Oral Carcinogenesis

口腔癌发生

基本信息

批准号：
7733911
负责人：
J Gutkind
金额：
$ 182.27万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/7733911
关键词：
Address Affect Alcohols Alleles Animal Model Animals Apoptotic Architecture Area Behavior Bioinformatics Cell Cycle Regulation Cell Maintenance Cells Cessation of life Chemopreventive Agent Clinical Complex Cytokeratin DNA Depth Detection Development Differentiation Antigens Disease Disease Progression Disease regression Dissection Dose Environment Epidermal Growth Factor Receptor Epigenetic Process Epithelial Event Evolution Exhibits Formalin Freezing Future Gene Proteins Genes Genetic Genomics Glucose Transporter Goals Growth Head and Neck Cancer Head and Neck Squamous Cell Carcinoma Head and neck structure Human In Vitro Institution International Knowledge Lesion Lip structure Lipids Malignant - descriptor Malignant Neoplasms Mass Spectrum Analysis Modality Molecular Molecular Profiling Monitor Mouth Neoplasms Mus Nature Neoplasm Metastasis Numbers Oncogenes Oncogenic Oral Oral cavity Oral mucous membrane structure PTEN gene Paraffin Embedding Pathway interactions Patients Pattern Pharmacology Pharyngeal structure Phospho-Specific Antibodies Phosphoric Monoester Hydrolases Phosphotransferases Population Procedures Property Protein-Serine-Threonine Kinases Proteins Proteome Proteomics Research Resistance 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 Stromal Cells System TP53 gene Techniques Testing Tetracycline Tetracyclines Therapeutic Tissue Microarray Tissue Sample Tissues Tobacco use Treatment Effectiveness Vascular Endothelial Growth Factors Xenograft procedure angiogenesis base cancer cell cancer type cell growth density improved in vivo laser capture microdissection malignant mouth neoplasm migration mutant neoplastic neoplastic cell novel novel strategies novel therapeutics oral carcinogenesis prevent programs protein degradation protein expression therapeutic target tumor tumor progression tumor xenograft tumorigenesis

项目摘要

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 involved in 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. Tumor cells are the primary target for rapamycin in HNSCC - A retro-inhibition approach. Although blockade of mTOR by rapamycin promotes the rapid regression of HNSCC-tumor xenografts in mice, rapamycin does not affect the growth of HNSCC cells in vitro, thus raising the possibility that, as for other cancer types, rapamycin may not target cancer cells directly but may instead act based on its anti-angiogenic properties. To assess whether the cancer cell-autonomous actions of rapamycin contributes to its antitumoral effects, we utilized a retro-inhibition approach consisting in the expression of a rapamycin-resistant form of mTOR (mTOR-RR) in HNSCC cells. Host-derived endothelial and stromal cells retained wild-type mTOR alleles, hence remaining rapamycin-sensitive. Expression of mTOR-RR prevented the decrease in pS6 levels caused by rapamycin through mTOR in HNSCC cells but not in stromal cells, and rendered HNSCC xenografts resistant to the antitumoral activity of rapamycin. This novel reverse-pharmacology strategy also enabled monitoring the direct consequences of inhibiting mTOR in cancer cells within the complex tumor micro-environment, which revealed that mTOR controls the expression of HIF-1, VEGF and a glucose transporter, Glut-1, in HNSCC cells. These findings indicate that HNSCC cells are the primary target of rapamycin in vivo, and provide evidence that its anti-angiogenic effects may represent a downstream consequence of mTOR inhibition in cancer cells. 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. 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. Animal models to study the role of the Akt-mTOR pathway in SCC: The emerging studies supporting a role for the Akt-mTOR pathway in HNSCC development prompted us to direct our efforts, and focus on the development of animal models in which we could conditionally activate this signaling route. Indeed, stimulation of PI3K-Akt-mTOR by the epithelial-specific deletion of Pten, a PIP3 lipid phosphatase, was sufficient to cause multiple hyperproliferative and tumor lesions in the skin and mouth and resulted in early lethality. This suggests that decreased PTEN activity in humans, which is a frequent genetic or epigenetic event in HNSCC, may contribute to tumor development. Remarkably, the prolonged administration of low doses of rapamycin promoted the rapid regression of advanced mucocutaneous lesions, and was sufficient to prevent their development if initiated before disease manifestation, underscoring the key role of mTOR for tumor progression in squamous epithelium.

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)，这是一种参与能量传感和生长促进信号通路的非典型激酶。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。肿瘤细胞是雷帕霉素治疗 HNSCC 的主要靶点 - 一种逆转录抑制方法。虽然雷帕霉素阻断 mTOR 可促进小鼠 HNSCC 肿瘤异种移植物的快速消退，但雷帕霉素在体外并不影响 HNSCC 细胞的生长，因此增加了一种可能性，即对于其他癌症类型，雷帕霉素可能不会直接靶向癌细胞，而是直接靶向癌细胞。相反，可能会根据其抗血管生成特性发挥作用。为了评估雷帕霉素的癌细胞自主作用是否有助于其抗肿瘤作用，我们采用了一种逆转录抑制方法，该方法包括在 HNSCC 细胞中表达雷帕霉素耐药形式的 mTOR (mTOR-RR)。宿主来源的内皮细胞和基质细胞保留了野生型 mTOR 等位基因，因此仍然对雷帕霉素敏感。 mTOR-RR 的表达阻止了 HNSCC 细胞中雷帕霉素通过 mTOR 引起的 pS6 水平下降，但在基质细胞中则不然，并使 HNSCC 异种移植物对雷帕霉素的抗肿瘤活性产生抗性。这种新颖的反向药理学策略还能够监测复杂肿瘤微环境中癌细胞中抑制 mTOR 的直接后果，这表明 mTOR 控制 HNSCC 中 HIF-1、VEGF 和葡萄糖转运蛋白 Glut-1 的表达细胞。这些发现表明 HNSCC 细胞是雷帕霉素体内的主要靶标，并提供证据表明其抗血管生成作用可能代表癌细胞中 mTOR 抑制的下游结果。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。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。研究 Akt-mTOR 通路在 SCC 中的作用的动物模型：支持 Akt-mTOR 通路在 HNSCC 发展中的作用的新兴研究促使我们努力指导我们的工作，并重点关注动物模型的开发，在这些模型中我们可以有条件地开发动物模型。激活该信号路由。事实上，Pten（一种 PIP3 脂质磷酸酶）的上皮特异性缺失刺激 PI3K-Akt-mTOR 足以引起皮肤和口腔的多种过度增殖和肿瘤病变，并导致早期死亡。这表明人类 PTEN 活性降低（这是 HNSCC 中常见的遗传或表观遗传事件）可能有助于肿瘤的发展。值得注意的是，长期给予低剂量雷帕霉素促进了晚期粘膜皮肤病变的快速消退，并且如果在疾病表现之前开始就足以阻止其发展，这强调了 mTOR 在鳞状上皮肿瘤进展中的关键作用。

项目成果

期刊论文数量（38）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Rac1 is required for epithelial stem cell function during dermal and oral mucosal wound healing but not for tissue homeostasis in mice.

DOI：
10.1371/journal.pone.0010503
发表时间：
2010-05-06
期刊：
PloS one
影响因子：
3.7
作者：
Castilho RM;Squarize CH;Leelahavanichkul K;Zheng Y;Bugge T;Gutkind JS
通讯作者：
Gutkind JS

Antitumor activity of UCN-01 in carcinomas of the head and neck is associated with altered expression of cyclin D3 and p27(KIP1).

DOI：
发表时间：
2002-11
期刊：
Clinical cancer research : an official journal of the American Association for Cancer Research
影响因子：
0
作者：
V. Patel;T. Lahusen;C. Leethanakul;T. Igishi;M. Kremer;L. Quintanilla‐Martinez;J. Ensley;E. Sausville;J. Gutkind;A. Senderowicz
通讯作者：
V. Patel;T. Lahusen;C. Leethanakul;T. Igishi;M. Kremer;L. Quintanilla‐Martinez;J. Ensley;E. Sausville;J. Gutkind;A. Senderowicz

Epidermal growth factor receptor-independent constitutive activation of STAT3 in head and neck squamous cell carcinoma is mediated by the autocrine/paracrine stimulation of the interleukin 6/gp130 cytokine system.

DOI：
发表时间：
2003-06
期刊：
Cancer research
影响因子：
11.2
作者：
V. Sriuranpong;Jong In Park;Panomwat Amornphimoltham;V. Patel;B. Nelkin;J. Gutkind
通讯作者：
V. Sriuranpong;Jong In Park;Panomwat Amornphimoltham;V. Patel;B. Nelkin;J. Gutkind

Oral and pharyngeal epithelial keratinocyte culture.