Fibronectin Mechanics and Signaling in Epithelial to Mesenchymal Transition

上皮间质转化中的纤连蛋白力学和信号传导

• 批准号: 9195389
• 负责人: Lauren A. Griggs
• 金额: $ 4.31万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195389
• 关键词: Adhesions; Affect; Algorithms; Apoptosis; Area; Automobile Driving; Belief; Binding; Binding Sites; Blocking Antibodies; Breast Epithelial Cells; Caliber; Cancer Biology; Carcinoma; Cell Culture Techniques; Cell Wall; Cell model; Cell surface; Cells; Chemicals; Complex; Cultured Cells; Data; Development; Disease; Embryonic Development; Epithelial; Epithelial Cells; Event; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Fibrosis; Generations; Growth; Height; Heparin Binding; Human; Immunofluorescence Immunologic; Knowledge; Location; Malignant Neoplasms; Mammary Gland Parenchyma; Mammary Neoplasms; Mechanical Stress; Mechanics; Mediating; Mesenchymal; Messenger RNA; Methodology; Molecular; Neoplasm Metastasis; Organ; Pathology; Patient-Focused Outcomes; Pattern; Phenotype; Printing; Process; Protein Fragment; Proteins; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Stress; Surface; System; Tissues; Traction; Transforming Growth Factors; Translating; Up-Regulation; Western Blotting; Wound Healing; Writing; base; cell assembly; combat; cytokine; driving force; epithelial to mesenchymal transition; fibrillogenesis; image processing; interest; latent TGF-beta binding protein; mRNA Expression; malignant breast neoplasm; monolayer; novel; polyacrylamide gels; polydimethylsiloxane; protein expression; response; targeted cancer therapy; transdifferentiation; tumor microenvironment; tumor progression

PROJECT SUMMARY Epithelial to Mesenchymal Transition (EMT) is a process by which a distinct change in the phenotype and function of epithelial cells causes them to convert into mesenchymal cells. EMT is involved in facilitating the progression of breast cancer to an invasive disease. Therefore, there is a strong need to fully understand the mechanism for the induction of EMT. Recent advances point to the fact that EMT is controlled by a combination of growth factors (gfs) and substrate stiffness. Transforming Growth Factor- (TGF-), a gf known to induce EMT in breast cancer formation, induces EMT on rigid surfaces and apoptosis on compliant surfaces. It is our belief that a combination of mechanical signals, gf signals, and the type of extracellular matrix (ECM) proteins assembled by cells together drive the process of EMT. This research will focus on the ECM protein fibronectin (FN), which assembles into elastic, insoluble fibrils. FN’s ability to serves as a gf delivery system along with its assembly by cell-generated forces, which become larger on stiffer surfaces, led us to examine the following hypothesis: increased tissue stiffness drives FN assembly, which exposes cryptic binding sites for various gfs, such as TGF-β1, and creates a high concentration of these gfs at the cell surface, which in turn drives EMT. In this project we will investigate three aims: (1) evaluate the effect of inhibiting FN fibrillogenesis and GF localization on TGF-β1-induced EMT, (2) probe the role of gf tethering to the FN matrix in spatial patterning of EMT, and (3) assess the effect of varying substrate rigidity in the absence of FN assembly on the generation of cellular traction forces in epithelial monolayers. FN assembly will be inhibited with a protein fragment of the bacterial cell wall protein adhesion F1, which has previously been shown to inhibit FN fibril assembly without altering FN expression. The correlation between FN fibril assembly and EMT marker presence will be observed qualitatively through immunofluorescence. Protein expression will be quantified via Western blotting, and mRNA expression will be determined with RT-PCR. FN fibril area and gf co-localization will be quantified with a self- written image processing algorithm. Microcontact-printed patterns will be generated from ECM protein coated polydimethylsiloxane stamps. Varying substrate rigidities will be obtained by preparing polyacrylamide gels with elastic moduli ranging from 0.4 kPa to 60 kPa, and microfabricated pillar arrays will be produced with 2 micron diameters and heights varying from 5 to 15 microns. These substrate stiffnesses represent the range from native breast tissue to fibrotic tissue. The knowledge gained from this study will elucidate how physical changes within the breast tumor microenvironment regulate cancer biology. By establishing a connection between FN assembly and the misregulation of EMT in cancer progression, we hope to potentially identify novel targets for cancer therapy.

项目总结 上皮向间充质转化(EMT)是一种表型和细胞表型发生明显变化的过程。 上皮细胞的功能导致它们转化为间充质细胞。EMT参与了促进 乳腺癌进展为浸润性疾病。因此，我们迫切需要充分了解 EMT的诱导机制。最近的进展表明，EMT是由一种组合控制的 生长因子(GFS)和衬底硬度。转化生长因子-(转化生长因子-)，一种已知的诱导 EMT在乳腺癌的形成中，诱导刚性表面的EMT和顺应性表面的细胞凋亡。这是我们的 认为机械信号、纤维蛋白信号和细胞外基质(ECM)蛋白的组合 细胞组装在一起驱动着EMT的进程。本研究将重点研究细胞外基质蛋白纤维连接蛋白。 (FN)，它组装成有弹性的、不溶于水的纤维。FN的能力作为GF交付系统与其 通过细胞生成的力进行组装，在更坚硬的表面上会变得更大，这让我们研究了以下几点 假设：增加的组织硬度驱动FN组装，这暴露了各种GFS的隐蔽结合部位， 例如转化生长因子-β-1，并在细胞表面产生高浓度的这些GFS，进而驱动EMT。在……里面 本课题主要研究三个方面的内容：(1)评价中药对FN和GF的抑制作用 在转化生长因子-β-1诱导的子宫内膜上皮细胞转化中的定位；(2)探讨GFs与FN基质的连接在FN细胞空间构型中的作用 EMT，以及(3)评估在没有FN组装的情况下改变底物刚性对产生FN的影响 上皮单分子层细胞牵引力。FN组装将被FN的蛋白片段所抑制 细菌细胞壁蛋白黏附F1，以前已被证明在没有FN的情况下抑制FN纤维组装 正在更改FN表达式。FN纤维组装和EMT标记存在的相关性将被观察到 通过免疫荧光定性。蛋白质的表达将通过Western blotting进行量化，而mRNA 用RT-PCR检测基因的表达。Fn原纤维面积和gf共定位将用自身 编写的图像处理算法。微接触印刷图案将由ECM蛋白涂层生成 聚二甲基硅氧烷邮票。通过用以下物质制备聚丙烯酰胺凝胶可获得不同的底物刚性 弹性模数从0.4千帕到60千帕，以及微细加工的柱子阵列将被生产出2微米 直径和高度从5微米到15微米不等。这些衬底硬度代表了从天然的 从乳房组织到纤维组织。从这项研究中获得的知识将阐明体内的物理变化 乳腺肿瘤微环境调节肿瘤生物学。通过在FN程序集之间建立连接 以及EMT在癌症进展中的错误调控，我们希望潜在地识别癌症的新靶点 心理治疗。