SFRP2 induces a mesenchymal subtype transition by suppression of SOX2 in glioblastoma

Abstract

Intratumoral heterogeneity is a characteristic of glioblastomas that contain an intermixture of cell populations displaying different glioblastoma subtype gene expression signatures. Proportions of these populations change during tumor evolution, but the occurrence and regulation of glioblastoma subtype transition is not well described. To identify regulators of glioblastoma subtypes we utilized a combination of in vitro experiments and in silico analyses, using experimentally generated as well as publicly available data. Through this combined approach SOX2 was identified to confer a proneural glioblastoma subtype gene expression signature. SFRP2 was subsequently identified as a SOX2-antagonist, able to induce a mesenchymal glioblastoma subtype signature. A subset of patient glioblastoma samples with high SFRP2 and low SOX2 expression was particularly enriched with mesenchymal subtype samples. Phenotypically, SFRP2 decreased tumor sphere formation, stemness as assessed by limiting dilution assay, and overall cell proliferation but increased cell motility, whereas SOX2 induced the opposite effects. Furthermore, an SFRP2/non-canonical-WNT/KLF4/PDGFR/phospho-AKT/SOX2 signaling axis was found to be involved in the mesenchymal transition. Analysis of human tumor tissue spatial gene expression patterns showed distinct expression of SFRP2- and SOX2-correlated genes in vascular and cellular areas, respectively. Finally, conditioned media from SFRP2 overexpressing cells increased CD206 on macrophages. Together, these findings present SFRP2 as a SOX2-antagonist with the capacity to induce a mesenchymal subtype transition in glioma cells located in vascular tumor areas, highlighting its role in glioblastoma tumor evolution and intratumoral heterogeneity.

Altered cytoskeletal status in the transition from proneural to mesenchymal glioblastoma subtypes

“…Previous studies have shown that based on its gene expression signature, cell line U-2982MG can be stratified as a mesenchymal subtype glioblastoma 19 . In contrast, cell line U-2987MG shows a proneural signature, because of high expression of the transcription factors SOX2 and PROX1 19 , 29 . Transcriptome analysis by RNAseq or gene arrays reported previously indicated that two separate groups of glioblastoma cell lines can be distinguished 19 , 25 .…”

“…Examples of such genes are GREM1 , FN1 , LUM , SPARC , and COL1A1 30 – 34 . The transcription factor SOX2 has been shown to regulate glioblastoma gene expression, to shift the cells from proneural subtype to mesenchymal subtype, and SOX2 can be suppressed by SFRP2 29 . To understand more about the critical factors that are decisive for the two major glioblastoma subtypes, we used two cell lines that were produced in an earlier study: cell line U-2982MG with stable overexpression of SOX2 (U-2982MG/SOX2), and cell line U-2987MG with stable overexpression of SFRP2 (U-2987MG/SFRP2).…”

“…U-2982MG and U-2987MG/SFRP2), two cell types with proneural subtype gene expression signature (i.e., U-2982MG/SOX2, and U-2987MG), and two transfection control cell lines (i.e., U-2982MG/YFP and U-2987MG/YFP) (Fig. 3 f) 29 . Cell line U-2982MG/SOX2 had more cells with actin arcs and fewer lamellipodia (57%, 37% of the cells, respectively), as compared to cell line U-2982MG (35%, 61%, respectively) (Fig.…”

“…Interestingly, re-expression of SOX2 in the mesenchymal type cell line U-2982MG resulted in regression of the phenotype into an epithelioid cell type, based on the cytoskeletal characteristics and migratory behavior. Expression of SFRP2 in cell line U-2987MG resulted in transition to a mesenchymal phenotype with a more specific cytoskeletal organization increasing collective migration and invasion, as SFRP2 has been shown to act as a SOX2 antagonist in glioblastoma cells 29 . In conclusion, elucidating different mechanisms in migration and invasion as well as the potential of SOX2 re-expression to reverse the mesenchymal subtype into a more proneural subtype, should thus open a strategy to alternative treatments in glioblastoma.…”

“…Cell lines U-2975MG, U-2987MG, U-2995MG, and U-2997MG were diagnosed as grade 3–4 according to the World Health Organization, and cell lines U-2982MG and U-2990MG were diagnosed as grade 4 26 . Cell lines U-2982MG/YFP, U-2982MG/SOX2, U-2987MG/YFP, and U-2987MG/SFRP2 were establish by transduction of SOX2 or SFRP2 overexpression lentivirus vectors into cell lines U-2982MG and U-2987MG, respectively 29 . YFP was introduced into both cell lines U-2982MG and U-2987MG in the same way, as controls.…”