remains quite stable, but is still frequently over-represented in Central and Eastern Europe, including Poland. A typical LSCC patient is a male over 60 years old. However, there is a noticeable rise in incidence of LSCC among the younger population (so-called young adults) aged 18–45 years. The overall 5-year survival rates of LSCC patients oscillate around 50% and have not been significantly improved since decades. Laryngeal carcinoma is still poorly understood. There are still no effective markers for diagnostic and prognostic tests, rapid identification of the disease or individual treatment.
Fanconi anemia (FA) describes a genetic syndrome, featuring diverse phenotypical and cellular abnormalities. It is characterized by hypersensitivity to DNA damaging crosslinking agents such as melphalan, cisplatin and mitomycin C (MMC), which cause mainly interstrand DNA cross-links (ICLs). ICLs represent highly toxic DNA damage which bind covalently both strands and stalk replication and require sophisticated mechanism of their removal. Impairment to repair ICLs results in a myriad of congenital malformations, varying between patients, displaying a spectrum of phenotypes from mild to severe. Numerous clinical data report that FA patients demonstrate a high incidence of aggressive forms of squamous cell carcinoma at a young age especially located in oral cavity and neck sites (HNSCC). The estimated risk is elevated 700– 1400 times as compared to non-FA population and is far beyond the cumulative risk effect of environmental factors. Importantly, defects in FA/BRCA pathway are present sporadically in general non-FA population which might be associated with susceptibility to cancer. The literature indicates genetic similarity between FA patients who develop HNSCC and sporadic HNSCC cases. Therefore, it is likely that alterations of the FA/BRCA pathway found in FA/HNSCCs correspond to genetic changes in sporadic HNSCCs and both diseases probably share same clinical features. It should be emphasized, that up to now there are only few reports considering the role of FA/ BRCA genes in head and neck tumors, and, to our knowledge, none of them has focused on cancer of the larynx. In order to meet this issue the main objective of this dissertation was the identification of genes from the FA/BRCA DNA repair pathway differentially regulated in laryngeal carcinoma specimens. Specific goals comprised also characterization of selected FA/BRCA genes at different molecular levels in larynx cancer, which included: promoter DNA methylation, DNA copy number, mRNA expression, protein level measure, mutational screens and miRNA expression. The last purpose was to verify the DNA damage and DNA
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
repair capacity in selected laryngeal cancer cell lines with altered FANCA gene after ICLs induction by cisplatin. The study group included 21 LSCC cell lines, 101 DNAs from primary LSCC tumors (64 fresh frozen and 37 archival isolated from FPPE), 55 DNAs from healthy margins from larynx tumor borders and 35 RNA from primary LSCCs. The control group consisted of DNAs from buccal swabs of 10 healthy donors, 20 DNA samples of whole blood from healthy donors, 4 cell lines cultured from fibroblasts, RNAs from commercial normal bronchial/tracheal epithelial cells and normal human tracheal cells and proteins isolated from healthy larynx tissue and whole blood donors. The methods used entailed pyrosequencing to analyze DNA methylation, data from microarrays for DNA copy number (Agilent 44K and 244K), mRNA expression (Affymetrix U133 plus 2.0) and verified by real-time quantitative PCR (RT-qPCR), Next Generation Sequencing (NGS) on MiSeq Illumina platform to determine potential variants of the FANCA coding and splicing sites sequences, miRNA expression microarray (Agilent Human miRNA expression 60K based on miRBase 18.0), Western immunoblotting for FANCA and FANCD2 detection and finally comet assay to assess DNA damage test, according to the FANCA protein level in selected LSCC cell lines. As a result the genes FANCA, FANCB, BRCA1 and BRCA2 showed significantly altered (t-test, p < 0.01) DNA methylation levels in LSCC cell lines in comparison to buccal swab samples used as controls and thus were further investigated in a group of 64 primary LSCC samples. For the FANCA gene the LSCC primary tumor group was furthermore extended to 77 samples. FANCA was the only gene where DNA methylation level was significantly altered (t-test, p < 0.0005) in all of the analyzed LSCC groups (Fig. 1). Therefore, the FANCA gene was
Fig. 1 – Comparison of FANCA DNA methylation levels between the studied groups
251
the only subjected to further analyses. Additionally, status of FANCA methylation was verified in healthy margin of the larynx tumor samples was similar to larynx tumor samples. Further, the analysis of 77 primary LSCC tumors points to significant differences ( p = 0.004) between mean survival time counted in a 5 year follow up frame with regard to quartiles of the methylation. Estimated mean overall survival rates in the quartile with the lowest FANCA methylation were nearly twofold better for the quartile with the highest FANCA methylation. Moreover, an arbitrary cut-off for FANCA methylation was established as the lowest level of methylation from non-cancerous control group minus 2 SD and, thus, as denoted as 60% of methylation. Hence, samples displaying methylation below the cut-off point were hypomethylated. Patients displaying hypomethylated FANCA showed a significantly longer overall mean survival time (41.9 months) as compared those with ‘‘normal’’ FANCA DNA methylation levels (22.7 months) ( p < 0.001) (Fig. 2). DNA methylation of LINE-1 was significantly lower in both groups as compared to controls ( p < 0.0001). The comparison of these data with DNA methylation results from FANCA gene showed a very significant correlation (Pearson's test, r = 0.81, p = 0.0006) between LSCC cell lines. Moreover, there was a similar trend for primary LSC tumors (r = 0.352, p = 0.004). In previous studies on head and neck cancers we used high resolution array-CGH 44K and 244K platforms to screen 13 LSCC cell lines for copy number alterations. Most of the analyzed genes are located within chromosomal regions with balanced copy number ranges (0.4 to 0.4 log2 ratio) excluding copy number gene alterations such as deletions or amplifications. A panel of 12 LSCC cell lines, 5 primary larynx tumor samples and 3 non-cancerous controls have been subjected to microarray expression analysis (GeneChip Affymetrix U133 plus 2.0) within our previous project. Six genes have showed significant overexpression FANCA, FANCB, BRCA1, FANCI, FANCL and BRIP1 ( p < 0.05). Based on the DNA methylation profiling and microarray expression analysis of 14 FA/BRCA pathway genes we focused on FANCA. qRT-PCR showed overexpression of the gene in majority of the samples compared to controls ( p = 0.0005). miRNA expression profiling has pointed at the miR-940, -374b, -1246 and -1290 as the only differentially expressed ( p < 0.05) and so far unreported in HNSCCs. The miR-940 targets the FANCA and upregulation of miR-940 could affect FANCA function. The FANCA gene is the most frequently mutated gene among FA patients and thus the complementation group A is the most common (66% of diagnosed FA cases). Sequencing of FANCA exons by the next generation sequencing (NGS) has delineated 12 different exonic variants (SNPs), 4 variants found in 30 UTR and 46 variants found in intronic sequences were identified. Nine of them, Chr16:89871832, T/C, 89871831, C/T, 89858543, C/T, 89858541, A/T, 89871835 A/G, 89859022, A/ T, 89858501 T/C, 89871830 G/A, 89871672C/T have not been annotated in any SNP databases. The polyphen2 pointed at 2 SNPs (rs11646374:A (and rs17233497:A) of being possibly damaging. Further, both SNPs were verified by SIFT and only SNP rs17233497 was indicated as damaging. These results could address the Western blot
252
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
Fig. 2 – Kaplan–Meier survival curve shows a significant difference ( p = 0.001) in the 5-year overall survival rate between the patient group below established cut off (60%) for FANCA DNA methylation (red line) and the group above the cut off (green line)
protein profile for UT-SCC-22 where only a very low FANCA protein signal was present. A diverse protein expression levels were detected. Cell lines UT-SCC-117, -106B, -107 and -116 show the strongest FANCA protein signal as normalized with the GAPDH. In the contrary, cell line UT-SCC-6A, -22, -23 and -29 and -57 were shown to have the weakest protein signal. Noteworthy, normal control (human larynx tissue lysate; Abcam, UK) displayed no FANCA signal, which was in concordance with mRNA expression and DNA methylation data. Further, both fractions (L and S) of FANCD2 were found only in cell line UT-SCC-34 and 50. Laryngeal cancer cell lines tested by the comet assay represented diverse FANCA protein levels measured by Western blot (UT-SCC-117 – FANCA +/+, UT-SCC-6A – FANCA /, UT-SCC-113 – FANCA +/ and UT-SCC-108 – FANCA +/). All cell lines demonstrated different levels of nuclei DNA expansion what directly corresponded to DNA damage level. The presence/absence of FANCA protein was correlated with DNA damage level after 1 h of cisplatin incubation and correlated with DNA repair capacity after additional 2 h incubation in fresh medium. Summarizing, the studies herein show DNA hypomethylation of the FANCA promoter, followed by overexpression of FANCA mRNA in LSCC. Hypomethylation was associated with better overall survival among LSCC patients, however this effect could have been distorted by other factors. There were no damaging mutations except SNPs found in the FANCA coding sequence of the analyzed samples and thus it
was excluded as possible reason of deregulation. By contrast, miR-940 could be an epigenetic factor modulating FANCA transcription. The FANCA protein levels suggest its expression in a portion of LSCC cell lines. Nonetheless, the lack of FANCD2 monoubiquitination indicates inactivity of the FA/BRCA pathway in most of the analyzed LSCC cell lines. Notwithstanding, the FANCA protein level was associated with DNA repair potential in the few cell lines treated with a cross-linking agent (cisplatin). Thus, an alternative and independent role of FANCA protein can probably function apart from the rest of the FA pathway.
Conflict of interest/Konflikt interesu None declared.
Financial support/Finansowanie Scholarship within the project ‘‘Scholarship support for Ph.D. students specializing in major strategic for Greater Poland development’’, Sub-measure 8.2.2 Human Capital Operational Programme, co-financed by European Union under the European Social Fund in 2011/2012 and 2012/ 2013 editions The National Science Centre grant no. 2012/05/N/NZ2/ 02245
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
Collaborative Experimental Scholarship For Central & Eastern Europe – Federation of European Biochemical Societies (FEBS) Short-Term Fellowship – European Molecular Biology Organization (EMBO).
Ethics/Etyka The work described in this article has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals.
253
Marcin Szaumkessel Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland *Correspondence to: Instytut Genetyki Człowieka Polskiej Akademii Nauk, ul. Strzeszyńska 32, 60-479 Poznań, Poland. Tel.: +48 61 657 91 00; fax: +48 61 823 32 35. E-mail address: [email protected] Received: 15.10.2014 Accepted: 21.10.2014 Available online: 31.10.2014 http://dx.doi.org/10.1016/j.ppotor.2014.10.005 2084-5308/
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/ppotor
Summary of Doctoral Thesis/Streszczenie pracy doktorskiej
The analysis of Fanconi anemia/BRCA pathway genes in laryngeal carcinoma
Keywords: Fanconi anemia/BRCA pathway DNA repair Laryngeal carcinoma DNA methylation microRNA mRNA level FANCA gene
PhD dissertation Poznań 24.06.2014 Promoters: Prof. dr Krzysztof Szyfter Institute of Human Genetics, Polish Academy of Sciences, Poznań Prof. dr Med. Reiner Siebert Institute of Human Genetics Christian-Albrechts-University Kiel University Hospital Schleswig-Holstein Campus Kiel, Germany Reviewers: Prof. dr Wanda Baer-Dubowska Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań Prof. dr Barbara Tudek Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw Institute of Genetics and Biotechnology, University of Warsaw
The majority of head and neck cancers (HNSCCs) in Poland are located in the larynx. During the last decades, the number of diagnosed laryngeal cancer cases in Europe remains quite stable, but is still frequently over-represented in Central and Eastern Europe, including Poland. A typical LSCC patient is a male over 60 years old. However, there is a noticeable rise in incidence of LSCC among the younger population (so-called young adults) aged 18–45 years. The overall 5-year survival rates of LSCC patients oscillate around 50% and have not been significantly improved since decades. Laryngeal carcinoma is still poorly understood. There are still no effective markers for diagnostic and prognostic tests, rapid identification of the disease or individual treatment.
Fanconi anemia (FA) describes a genetic syndrome, featuring diverse phenotypical and cellular abnormalities. It is characterized by hypersensitivity to DNA damaging crosslinking agents such as melphalan, cisplatin and mitomycin C (MMC), which cause mainly interstrand DNA cross-links (ICLs). ICLs represent highly toxic DNA damage which bind covalently both strands and stalk replication and require sophisticated mechanism of their removal. Impairment to repair ICLs results in a myriad of congenital malformations, varying between patients, displaying a spectrum of phenotypes from mild to severe. Numerous clinical data report that FA patients demonstrate a high incidence of aggressive forms of squamous cell carcinoma at a young age especially located in oral cavity and neck sites (HNSCC). The estimated risk is elevated 700– 1400 times as compared to non-FA population and is far beyond the cumulative risk effect of environmental factors. Importantly, defects in FA/BRCA pathway are present sporadically in general non-FA population which might be associated with susceptibility to cancer. The literature indicates genetic similarity between FA patients who develop HNSCC and sporadic HNSCC cases. Therefore, it is likely that alterations of the FA/BRCA pathway found in FA/HNSCCs correspond to genetic changes in sporadic HNSCCs and both diseases probably share same clinical features. It should be emphasized, that up to now there are only few reports considering the role of FA/ BRCA genes in head and neck tumors, and, to our knowledge, none of them has focused on cancer of the larynx. In order to meet this issue the main objective of this dissertation was the identification of genes from the FA/BRCA DNA repair pathway differentially regulated in laryngeal carcinoma specimens. Specific goals comprised also characterization of selected FA/BRCA genes at different molecular levels in larynx cancer, which included: promoter DNA methylation, DNA copy number, mRNA expression, protein level measure, mutational screens and miRNA expression. The last purpose was to verify the DNA damage and DNA
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
repair capacity in selected laryngeal cancer cell lines with altered FANCA gene after ICLs induction by cisplatin. The study group included 21 LSCC cell lines, 101 DNAs from primary LSCC tumors (64 fresh frozen and 37 archival isolated from FPPE), 55 DNAs from healthy margins from larynx tumor borders and 35 RNA from primary LSCCs. The control group consisted of DNAs from buccal swabs of 10 healthy donors, 20 DNA samples of whole blood from healthy donors, 4 cell lines cultured from fibroblasts, RNAs from commercial normal bronchial/tracheal epithelial cells and normal human tracheal cells and proteins isolated from healthy larynx tissue and whole blood donors. The methods used entailed pyrosequencing to analyze DNA methylation, data from microarrays for DNA copy number (Agilent 44K and 244K), mRNA expression (Affymetrix U133 plus 2.0) and verified by real-time quantitative PCR (RT-qPCR), Next Generation Sequencing (NGS) on MiSeq Illumina platform to determine potential variants of the FANCA coding and splicing sites sequences, miRNA expression microarray (Agilent Human miRNA expression 60K based on miRBase 18.0), Western immunoblotting for FANCA and FANCD2 detection and finally comet assay to assess DNA damage test, according to the FANCA protein level in selected LSCC cell lines. As a result the genes FANCA, FANCB, BRCA1 and BRCA2 showed significantly altered (t-test, p < 0.01) DNA methylation levels in LSCC cell lines in comparison to buccal swab samples used as controls and thus were further investigated in a group of 64 primary LSCC samples. For the FANCA gene the LSCC primary tumor group was furthermore extended to 77 samples. FANCA was the only gene where DNA methylation level was significantly altered (t-test, p < 0.0005) in all of the analyzed LSCC groups (Fig. 1). Therefore, the FANCA gene was
Fig. 1 – Comparison of FANCA DNA methylation levels between the studied groups
251
the only subjected to further analyses. Additionally, status of FANCA methylation was verified in healthy margin of the larynx tumor samples was similar to larynx tumor samples. Further, the analysis of 77 primary LSCC tumors points to significant differences ( p = 0.004) between mean survival time counted in a 5 year follow up frame with regard to quartiles of the methylation. Estimated mean overall survival rates in the quartile with the lowest FANCA methylation were nearly twofold better for the quartile with the highest FANCA methylation. Moreover, an arbitrary cut-off for FANCA methylation was established as the lowest level of methylation from non-cancerous control group minus 2 SD and, thus, as denoted as 60% of methylation. Hence, samples displaying methylation below the cut-off point were hypomethylated. Patients displaying hypomethylated FANCA showed a significantly longer overall mean survival time (41.9 months) as compared those with ‘‘normal’’ FANCA DNA methylation levels (22.7 months) ( p < 0.001) (Fig. 2). DNA methylation of LINE-1 was significantly lower in both groups as compared to controls ( p < 0.0001). The comparison of these data with DNA methylation results from FANCA gene showed a very significant correlation (Pearson's test, r = 0.81, p = 0.0006) between LSCC cell lines. Moreover, there was a similar trend for primary LSC tumors (r = 0.352, p = 0.004). In previous studies on head and neck cancers we used high resolution array-CGH 44K and 244K platforms to screen 13 LSCC cell lines for copy number alterations. Most of the analyzed genes are located within chromosomal regions with balanced copy number ranges (0.4 to 0.4 log2 ratio) excluding copy number gene alterations such as deletions or amplifications. A panel of 12 LSCC cell lines, 5 primary larynx tumor samples and 3 non-cancerous controls have been subjected to microarray expression analysis (GeneChip Affymetrix U133 plus 2.0) within our previous project. Six genes have showed significant overexpression FANCA, FANCB, BRCA1, FANCI, FANCL and BRIP1 ( p < 0.05). Based on the DNA methylation profiling and microarray expression analysis of 14 FA/BRCA pathway genes we focused on FANCA. qRT-PCR showed overexpression of the gene in majority of the samples compared to controls ( p = 0.0005). miRNA expression profiling has pointed at the miR-940, -374b, -1246 and -1290 as the only differentially expressed ( p < 0.05) and so far unreported in HNSCCs. The miR-940 targets the FANCA and upregulation of miR-940 could affect FANCA function. The FANCA gene is the most frequently mutated gene among FA patients and thus the complementation group A is the most common (66% of diagnosed FA cases). Sequencing of FANCA exons by the next generation sequencing (NGS) has delineated 12 different exonic variants (SNPs), 4 variants found in 30 UTR and 46 variants found in intronic sequences were identified. Nine of them, Chr16:89871832, T/C, 89871831, C/T, 89858543, C/T, 89858541, A/T, 89871835 A/G, 89859022, A/ T, 89858501 T/C, 89871830 G/A, 89871672C/T have not been annotated in any SNP databases. The polyphen2 pointed at 2 SNPs (rs11646374:A (and rs17233497:A) of being possibly damaging. Further, both SNPs were verified by SIFT and only SNP rs17233497 was indicated as damaging. These results could address the Western blot
252
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
Fig. 2 – Kaplan–Meier survival curve shows a significant difference ( p = 0.001) in the 5-year overall survival rate between the patient group below established cut off (60%) for FANCA DNA methylation (red line) and the group above the cut off (green line)
protein profile for UT-SCC-22 where only a very low FANCA protein signal was present. A diverse protein expression levels were detected. Cell lines UT-SCC-117, -106B, -107 and -116 show the strongest FANCA protein signal as normalized with the GAPDH. In the contrary, cell line UT-SCC-6A, -22, -23 and -29 and -57 were shown to have the weakest protein signal. Noteworthy, normal control (human larynx tissue lysate; Abcam, UK) displayed no FANCA signal, which was in concordance with mRNA expression and DNA methylation data. Further, both fractions (L and S) of FANCD2 were found only in cell line UT-SCC-34 and 50. Laryngeal cancer cell lines tested by the comet assay represented diverse FANCA protein levels measured by Western blot (UT-SCC-117 – FANCA +/+, UT-SCC-6A – FANCA /, UT-SCC-113 – FANCA +/ and UT-SCC-108 – FANCA +/). All cell lines demonstrated different levels of nuclei DNA expansion what directly corresponded to DNA damage level. The presence/absence of FANCA protein was correlated with DNA damage level after 1 h of cisplatin incubation and correlated with DNA repair capacity after additional 2 h incubation in fresh medium. Summarizing, the studies herein show DNA hypomethylation of the FANCA promoter, followed by overexpression of FANCA mRNA in LSCC. Hypomethylation was associated with better overall survival among LSCC patients, however this effect could have been distorted by other factors. There were no damaging mutations except SNPs found in the FANCA coding sequence of the analyzed samples and thus it
was excluded as possible reason of deregulation. By contrast, miR-940 could be an epigenetic factor modulating FANCA transcription. The FANCA protein levels suggest its expression in a portion of LSCC cell lines. Nonetheless, the lack of FANCD2 monoubiquitination indicates inactivity of the FA/BRCA pathway in most of the analyzed LSCC cell lines. Notwithstanding, the FANCA protein level was associated with DNA repair potential in the few cell lines treated with a cross-linking agent (cisplatin). Thus, an alternative and independent role of FANCA protein can probably function apart from the rest of the FA pathway.
Conflict of interest/Konflikt interesu None declared.
Financial support/Finansowanie Scholarship within the project ‘‘Scholarship support for Ph.D. students specializing in major strategic for Greater Poland development’’, Sub-measure 8.2.2 Human Capital Operational Programme, co-financed by European Union under the European Social Fund in 2011/2012 and 2012/ 2013 editions The National Science Centre grant no. 2012/05/N/NZ2/ 02245
polski przeglĄd otorynolaryngologiczny 3 (2014) 250–253
Collaborative Experimental Scholarship For Central & Eastern Europe – Federation of European Biochemical Societies (FEBS) Short-Term Fellowship – European Molecular Biology Organization (EMBO).
Ethics/Etyka The work described in this article has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals.
253
Marcin Szaumkessel Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland *Correspondence to: Instytut Genetyki Człowieka Polskiej Akademii Nauk, ul. Strzeszyńska 32, 60-479 Poznań, Poland. Tel.: +48 61 657 91 00; fax: +48 61 823 32 35. E-mail address: [email protected] Received: 15.10.2014 Accepted: 21.10.2014 Available online: 31.10.2014 http://dx.doi.org/10.1016/j.ppotor.2014.10.005 2084-5308/