Dual-specificity phosphatase (DUSP6) in human glioblastoma: epithelial-to-mesenchymal transition (EMT) involvement

Erika Di Zazzo,Samantha Messina,Candida Zuchegna,Bruno Moncharmont

doi:10.1186/s13104-020-05214-y

Abstract

ObjectiveGlioblastoma (GBM) is the most aggressive and common form of primary brain cancer. Survival is poor and improved treatment options are urgently needed. Dual specificity phosphatase-6 (DUSP6) is actively involved in oncogenesis showing unexpected tumor-promoting properties in human glioblastoma, contributing to the development and expression of the full malignant and invasive phenotype. The purpose of this study was to assess if DUSP6 activates epithelial-to-mesenchymal transition (EMT) in glioblastoma and its connection with the invasive capacity.ResultsWe found high levels of transcripts mRNA by qPCR analysis in a panel of primary GBM compared to adult or fetal normal tissues. At translational levels, these data correlate with high protein expression and long half-life values by cycloheximide-chase assay in immunoblot experiments. Next, we demonstrate that DUSP6 gene is involved in epithelial-to-mesenchymal transition (EMT) in GBM by immunoblot characterization of the mesenchymal and epithelial markers. Vimentin, N-Cadherin, E-Cadherin and fibronectin were measured with and without DUSP6 over-expression, and in response to several stimuli such as chemotherapy treatment. In particular, the high levels of vimentin were blunted at increasing doses of cisplatin in condition of DUSP6 over-expression while N-Cadherin contextually increased. Finally, DUSP6 per se increased invasion capacity of GBM. Overall, our data unveil the DUSP6 involvement in invasive mesenchymal-like properties in GBM.

Highlights

Dual specificity phosphatase-6 (DUSP6) plays a pro-oncogenic role in cancers such as human glioblastoma, thyroid carcinoma, breast cancer, and acute myeloid leukemia [1,2,3,4]
Quantitative transcriptional analysis of DUSP6 was assessed by RT-qPCR measuring high mRNA levels in a panel of twenty primary glioblastomas (GBM, WHO grade 4) (Fig. 1b) by quantifying the mRNA fold-induction over Normal Human Astrocytes (NHA) and Neural Stem Cells (NSC) which specify distinct glioblastoma subtype [17,18,19,20]
Human long-term cultures U87MG, U251MG and T98G displayed high mRNA levels compared to primary samples of GBM (Fig. 1c)

Summary

Results

Quantitative transcriptional analysis of DUSP6 was assessed by RT-qPCR measuring high mRNA levels in a panel of twenty primary glioblastomas (GBM, WHO grade 4) (Fig. 1b) by quantifying the mRNA fold-induction over Normal Human Astrocytes (NHA) and Neural Stem Cells (NSC) which specify distinct glioblastoma subtype [17,18,19,20]. Human long-term cultures U87MG, U251MG and T98G displayed high mRNA levels compared to primary samples of GBM (their values differ by several orders of magnitude) (Fig. 1c). Unstimulated primary GBM (Panel a) and long-term cultures (Panel b) show high protein levels in both primary and long-term glioblastoma as assayed by semi-quantitative Western blot analysis (Fig. 2a, b). In contrast to previously published data [21] we report long half-life in both cell lines U87MG and U251 (respectively more than 1 h and up to 5 h) These results agree with the stable endogenous protein exerting oncogenic properties in cancers. We assayed protein endogenous levels of Vimentin, N-Cadherin, E-Cadherin, Fibronectin in U87MG upon several stimuli (serum addition, serum deprivation, EGF and cisplatin) by Western Blot with specific antibodies (Fig. 3a). We here report that DUSP6 increased the invasion capacity of the glioma U87MG cells (Fig. 3c) compared to MOCK cells (naïve U87MG) and adenoviral empty vector as negative control (TRK)

Introduction

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