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.

理解口腔癌的基因组和蛋白质组学方法 尽管众所周知，HNSCC的危险因素，例如酒精和烟草消费，但仍未完全了解造成这种恶性肿瘤的分子机制。我们已经使用了许多新型方法来研究HNSCC中的基因和蛋白质表达谱。我们已经表明，激光捕获显微解剖（LCM）可用于从异源肿瘤样品中采购特定的细胞群，并且可以有效地使用LCM所致的材料来提取RNA，DNA和蛋白质。我们已经与其他研究机构合作，通过组合LCM，基因阵列和蛋白质组学平台对HNSCC进行基因和蛋白质表达分析。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组织的全蛋白质组分析。最终，HNSCC细胞中存在的分子的异常表达和活性是导致其恶性行为的原因。因此，我们最近进行了一致的努力，以研究HNSCC肿瘤发育过程中蛋白质表达谱。在先前的研究中，我们已经研究了冷冻HNSCC样品中的蛋白质表达谱，但我们现在探索了标准的福尔马林固定石蜡包裹（FFPE）组织进行蛋白质组学分析的使用，因为它们可以保留组织结构，并可能反映出蛋白质组成更好，因为它存在于体内。我们将LCM与蛋白质提取程序和高度敏感的质谱技术结合在一起，以鉴定在正常口服鳞状上皮和HNSCC中表达的蛋白质，并显示出不同的分化模式。质谱和生物信息学分析导致​​鉴定出大量分子，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，基质降解以及具有肿瘤抑制和致癌基因的蛋白质的蛋白质。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。综上所述，结合FFPE组织的LCM和深入蛋白质组学分析的能力提供了有关在正常鳞状上皮和HNSCC进展过程中表达的蛋白质性质的宝贵信息。 HNSCC中的信号网络失调：基于新型机制的HSNCC处理方法 考虑到他们的总体5年生存率相对较低（50％），并且在过去3年中没有太大改善，因此迫切需要针对HNSCC患者的新治疗选择。 有关负责HNSCC进展的放松管制分子机制的性质的新兴信息提供了探索基于HNSCC的新机制治疗方法的可能性。例如，我们已经观察到丝氨酸 - 硫代激酶Akt的持续激活是HNSCC的常见事件，并且其阻断抑制了肿瘤细胞的生长。在活性受AKT调节的许多分子中，我们对雷帕霉素（MTOR）的哺乳动物靶标进行了焦点，这是一种非典型激酶，它位于能量感应和促进信号传导途径之间的十字路口。我们已经表明，大量的HNSCC临床样品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，并且通过使用Rapamycin抑制MTOR会导致PS6和Apoptotic tamby tamby cacccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccced cacccccccccin cacccccccin tamby caccccccccccccin的快速降低。这些努力将AKT-MTOR途径确定为HNSCC的潜在治疗靶点。 头部和颈部组织阵列倡议：Akt-MTOR信号网络的解剖。作为评估HNSCC患者临床标本中信号通路激活的表达模式和状态的一种方法，我们建立了头颈癌组织阵列倡议，这是一个旨在开发高密度口服特异性HNSCC组织微阵列的国际财团。通过使用检测信号分子激活状态的磷酸特异性抗体的新闻信息表明，Akt/mTOR途径在HNSCC中经常被激活，但独立于EGFR的激活或突变体p53的检测。实际上，我们确定了在没有EGFR激活的情况下显示出活跃Akt和MTOR的大量组织样品。此外，我们还确定了一小部分患者亚组，其中MTOR途径被激活而不是AKT，这表明存在非AKT非依赖性的信号途径刺激MTOR。这些发现提供了有关HNSCC失调信号网络的性质的重要信息，并且还可能为未来选择的HNSCC患者的选择提供理由，这些HNSCC患者可能会受益于新型基于机制的疗法。 口腔恶性肿瘤的动物模型 HNSCC研究领域的一个主要局限性是动物模型的可用性有限，以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。我们一直在使用最近开发的四环素诱导系统来表达皮肤，口服粘膜和其他条件动物系统中的癌基因，以探索最近确定的非调节分子机制对SCC进展的贡献。 RAC1的需求将卵泡与流体间上皮干细胞区分开。许多癌症似乎是由于居民，组织特异性干细胞或已恢复类似干细胞特征的细胞中遗传改变的积累而产生的。这促使我们开始努力识别潜在的口腔上皮干细胞。目前尚不清楚口服和室间上皮干细胞是否与表征较好的毛囊干细胞不同。由于卵泡干细胞需要从头发的凸起区域迁移到灯泡以开始新的头发周期，因此我们假设破坏上皮细胞迁移的分子可能有助于区分这两个干细胞群体。在这方面，小的GTPase Rac1在细胞运动中起着核心作用。因此，在最近的一项研究中，我们从鳞状上皮有条件地删除了Rac1。令人惊讶的是，我们观察到，尽管由于头发发育缺陷而在皮肤和口服粘膜中缺乏Rac1表达的小鼠表现出脱发，但它们在毛皮间皮肤和口服上皮中没有显示任何重大改变。此外，新生小鼠上皮细胞的Rac1切除导致其居住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，这些发现支持了新兴观点，即口腔粘膜和表皮与与毛囊再生的干细胞区室保持不同，并且这些独特的干细胞种群可以通过对RAC1功能的需求来区分分子。