Elsevier

European Journal of Cancer

Volume 38, Issue 12, August 2002, Pages 1585-1592
European Journal of Cancer

Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma

https://doi.org/10.1016/S0959-8049(01)00422-1Get rights and content

Abstract

Studies of allelic imbalance and suppression of tumourigenicity have consistently suggested that the short arm of chromosome three (3p) harbours tumour suppressor genes (TSGs) whose inactivation leads to the development of various types of neoplasia including head and neck squamous cell carcinoma (HNSCC). Previously, we defined a critical minimal region of 120kb at 3p21.3 that contains overlapping homozygous deletions in lung and breast tumour lines and isolated eight genes from the minimal region. Mutation analysis in a large panel of lung and breast cancers revealed only rare mutations, but the majority of lung tumour lines showed loss of expression for one of the eight genes (RASSF1A) due to hypermethylation of a CpG island in the promoter region of RASSF1A. We found RASSF1A to be methylated in the majority of lung tumours, but to a lesser extent in breast and ovarian tumours. In order to define the role of 3p TSGs, in particular RASSF1A in HNSCC, we (a) analysed 43 primary HNSCC for allelic loss in regions proposed to contain 3p TSGs (3p25-26, 3p24, 3p21-22, 3p14 and 3p12), (b) analysed 24 HNSCC for evidence of RASSF1A methylation and (c) undertook mutation analysis of RASSF1A in HNSCC. We found that 81% of HNSCC showed allele loss at one or more 3p markers, 66% demonstrated loss for 3p21.3 markers and 56% showed allelic losses at 3p12 loci. Thus, 3p loss is common in HNSCC and extensive 3p loss occurs even in early stage tumours. RASSF1A promoter region hypermethylation was found in 17% (4/24) of the sporadic HNSCC, but RASSF1A mutations were not identified. Furthermore, we found RASSF1A methylation to be significantly higher in poorly differentiated then in moderate to well differentiated HNSCC (P=0.0048). Three of the four tumours showing RASSF1A methylation also underwent 3p21.3 allelic loss, hence RASSF1A behaves as a classical TSG (two hits, methylation and loss). One tumour with RASSF1A methylation had retention of markers at 3p providing further evidence of specific inactivation of RASSF1A as a critical step in some HNSCC. Although the frequency of 3p21.3 allele loss was substantially higher than that of RASSF1A methylation this does not necessarily suggest that other genes from 3p21.3 are also implicated in HNSCC, as 3p21.3 LOH was invariably found with LOH at other 3p loci. Thus, the presence of 3p21.3 allele loss without RASSF1A methylation might reflect a propensity for 3p21.3 loss to occur as a secondary consequence of large 3p deletions targeted at other 3p TSG regions. Furthermore, in the presence of homozygous inactivation of other 3p TSGs, RASSF1A haploinsufficiency might be sufficient to promote tumourigenesis in many HNSCC.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is a common neoplasm associated with exposure to tobacco and alcohol. It accounts for up to 5% of all malignancies in the Western world [1]. There are major geographical differences in the incidence of HNSCC, in the Far East and the Indian subcontinent up to 40% of malignancies occur in the head and neck region [2]. Recent evidence from epidemiological studies suggest an increased incidence and mortality of head and neck cases particularly in developed countries with young males being the worst affected. Changes in tobacco and alcohol habits seem to be the most likely explanation for the trend.

The activation of oncogenes and inactivation of tumour suppressor genes (TSGs) are mechanisms that play pivotal roles in the multistep molecular process of tumourigenesis. Chromosomal arms 1p, 3p, 4, 2q, 8p, 9p, 11q, 13q, 17p, 18q (3, 4, 5, 6, 7, reviewed in Ref. [8]) show high levels of allelic losses in HNSCC. These regions of high levels of loss of heterozygosity (LOH) have been corroborated by cytogenetic and comparative genome hybridisation studies in many cases.

Deletions of 3p are also frequent in many other common sporadic cancers including lung, breast, kidney, cervical and ovarian cancer (reviewed in Ref. [9]). Regions which have been consistently implicated as harbouring 3p HNSCC TSGs mapped by LOH to a number of loci— namely 3p25, 3p21.3, 3p14.2 and 3p12 7, 10 and the multiplicity of sites implies the possibility of multiple TSGs which may either operate singly or in concert, depending on the carcinogen involved. These regions are in part concordant with some other sites of intensive investigation in lung, breast and other cancers. The von Hippel–Lindau (VHL) TSG at 3p25 11, 12 has been excluded as a candidate HNSCC TSG using mutation and methylation studies 7, 13. The region at 3p21.3 is thought to harbour at least two TSGs. One region (LCTSGR1) is defined by overlapping homozygous deletions in a breast tumour and in three small cell lung cancer tumour lines with a 120kb minimal commonly deleted region 14, 15, 16. Eight genes have been isolated from the minimal region, but none shows frequent mutations in lung or breast tumours [16]. Very recently, we and others have shown that one of the eight genes from the 120kb minimal region (RASSF1A) is silenced by hypermethylation of a CpG island in its promoter region in lung and breast tumours and tumour lines 17, 18, 19. The other region is telomeric to the first region and contains a 800kb homozygous deletion in one lung tumour line [20]. FHIT [21], a putative TSG which resides at the 3p14.2 FRA3B fragile site, has for a while been thought of as a strong candidate in multiple cancer types and in HNSCC has been found to demonstrate homozygous deletions and aberrant transcripts [22] in cell lines. In addition, a 3p12 region (LCTSGR2) is implicated as a candidate TSG interval by the finding of overlapping homozygous deletions in two small cell lung cancer lines and in one breast tumour line and contains the DUTT1 candidate gene [23].

The functional relevance of 3p in HNSCC is demonstrated by suppression of tumorigenicity studies which have suggested 3p21.3 and 3p21.3-p21.2/3p25 as regions containing TSGs in nasopharyngeal carcinoma [24] and in oral cancer [25], respectively. Studies examining the relationship between tumour stage and grade with LOH rate have suggested an accumulation of aberrations with clinical progression and 3p loss has also been shown to be an early event in oral tumorigenesis 26, 27. Similar findings have been published for lung cancer [28]. The fact that smoking is a risk factor of such dominant importance in both HNSCC and lung cancer means that lung cancer TSG regions should be carefully tested to determine whether they harbour genes that are critical in both of these tumour types.

In order to investigate the role of 3p TSGs in the pathogenesis of HNSCC, we performed detailed studies of 3p allelic loss in primary HNSCC using microsatellite markers spread across 3p and including recently developed markers from within regions LCTSGR1 at 3p21.3 and LCTSGR2 at 3p12, regions that have been implicated in other common cancers including lung. We also analysed the methylation and mutation status of a newly identified TSG from region 3p21.3 (RASSF1A), which we have previously reported to be hypermethylated in the majority of lung tumours.

Section snippets

Clinical material

Tumour tissue samples and patient-matched normal blood or mucosa were collected at the Queen Elizabeth Medical Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK. Amongst the HNSCC, there were 23 laryngeal, 13 pharyngeal, five from the oral cavity and two paranasal sinuses. Histological grade: eight were well differentiated, 19 were moderately differentiated, 13 were poorly differentiated and for three HNSCC the grade was unknown. Clinical Stage: one was stage 1, nine were

3p LOH analysis in head and neck tumours

We determined the frequency, extent and patterns of 3p loss in 43 HNSCC using 23 polymorphic microsatellite markers spanning 3p, but targeted to regions containing known or putative 3p TSGs (Fig. 1). One tumour showed microsatellite instability for the majority of the 3p markers analysed. 34 tumours (81%) showed LOH for at least one of the 3p loci tested. 15 tumours showed LOH of all informative markers (indicating a complete deletion of 3p), eight tumours showed no loss for any 3p informative

Discussion

Loss of 3p occurs commonly in head and neck tumours. Previous studies in HNSCC (and comparable studies in lung cancer) have consistently indicated that this loss is maximal at 3p25, 3p21.3, 3p14.2 and 3p12 7, 10, 31. In order to further define the pattern and extent of 3p loss, we performed fine deletion mapping in a large set of head and neck primary tumours by using 23 microsatellite markers encompassing the entire 3p arm, but focusing on regions that have previously been localised as strong

Acknowledgements

This work is supported, in part, by the Association for International Cancer Research, Cancer Research Campaign and Get A-Head charity. R.P.H. is a recipient of a fellowship from Royal College of Surgeons of England (Newman fellowship). M.J.K. is a recipient of a Medical Research Council/Royal College of Surgeons of England Joint Special Training Fellowship. S.H. is supported by Portuguese Foundation for Science and Technology. A.M. is supported by the University of Antioquia, Medellin,

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