DNA repair gene ERCC2 polymorphisms and risk of squamous cell carcinoma of the head and neck

Introduction

Genetic aberrations of DNA repair enzymes are known to be common events and to be associated with different cancer entities. Aim of the following study was to analyze the genetic association of single nucleotide polymorphisms (SNP) of the DNA repair genes with the risk of squamous cell carcinoma of the head and neck (HNSCC).

Materials and methods

Genetic variants ERCC2 Lys751Gln (rs13181), ERCC2 Asp312Asn (rs1799793), XRCC1 Arg194Trp (rs1799782); XRCC1 Gln399Arg (rs25487), XRCC1 Arg280His (rs25489) and XRCC3 Thr241Met (rs861539) were analyzed in a primary study group comprising 169 patients with histologically confirmed HNSCC and 463 healthy control subjects. Polymorphisms associated with HNSCC were furthermore analyzed in an independent replication study including 125 HNSCC.

Results

Only the ERCC2 751 Gln/Gln genotype was associated with HNSCC in the primary study (p = 0.033) and in the replication study (p = 0.023), resulting in an overall odds ratio of 0.54 (95% confidence interval 0.35–0.92; p = 0.006).

Conclusion

Carriers of the homozygous ERCC2 751 Gln/Gln genotype may be at lower risk for HNSCC.     

Frequent allelic imbalance and loss of protein expression of the DNA repair gene hOGG1 in head and neck squamous cell carcinoma

Reactive oxygen species produced by aerobic cellular metabolism or through exposure to environmental carcinogens can cause oxidative DNA damage by generating DNA base lesions and strand breakage. Prime among these base lesions is the conversion of guanine to 8-oxoguanine. Among 20 or so oxidative DNA base lesions, 8-oxoguanine is the most abundant and is critical in terms of mutagenesis because it is capable of mispairing with adenine, which, if not sufficiently repaired, may lead to G:C to T:A transversion upon DNA replication. The gene encoding human 8-oxoguanine DNA glycosylase 1 (hOGG1), capable of excision repair of 8-oxoguanine, has been recently cloned, characterized, and mapped to the short arm of chromosome 3 (3p25-26), a region showing frequent loss of heterozygosity (LOH) in head and neck squamous cell carcinoma (HNSCC). In the present study, we developed a tissue microdissection approach designed for use with formalin-fixed, paraffin-embedded specimens which is capable of detecting and characterizing the hOGG1 allelic loss using two highly informative, intragenic single nucleotide polymorphisms. Among 45 cases of HNSCC, 18 cases were informative. We analyzed these 18 cases and found that 11 showed evidence of hOGG1 allelic loss. By immunohistochemical staining on a total of 71 HNSCC cases using a commercially available anti-hOGG1 antibody, we showed that hOGG1 gene expression was markedly suppressed in up to 38% of the cases. The frequent allelic imbalance and suppression of the hOGG1 gene thus imply that repair for oxidative DNA damages may be relevant in future studies on head and neck squamous carcinogenesis.     

Methylation, a major mechanism of p16/CDKN2 gene inactivation in head and neck squamous carcinoma.

We studied 11 head and neck squamous carcinoma (HNSC) cell lines and 46 primary tumors for p16 gene status by protein, mRNA, and DNA genetic/epigenetic analyses to determine the incidence, the mechanism(s), and the potential biological significance of its inactivation. Of the 11 cell lines, only 1 showed intact p16 and 10 lacked its protein and mRNA; DNA analysis of these 10 cell lines showed 2 homozygous deletions, 6 methylations at exon 1 and 2, and 2 with no detectable abnormalities. In primary tumors, 16 (34.7%) of the 46 showed detectable p16 protein and mRNA; of these, 12 had no DNA abnormalities and 4 had only exon 2 methylation. Loss of p16 expression was found in three tumors with concurrent mutation at exon 2 and methylation at exon 2 (two) and both 1 and 2 (one). Of the 30 tumors that lacked p16 protein, 27 also lacked mRNA, 1 had detectable p16 mRNA, and 2 failed RT-PCR amplification. Twenty-two of the thirty tumors showed DNA alterations and eight manifested no abnormalities; DNA alterations comprised 6 homozygous deletions, 2 concurrent mutations and methylation of exon 2, and 13 with methylation at exon 1 and exons 1 and 2 (12 with methylation only and 1 with mutation) at exon 1. Except for patients' gender (P = 0.02), no significant correlation between p16 and clinicopathological factors was observed. We conclude that in HNSC 1) intragenic p16 alterations are infrequent events, 2) methylation of exon 1 constitutes a common mechanism in silencing the p16 gene, 3) p16 inactivation may play an important role in the early development and progression of HNSC, and 4) no association between p16 alterations and conventional clinicopathological factors was noted in this cohort.     

Microsatellite instability in esophageal squamous cell carcinoma is not associated with hMLH1 promoter hypermethylation

To test whether a subset of esophageal squamous cell carcinomas (SCC) develop through a deficiency in DNA mismatch repair, we examined microsatellite instability (MSI) using 11 microsatellite markers including BAT-26, hMLH1 protein expression by immunohistochemistry, and methylation status of the hMLH1 promoter by methylation-specific polymerase chain reaction (MSP). p53 mutations were also investigated. Microsatellite instability at one or more loci was observed in 40% (12/30) of esophageal SCC tumor samples, although only one of these tumors was categorized as high-frequency MSI (MSI-H) and none showed BAT-26 instability. While immunohistochemistry revealed decreased hMLH1 protein expression in 27% (8/30) of the tumors, hMLH1 promoter hypermethylation was not observed. Absence of hMLH1 protein expression was relatively common in well-differentiated (keratinizing-type) esophageal SCC, but was not associated with hMLH1 promoter hypermethylation. p53 mutation was detected in 37% (11/30) and loss of heterozygosity (LOH) in 90% (27/30) of esophageal SCC samples. Our results suggested that most esophageal SCC develop through defects in tumor suppressor genes (i.e. the suppressor pathway), and that MSI in esophageal SCC probably represent random replication errors rather than being associated with DNA mismatch repair deficiency.     

DNA ploidy and chromosomal alterations in head and neck squamous cell carcinoma

In head and neck squamous cell carcinomas (HNSCC) the prognostic factors that are routinely considered when deciding therapeutic strategies are still stage and site of the primary tumour, and the presence of nodal or distant metastases. However, it is recognised that these clinical predictors are limited since they do not satisfactorily reflect the biological behaviour of the individual tumour. With the evolving understanding of the genetic and molecular basis of human malignancies, there are an increasing number of factors being claimed to provide prognostic information even in HNSCC. Here we review own and published data on DNA ploidy, karyotyping and molecular cytogenetic changes and its relevance in HNSCC carcinogenesis. The survey suggests that the induction of aneuploidy is a very early event in tumour development being detectable already in non-dysplastic leukoplakia and highly predictive for the subsequent development of a carcinoma. Moreover, specific chromosomal imbalances are associated with different stages of cancer progression and patient's survival, which we have compiled into a progression model of HNSCC.     

MHC class I chain-related gene a diversity in head and neck squamous cell carcinoma

Many immune-related genes are located within the human leukocyte antigen (HLA) region on chromosome 6. The MHC class I chain-related gene A (MICA), located centromeric of HLA-B, is involved in the innate and adaptive immune response through activation of NK and T cells. Differences of MICA transmembrane repeat lengths have been associated with diseases and expression is observed on epithelial tumors. Head and neck squamous cell carcinoma (HNSCC) is an epithelial tumor. In the present study we evaluated the MICA repeat length diversity in relation to MICA expression in Dutch HNSCC patients. MICA short tandem repeat analysis indicated a significant decrease in the frequency for the MICA-A9 repeat in patients diagnosed with oral cavity squamous cell carcinoma (SCC) but not in patients with SCC in the hypoharynx, larynx, or oropharynx. Interestingly, the majority of patients expressed MICA as observed with immunohistochemical staining whereas no soluble MICA was detected in patients' sera by enzyme-linked immunosorbent assay. In conclusion, the length of the MICA transmembrane repeats in Dutch HNSCC patients does not influence the MICA expression on tumor cells.     

Promoter methylation status of the DNA repair genes hMLH1 and MGMT in gastric carcinoma and metaplastic mucosa.

Hypermethylation of CpG islands in the promoter region is associated with the silencing of a variety of tumor suppressor genes. DNA repair genes human Mut L homologue 1 (hMLH1) and O(6)-methylguanine-DNA methyltransferase (MGMT) have been shown to be hypermethylated in certain carcinomas. We studied DNA methylation of CpG islands in hMLH1 and MGMT in 50 gastric carcinomas and 10 intestinal metaplastic mucosa samples. We analyzed the methylation status of hMLH1 and MGMT using methylation-specific polymerase chain reaction and DNA sequencing analysis. We measured protein levels of hMLH1 using Western blot and immunohistochemical analysis. CpG island hypermethylation of hMLH1 and MGMT was detected in 11 (22%) and 8 (16%) of the 50 gastric tumors, respectively. Hypermethylation of the promoter was more common in intestinal-type gastric carcinomas than in poorly diffuse-type gastric carcinomas (p = 0.016 and 0.021, respectively; Fisher's exact test). However, hMLH1 promoter hypermethylation did not coincide with MGMT promoter hypermethylation except in 1 patient. Hypermethylation of the hMLH1 promoter but not the MGMT promoter occurred in intestinal metaplastic mucosae. Immunohistochemical analysis revealed a corresponding reduction in hMLH1 protein expression in some of the intestinal metaplastic mucosae. Our results suggest that at least two types of promoter methylation participate in the development of gastric carcinoma. Tumor-specific promoter hypermethylation of hMLH1 may be an early event in carcinogenesis in the stomach.     

Amplification and overexpression of the cyclin D1 gene in head and neck squamous cell carcinoma

Aims—To determine cyclin D1 gene amplification and expression levels in head and neck squamous cell carcinoma (HNSCC) patients.     

Tid1 functions as a tumour suppressor in head and neck squamous cell carcinoma

Human tumourous imaginal disc (Tid1), a human homologue of the Drosophila tumour suppressor protein Tid56, is involved in multiple intracellular signalling pathways such as apoptosis, cell proliferation, and cell survival. Here, we investigated the anti‐tumourigenic activity of Tid1 in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Firstly, the clinical association between Tid1 expression and progression of HNSCC was explored. It was found that expression of Tid1 was negatively associated with tumour status, recurrence, and survival prognosis using immunohistochemical analysis of primary HNSCC patient tumour tissue. Secondly, ectopic expression of Tid1 in HNSCC cells was shown to significantly inhibit cell proliferation, migration, invasion, anchorage‐independent growth, and xenotransplantation tumourigenicity. Thirdly, we showed that overexpression of Tid1 attenuated EGFR activity and blocked the activation of AKT in HNSCC cells, which are known to be involved in the regulation of survival in HNSCC cells. On the other hand, ectopic expression of constitutively active AKT greatly reduced apoptosis induced by Tid1 overexpression. Together, these findings suggest that Tid1 functions as a tumour suppressor in HNSCC tumourigenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Differential targets of CpG island hypermethylation in primary and metastatic head and neck squamous cell carcinoma (HNSCC)

Head and neck squamous cell carcinomas (HNSCC) often metastasise to the cervical lymph nodes. It is known for HNSCC as well as other cancers that progression from normal tissue to primary tumour and finally to metastatic tumour is characterised by an accumulation of genetic mutations. DNA methylation, an epigenetic modification, can result in loss of gene function in cancer, similar to genetic mutations such as deletions and point mutations. We have investigated the DNA methylation phenotypes of both primary HNSCC and metastatic tumours from 13 patients using restriction landmark genomic scanning (RLGS). With this technique, we were able to assess the methylation status of an average of nearly 1300 CpG islands for each tumour. We observed that the number of CpG islands hypermethylated in metastatic tumours is significantly greater than what is found in the primary tumours overall, but not in every patient. Interestingly, the data also clearly show that many loci methylated in a patient's primary tumour are no longer methylated in the metastatic tumour of the same patient. Thus, even though metastatic HNSCC methylate a greater proportion of CpG islands than do the primary tumours, they do so at different subsets of loci. These data show an unanticipated variability in the methylation state of loci in primary and metastatic HNSCCs within the same patient. We discuss two possible explanations for how different epigenetic events might arise between the primary tumour and the metastatic tumour of a person.     

错配修复基因hMLH1在雌激素诱导结肠癌细胞凋亡中的作用

 摘 要 目的：探讨错配修复基因hMLH1在雌激素诱导结肠癌细胞凋亡中的作用。方法：将含野生型hMLH1-cDNA的质粒转入hMLH1缺陷的人结肠癌细胞株HCT116,在不同浓度的雌激素干预下,分别以流式细胞仪和活细胞计数试剂盒（cell counting kit-8）法检测转染后细胞的凋亡率和活性。结果：与空载组相比,转染hMLH1后雌激素能明显诱导HCT116细胞活性降低,且凋亡率增加。结论:hMLH1参与雌激素诱导的结肠癌细胞株HCT116凋亡。     

Thermoradiotherapy in human head and neck squamous cell carcinoma cell lines

Thermoradiosensitivity of 8 cell lines of head and neck squamous cell carcinoma (HO-1-u-1, HSC2, HSC3, HSC4, SAS, KB, Hep2, and Ca9-22) was investigated. The differences of radiosensitivity between the cell line with the highest radiosensitivity and the cell line with the lowest radiosensitivity were 1.7-, 7.7-, and 41-fold at 2, 6 and 8 Gy, respectively. The differences between the cell line with the highest thermosensitivity and the cell line with the lowest thermosensitivity were 2.4-, 6.2- and 34.4-fold at 43 degrees C for 40, 60 and 100 min, and 2.6-, 4.9- and 127-fold at 44 degrees C for 20, 30 and 50 min, respectively. These findings indicated that there were large differences in both radiosensitivity and thermosensitivity among the 8 cell lines. There was a negative relationship between radiosensitivity and thermosensitivity (43 degrees C: r=-0.600, 44 degrees C: r=-0.848) in 7 of 8 cell lines, the exception being the HSC4 cell line, which was resistant to both therapies. Four of the 8 cell lines at 43 degrees C and 5 at 44 degrees C in the radiotherapy combined with thermotherapy showed actual survival rates smaller than the theoretical survival rates. Thus, thermoradiotherapy was deemed effective in the head and neck carcinoma cell lines, although 1 of 8 cell lines was resistant to both radiotherapy and thermotherapy.     

Amplification and overexpression of PPFIA1, a putative 11q13 invasion suppressor gene, in head and neck squamous cell carcinoma

Chromosomal amplifications of the 11q13 genomic region are frequent in head and neck squamous cell carcinoma (HNSCC). To identify novel 11q13 amplification targets, we integrated high-resolution array-based comparative genomic hybridization and Affymetrix gene-expression profiling of eight HNSCC cell lines. We found that PPFIA1 was the highest upregulated gene in the 11q13 amplicon of HNSCC cell lines when compared with HNSCC lines without 11q13 amplification and confirmed the upregulation of PPFIA1 in primary HNSCCs by real-time PCR. Using siRNA knockdown, we investigated PPFIA1 function in three HNSCC lines using both in vitro invasion assays and wound-healing assays. Surprisingly, we found that cancer cells become more invasive when the PPFIA1 protein levels were reduced, suggesting that PPFIA1 may act as an invasion inhibitor in HNSCC. This unexpected result suggests that the 11q13 amplicon may comprise both positive and negative regulators involved in HNSCC. Our study is the first to evaluate the role of PPFIA1 in head and neck carcinogenesis and suggests a potential link between PPFIA1 activity and cell-extracellular matrix interactions. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Induced expression of syndecan-1 in the stroma of head and neck squamous cell carcinoma.

Syndecan-1 (CD138), a cell-surface heparan sulfate proteoglycan, is involved in cell-cell, cell-matrix interaction and growth factor binding. Loss of expression of syndecan-1 in tumor cells leads to decreased intercellular cohesion, increased potential for tumor invasiveness, and metastatic spread. Furthermore, induction of syndecan-1 expression in the tumor stroma has been postulated to promote tumor angiogenesis via its binding to growth factors such as basic fibroblast growth factor. Although syndecan-1 expression within tumor cells has been investigated in head and neck squamous cell carcinoma, stromal expression has not been studied in detail. We analyzed 38 cases of head and neck squamous cell carcinoma by immunohistochemical staining for syndecan-1 expression within the stroma. The expression of syndecan-1 within tumor cells of various histologic grades of differentiation, squamous cell carcinoma in situ cells, and benign squamous epithelium was also determined. Variable levels of diminished syndecan-1 expression were noted within the dysplastic cells of 9 of 16 (60%) squamous cell carcinoma in situ lesions and in all 38 (100%) invasive squamous cell carcinoma. In general, higher levels of syndecan-1 expression were observed in the well-differentiated tumors, in contrast to significant reduction of expression seen in poorly differentiated tumors. Syndecan-1 expression was observed within the stroma (in fibroblasts) surrounding infiltrating carcinoma cells in 28 of 38 (74%) cases. The intensity of syndecan-1 staining within the stroma showed generally an inverse correlation with the degree of tumor cell differentiation. Syndecan-1 expression was not detected in the stroma beneath normal squamous epithelium or adjacent to areas of squamous cell carcinoma in situ. We conclude that induced expression of syndecan-1 in the stroma surrounding tumor cells of invasive head and neck squamous cell carcinoma is a frequent event. The increased stromal syndecan-1 expression, coupled with its loss from the surface of carcinoma cells, may contribute to tumor cell invasion and the development of metastases.     

Gene amplification and gene dosage in cell lines derived from squamous cell carcinoma of the head and neck.

Gene amplification and related alterations in gene dosage were analyzed in a series of 34 cell lines derived from different human head and neck squamous cell carcinomas (SCCHN). INT2 gene amplification was observed in 62%, MYC gene amplification in 24%, and EGFR gene amplification in 21% of the cell lines. There was a strong correlation between EGFR gene amplification and increased copies of the ERBB2 gene on chromosome 17, suggesting a synergistic selection for these two genes either during cancer progression or in culture. Two abnormalities showed a significant correlation with clinical course: MYC gene amplification showed an inverse correlation with tumor recurrence (r = -0.44, p = 0.01), and a small increase in MYCL gene copies on chromosome I correlated with the presence of metastases (r = 0.61, p = 0.001). This altered MYCL gene dosage might represent a chromosome translocation rather than true gene amplification. In addition to gene amplification, 79% of the cell lines had increased copies of chromosome 8. Comparison of the cell lines with several of the corresponding primary tumors demonstrated that most gene amplifications were already present in the primary tumors, although some appeared de novo in cell culture. These studies indicate that gene amplification, especially of INT2, is a prominent abnormality in head and neck squamous cell cancer. Aneuploidy and chromosomal lesions other than gene amplification were also found to alter the dosage of several oncogenes specifically.