Pilot Project 2

试点项目2

• 批准号: 9353745
• 负责人: Gayathri Devi
• 金额: $ 3.12万
• 依托单位: DUKE UNIVERSITY
• 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/9353745
• 关键词: Adult; African American; Age; Apoptosis; Archives; Automobile Driving; Biological; Biological Assay; Breast Cancer Cell; CRISPR/Cas technology; Cancer Biology; Cancer Control; Cancer Model; Cancer Patient; Cancer cell line; Cell Line; Cell Proliferation; Cell model; Cells; Cellular biology; Cessation of life; Clinic; Collaborations; Data; Development; Disadvantaged; Disease; Down-Regulation; Drug resistance; Embryonic Development; Erinaceidae; Exhibits; FRAP1 gene; GLI gene; Gene Amplification; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Growth; Human; Immunohistochemistry; Incidence; Income; Joints; Knock-out; Lead; Link; Malignant Neoplasms; Mediating; Minority; Mission; Modeling; Molecular; Outcome; Pathway interactions; Patients; Pharmacology; Phenotype; Pilot Projects; Population-Based Registry; Postdoctoral Fellow; Prevalence; Public Health; Quantitative Reverse Transcriptase PCR; Race; Relapse; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Signal Transduction; Testing; Therapeutic; Time; Tissue Sample; Translating; Translational Research; Underrepresented Minority; United States National Institutes of Health; Validation; Woman; Work; Xenograft procedure; anticancer research; cancer cell; cancer health disparity; cancer therapy; cell growth; cell motility; clinical practice; differential expression; genetic signature; genetically modified cells; health care availability; inflammatory breast cancer; inhibitor/antagonist; innovation; insight; knock-down; malignant breast neoplasm; migration; mortality; novel; novel strategies; novel therapeutic intervention; novel therapeutics; programs; repository; response; screening; small hairpin RNA; therapeutic target; transcription factor; transcriptome sequencing; tumor; tumorigenesis

PROJECT ABSTRACT Inflammatory breast cancer (IBC), one of the most lethal forms of breast cancer, accounts for ~15% of all breast cancer deaths. IBC is particularly devastating in disadvantaged minority women who have both a higher incidence and poorer survival. Gene expression profiling of IBC tumors has identified the hedgehog(Hh)/GLI1 pathway as higher in patients with aggressive and relapsing IBC. We have previously demonstrated that GLI1 is highly expressed in IBC cells derived from an aggressive primary IBC tumor, and that down-regulation of GLI1 in these cells decreased cell proliferation, increased apoptosis and reduced migration. The objective of the proposed research is to identify those genes that are critical for GLI activation and function, including those that are both up- and downstream in of GLI1, to provide insight into the molecular mechanisms underlying or driving IBC. The hypothesis to be explored is that GLI1 and the genes that it regulates promote the growth and survival of IBC, and that identifying these genes may lead to the elucidation of novel targets. The long-term goal is to inform novel approaches for anti-tumor efficacy in IBC. This objective will be met by applying an innovative approach that includes utilization of high-throughput transcriptome sequencing (RNA-Seq), genetically engineered cell lines and human IBC tissue samples. This will become accomplished by the following Aims: 1) To identify gene expression differences between low and high GLI1 expressing IBC models including drug resistant and GLI1-antagonist treated IBC models. Differences in gene expression patterns will be determined by high-throughput transcriptome sequencing (RNA-Seq). 2) To confirm and validate those gene expression differences by qRT-PCR analysis including a comparison of IBC and non-IBC cell lines. To assess overlap of those differentially expressed genes with African American patient derived non-IBC cell lines. We will assess GLI1 expression and the expression of a subset of differentially expressed target genes identified in Aim 1 in archival IBC patient and control tissue samples accessed from the Duke Biospecimen Repository and Processing Core by immuno-histochemistry, and gene expression analysis. To define functional roles for these target genes, a subset will be knocked down by shRNA or knocked out using CRISPR/Cas9 and effects on IBC growth and survival will be determined. 3) Selective pharmacological inhibitors will be used to assess the role of previously identified pathways in activating GLI1 in IBC and non- IBC. We assess effects on IBC cell biology in a panel of functional and phenotypic assays. This proposed research will further the collaboration between Duke and NCCU on IBC and will support the development of an under-represented minority postdoctoral fellow performing research focused on transcriptional profiling of GLI1 activation pathways in IBC. Completion of these Aims will provide mechanistic insights into the consequences of GLI1 activation and antagonism in IBC, the downstream pathways involved and inform novel approaches for anti-tumor efficacy in IBC with the long term goal to translate new therapies for IBC into the clinic.

项目摘要 炎症性乳腺癌(IBC)是最致命的乳腺癌之一，约占所有乳腺癌的15% 乳腺癌死亡人数。IBC在弱势少数族裔女性中尤其具有破坏性，她们既有更高的 发病率和较差的存活率。IBC肿瘤的基因表达谱鉴定为Hedgehog(HH)/GLI1 在侵袭性和复发性IBC患者中，这一途径较高。我们之前已经证明了GLI1 在侵袭性原发IBC肿瘤来源的IBC细胞中高表达，并且下调 这些细胞中的Gli1抑制了细胞的增殖，增加了细胞的凋亡率，减少了迁移。的目标是 这项拟议的研究旨在确定那些对GLI激活和功能至关重要的基因，包括 都在GLI1的上游和下游，以深入了解 开着IBC。有待探索的假说是GLI1及其调控的基因促进生长和 IBC的生存，识别这些基因可能会导致新的靶点的阐明。长期的 目的是为IBC抗肿瘤疗效的新方法提供信息。这一目标将通过应用 创新的方法，包括利用高通量转录组测序(RNA-Seq)， 基因工程细胞系和人类IBC组织样本。这将由 目的如下：1)确定低表达和高表达GLI1的IBC模型之间的基因表达差异 包括耐药模型和GLI1拮抗剂处理的IBC模型。基因表达模式的差异将 通过高通量转录组测序(RNA-Seq)确定。2)确认和验证 QRT-PCR分析基因表达的差异，包括IBC和非IBC细胞系的比较。至 评估这些差异表达基因与非裔美国人患者来源的非IBC细胞的重叠 台词。我们将评估GLI1的表达和差异表达的靶基因的子集的表达 在从Duke Biospecimen获取的IBC患者和对照组织样本的AIM 1中进行鉴定 通过免疫组织化学和基因表达分析获得存储库和处理核心。要定义 这些靶基因的功能角色，其中一个子集将被shRNA敲除或使用 CRISPR/CAS9以及对IBC生长和存活的影响将被确定。3)选择性药理 抑制剂将用于评估先前发现的途径在激活IBC和非IBC中GLI1中的作用 英国广播公司。我们通过一组功能和表型分析来评估对IBC细胞生物学的影响。这项建议 研究将进一步促进杜克大学和NCCU在IBC方面的合作，并将支持开发 少数族裔博士后专注于GLI1转录图谱的研究 IBC中的激活途径。完成这些目标将提供对后果的机械性洞察 IBC中GLI1的激活和拮抗，涉及的下游通路，并提供新的方法 IBC的抗肿瘤疗效，长期目标是将IBC的新疗法转化为临床。