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

间充质细胞的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沿，导致形成被称为板状脂膜的宽而平的突起(1)。细胞迁移也依赖于确定的细胞极性的建立和维持；我们假设，这可能部分是通过配体驱动的内吞作用和指导迁移的受体(例如酪氨酸激酶受体，G蛋白偶联受体)的内吞作用来实现的。为了支持这一点，文献中有证据表明依赖于和不依赖于细胞骨架蛋白的内吞作用在迁移中起作用(3-13)。快速亲内素介导的内吞作用(FMEE)是在迁移过程中调节细胞极性的一种途径。FEME是一种不依赖于内吞蛋白的内吞作用机制，它是在细胞的前沿触发对连接和结合的反应15).这个过程涉及到亲内蛋白A(在此称为亲内素)蛋白的活性，这是包含BAR结构域的蛋白质的一个亚家族(14)。Feme在细胞前沿的丰富定位表明这一途径在细胞迁移中发挥了作用。为了支持这一点，在上皮和内皮细胞系中，抑制内亲和素导致这些细胞的迁移受阻(14，16)。此外，已证明女性启动复合体的一些成分，包括LPD，CIP4，FBP7和SHIP2，促进乳腺癌的侵袭和转移(17-20)。这些发现可能暗示女性在乳腺癌细胞迁移和入侵中的作用。将乳腺癌分为10个分子亚型，突出了Nance Horan综合征样2(NHSL2)是第二亚型中突变最多的第五大基因，也是该亚型中唯一突变频率显示亚型特异性关联的基因(21)。这一发现表明NHSL2在这一亚型乳腺癌的发病机制中发挥了作用。NHSL2是Nance Horan综合征(NHS)和Nance Horan综合征样蛋白1(NHSL1)(22)蛋白家族的一部分，对NHS的研究表明它在肌动蛋白重塑的调节中发挥作用，并包含一个功能性的WAVE同源结构域(WHD)，促进与SCAR/WAVE复合体的相互作用。SCAR/WAVE本身通过激活Arp2/3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，NHSL1通过两个Abi结合位点(23)负向调节SCAR/WAVE复合体的迁移。这些结合位点和上述WHD在所有三个NHS蛋白和唯一的果蝇同源基因GukH中保守，暗示该家族在5迁移(22，23)中发挥作用。我们以前观察到了NHSL1和NHSL2的囊泡定位(23个，未发表)，这表明这些蛋白在内体运输中发挥了作用。NHSL2与亲内素A3共同定位于这些囊泡的一个子集。此外，在GFP TRAP实验中，我们发现NHSL2与所有三种GFP标记的亲内素蛋白都有免疫共沉淀。这种相互作用可能指向NHSL2在FEME中的作用。我们感兴趣的是确定NHSL2是否调节女性，以及这是否可能作为NHSL2调节乳腺癌细胞迁移和侵袭的机制。我们假设NHSL2通过对抗极化突起和通过快速内吞蛋白介导的内吞作用(FEME)的定向迁移来抑制乳腺癌细胞的迁移和侵袭。为了解决这一假说，我们设计了以下目的：1.研究NHSL2在快速内毒素介导的内吞作用中的作用。2.探索NHSL2在乳腺癌细胞定向2D迁移中的作用。3.确定NHSL2是否调节乳腺癌细胞的3D侵袭。

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.