Serglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined. Overexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining. SRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation. Proteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.

中文翻译：

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蛋白聚糖Serglycin通过其糖胺聚糖与CD44的相互作用促进非小细胞肺癌细胞迁移

先前被认为是分泌性颗粒的储存过程中所涉及的细胞内蛋白聚糖的Serglycin（SRGN），最近已在几种实体瘤中被上调。先前我们已经表明，非小细胞肺癌（NSCLC）中的SRGN以CD44依赖性方式促进恶性表型，并且SRGN的表达增加预示了原发性肺腺癌的不良预后。但是，底层机制仍有待定义。使用伤口愈合和博登室迁移试验，进行了过表达，敲除和敲除方法，以评估SRGN在细胞运动中的作用。通过定点诱变或软骨素酶处理产生了没有糖胺聚糖（GAG）修饰的SRGN。采用液相色谱/串联质谱法对SRGN GAGs的二糖组成和硫酸化程度进行定量分析。进行蛋白质印迹和免疫共沉淀分析以确定目的蛋白的表达和相互作用。通过免疫荧光染色监测肌动蛋白的细胞骨架组织。由NSCLC细胞表达的SRGN易于以高度糖基化的形式分泌到细胞外基质中，并主要与硫酸软骨素（CS）-GAG链相连，而在较小程度上与硫酸肝素（HS）相连。CS-GAG部分充当SRGN与肿瘤细胞表面CD44结合的结构基序，并促进细胞迁移。没有CS-GAG修饰的SRGN无法与CD44相互作用，并且失去了促进细胞迁移的能力。SRGN / CD44相互作用通过Src介导的Paxillin磷酸化和Paxillin / FAK粘附复合物的分解而促进粘着斑更新，从而促进细胞迁移。作为支持，Src活性的耗尽或CS-GAG的去除可有效阻止SRGN介导的Src活化和细胞迁移。SRGN还通过诱导由RAC1和CDC42激活介导的细胞骨架重组，同时伴随着片状脂膜增多症和丝状伪足的形成增加，促进细胞迁移。蛋白聚糖SRGN通过其GAG基序与CD44结合促进NSCLC细胞迁移。SRGN / CD44相互作用诱导Rho家族GTPase介导的细胞骨架重组，并促进Src介导的粘着粘附更新，从而导致细胞迁移增加。