Molecular introduction to head and neck cancer (HNSCC) carcinogenesis
Introduction
Oral cancer is one of the few cancer types where it is possible to obtain biopsies at all stages of the disease and study the genetic progression of tumorigenesis to metastasis. The pioneering work on the characterization of genetic alterations of colorectal cancer by Fearon and Vogelstein has become a paradigm for other human neoplasias (Diagram 1).1 It is now proposed that HNSCC follows a similar genetic progression in its development from premalignant lesions such as leukoplakia, dysplasia, erythroplakia and lichen planus. The precise nature of genetic alterations occurring at each step is still unclear but Califano et al. 1996 have described a preliminary HNSCC molecular progression model from benign to the invasive state with microsatellite analysis for allelic loss at 10 major chromosomal loci. The spectrum of chromosomal loss progressively increased at each histopathological step from benign hyperplasia to dysplasia to carcinoma in situ to invasive cancer (Diagram 2).2 Current established view on HNSCC formation requires the following three genetic alterations. The alterations are the p53 tumour suppressor protein, inactivation of the cyclin dependent kinase inhibitor p16 and overexpression of epidermal growth factor receptor (EGFR) in 40%,3 70%,4 and 90%5 of all oral cancers, respectively. With the advent of cDNA profiling of many thousands of genes in a single assay, a wide range of molecular markers are currently being identified that can distinguish patients' prognosis preoperatively. Traditionally, carcinogenesis and tumour progression has been attributed to alterations in oncogenes or tumour suppressor genes. This review will focus on the various established types of genetic alterations in HNSCC.
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Hereditary
There is now increasing epidemiological evidence from case control studies of HNSCC patients that a family history of HNSCC is a risk factor. One matched case-control study using first-degree relatives of patients with new HNSCC, and first-degree relatives of the patients' spouses as controls, demonstrated an increased relative risk of 3.5 in association with a positive family history.6 Another large age and sex-matched study adjusting for alcohol consumption showed a similar relative risk of
The oncogenes
Oncogenes encode proteins that result in abnormal cell growth or tumorigenesis when overexpressed or mutated. The majority of these oncogenes are growth factors or growth factor receptors (hst-1, int-2, EGFR/erbB, c-erbB-2/Her-2, sis), intracellular signal transducers (ras, raf, stat-3), transcription factors (myc, fos, jun, c-myb), cell cycle regulators (Cyclin D1) and those involved in apoptosis (bcl-2, Bax). These growth regulatory pathways can be altered by gene mutation,8 chromosomal
Growth factor and receptors
EGFR (epidermal growth factor receptor) and its ligands have been studied extensively in HNSCC.12., 13., 14., 15. The m-RNA for EGFR was elevated in 69-fold in 92% of tumours when compared with normal mucosa.5 Overexpression of c-erbB-2 (Her-2), which is an EGFR like oncogene located on chromosome 17, has been observed in 75% of HNSCC patients and correlated to shorter survival.16
Chromosome 11q13 amplification
Fractional or entire DNA loss of chromosome 3p was found in 97% and amplifications of 11q13 region in 70% of primary HNSCC tumours.17 Amplification and rearrangements of the 11q13 region in primary HNSCC tumours has been reported in between 30–60% of cases using Southern hybridisation analysis.18., 19., 20. Amplification of the 11q13 region was correlated with high grade, late stage, aneuploid tumours,21., 22. poor prognosis,23 recurrence and distant metastasis.24
The oncogenes present in 11q13
The cell cycle regulator genes
Cylin D1 gene regulates initiation of DNA synthesis and G1/S transition of cells. It was initially identified as the bcl-1 gene at chromosome 11q13, at the site of translocation t(11:14) (q13:q32) in B-cell malignancies.27 Amplification of Cyclin D1 gene was reported in 34% of HNSCC.21., 24. The dysregulation of cyclin D1 expression has been shown to occur early during the tumorigenesis process in HNSCC and enables subsequent cyclin D1 gene amplification.28 The overall 5-year survival of HNSCC
Transcription factors
Saranath et al. in30 1989 studied 23 primary HNSCC and observed a 5- to 10-fold amplification of one or more of c-myc, N-myc, Ki-ras and N-ras oncogenes in 56% of the tumour tissue samples, with these oncogenes not being amplified in the peripheral blood cells of the same patients. L-myc and H-ras were not amplified in any of the samples. The oncogene amplifications seemed to be associated with advanced stages of squamous cell carcinomas, with the ras and myc family oncogenes being amplified in
Intracellular transducers
Up-regulation of EGFR occurs early in squamous cell carcinogenesis and is critical for the loss of growth control in a variety of human cancers, including HNSCC. The Jak/Stat signalling pathway transmits signals from many cytokines and growth factor receptors to target genes in the nucleus. In HNSCC cells culture, EGFR stimulation initiates signaling via persistent activation of selective STAT proteins. Grandis et al. 2000 were first to provide evidence that constitutively activated Stat-3 is
The Tumour suppressor genes and proteins
Tumour suppressor genes serve as transducers of negative growth signals.37 These genes are involved in cell cycle regulation including cell cycle arrest and apoptosis. Tumour suppressor genes can be altered in their functions by several mechanisms including point mutations, deletions or binding with cellular and viral proteins.37., 38.
Alterations in the p53 gene expression
The p53 gene is located on the short arm of chromosome 17p13.1 and encodes a 53 kDa nuclear phosphoprotein that maintains genome stability. It regulates cell cycle progression, cellular differentiation, DNA repair and apoptosis.39 Mutations of the p53 protein are the most frequent genetic alterations found in human malignancies.40
Usually, one of the p53 alleles is lost through a deletion, and the other is mutated. Thus, the tumour cells are, for all practical purposes, homozygous for the loss
Tumour suppressor gene p16 (CDKN2A, MTS1-multiple tumour suppressor 1)
The p16 protein is a tumour suppressor protein of 156 amino acids; 16.5 kDa in size and the gene, called CDKN2A (cyclin-dependent kinase inhibitor 2A) is located at 9p21.64 It interacts strongly with CDK4 and CDK6 and inhibits its ability to interact with Cyclin D and function as a negative regulator of proliferation of normal cells.65 This effect is mediated through the retinoblastoma protein (Rb), another tumour suppressor.
The Rb protein is found in the nucleus of cells. When active
Regulation of apoptosis
Bax, Bcl-2, and p53 proteins are involved in the regulation of apoptosis and have been reported to correlate with prognosis in several tumour types. Over-expression of Bcl-2 and loss of Bax expression is significantly associated with poor prognosis. High apoptosis was significantly associated with high Bax expression and highly differentiated tumours.71
Other tumour suppressor genes associated with HNSCC
The p15 gene has been designated MTS2 ‘multiple tumour suppressor 2’ (CDKN2B). It is deleted in 10–50% of HNSCC.72 It is thought that loss of 9p is an early event in the development of HNSCC.2 Loss of heterozygosity (LOH) at 9p21-22 was reported in 72% of 29 HNSCC cases studied.73
The p21 gene is mapped to chromosome 6p21. The p21 tumour suppressor protein was found to inactivate cyclin E-cdk-2 and cyclin D1, D2, D3- cdk-4 complexes components of the regulatory kinases that target pRB for
Angiogenesis
Tumour growth is associated with elevated cellular activities and increased blood supply is crucial for its continuing development. The process of angiogenesis is in itself a multi-step process that appears to be regulated by both stimulatory and inhibitory factors.77 Steps critical to successful neovascularization include degradation of the extracellular matrix, endothelial cell proliferation, migration and remodelling of extracellular matrix.
Angiogenesis has been linked to increased
Acknowledgements
The authors would like to acknowledge and thank the following funding bodies: Queensland Cancer Fund Clinical Fellowship Garnett-Passe and Rodney Williams Memorial Foundation Project Grant University of Queensland Graduate School Scholarship.
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