RSU1对MEK-ERK信号通路的调控作用及其机制研究

细胞-细胞外基质（extracellular matrix，ECM）黏附是细胞辨别周围环境变化的重要途径，细胞通过细胞-基质黏附感知来自于细胞微环境的变化，特别是胞外基质的变化，进而通过胞内信号传导调整自身的形态、运动、生长、分裂、凋亡和分化等。细胞脱离细胞外基质可引起胞内信号传导的变化，进而抑制细胞增殖、激活细胞凋亡信号，并最终引起细胞“失巢凋亡”。细胞失巢凋亡是组织形成并行使正常功能的重要机制之一，而肿瘤细胞也由于具有抗失巢凋亡的能力而变得具有很强的侵袭性。

整合素（integrin）家族蛋白是细胞外基质的重要受体，在细胞与细胞外基质相互作用的过程中起着重要的连接作用。整合素受体与配体结合促进整合素的激活，其胞内结构域可以招募超过180种不同的胞内蛋白到其胞内临近细胞膜的位置，形成黏着斑（focal adhesion）。黏着斑是集结整合素-配体结合信号、诱发下游信号传导的胞内信号枢纽。 ILK是整合素信号传递的关键分子，可以和parvin、PINCH形成ILK-PINCH-parvin三元复合物（简称IPP复合物）。Ras 抑制蛋白1（Ras suppressor 1，RSU1）是进化上高度保守的黏着斑蛋白，通过与 PINCH1结合定位于黏着斑。

细胞脱离细胞外基质将引起一系列胞内信号如MEK-ERK信号通路的改变，然而相关的分子机制仍有待进一步阐明。本论文发现整合素信号关键分子RSU1的缺失增强了MEK/ERK的磷酸化水平，并且RSU1缺失后细胞的增殖也显著增加。另外，黏着斑蛋白RSU1的缺失也抑制了细胞的伸展、迁移和侵袭等细胞运动过程。本论文进一步的研究发现，RSU1参与负调节细胞脱离细胞外基质即细胞失巢时MEK-ERK信号通路的活性。当细胞失巢时，RSU1的缺失抑制了细胞失巢导致的MEK-ERK通路活性的下调。PHB2是RSU1的新的互作蛋白，PHB2定位于细胞膜的脂筏，并正调节Ras-Raf-MEK-ERK信号通路。本论文采用免疫共沉淀、pull-down、FRET等方法证明了RSU1与PHB2的相互作用，并且发现在细胞失巢时RSU1-PHB2互作增强。此外，与PHB2一样，RSU1也可以分布在细胞膜的脂筏，而且在细胞失巢时RSU1在脂筏的量有所增多，同时RSU1-PHB2的互作也增强。本论文还进一步研究了RSU1-PHB2互作，分别阐明了参与二者互作的各自的结构域。其中，PHB2的氨基酸（aa 150-206）片段与RSU1有较强的结合力，可以作为发挥显性抑制（dominant negative，DN）的片段抑制细胞内RSU1与全长PHB2互作，同时也抑制了“悬浮” 条件下培养细胞的MEK/ERK磷酸化水平的下调。这说明PHB2 DN片段与RSU1缺失一样，能够抑制因细胞脱离胞外基质而引起的MEK-ERK信号通路失活，这就进一步证明了RSU1-PHB2互作在细胞失巢时调节MEK-ERK信号通路。另外，过表达不和PHB2结合的RSU1-C末端片段则不能影响细胞失巢时对MEK-ERK信号通路活性的调节。由于RSU1通过与PINCH1互作而定位于黏着斑，PINCH1也能调控MEK-ERK信号通路，本论文发现RSU1在PINCH1调控MEK-ERK信号通路中发挥作用。此外，本论文还利用不和PINCH1结合的RSU1-Y140K突变体验证了RSU1-PHB2信号轴对MEK-ERK通路的调节不依赖于RSU1和PINCH1的互作。综上所述，本论文提出“RSU1-PHB2”信号轴调控细胞失巢时MEK-ERK信号通路的分子机制，并进一步在3D条件培养细胞中验证了这一机制，揭示了RSU1-PHB2信号轴对体内细胞MEK-ERK信号通路的调节作用。

本论文的研究全面论证了细胞-基质黏附调控MEK-ERK信号通路的“RSU1-PHB2 信号轴”分子机制，完善了对整合素信号通路调控癌细胞迁移和侵袭的认识，也为探索癌症研究和治疗提供了潜在的新思路和新靶点。

Cell-extracellular matrix (Cell-ECM) adhesion is crucial to tissue organization and cellular communication. Cells can perceive the environmental changes through cell-extracellular matrix interaction, which leads to changes of cell behavior such as cell morphology, cell migration, cell proliferation, cell division, cell differentiation, cell apoptosis and so on. It is known that Cell-ECM detachment induces alterations of many intracellular signaling pathways, which inhibit cell proliferation and activate apoptosis, resulting in cell death known as “anoikis”. Anoikis is critical to tissue homeostasis, and cancer cells can become more aggressive by gaining resistance to anoikis.

Integrin family proteins are critical ECM receptors on the cell membrane, and play key roles in mediating Cell-ECM interaction as well as sensing and transducing signals derived from ECM. Upon activation by binding to the ECM ligands, integrin recruits more than 180 intracellular proteins to the cell membrane through its intracellular domain. ILK, as a key signaling protein in integrin signaling, can form a ternary complex with parvin and PINCH known as IPP complex. Ras suppressor (RSU1) is another evolutionarily conserved focal adhesion.  It has been demonstrated that PINCH interacts with RSU1, which recruits RSU1 to focal adhesion.

How Cell-ECM detachment is linked to down-regulation of MEK-ERK signaling is however, incompletely understood. In this thesis, I found that RSU1-deficiency promotes MEK-ERK activation cell proliferation. Moreover, RSU1-deficiency inhibits cell spreading, cell migration and invasion. It was revealed that RSU1 is a negative regulatory factor of MEK-ERK signaling upon cell-ECM detachment. RSU1-deficiency inhibits cell-ECM detachment-induced decreases of MEK-ERK activation. Mechanistically, we have identified PHB2 as a new binding partner of RSU1, which regulates MEK-ERK activation positively on lipid rafts. Using co-immunoprecipitation, pull-down, and FRET assays, I have proved the interaction between RSU1 and PHB2. Interestingly, the RSU1-PHB2 interaction is enhanced upon cell-ECM detachment. It was also revealed that RSU1 is distributed on lipid rafts and the association of RSU1 with lipid rafts is increased upon cell-ECM detachment. In addition, I have mapped a major RSU1-binding site to PHB2 amino acids 150-206 in the C-terminal region of the PHB domain. This PHB2 (aa 150-206) fragment, which shows higher affinity to RSU1 than full-length PHB2 to RSU1, plays a dominant negative role by disrupting the interaction between RSU1 and PHB2. Overexpression of the PHB2 (aa 150-206) fragment, like RSU1-deficiency, inhibits the decreases of MEK-ERK activation induced by cell-ECM detachment, while overexpression of RSU1-C-term, which is unable to interact with PHB2, could not mediate MEK-ERK activation. Together, these results strengthen the role of RSU1-PHB2 interaction in regulating MEK-ERK activation. It is known that RSU1 is recruited to focal adhesion via interaction with PINCH1. PINCH1 is implicated in regulation of the MEK-ERK signaling. Therefore, the role of RSU1in PINCH1-mediated regulation of the MEK-ERK signaling pathway was also investigated. It was revealed that RSU1 is indeed involved in the suppression of PINCH1-mediated MEK-ERK activation. Moreover, using the RSU1-Y140K mutant, which is unable to interact with PINCH1, I have demonstrated that the MEK-ERK signaling mediated by RSU1-PHB2 axis is independent of RSU1-PINCH1 interaction. In conclusion, this thesis shows a mechanism of RSU1-PHB2 axis by which MEK-ERK activation is regulated, which is also supported by cells grown in 3D culture.

This thesis shows explicitly how RSU1-PHB2 axis regulated MEK-ERK activation, shedding light on our understanding of integrin signaling and cancer development and metastasis. This may also provide potential new therapeutic targets for cancer treatment.

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