Oral Carcinogenesis: Studies With Human Gingival Keratin

口腔癌发生：人类牙龈角蛋白的研究

• 批准号: 6966498
• 负责人: MYUNG H PARK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966498
• 关键词: carcinogenesis; gene expression; head /neck neoplasm; human subject; keratinocyte; microarray technology; oral pharyngeal neoplasm

Carcinogenesis is a multi-step process involving the activation of cellular proto-oncogenes and inactivation of tumor suppressor genes. Epithelial cells undergo different stages of phenotypic and genotypic alterations and gradually acquire malignant growth characteristics. In spite of the relatively high frequency of oral cancers, little is known about the molecular events that lead to disease initiation and progression. Previously we reported that normal human oral keratinocytes (NHOK) can be induced to undergo terminal differentiation by high Ca2+ in culture. The capacity of the HPV 16 E6/E7 immortalized human oral keratinocytes (IHOK) to form cornified envelopes was significantly reduced compared to NHOK and correlated with the reduced induction of transglutaminase 1. Furthermore, IHOK cells were resistant to growth inhibition by TGF-beta, probably due to inactivation of the Rb pathway by HPV16 E7. In contrast to NHOK and IHOK, several HNSCC lines were totally incapable of induction of transglutaminase 1 and cornified envelope formation. The goal of our study is to understand the genetic and molecular changes that occur during HNSCC carcinogenesis. To this end we carried out gene expression studies of head and neck squamous cell carcinoma (HNSCC) lines to relate the changes in the gene expression profiles with phenotypic alterations. Global gene expression profiles of human head and neck squamous carcinoma cell lines : We have conducted microarray experiments using a NCI-Onco chip with 9700 cDNA clones for a systematic analyses of gene expression profiles in 25 HNSCC cell lines and IHOK. We used NHOK as a reference in this study to characterize gene expression changes associated with HNSCC carcinogenesis. Genes primarily involved in cell cycle regulation, oncogenesis, cell proliferation, differentiation, apoptosis and cell adhesion were widely altered in HNSCC cells. Genes consistently upregulated in most HNSCC cells included growth factors (EGF receptor pathway substrate 8, IGF binding proteins3 and -6), DNA binding proteins and transcription factors (topoisomerase II alpha, PCNA, replication protein 3), cell cycle regulatory genes (CDC20, CDC25B, serine/threonine kinase 6), oncogenes (DEK oncogene, ECT2, and stathmin1), TTK protein kinase, cytokines IL8 and TNFSF10, antiapoptotic gene survivin, and stress related gene DHFR. The most remarkable feature of the gene expression profiles of HNSCC was the coordinated pattern of downregulation of a wide range of genes involved in terminal differentiation (transglutaminase 1 and -3, SPRR1A and -B, SPRR3, various cytokeratins 1,5,6A, 7, 13, 14, 15, 16, and 17) and cell adhesion (annexins 1,2 and 3, cadherins 1 and 13, laminins alpha 3, beta 3 and gamma 2, integrins alpha 5 and beta 1). This finding is consistent with the previous observation of the loss of competency to form cornified cell envelopes in several HNSCC cell lines. These findings suggest that the loss of the cellular differentiation machinery represents a critical feature of HNSCC. Furthermore, hierarchical clustering analysis, as well as principal component analysis, revealed two distinct subtypes of gene expression patterns among the 26 cell lines, reflecting a degree of heterogeneity in HNSCC. By applying Significance Analysis of Microarrays, 136 genes were selected for being distinctively expressed between the two groups. Genes differentially expressed in the two subgroups include cell proliferation-related genes, IGFBP6, EGFR, and VEGFC; tumor suppression and apoptosis related genes such as Tp53, Tp63 as well as cell cycle regulators such as CCND1 and CCND2 (cyclins D1 and D2) suggesting that the two subgroups might have undergone different pathways of carcinogenesis. Group I, that seems to represent more malignant properties include Hep2, KB, HN8, HN22, HN13, HN19, UMSCC-22B and UMSCC-74B. Group II consists of 18 cell lines including IHOK, HN4, HN6, HN12, HN1117, HN26, HN30, HN31, PCII-15B, and UMSCC-11A, -11B, -14A, -14B, -14C, -17A, -17B, and -22A. It is interesting to note that the segregation of 11 HN series cell lines from this study is in total agreement with the subgrouping from the previous microarray analysis (6700 cDNA chip) done with a human cancer RNA pool as a reference. Cells in group I showed lower expression of proliferation genes and cell adhesion genes than group II cells, but stronger expression of anti-apoptotic genes. These findings suggest that group I cells represent a later stage of tumorigenesis than group II cells. This idea is also line with the finding that IHOK clustered with group II. We have searched for potential genes that may be responsible for the across the board loss of terminal differentiation in HNSCC cells. We have found that the expression of a putative tumor suppressor gene, KLF4, and of TGF beta receptors 1 and -2 was significantly reduced in HNSCC cells. The role of these genes and a potential of the TGF beta pathway signaling in the regulation of the zinc-finger transcription factor, KLF4, in oral carcinogenesis is under study.

癌的发生是一个多步骤的过程，涉及细胞原癌基因的激活和肿瘤抑制基因的失活。上皮细胞经历不同阶段的表型和基因型改变，并逐渐获得恶性生长特征。尽管口腔癌的发病率相对较高，但对导致疾病发生和进展的分子事件知之甚少。以前我们报道过正常人口腔角质形成细胞（NHOK）可以被高Ca ~（2+）诱导终末分化。与NHOK相比，HPV 16 E6/E7永生化的人口腔角质形成细胞（IHOK）形成角膜上皮包膜的能力显著降低，并且与转氨酶1的诱导降低相关。此外，IHOK细胞对TGF-β的生长抑制具有抗性，这可能是由于HPV 16 E7使Rb途径失活。与NHOK和IHOK相反，一些HNSCC系完全不能诱导转氨酶1和皮质包膜形成。我们研究的目的是了解HNSCC癌变过程中发生的遗传和分子变化。为此，我们进行了基因表达研究的头部和颈部鳞状细胞癌（HNSCC）线与表型改变的基因表达谱的变化。 人头颈部鳞状细胞癌细胞系的总体基因表达谱： 我们进行了微阵列实验，使用NCI-Onco芯片与9700 cDNA克隆的基因表达谱在25 HNSCC细胞系和IHOK的系统分析。在这项研究中，我们使用NHOK作为参考，以表征与HNSCC癌变相关的基因表达变化。主要涉及细胞周期调控、肿瘤发生、细胞增殖、分化、凋亡和细胞粘附的基因在HNSCC细胞中广泛改变。在大多数HNSCC细胞中持续上调的基因包括生长因子（EGF受体途径底物8、IGF结合蛋白3和-6）、DNA结合蛋白和转录因子（拓扑异构酶II α，PCNA，复制蛋白3），细胞周期调控基因（CDC 20、CDC 25 B、丝氨酸/苏氨酸激酶6）、癌基因（DEK癌基因、ECT 2和stathmin 1）、TTK蛋白激酶、细胞因子IL 8和TNFSF 10、抗凋亡基因生存素和应激相关基因DHFR。HNSCC基因表达谱最显著的特征是参与终末分化的广泛基因的协同下调模式（转氨酶1和-3，SPRR 1A和-B，SPRR 3，各种细胞角蛋白1，5，6A，7，13，14，15，16和17）和细胞粘附（膜联蛋白1、2和3，钙粘蛋白1和13，层粘连蛋白α 3、β 3和γ 2，整联蛋白α 5和β 1）。这一发现与先前在几种HNSCC细胞系中观察到的形成皮质细胞包膜的能力丧失一致。这些发现表明细胞分化机制的丧失是HNSCC的一个重要特征。此外，层次聚类分析，以及主成分分析，揭示了两个不同的亚型的基因表达模式之间的26个细胞系，反映了一定程度的异质性，在HNSCC。应用基因芯片显著性分析，筛选出136个基因在两组间差异表达。在两个亚组中差异表达的基因包括细胞增殖相关基因IGFBP 6、EGFR和VEGFC;肿瘤抑制和凋亡相关基因如Tp 53、Tp 63以及细胞周期调节因子如CCND 1和CCND 2（细胞周期蛋白D1和D2），这表明两个亚组可能经历了不同的致癌途径。组I，似乎代表更恶性的性质，包括Hep 2、KB、HN 8、HN 22、HN 13、HN 19、USCC-22 B和USCC-74 B。II组由18种细胞系组成，包括IHOK、HN 4、HN 6、HN 12、HN 1117、HN 26、HN 30、HN 31、PCII-15 B和USCC-11 A、-11 B、-14 A、-14 B、-14 C、-17 A、-17 B和-22 A。值得注意的是，本研究中11个HN系列细胞系的分离与以前用人癌RNA库作为参考进行的微阵列分析（6700 cDNA芯片）的亚组完全一致。I组细胞增殖相关基因和细胞粘附相关基因的表达低于II组细胞，但抗凋亡相关基因的表达高于II组细胞。这些发现表明，I组细胞代表比II组细胞更晚的肿瘤发生阶段。这一想法也与IHOK与第II类聚类的发现一致。 我们已经寻找了可能导致HNSCC细胞终末分化全面丧失的潜在基因。我们已经发现，一个假定的肿瘤抑制基因，KLF 4，和TGF β受体1和-2的表达显着减少在HNSCC细胞。这些基因的作用和潜在的TGF β通路信号在锌指转录因子KLF 4的调节，在口腔癌的发生正在研究中。