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

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.

头颈部鳞状细胞癌（Squamous cell carcinoma of the head and neck, HNSCC）是发达国家中第六大常见癌症。绝大多数这类恶性肿瘤累及口腔、唇、喉及咽部的肿瘤性病变。本研究旨在阐明促成HNSCC发生的遗传改变，并利用相关知识开发预示恶性转化的分子标志物。我们预期这些工作将有助于口腔癌病变的早期检测，同时为这一毁灭性疾病发现新的药理学干预潜在靶点。研究HNSCC肿瘤进展不同阶段的基因表达谱，通常会受到这类肿瘤性病变显著异质性的限制。激光捕获显微切割（Laser Capture Microdissection, LCM）等新技术的突破，现已为特定细胞群体的基因与蛋白质表达分析提供了独特平台。我们团队近期开发了基于LCM的技术，从具有代表性的HNSCC队列及其匹配的正常组织中获取肿瘤性与表型正常的细胞。研究证实，激光辅助显微切割5000个细胞即可提取完整性良好的总RNA（14.7~18.6 ng），用于合成标记扩增的互补DNA（complementary DNA, cDNA）探针，随后可将其与排布有已知人类癌症相关cDNA的膜阵列进行杂交。通过该方法将HNSCC与正常组织进行对比，发现癌细胞的分化标志物（如细胞角蛋白）表达持续下调，而多种信号转导、细胞周期调控分子、生长及血管生成因子、基质降解蛋白酶的表达均出现上调。出乎意料的是，大多数HNSCC会过表达Wnt与Notch生长及分化调控系统的成员，这提示它们可能参与鳞状细胞癌变过程。此外，我们借助LCM技术启动了一项基因发现工作，包括从显微切割的HNSCC组织中构建cDNA文库。本研究的HNSCC组织队列包含口腔鳞状细胞癌及其匹配的正常组织。分离得到的RNA通过寡聚(dT)介导的逆转录合成平端双链cDNA，随后添加接头。使用带有尿嘧啶DNA糖苷酶（Uracil-DNA Glycosylase, UDG）兼容末端的接头特异性引物，通过PCR扩增这些cDNA，并将产物亚克隆至pAMP10克隆载体。在初始分析中，我们对6个文库各自的96个克隆进行了随机测序。结果显示，76%~96%的插入片段分别为匿名表达序列标签（Expressed Sequence Tag, EST）（25%~48%）、已知基因（9%~29%）或新序列（27%~51%），冗余度极低。上述发现表明，可从显微切割组织中构建高质量、具有代表性的cDNA文库，并由此鉴定出多个HNSCC特异性新基因。为着手解析HNSCC的分子基础，我们进一步使用强大的生物信息学工具分析了这6个显微切割文库。通过新开发的搜索引擎，我们发现这些文库[HN7（正常组织）与HN8（高分化浸润性癌）；HN9（正常组织）与HN10（原位癌）；HN11（正常组织）与HN12（中-低分化浸润性癌）]包含138个未知独特基因，以及大量未知非独特基因。从已知基因的现有信息中，我们也开始认识到该肿瘤类型独特的基因表达模式。后续我们通过LCM平台与高通量基因阵列技术进一步分析，获得了大量与鳞状癌细胞定植及生长相关的基因信息。此外，这些工作还发现了至少189个新基因，它们可能在HNSCC的发病机制中发挥作用，因此可作为该疾病早期检测的新型标志物以及药理学干预靶点。最后，所有数据与DNA克隆均已提交至公共领域，可供科学界开展进一步研究。