EGF/EGFR upregulates and cooperates with Netrin-4 to protect glioblastoma cells from DNA damage-induced senescence.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment. METHODS: Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis. RESULTS: Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients. CONCLUSIONS: This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.

Reduced gene expression of netrin family members in skin and sural nerve specimens of patients with painful peripheral neuropathies.

OBJECTIVE: To investigate the expression of axon guidance cues in skin and sural nerve biopsies of patients with polyneuropathies (PNP) as potential markers of nerve de- and regeneration and inflammation. METHODS: We prospectively recruited 88 patients with PNP and compared data between patient subgroups and healthy controls. All patients underwent skin punch and/or sural nerve biopsy at the lower leg and proximal thigh. We characterized gene expression profiles of netrin family members as target genes involved in neuronal de- and regeneration [netrin 1, deleted in colorectal cancer (DCC), uncoordinated5H2, neogenin 1 (NEO1), netrin G1, netrin G2] using quantitative real-time PCR. RESULTS: Gene expression of netrin 1 (p < 0.05 in proximal skin), DCC (p < 0.001 in distal skin), NEO1 (p < 0.05 in distal skin), netrin G1 (p < 0.05 in proximal and p < 0.01 in distal skin), and netrin G2 (p < 0.001 in distal skin) was lower in skin biopsies of patients with neuropathy compared to healthy controls. Gene expression of NEO1 (p < 0.05 in distal skin), netrin G2 (p < 0.05 in distal skin), and DCC (p < 0.05 in sural nerve) was lower in samples of patients with painful compared to painless PNP and also correlated positively with intraepidermal nerve fiber density. Skin and sural nerve gene expression of the investigated target genes did not differ between neuropathies of different etiologies. CONCLUSION: We show reduced cutaneous and neural axon guide expression, which may contribute to a dysregulation of nerve fiber de- and regeneration.