LY6K Promotes Glioblastoma Tumorigenicity via CAV-1-Mediated ERK1/2 Signaling Enhancement

Sastry, Namratha  G

doi:https://doi.org/10.21985/n2-db5p-9v07

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LY6K Promotes Glioblastoma Tumorigenicity via CAV-1-Mediated ERK1/2 Signaling Enhancement

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Background: Glioblastoma (GBM) tumors are the most malignant brain cancers and are characterized as Grade IV astrocytomas by the World Health Organization. GBM tumors can be classified into three molecular subtypes known as proneural, classical, and mesenchymal. In addition, GBM tumors also have a small population of cells known as glioma stem-like cells or GSCs, which can also be classified into subtypes that mirror clinical GBM. Gene expression array data revealed that Lymphocyte Antigen 6 Complex, Locus K (LY6K) may be a differentially expressed gene between the PN and MES subtypes within the GSC subpopulation. LY6K is a putative oncogene in various cancers, and elevated expression of LY6K is correlated with poor patient prognosis in GBM. The aim of our research is to advance our understanding of the mechanism by which LY6K contributes to GBM tumor biology. Methods: Bioinformatic data mining was used to investigate LY6K expression in relation to GBM clinical outcome. To understand the role of LY6K in GBM biology, we utilized patient-derived glioma stem-like cells (GSCs) and U87 glioma cells and conducted cell proliferation assays, tumor sphere forming frequency assays, and utilized orthotopic GBM xenograft models in immunocompromised mice. We also studied the mechanism that promotes LY6K-mediated GBM tumorigenicity by employing in vitro signaling assays, immunoblotting assays, and immunofluorescent staining. Finally, we analyzed the importance of methylation on LY6K gene expression by using combined bisulfite and restriction analyses and bisulfite sequencing. We also demonstrated the clinical significance of LY6K by using ionizing radiation treatment and evaluating the effect on GSC proliferation. Results: Increased expression of LY6K inversely correlates with survival of patients with GBM. Our results show that LY6K promotes tumorigenicity in GBM cells both in vitro and in vivo. The mechanism underlying this tumorigenic behavior is enhancement of ERK1/2 signaling. Interestingly, we observed that tumor-promoting LY6K-ERK1/2 signaling is mediated by the interaction of LY6K with caveolin-1, rather than through oncogenic receptor tyrosine kinase (RTK)-mediated signaling. Moreover, association of LY6K with the cell membrane is crucial for its tumorigenic functions. Finally, DNA methylation maintains LY6K silencing, and hypomethylation of the LY6K promoter increases its expression. In GSCs, ionizing radiation leads to demethylation of the LY6K promoter, thereby increasing LY6K expression and GSC resistance to radiation therapy (RT). Conclusion and Significance: Our study highlights the importance of the contribution of LY6K to GBM tumor biology and suggests LY6K as a potential membrane target for treating GBM. Although LY6K was reported as an oncogenic protein that promotes tumorigenicity in multiple types of cancers, its role in GBM and the underlying mechanism by which LY6K mediates oncogenic signaling are unknown. Here we determined oncogenic functions of LY6K in GBM and describe a novel mechanism by which membrane-anchored LY6K stimulates ERK1/2 signaling through its association with caveolin-1 (CAV-1). Additionally, LY6K expression is regulated by gene promoter methylation, and irradiation induces LY6K expression by demethylating the LY6K promoter. This is the first study to determine the role and mode of action of LY6K in GBM biology. Our study highlights the importance of LY6K in enhancing tumorigenicity in GBM and suggests LY6K as a potential target for reducing GBM resistance to ionizing radiation (IR). Key points 1. Elevated LY6K expression correlates with poor prognosis and tumorigenicity in GSCs and GBM cells. 2. LY6K functions by enhancing ERK1/2 signaling through interactions with CAV-1. 3. DNA methylation regulates LY6K. IR promotes LY6K promoter demethylation, thereby increasing LY6K expression. Importance of Study Although LY6K was previously implicated in tumorigenicity of multiple types of cancers including GBM, its role in GBM and the underlying mechanistic basis by which LY6K mediates tumor-promoting signaling are unknown. Here, we illustrate a novel signaling mechanism by which membrane-anchored LY6K enhances ERK1/2 signaling through its association with caveolin-1 to facilitate GBM tumorigenicity. Additionally, DNA methylation contributes to LY6K expression, and irradiation induces LY6K expression by promoter demethylation, resulting in enhanced radiation resistance in GSCs. This is the first study to investigate the function and underlying mechanism of LY6K in GBM tumor biology. Our study highlights LY6K as a potential targetable protein to promote radiation sensitivity in GBM.

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