Tumbarello David A, Andrews Melissa R, Brenton James D
Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge, United Kingdom.
University of St Andrews, School of Medicine, MBSB, North Haugh, St Andrews, United Kingdom.
PLoS One. 2016 Sep 13;11(9):e0162698. doi: 10.1371/journal.pone.0162698. eCollection 2016.
TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1-256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior.
已证明转化生长因子β诱导蛋白（TGFBI）通过一种调节微管稳定性的整合素受体介导机制，使卵巢癌细胞对紫杉醇的细胞毒性作用敏感。在此，我们确定TGFBI定位于间皮细胞衍生的细胞外基质（ECM）中的有组织的纤维状结构内。我们发现，通过短发夹RNA（shRNA）抑制富含半胱氨酸的酸性分泌蛋白（SPARC）的表达会减少TGFBI在间皮细胞衍生的ECM中的沉积，而不影响其整体蛋白表达或分泌。相反，SPARC的过表达增加了TGFBI的沉积。Met5a细胞系表达的SPARC-黄色荧光蛋白（YFP）融合构建体在细胞衍生的ECM中与TGFBI共定位。有趣的是，体外产生的SPARC能够从细胞裂解物中沉淀出TGFBI，这依赖于完整的SPARC羧基末端，体外结合试验证实了直接相互作用。SPARC的最后37个氨基酸被证明是TGFBI相互作用所必需的，而缺乏该区域（第1至256位氨基酸）的SPARC-YFP构建体的表达在ECM中不与TGFBI相互作用和共定位。此外，与对照间皮细胞衍生的ECM相比，当接种在缺乏SPARC的间皮细胞衍生的ECM上时，卵巢癌细胞的运动性降低，对化疗药物紫杉醇的反应减弱。总之，SPARC通过一种新的相互作用调节TGFBI的纤维状ECM沉积，随后影响癌细胞行为。
