Pattern of gene expression in squamous cell carcinoma of head and neck

Abstract

Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common cancer in the developed world. The vast majority of these malignancies involve neoplastic lesions in the oral cavity, lip, larynx, and pharynx. The goal of this study is to help elucidate the genetic changes contributing to HNSCC, and to use this knowledge to develop molecular markers heralding malignancy. We expect these efforts will facilitate the early detection of oral cancer lesion, as well as the discovery of novel potential targets for pharmacoligical intervention in this devastating disease. The ability to investigate gene expression profiles at different stages of tumor progression in HNSCC is usually limited by the remarkable heterogeneity of these neoplastic lesions. New technological breakthroughs, such as the development of laser capture microdissection (LCM), have now provided a unique platform for gene and protein expression analysis in specific cell populations. We have recently developed LCM-based techniques to procure neoplastic and phenotypically normal cells from representative sets of HNSCCs and their matching normal tissues. Indeed, we found that the laser assisted microdissection of 5,000 cells was sufficient to extract total RNA (14.7-18.6 ng) of high integrity for the synthesis of labeled amplified cDNA probes which could then be hybridized to membranes arrayed with known human cancer-related cDNAs. By this approach, HNSCCs were compared to normal tissues, and found that cancer cells exhibit a consistent decrease in expression of differentiation markers such as cytokeratins, and an increase in the expression of a number of signal transducing and cell cycle regulatory molecules, growth and angiogenic factors, and matrix degrading proteases. Unexpectedly, most of the HNSCCs overexpress members of the wnt and notch growth and differentiation regulatory systems, thus suggesting that they may contribute to squamous cell carcinogenesis. Furthermore, we took advantage of the LCM technology for the launching of a gene discovery effort, which involved the generation of cDNA libraries from microdissected HNSCC tissues. HNSCC tissue sets comprised oral squamous cell carcinomas and matching normal tissues. Isolated RNAs were used for the synthesis of blunt-ended, double strand cDNAs by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with UDG-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. For our initial analysis, ninety-six clones from each of the 6 libraries were randomly sequenced. Results indicated that 76-96% of the inserts represented either anonymous ESTs (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These findings indicated that high quality, representative cDNA libraries can be generated from microdissected tissues, and led to the identification of a number of novel HNSCC specific genes. In an effort to begin addressing the molecular basis of HNSCC, these 6 microdissection libraries were further analyzed using powerful bioinformatic tools. Using newly developed search engines, we found that these libraries [HN7 (normal) and HN8 (well differentiate invasive carcinoma); HN9 (normal) and HN10 (carcinoma in situ); HN11 (normal) and HN12 (moderate to poorly differentiated invasive carcinoma)] include 138 unknown unique genes, and a large number of unknown non-unique genes. From the available information on known genes, we have also begun to appreciate the unique pattern of gene expression in this tumor type. This was further analyzed using the LCM platform and high-throughput gene array technologies, which has provided a wealth of information on genes that are likely responsible for the establishment and growth of squamous carcinoma cells. Furthermore, these efforts led to the discovery of at least 189 novel genes, which may have a role in the pathogenesis of HNSCC, and thus may represent novel markers for early detection as well as targets for pharmacological intervention in this disease. Finally, all data and DNA clones have been deposited in the public domain, and are available for further investigation by our scientific community.