Dissecting the metastasis suppressor complex to identify colon cancer biomarkers

剖析转移抑制复合物以鉴定结肠癌生物标志物

• 批准号: 8509880
• 负责人: Sam Thiagalingam
• 金额: $ 21.36万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509880
• 关键词: Accounting; Apoptosis; Biological Assay; Biological Markers; Biological Models; Cancer Model; Candidate Disease Gene; Cell Culture Techniques; Cell Line; Cells; Chromosomes; Co-Immunoprecipitations; Colon Carcinoma; Colonic Neoplasms; Complex; Computer Simulation; Databases; Defect; Deletion Mutation; Development; Disease; Down-Regulation; Engineering; Epigenetic Process; Event; Exhibits; Fluorouracil; Follow-Up Studies; Frequencies; Gene Expression; Genes; Hypoxia; In Vitro; Link; Loss of Heterozygosity; MADH4 gene; MAP Kinase Gene; MAPK14 gene; MEKs; MMP9 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Meta-Analysis; Metastasis Suppression; Metastatic to; Modeling; Molecular; Molecular Profiling; Mus; Nature; Neoplasm Metastasis; Pathway interactions; Patients; Play; Prognostic Marker; Proteins; Proteomics; RNA Splicing; Reporter; Resistance; Role; SLC2A1 gene; Sampling; Severity of illness; Signal Transduction; Staging; Staining method; Stains; Testing; The Cancer Genome Atlas; Therapeutic; Time; Transcript; Tumor Suppressor Proteins; Vascular Endothelial Growth Factors; Western Blotting; Xenograft Model; adenoma; aerobic glycolysis; base; cancer cell; cofactor; colon cancer cell line; follow-up; hypoxia inducible factor 1; in vivo; knock-down; loss of function; migration; novel; outcome forecast; overexpression; programs; protein complex; public health relevance; research study; transcription factor; tumor; tumor progression

DESCRIPTION (provided by applicant): Loss of heterozygosity (LOH) analysis and follow up studies of sporadic colon cancer enabled us to show that SMAD4 is the primary target tumor suppressor, localized to the minimally lost region at chromosome 18q21-linked to an advanced stage disease. Subsequent studies by others have confirmed these initial observations and have established that the frequency of SMAD4 mutations/deletions increases as the cancer progresses from adenomas to the metastatic disease. Overall, these findings are consistent with loss of Smad4 function in metastatic colon cancer. In an effort to delineate a molecular basis for an association between SMAD4 deficiency and metastatic colon cancer, we have begun to use appropriately engineered model systems. Our preliminary studies showed that the SMAD4 defect was responsible for an increase in the levels of VEGF, overactivation of MEK-Erk and p38-MAPK auxiliary pathways, enhanced migration of colon cancer cells with a corresponding increase in MMP9, overexpression of GLUT1 under hypoxia, increased aerobic glycolysis and resistance to 5-FU-mediated apoptosis. We also found that overexpression of Smad4 in the model colon cancer cells with SMAD4 deficiency inhibited VEGF reporter activity and Smad4 physically interacts with specific transcription factors (TFs) such as HIF1? to potentially regulat metastatic progression of colon cancer. While the molecular characterizations are consistent with the notion that Smad4 may play a central role in forming a colon cancer metastasis suppressor complex consisting of various transcription factors and cofactors to collectively block colon cancer metastasis, direct targeting for loss of Smad4 function could only account for approximately 30% of tumors that harbor LOH at 18q21. Therefore, metastatic colon cancers that retained intact Smad4 could also progress through inactivation of the other components of the complex that suppresses the metastatic program. In this proposal, we will test the hypothesis that metastatic colon cancers which retain Smad4 exhibit alterations in the other components of the colon cancer metastasis suppressor complex. Thus, we predict that by unraveling the composition of the colon cancer metastasis suppressor complex, one would uncover novel prognostic biomarkers that are alternatively targeted for inactivation in metastatic colon cancer. Here, we outline a strategy using the model cell lines to isolate and characterize the components of the colon cancer metastasis suppressor complex consisting of Smad4 to (1) investigate the nature of the higher order protein complexes that are assembled and dissolved under the conditions of intact and defective Smad4 signaling using proteomic analysis to identify TFs and co-factors as candidate prognostic biomarkers; (2) examine if dysregulation of these other factors of the metastasis suppressor complex disrupts its functionality; and (3) determine if defects/deficiency in the alternate targets of Smad4 metastasis suppressor complex could serve as prognostic biomarkers for colon cancer. In summary, the proposed studies may unravel novel prognostic biomarkers for metastatic colon cancer and it could aid the development of personalized therapy.

描述（由申请人提供）：杂合性丢失（LOH）分析和散发性结肠癌的后续研究使我们能够证明 SMAD4 是主要靶标肿瘤抑制因子，定位于与晚期疾病相关的染色体 18q21 的最小丢失区域。其他人的后续研究证实了这些初步观察结果，并确定 SMAD4 突变/缺失的频率随着癌症从腺瘤进展为转移性疾病而增加。总体而言，这些发现与转移性结肠癌中 Smad4 功能的丧失一致。为了描绘 SMAD4 缺陷与转移性结肠癌之间关联的分子基础，我们已经开始使用适当设计的模型系统。我们的初步研究表明，SMAD4缺陷导致VEGF水平增加、MEK-Erk和p38-MAPK辅助通路过度激活、结肠癌细胞迁移增强（MMP9相应增加）、缺氧下GLUT1过度表达、有氧糖酵解增加和对5-FU介导的细胞凋亡的抵抗。我们还发现，SMAD4 缺陷模型结肠癌细胞中 Smad4 的过度表达抑制了 VEGF 报告基因活性，并且 Smad4 与特定转录因子 (TF) 如 HIF1? 发生物理相互作用。潜在地调节结肠癌的转移进展。虽然分子特征与 Smad4 可能在形成结肠癌转移抑制复合物中发挥核心作用的观点一致，该复合物由各种转录因子和辅助因子组成，共同阻止结肠癌转移，但直接靶向 Smad4 功能的丧失只能占 18q21 处含有 LOH 的肿瘤的大约 30%。因此，保留完整 Smad4 的转移性结肠癌也可能通过抑制转移程序的复合物的其他成分失活而进展。在本提案中，我们将测试保留 Smad4 的转移性结肠癌在结肠癌转移抑制复合物的其他成分中表现出改变的假设。因此，我们预测，通过揭示结肠癌转移抑制复合物的组成，人们将发现新的预后生物标志物，这些生物标志物可以选择性地针对转移性结肠癌的失活。在这里，我们概述了一种策略，使用模型细胞系来分离和表征由 Smad4 组成的结肠癌转移抑制复合物的成分，以（1）研究在完整和有缺陷的 Smad4 信号传导条件下组装和溶解的高级蛋白质复合物的性质，使用蛋白质组分析来识别 TF 和辅助因子作为候选预后生物标志物； (2) 检查转移抑制复合物的这些其他因素的失调是否会破坏其功能； (3) 确定是否 Smad4 转移抑制复合物替代靶标的缺陷/缺乏可以作为结肠癌的预后生物标志物。总之，拟议的研究可能会揭示转移性结肠癌的新预后生物标志物，并有助于个性化治疗的发展。