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Abstract

Epithelial membrane protein 3 (EMP3) is a small, N-glycosylated tetraspanin that is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), a highly aggressive brain tumor characterized by extreme intratumoral heterogeneity. Specifically, it is highly expressed in astrocytic- and mesenchymal-like cells that predominantly compose the receptor tyrosine kinase II/classical (RTK II/CL) and mesenchymal (MES) IDH-wt GBM bulk tumor subtypes, respectively. EMP3 has been implicated in various biological processes and is proposed to facilitate tumor development in IDH-wt GBM. However, its exact molecular functions, and how these relate to IDH-wt GBM molecular heterogeneity, remain unclear. By integrating protein-protein interaction screens with transcriptomics, phosphoproteomics, and functional characterization of CRISPR/Cas9-edited EMP3 knockout (EMP3 KO) cells, this study defined the receptor trafficking and membrane organizing functions of EMP3 in IDH-wt GBM cellular models that approximate the RTK II/CL and MES subtypes, respectively. To obtain initial insights on the subcellular context in which EMP3 could operate in, interactome mapping using BioID2-based proximity labeling coupled to mass spectrometry (MS) analysis was performed using RTK II/CL-like U-118 and MES-like LN-18 GBM cells. This approach identified several GBM-associated transmembrane receptors and trafficking regulators as novel, putative EMP3 interactors. Validation by co-immunoprecipitation and proximity ligation assays confirmed the RTK II/CL driver EGFR, the MES receptors CD44 and MET, the retromer components (TBC1D5, SNX1, and SNX2), the clathrin-coated vesicle protein CLINT1, and the EARP member VPS53 as bona fide EMP3 interacting partners. Furthermore, the analysis also identified potential cell type-specific and glycosylation-dependent interactors of EMP3. These included proteins involved in mitochondrial processes and nascent protein synthesis and transport in U-118 cells, as well as a Rho GTPase signaling subnetwork and mesenchymal RTKs in LN-18 cells.

Functional characterization of DK-MG and U-118 EMP3 KOs identified a novel EMP3-dependent mechanism by which EGFR activity could be sustained in these RTK II/CL-like cells. Loss of EMP3 enhanced epidermal growth factor (EGF)-induced EGFR degradation, an effect that correlated with increased EGFR trafficking to RAB7+ late endosomes. The degradation phenotype was rescued by overexpression of the novel EMP3 interactor and RAB7 GTPase-activating protein TBC1D5 in a manner that is dependent on TBC1D5’s catalytic activity. Transcriptomic analysis further revealed dysregulation of DNA replication, cell cycle, and EGFR tyrosine kinase inhibitor resistance upon EMP3 depletion in these cell lines. Phosphoproteomic analysis of DK-MG and U-118 EMP3 KOs also indicated inhibition of downstream EGFR effector AKT1, as well as distal inhibition of cyclin-dependent kinases, most significantly CDK2. These signaling defects translated into cellular phenotypes, as EMP3 KO cells exhibited reduced cellular proliferation, blunted mitogenic response to the EGFR ligand EGF, and increased sensitivity to targeted EGFR inhibition by osimertinib. The positive correlation between EMP3 and EGFR was further reflected in clinical data from The Cancer Genome Atlas (TCGA), which indicated higher total and phosphorylated EGFR levels in GBMs with high EMP3 expression. Taken together, the findings imply that EMP3 may help maintain EGFR signaling in RTK II/CL tumors.

On the other hand, characterization of LN-18 EMP3 KOs revealed EMP3’s potential role in maintaining CD44 and MET activity in MES-like cells. Specifically, loss of EMP3 impaired the transcription of genes that are dependent on CD44 and the Rho GTPase signaling effector and CD44 downstream target IQGAP1. EMP3-dependent activation of CD44/IQGAP1 signaling was further hypothesized to be mediated by PAK1, an EMP3 interactor that is known to also interact with and activate IQGAP1. Consistent with impaired CD44/IQGAP1 signaling, EMP3 KOs displayed reduced mitogenic response to the CD44 ligand hyaluronic acid (HA). At the same time, loss of EMP3 also abrogated the complex formation between the standard CD44 isoform, CD44s, and MET. This coincided with reduced MET membrane presentation as revealed by MS-based cell surface proteome analysis. Reduced EMP3-dependent CD44s-MET complex formation is presumed to cause MET signaling defects, as evidenced by the reduced transcription of genes regulated by the downstream MET effector ERK and impaired mitogenic response of EMP3 KOs to the MET ligand hepatocyte growth factor (HGF). Collectively, the findings indicate the EMP3 may organize the cytosolic and transmembrane interactions of CD44 and MET, and by doing so facilitate HA- and HGF-induced signaling.

In conclusion, this study identifies EMP3 to be a multifunctional protein with context-dependent and cell type-specific functions in IDH-wt GBM. In RTK II/CL-like DK-MG and U-118 cells, EMP3 facilitates EGFR signaling by restricting receptor degradation, while in MES-like LN-18 cells, EMP3 organizes signal transduction complexes required for CD44 and MET signaling. This dual trafficking and membrane organizing function highlights how moonlighting proteins like EMP3 could further contribute to the underlying subcellular functional diversity of tumor cells in IDH-wt GBMs. Additionally, it exemplifies how a non-oncogene dependency may ultimately contribute to the maintenance of oncogenic signaling and development of therapeutic resistance. Altogether, these findings clarify the role of EMP3 in IDH-wt GBMs and provide the foundation for future mechanistic and therapeutic investigations into this protein.