Fibronectin Mechanics and Signaling in Epithelial to Mesenchymal Transition

项目摘要 上皮细胞向间充质细胞转化（EMT）是一个过程，通过该过程，表型和 上皮细胞的功能使它们转化为间充质细胞。EMT参与促进 乳腺癌进展为侵袭性疾病。因此，有必要充分了解 诱导EMT的机制。最近的进展指出，EMT是由一个组合控制的事实， 生长因子（GF）和基质硬度。转化生长因子-TGF-β 1（TGF-β 2），一种已知诱导 乳腺癌形成中的EMT诱导刚性表面上的EMT和柔顺表面上的细胞凋亡。是我们 相信机械信号、gf信号和细胞外基质（ECM）蛋白的类型的组合 由细胞组装在一起驱动EMT的过程。本研究将集中在ECM蛋白纤连蛋白 (FN)，其组装成弹性的、不溶性的原纤维。FN的能力，作为一个gf输送系统，沿着与其 通过细胞产生的力进行组装，这种力在更硬的表面上变得更大，这使我们研究了以下问题 假设：增加的组织硬度驱动FN组装，其暴露各种GF的隐蔽结合位点， 如TGF-β1，并在细胞表面产生高浓度的这些gfs，这反过来又驱动EMT。在 本课题主要研究三个方面的问题：（1）评价抑制FN纤维形成和GF 定位于TGF-β1诱导的EMT，（2）探讨连接到FN基质的GF在空间模式中的作用， EMT，和（3）评估在不存在FN组装的情况下改变基底刚度对产生FN的影响。 上皮细胞单层中的细胞牵引力。FN组装将被抑制与蛋白片段的 细菌细胞壁蛋白粘附F1，其先前已显示抑制FN原纤维组装， 改变FN表达。将观察FN原纤维组装和EMT标记物存在之间的相关性 通过免疫荧光定性。蛋白质表达将通过蛋白质印迹法定量，mRNA表达将通过免疫印迹法定量。 将用RT-PCR测定表达。FN原纤维面积和gf共定位将用自身免疫荧光定量。 编写的图像处理算法。微接触印刷图案将由ECM蛋白包被的 聚二甲基硅氧烷印模。通过制备聚丙烯酰胺凝胶， 弹性模量范围从0.4 kPa到60 kPa，并且微制造的柱阵列将产生2微米 直径和高度从5到15微米不等。这些基材硬度代表了从天然的 乳腺组织转移到纤维化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癌症生物学。通过在FN组件之间建立连接， 以及EMT在癌症进展中的错误调节，我们希望潜在地确定癌症的新靶点。 疗法

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.