Role of the Nance Horan Syndrome protein family in breast cancer invasion

南斯霍兰综合征蛋白家族在乳腺癌侵袭中的作用

• 批准号: 2290132
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2290132
• 关键词: Role; Nance; Horan; Syndrome; protein

Mesenchymal cell migration is dependent on polarised polymerisation of actin in the direction of movement.Actin polymerisation preferentially occurs at the leading edge of the migrating cell resulting in the formation of broad, flat protrusions known as lamellipodia (1).Cell migration is also dependent on establishment and maintenance of a defined cellular polarity; i.e. a leading edge and a trailing edge(2).We hypothesise that this may be achieved, in part, through ligand-driven endocytosis and endosomal trafficking of receptors (e.g. Tyrosine kinase receptors, G-protein coupled receptors) thatdirect migration.In support of this, there is evidence in the literature for a role of both clathrin-dependent and clathrin-independent endocytosis pathways in migration (3-13).Fast-endophilin mediated endocytosis (FEME) is one pathway which may regulate cellular polarity during migration.FEME is a clathrin-independent mechanism of endocytosis that is triggered at the leading edge of cells in response to ligand binding(14, 15).This process involves the activity of the Endophilin A (hereby referredto as Endophilin) proteins, a subfamily of BAR domain containing proteins (14).The enriched localisation of FEME to the leading edge of the cellindicatesa role for this pathway in cell migration. In support of this, knockdown of endophilin, in both epithelial and endothelial cell lines, results in impaired migration of these cells(14, 16).Furthermore, a number of the components of the FEME priming complex, including Lpd, CIP4, FBP7 and SHIP2, have been shown to promote invasion and metastasis of breast cancer(17-20). These findings may implicate a role for FEME in breast cancercell migration and invasion.A classification of breast cancer into 10 molecular subtypes has highlighted Nance Horan Syndrome-like 2 (NHSL2) as the 5th most highly mutated gene in subtype 2 and the only gene in this subtype where mutation frequency shows a subtype-specific association(21). This finding suggests a role for NHSL2 in the pathogenesis of this subtype of breast cancer. NHSL2 is part of a protein family withtwo other proteins: Nance Horan Syndrome (NHS) and Nance Horan Syndrome-like 1 (NHSL1)(22).Work on NHS has shown that it plays a role in the regulation of actin remodelling and contains a functional WAVEhomology domain (WHD), which facilitates interaction with the Scar/WAVE complex. Scar/WAVE itself functions to promote cell migration through the activation of Arp2/3 mediated actin polymerisation (22).Recently, the Krause group has also shown that NHSL1 negatively regulates migration through the Scar/WAVE complex via two Abi binding sites (23). These binding sites and the above WHD are conserved amongst all three NHS proteins and the sole Drosophila orthologue GukH, implicating a role for this family in 5migration (22, 23). We have previously observed a vesicular localisation of both NHSL1 and NHSL2 (23, and unpublished), suggesting a role for these proteins in endosomal trafficking. NHSL2 specifically co-localises with endophilin A3 at a subset of these vesicles. Furthermore, we have found that NHSL2 co-immunoprecipitateswith all three GFP-tagged Endophilin proteins inGFP trap experiments. This interaction may point to a role of NHSL2 in FEME.We are interested to establish whether NHSL2 regulates FEME, and whether this may serve as a mechanism by which NHSL2 regulates breast cancer cell migration and invasion.We hypothesise that NHSL2 inhibits breast cancer cell migration and invasion by counteracting polarised protrusions and directional migration through the fast endophilin-mediated endocytosis (FEME) pathway.To address this hypothesis, we have devised the following aims:1.To investigate the role of NHSL2 in fast endophilin-mediated endocytosis (FEME).2.To explore the function of NHSL2 in directed 2D migration of breast cancer cells.3.To establish whether NHSL2 regulates 3D breast cancer cell invasion.

间充质细胞迁移依赖于肌动蛋白在运动方向上的极化聚合。肌动蛋白聚合优先发生在迁移细胞的前缘，导致形成宽而平的突起，称为板状伪足（1）。细胞迁移也依赖于确定的细胞极性的建立和维持。即前缘和后缘（2）。我们假设这可能部分通过配体驱动的内吞作用和受体的内体运输来实现（例如酪氨酸激酶受体、G蛋白偶联受体）。为了支持这一点，在文献中有证据表明网格蛋白依赖性和网格蛋白非依赖性内吞途径在迁移中的作用（3-13）。快速内啡肽介导的内吞作用（FEME）FEME是一种不依赖网格蛋白的内吞作用机制，其在细胞的前沿响应于配体结合而被触发（14，15）.这一过程涉及的活动的Endophilin A（在此称为Endophilin）蛋白质，含BAR结构域蛋白质的亚家族（14）FEME富集定位于Cellindicatesa的前缘，这是该途径在细胞迁移中的作用。为了支持这一点，上皮细胞系和内皮细胞系中嗜内蛋白的敲低导致这些细胞的迁移受损（14，16）此外，FEME引发复合物的许多组分，包括Lpd、CIP 4、FBP 7和SHIP 2，已显示促进乳腺癌的侵袭和转移（17-20）。这些发现可能暗示FEME在乳腺癌细胞迁移和侵袭中的作用。将乳腺癌分为10种分子亚型突出显示Nance Horan综合征样2（NHSL 2）是亚型2中第5大高度突变的基因，也是该亚型中突变频率显示亚型特异性关联的唯一基因（21）。这一发现表明NHSL 2在该亚型乳腺癌的发病机制中的作用。NHSL 2是与另外两种蛋白质Nance Horan综合征（NHS）和Nance Horan综合征样1（NHSL 1）（22）的蛋白质家族的一部分。对NHS的研究表明，它在肌动蛋白重塑的调节中起作用，并含有功能性WAVE同源结构域（WHD），其促进与瘢痕/WAVE复合物的相互作用。Scar/WAVE本身通过激活Arp 23介导的肌动蛋白聚合来促进细胞迁移（22）。最近，Krause小组还表明，NHSL 1通过两个Abi结合位点负调节通过Scar/WAVE复合物的迁移（23）。这些结合位点和上述WHD在所有三种NHS蛋白和唯一的果蝇直向同源物GukH中是保守的，暗示了该家族在迁移中的作用（22，23）。我们先前已经观察到NHSL 1和NHSL 2的囊泡定位（23，未发表），表明这些蛋白在内体运输中的作用。NHSL 2特异性地与这些囊泡的子集处的内亲蛋白A3共定位。此外，在GFP诱捕实验中，我们发现NHSL 2与所有三种GFP标记的Endophilin蛋白共免疫沉淀。这种相互作用可能指向NHSL 2在FEME中的作用。我们有兴趣确定NHSL 2是否调节FEME，我们假设NHSL 2通过快速内嗜蛋白介导的内吞作用（FEME）抑制极化突起和定向迁移，从而抑制乳腺癌细胞的迁移和侵袭。为了验证这一假设，我们设计了以下目的：1.研究NHSL 2在快速内嗜蛋白介导的内吞作用（FEME）中的作用; 2.研究NHSL 2在乳腺癌细胞定向二维迁移中的作用; 3.确定NHSL 2是否调控乳腺癌细胞的三维侵袭。