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Serglycin proteoglycan is required for multiple myeloma cell adhesion, in vivo growth, and vascularization.

ABSTRACT: Recently, it was discovered that serglycin, a hematopoietic cell proteoglycan, is the major proteoglycan expressed and constitutively secreted by multiple myeloma (MM) cells. High levels of serglycin are present in the bone marrow aspirates of at least 30% of newly diagnosed MM patients. However, its contribution to the pathophysiology of MM is unknown. Here, we show that serglycin knockdown (by ?85% compared with normal levels), using lentiviral shRNA, dramatically attenuated MM tumor growth in mice with severe combined immunodeficiency. Tumors formed from cells deficient in serglycin exhibited diminished levels of hepatocyte growth factor expression and impaired development of blood vessels, indicating that serglycin may affect tumor angiogenesis. Furthermore, knockdown of serglycin significantly decreased MM cell adhesion to bone marrow stromal cells and collagen I. Even though serglycin proteoglycan does not have a transmembrane domain, flow cytometry showed that serglycin is present on the MM cell surface, and attachment to the cell surface is, at least in part, dependent on its chondroitin sulfate side chains. Co-precipitation of serglycin from conditioned medium of MM cells using a CD44-Fc chimera suggests that CD44 is the cell surface-binding partner for serglycin, which therefore may serve as a major ligand for CD44 at various stages during myeloma progression. Finally, we demonstrate that serglycin mRNA expression in MM cells is up-regulated by activin, a predominant cytokine among those increased in MM patients with osteolytic lesions. These studies provide direct evidence for a critical role for serglycin in MM pathogenesis and show that targeting serglycin may provide a novel therapeutic approach for MM.

SUBMITTER: Purushothaman A

PROVIDER: S-EPMC3937626 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Serglycin proteoglycan is required for multiple myeloma cell adhesion, in vivo growth, and vascularization.

Purushothaman Anurag A Toole Bryan P BP

The Journal of biological chemistry 20140108 9

Recently, it was discovered that serglycin, a hematopoietic cell proteoglycan, is the major proteoglycan expressed and constitutively secreted by multiple myeloma (MM) cells. High levels of serglycin are present in the bone marrow aspirates of at least 30% of newly diagnosed MM patients. However, its contribution to the pathophysiology of MM is unknown. Here, we show that serglycin knockdown (by ∼85% compared with normal levels), using lentiviral shRNA, dramatically attenuated MM tumor growth in ...[more]

PMID: 24403068

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Project description:BACKGROUND: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. METHODS: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. RESULTS: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. CONCLUSIONS: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.

Project description:BACKGROUND:Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment. METHODS:N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells. RESULTS:GRP78 KD led to concomitant downregulation of N-cad in both tumors types. In PC3 cells, GRP78 KD significantly decreased E-cadherin (E-cad) expression likely associated with the induction in TGF-?1 expression. Furthermore, GRP78 KD also triggered drastic changes in PC3 cells morphology and decreased their adhesion to osteoblasts (OSB) dependent, in part, to the reduced N-cad expression. CONCLUSION:This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche.

Dataset Information

Serglycin proteoglycan is required for multiple myeloma cell adhesion, in vivo growth, and vascularization.

Publications

Serglycin proteoglycan is required for multiple myeloma cell adhesion, in vivo growth, and vascularization.

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