BBSRC Case Studentship- The role of the chemokine CXCL12 in cell-matrix interactions and ECM remodelling in biological processes

背景：趋化因子CXCL 12与其CXCR 4受体相互作用的机制多种多样。这两个基因在进化中都是高度保守的--敲除小鼠的受体或配体都会导致胚胎死亡。通过其CXCR 4受体的信号传导，CXCL 12参与许多基本过程，如生殖细胞迁移，细胞-细胞相互作用和血管生成（1，2）。CXCL 12也是恶性疾病中的重要趋化因子，由许多癌症中的恶性细胞和/或宿主细胞产生（3，4）。主要申请人的实验室已经产生了CXCL 12作为恶性细胞的存活因子的证据;是这些细胞中自分泌信号网络的主要介质;参与肿瘤新血管形成以及肿瘤和基质之间的相互作用（5-8）。在这个项目中，我们将研究我们未发表的数据，这些数据将人类组织中CXCL 12基因表达水平与细胞-基质相互作用和细胞外基质ECM重塑相关的途径和过程紧密联系起来。简而言之，我们询问了来自245个卵巢癌活检组织的Affytron基因表达阵列数据集，以找到其表达与CXCL 12 mRNA水平相关的基因（Kulbe等人未发表的数据）。高CXCL 12 mRNA水平与细胞基质相互作用和ECM重塑相关的途径/过程以及与趋化性途径的预期关联之间存在显著关联。与高CXCL 12 mRNA（α>0.70）显著相关的个体基因包括ECM蛋白、蛋白酶及其抑制剂。在该分析中鉴定的ECM基因与在化学抗性卵巢癌中上调的ECM基因之间存在显著重叠，表明CXCL 12可能是这种抗性特征的关键驱动因素（9）。假设：在正常组织和病理状态下，CXCL 12通过调节参与细胞-基质相互作用和ECM重塑的一组特定基因来调节细胞迁移。目的：本项目的总体目标是分析CXCL 12在细胞-基质相互作用和ECM重塑中的作用。我们将在以下实验方法中使用基因表达阵列数据集、永生化卵巢表面上皮细胞和卵巢癌细胞系：确认并扩展将CXCL 12水平与细胞基质相互作用和细胞外基质重塑B联系起来的基因关联研究。研究CXCL 12在上皮细胞和细胞外基质或间皮细胞单层之间相互作用中的作用。C.研究CXCL 12在上皮细胞生长和存活中的作用。D.研究CXCL 12在卵巢癌化疗耐药性中的作用。学生将获得以下技术方面的专业知识：组织培养; RNA制备和实时RT-PCR; shRNA技术和基因转染;细胞因子释放的多重测定;细胞增殖和细胞死亡测定; Affyphase和生物信息学。预期成果：CXCL 12在发育、造血、炎症和恶性肿瘤过程中刺激正常和转化细胞的迁移。基于初步的数据，我们认为，这是通过调节细胞：基质相互作用和ECM重塑。从这个项目中获得的知识可能在细胞生物学中具有广泛的意义。参考文献1. M. Doitsidou等人，Cell 111，647（2002）。2. A.阿尤蒂岛J. Webb，C. Bleul，T.施普林格，J.C. Gutierrez-Ramos，J Exp Med 185，111（1997）. 3. F. 1999年，《癌症生物学研讨会》，第14卷，第171页（2004年）。4. J. A.伯格，T。J. Kipps，Blood 107，1761（2006）. 5. C. Scotton，D. Milliken，J. Wilson，S. Raju，F. 1999年，《英国癌症杂志》，第85卷，第891页，2001年。6. D.米利肯角Scotton，S. Raju，F. J. Wilson，Clin Cancer Res 8，1108（2002）。7. H. Kulbe，T. Hagermann，P. W. Szlosarek，F. R.约翰·威尔Wilson，Cancer Res 65，10355（2005）. 8. H. Kulbe等人，Cancer Res 67，585（2007）. 9. A. A. Jazaeri等人

Background: The mechanisms underlining the interaction of the chemokine CXCL12 with its CXCR4 receptor are diverse. Both genes are highly conserved in evolution - knocking out either receptor or ligand in mice leads to embryonic lethality. Via signalling through its CXCR4 receptor, CXCL12 is involved in many essential processes such germ cell migration, cell-cell interactions and angiogenesis (1, 2). CXCL12 is also an important chemokine in malignant disease being produced by malignant and/or host cells in many cancers (3, 4). The lab of the Principal Applicant has generated evidence that CXCL12 acts as a survival factor for malignant cells; is a major mediator of an autocrine signalling network in these cells; is involved in tumour neovascularisation as well as in interactions between tumour and stroma (5-8). In this project we will investigate our unpublished data that strongly link CXCL12 gene expression levels in human tissue with pathways and processes associated with cell - matrix interactions and extra-cellular matrix, ECM, remodelling. In brief, we interrogated Affymetrix gene expression array datasets from 245 ovarian cancer biopsies to find genes whose expression correlated with levels of CXCL12 mRNA (Kulbe et al unpublished data). There was a significant association between high CXCL12 mRNA levels and pathways/processes associated with cell matrix interactions and ECM remodelling as well as expected associations to chemotaxis pathways. Individual genes significantly associated with high CXCL12 mRNA (cc>0.70) included ECM proteins, proteases and their inhibitors. There was significant overlap between ECM genes identified in this analysis and those up-regulated in chemoresistant ovarian cancer, suggesting that CXCL12 may be a key driver for this resistance signature (9). Hypothesis: In normal tissues and in pathological states, CXCL12 regulates cell migration by modulating a specific set of genes involved in cell-matrix interactions and ECM remodelling. Aims: The overall aim of this project is to analyze the role of CXCL12 in cell-matrix interactions and ECM remodelling. We will use gene expression array datasets, immortalised ovarian surface epithelial cells and cell lines from ovarian cancer in the following experimental approaches: A. Confirm and extend gene association studies that link CXCL12 levels with cell matrix interactions and extra-cellular matrix remodelling B. Investigate a role for CXCL12 in interactions between epithelial cells and the extra-cellular matrix or mesothelial cell monolayers. C. Investigate a role for CXCL12 in epithelial cell growth and survival. D. Investigate a role for CXCL12 in chemoresistance in ovarian cancer. The student will gain expertise in the following techniques: Tissue culture; RNA preparation and real-time RT-PCR; shRNA techniques and gene transfection; multiplex assays for cytokine release; cell proliferation and cell death assays; Affymetrix and bioinformatics. Expected outcomes: CXCL12 stimulates the migration of both normal and transformed cells during development, haematopoiesis, inflammation and malignancy. Based on preliminary data we suggest that one way this is achieved is via regulation of cell:matrix interactions and ECM remodelling. Knowledge gained from this project may have wide significance in cell biology. References 1. M. Doitsidou et al., Cell 111, 647 (2002). 2. A. Aiuti, I. J. Webb, C. Bleul, T. Springer, J. C. Gutierrez-Ramos, J Exp Med 185, 111 (1997). 3. F. Balkwill, Seminars in Cancer Biology 14, 171 (2004). 4. J. A. Burger, T. J. Kipps, Blood 107, 1761 (2006). 5. C. Scotton, D. Milliken, J. Wilson, S. Raju, F. Balkwill, Br J Cancer 85, 891 (2001). 6. D. Milliken, C. Scotton, S. Raju, F. Balkwill, J. Wilson, Clin Cancer Res 8, 1108 (2002). 7. H. Kulbe, T. Hagermann, P. W. Szlosarek, F. R. Balkwill, J. L. Wilson, Cancer Res 65, 10355 (2005). 8. H. Kulbe et al., Cancer Res 67, 585 (2007). 9. A. A. Jazaeri et al.