Oral Carcinogenesis

口腔癌发生

• 批准号: 7593368
• 负责人: J Gutkind
• 金额: $ 180.45万
• 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/7593368
• 关键词: Address; Affect; Alcohols; Alopecia; Animal Model; Animals; Apoptotic; Architecture; Area; Behavior; Bioinformatics; Cell Cycle Regulation; Cell Maintenance; Cells; Cessation of life; Chemopreventive Agent; Clinical; Cytokeratin; DNA; Depth; Detection; Development; Differentiation Antigens; Disease Progression; Disease regression; Dissection; Epidermal Growth Factor Receptor; Epidermis; Epithelial; Epithelial Cells; Event; Evolution; Excision; Exhibits; Formalin; Freezing; Future; Gene Proteins; Genes; Genetic; Genomics; Goals; Growth; Hair; Hair follicle structure; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Head and neck structure; Institution; International; Knowledge; Lesion; Lip structure; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Modality; Molecular; Molecular Profiling; Monomeric GTP-Binding Proteins; Mouth Neoplasms; Mus; Mutation; Natural regeneration; Nature; Neoplasm Metastasis; Newborn Infant; Numbers; Oncogenes; Oncogenic; Oral; Oral cavity; Oral mucous membrane structure; Paraffin Embedding; Pathway interactions; Patients; Pattern; Pharyngeal structure; Phospho-Specific Antibodies; Phosphotransferases; Play; Population; Procedures; Protein-Serine-Threonine Kinases; Proteins; Proteome; Proteomics; Research; Ribosomal Protein S6; Risk Factors; Role; Route; Sampling; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Sirolimus; Skin; Specimen; Squamous Epithelium; Standards of Weights and Measures; Stem cells; Subgroup; System; TP53 gene; Techniques; Testing; Tetracycline; Tetracyclines; Therapeutic; Tissue Microarray; Tissue Sample; Tissues; Tobacco use; Treatment Effectiveness; angiogenesis; base; cell growth; cell motility; density; improved; in vivo; laser capture microdissection; malignant mouth neoplasm; migration; mutant; neoplastic; neoplastic cell; novel; novel strategies; novel therapeutics; oral carcinogenesis; oral cavity epithelium; programs; protein degradation; protein expression; reconstitution; therapeutic target; tumor; tumor xenograft; tumorigenesis

Genomic and proteomic approaches to understand oral cancer Although risk factors for HNSCC, such as alcohol and tobacco consumption, are well recognized, the molecular mechanisms responsible for this malignancy are still not fully understood. We have used a number of novel approaches to investigate gene and protein expression profiles in HNSCC. We have shown that laser capture microdissection (LCM) can be used to procure specific cell populations from heterogenous tumor samples, and that LCM-procured material can be used effectively to extract RNA, DNA, and proteins. We have teamed up with other research institutions to conduct gene and protein expression analysis of HNSCC by combining LCM, gene arrays, and proteomic platforms. These efforts have already provided a wealth of information about the distinctive pattern of gene and protein expression in HNSCC. Proteome-wide analysis of HNSCC tissues using LCM and mass spectrometry. Ultimately, the aberrant expression and activity of molecules present in HNSCC cells are responsible for their malignant behavior. Thus, we have recently made a concerted effort to investigate protein expression profiles during HNSCC tumor development. While in prior studies we have investigated protein expression profiles in frozen HNSCC samples, we now explored the use of standard formalin-fixed paraffin-embedded (FFPE) tissues for proteomic analysis, as they preserve the tissue architecture and may reflect better the protein composition as it exists in vivo. We combined LCM with protein extraction procedures and highly sensitive mass spectrometry techniques to identify proteins expressed in normal oral squamous epithelium and HNSCCs displaying distinct differentiation patterns. Mass spectrometry and bioinformatic analysis led to the identification of large numbers of molecules, including cytokeratins, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. Taken together, the ability to combine LCM and in-depth proteomic analysis of FFPE tissues provided valuable information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression. Dysregulated signaling networks in HNSCC: novel mechanism-based approaches for HSNCC treatment There is an urgent need for new treatment options for HNSCC patients, considering that their overall 5-year survival is relatively low (50%) and has not improved much over the past 3 decades. The emerging information on the nature of the deregulated molecular mechanisms responsible for HNSCC progression has provided the possibility of exploring new mechanisms-based therapeutic approaches for HNSCC. For example, we have observed that persistent activation of the serine-threonine kinase Akt is a frequent event in HNSCC, and that its blockade inhibits tumor cell growth. Among the many molecules whose activity is regulated by Akt, we have made a focal effort on the mammalian target of rapamycin (mTOR), an atypical kinase that seats at the cross-road between energy sensing and growth promoting signaling pathways. We have shown that a high fraction of HNSCC clinical samples exhibit high levels of phosphorylated ribosomal S6 protein (pS6), the most downstream target of the Akt-mTOR pathway, and that inhibition of mTOR by the use of rapamycin causes the rapid decrease in the level of pS6 and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. These efforts have identified the Akt-mTOR pathway as a potential therapeutic target for HNSCC. The Head and Neck Tissue Array Initiative: Dissection of the Akt-mTOR signaling network. As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from HNSCC patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high density oral specific HNSCC tissue microarray. Emerging information by the use of phospho-specific antibodies detecting the activated state of signaling molecules indicates that the Akt/mTOR pathway is frequently activated in HNSCC, but independently from the activation of EGFR or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of EGFR activation. Furthermore, we have also identified a small subgroup of patients in which the mTOR pathway is activated but not Akt, suggesting the existence of an Akt-independent signaling route stimulating mTOR. These findings provide important information about the nature of the dysregulated signaling networks in HNSCC, and may also provide the rationale for the future selection of HNSCC patients that may benefit from novel mechanism-based therapies. Animal models for oral malignancies A major limitation in the area of HNSCC research is the limited availability of animal models to test the validity of current genetic paradigms of tumorigenesis, and to explore the effectiveness of treatment modalities or chemopreventive approaches. We have been using our recently developed tetracycline-inducible system to express oncogenes in the skin and oral mucosa and other conditional animal systems to explore the contribution of recently identified dysregulated molecular mechanism to SCC progression. Requirement of Rac1 distinguishes follicular from interfollicular epithelial stem cells. Many cancers appear to arise from the accumulation of genetic alterations in resident, tissue-specific stem cells, or in cells that have regained stem-cell like features. This prompted us to initiate an effort to identify potential oral epithelial stem cells. It is still unclear whether oral and interfollicular epithelial stem cells are distinct from the better characterized hair-follicle stem cells. As follicular stem cells need to migrate from the bulge region of the hair to the bulb to start a new hair cycle, we hypothesized that disrupting molecules involved in epithelial cell migration may help distinguish these two stem cell populations. In this regard, the small GTPase Rac1 plays a central role in cell motility. Thus, in a recent study we conditionally deleted Rac1 from squamous epithelium. Surprisingly, we observed that although mice lacking Rac1 expression in the skin and oral mucosa exhibit alopecia due to the defective hair development, they do not display any major alterations in their interfollicular skin and oral epithelium. Furthermore, Rac1 excision from newborn mice epithelial cells results in the inability of their residing stem cells to reconstitute hair follicles, but they can nonetheless regenerate a healthy epidermis. Together, these findings supported the emerging view that the oral mucosa and epidermis are maintained by different stem cell compartments than those regenerating the hair follicles, and that these distinct stem cell populations can be distinguished molecularly by their requirement for Rac1 function.

了解口腔癌的基因组学和蛋白质组学方法 尽管 HNSCC 的危险因素（例如饮酒和吸烟）已得到广泛认可，但导致这种恶性肿瘤的分子机制仍不完全清楚。我们使用了许多新方法来研究 HNSCC 中的基因和蛋白质表达谱。我们已经证明，激光捕获显微切割 (LCM) 可用于从异质肿瘤样本中获取特定细胞群，并且 LCM 获取的材料可有效用于提取 RNA、DNA 和蛋白质。我们与其他研究机构合作，结合LCM、基因芯片和蛋白质组平台进行HNSCC的基因和蛋白表达分析。这些努力已经提供了有关 HNSCC 中基因和蛋白质表达的独特模式的大量信息。 使用 LCM 和质谱法对 HNSCC 组织进行全蛋白质组分析。最终，HNSCC 细胞中分子的异常表达和活性是其恶性行为的原因。因此，我们最近齐心协力研究 HNSCC 肿瘤发展过程中的蛋白质表达谱。虽然在之前的研究中，我们研究了冷冻 HNSCC 样本中的蛋白质表达谱，但我们现在探索使用标准福尔马林固定石蜡包埋 (FFPE) 组织进行蛋白质组分析，因为它们保留了组织结构，并且可以更好地反映蛋白质组成，如它存在于体内。我们将 LCM 与蛋白质提取程序和高灵敏度质谱技术相结合，以鉴定在正常口腔鳞状上皮和表现出不同分化模式的 HNSCC 中表达的蛋白质。质谱和生物信息分析导致大量分子的鉴定，包括细胞角蛋白、分化标记物以及参与干细胞维持、信号转导、迁移、细胞周期调节、血管生成、基质降解的蛋白质以及具有肿瘤抑制和致癌作用的蛋白质潜在的。使用 HNSCC 组织切片和组织微阵列中的免疫组织化学研究进一步验证了代表性蛋白质。总而言之，将 LCM 和 FFPE 组织的深入蛋白质组学分析相结合的能力提供了有关正常鳞状上皮和 HNSCC 进展过程中表达的蛋白质性质的宝贵信息。 HNSCC 信号网络失调：基于机制的 HSNCC 治疗新方法 考虑到 HNSCC 患者的总体 5 年生存率相对较低（50%），并且在过去 30 年来没有太大改善，因此迫切需要新的治疗方案。 关于导致 HNSCC 进展的失调分子机制性质的新信息为探索新的基于机制的 HNSCC 治疗方法提供了可能性。例如，我们观察到丝氨酸-苏氨酸激酶 Akt 的持续激活是 HNSCC 中的常见事件，并且其阻断会抑制肿瘤细胞的生长。在众多活性受 Akt 调节的分子中，我们重点关注哺乳动物雷帕霉素靶标 (mTOR)，这是一种位于能量传感和生长促进信号通路之间十字路口的非典型激酶。我们已经证明，大部分 HNSCC 临床样本表现出高水平的磷酸化核糖体 S6 蛋白 (pS6)，这是 Akt-mTOR 途径的最下游靶标，并且使用雷帕霉素抑制 mTOR 会导致pS6 水平和 HNSCC 肿瘤异种移植物凋亡，从而导致肿瘤消退。这些努力已将 Akt-mTOR 通路确定为 HNSCC 的潜在治疗靶点。 头颈组织阵列计划：Akt-mTOR 信号网络的剖析。作为评估 HNSCC 患者临床标本中信号通路的表达模式和激活状态的方法，我们建立了头颈癌组织阵列计划，这是一个旨在开发高密度口腔特异性 HNSCC 组织微阵列的国际联盟。使用磷酸化特异性抗体检测信号分子激活状态的新信息表明，Akt/mTOR 通路在 HNSCC 中经常被激活，但与 EGFR 的激活或突变 p53 的检测无关。事实上，我们鉴定了一大组在没有 EGFR 激活的情况下显示出活性 Akt 和 mTOR 的组织样本。此外，我们还发现一小部分患者的 mTOR 通路被激活，但 Akt 未被激活，这表明存在刺激 mTOR 的不依赖于 Akt 的信号通路。这些发现提供了有关 HNSCC 信号网络失调性质的重要信息，也可能为未来选择可能受益于基于新机制的疗法的 HNSCC 患者提供依据。 口腔恶性肿瘤动物模型 HNSCC 研究领域的一个主要限制是动物模型的可用性有限，无法测试当前肿瘤发生遗传范式的有效性，并探索治疗方式或化学预防方法的有效性。我们一直在使用我们最近开发的四环素诱导系统在皮肤和口腔粘膜和其他条件动物系统中表达癌基因，以探索最近发现的失调分子机制对鳞状细胞癌进展的贡献。 Rac1 的需要可区分滤泡和滤泡间上皮干细胞。许多癌症似乎是由常驻组织特异性干细胞或已恢复干细胞样特征的细胞中基因改变的积累引起的。这促使我们开始努力鉴定潜在的口腔上皮干细胞。目前尚不清楚口腔和毛囊间上皮干细胞是否与特征更好的毛囊干细胞不同。由于毛囊干细胞需要从头发的隆起区域迁移到球茎以开始新的毛发周期，我们假设破坏参与上皮细胞迁移的分子可能有助于区分这两种干细胞群。在这方面，小 GTPase Rac1 在细胞运动中发挥着核心作用。因此，在最近的一项研究中，我们有条件地从鳞状上皮中删除了 Rac1。令人惊讶的是，我们观察到，虽然皮肤和口腔粘膜缺乏 Rac1 表达的小鼠由于毛发发育缺陷而表现出脱发，但它们的毛囊间皮肤和口腔上皮没有表现出任何重大改变。此外，从新生小鼠上皮细胞中切除 Rac1 会导致其驻留的干细胞无法重建毛囊，但它们仍然可以再生健康的表皮。总之，这些发现支持了新的观点，即口腔粘膜和表皮由与毛囊再生的干细胞区室不同的干细胞区室维持，并且这些不同的干细胞群可以通过它们对 Rac1 功能的需求进行分子区分。