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Dual-Specificity Phosphatase (DUSP6) Zuchegna et al. BMC Res Notes (2020) 13:374 https://doi.org/10.1186/s13104-020-05214-y BMC Research Notes RESEARCH NOTE Open Access Dual-specifcity phosphatase (DUSP6) in human glioblastoma: epithelial-to-mesenchymal transition (EMT) involvement Candida Zuchegna1, Erika Di Zazzo2, Bruno Moncharmont2 and Samantha Messina3* Abstract Objective: Glioblastoma (GBM) is the most aggressive and common form of primary brain cancer. Survival is poor and improved treatment options are urgently needed. Dual specifcity phosphatase-6 (DUSP6) is actively involved in oncogenesis showing unexpected tumor-promoting properties in human glioblastoma, contributing to the develop- ment 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. Results: We 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 epithe- lial markers. Vimentin, N-Cadherin, E-Cadherin and fbronectin were measured with and without DUSP6 over-expres- sion, 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. Keywords: Dual-specifcity phosphatase (DUSP6), Glioblastoma, Epithelial-to-mesenchymal transition (EMT) Introduction levels of cellular detachment after stimulation with EGF DUSP6 plays a pro-oncogenic role in cancers such as and an increased propensity to form colonies in soft agar. human glioblastoma, thyroid carcinoma, breast cancer, Surprisingly, mouse xenograft tumors expressing DUSP6 and acute myeloid leukemia [1–4]. Particularly, DUSP6 grew signifcantly faster than controls thus refect- is upregulated in human glioblastoma where its over- ing these changes in cell adhesion and morphology [1]. expression induces reduction in proliferation rate. Cell Moreover, overexpression of DUSP6 has also been iden- morphology exhibits a more fattened appearance, lower tifed in a subset of mouse melanoma cell lines, where it is associated with enhanced anchorage-independent growth and invasive capacity [5] and its overexpression *Correspondence: [email protected]; samantha. in papillary thyroid carcinoma (PTC) is associated with [email protected] 3 Department of Science, Roma Tre University, Viale Guglielmo Marconi increased cell migration and invasion [2, 6]. Finally, in 446, 00146 Rome, Italy acute lymphoblastic leukemia (ALL) DUSP6 acts as pro- Full list of author information is available at the end of the article oncogenic phosphatase in pre-B cell transformation [7]. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zuchegna et al. BMC Res Notes (2020) 13:374 Page 2 of 8 Glioblastoma (GBM) is the most common and malig- cycloheximide-chase assay experiments the cells were nant type of primary brain tumor with relevant invasive treated with 20 µg/mL CHX in complete medium and and resistant properties [8]. Te involvement of epithe- then lysed at the indicated times as described in the fg- lial-to-mesenchymal transition (EMT) has been exten- ures’ legends. sively investigated in glioblastoma although the real relevance of this program in malignant glioma is still controversial. A number of preclinical studies have been Western Blot analysis launched to target the process considering the critical Cultured U87-MG and U251-MG cells were washed with role played by EMT on GBM dissemination, resistance PBS and lysed for 15 min in ice-cold Radioimmunopre- to apoptosis and cancer stemness maintenance [9–11]. cipitation (RIPA) bufer (1% Triton X-100, 0.5% deox- Of note, DUSP6 is involved in epithelial-to-mesenchymal ycholate-DOC), 0.1% sodium dodecyl sulphate (SDS), transition (EMT) in epithelial cancers such as breast and 50 mM Tris pH 7.6, 150 mM NaCl, 1 mM phenyl-methyl- endometrial adenocarcinoma [12, 13]. Moreover, GBMs sulfonyl fuoride (PMSF), 1 mg/mL aprotinin, leupeptin mesenchymal subtype are characterized by an elevated and pepstatin. Cell lysates were clarifed at 12,000 rcf for invasive potential and, of note, the most commonly used 30 min at 4 °C and the cytosolic fraction was immedi- glioma cell lines (i.e. U87MG and U251) also present a ately subjected to protein determination using a Bradford predominant mesenchymal signature [14] with elevation colorimetric assay (Bio-Rad Laboratories Inc., Hercules, in mesenchymal markers [15]. Te aim of this study was CA, USA). DUSP6 was detected with a ‘home-made’ rab- to assess the importance of DUSP6 gene in epithelial-to- bit polyclonal specifc antibody against DUSP6 (Lennart- mesenchymal transition in GBM in correlation with its son’s lab). Monoclonal anti-α-tubulin as loading control, invasive capacity. anti-phospho-ERK and anti-Fibronectin were purchased by Sigma-Aldrich (St Louis, MO, USA). Anti-Vimentin Main text was purchased from Millipore and anti-ERK was pur- Methods chased from Cell Signaling Technology. Anti N-Cadherin Cell cultures and anti-E-Cadherin were purchased from Santa Cruz Astrocytoma primary (WHO grade IV) GBM#1; Biotechnology. Goat anti-Mouse IgG (H + L) Highly GBM#10 etc. were established from tumor specimens Cross-Adsorbed Secondary Antibody, HRP (A16078) e of patients and cultured as described [1]. NHA (Nor- Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed mal Human Astrocytes) and NSC (Neural Stem Cells) Secondary Antibody, HRP (A16119) Termofsher Scien- were purchased from Cambrex (Corporate, NJ 07073, tifc. ECL detection kit from Amersham GE-Healthcare. USA) and grown according to the manufacturer’s instructions. Normal Human Astrocytes were used as Real-time quantitative PCR reference because of the presumed similarity between astrocytes and the cell-of-origin from which glioblas- A quantitative assay for Human DUSP6 mRNA (seq ref toma develops, both adult (NHA) and foetal (Primary NM_001946.4) expression was established using StepO- ™ ™ Fetal Normal Neural Stem Cells from SVZ). U87-MG nePlus Real-Time PCR System (Applied Biosystems ) ™ ™ (human GBM–astrocytoma) cell line was purchased using PowerUp SYBR Green Master Mix (Applied ™ from the bank of biological material Interlab Cell Line Biosystems ) using the following program: 95 °C/10 min Collection (Genova, Italy). Human breast cell cultures ×1 cycle; 95 °C/15 s, 63 °C/90 s, ×5 cycles; 95 °C/15 s, MCF7, MCF10A and MDA231 cell lines were pur- 60 °C/90 s, ×35 cycles. All reactions were normalized chased from the American Type Culture Collection with the housekeeping gene for 18S. PCR oligo-primers ′ (ATTC, LGC Standards s.r.l, Italy). Cells were cultured were: Human DUSP6 forward primer 5 -CgAggAC- ′ ′ at 37 °C in 5% CO in DMEM with high glucose plus CgggACCgCTT CAC C-3 and reverse primer 5 -CCgA- 2 ′ 10% (v/v) fetal bovine serum (FBS, Euroclone, Milan, gATggggATTTgCTTgTATT-3 generating a 543 bp ′ Italy), penicillin–streptomycin (100 U/mL, Euroclone) fragment; Human 18S forward primer 5 -gACCgATgTA- ′ ′ and l-glutamine (2 mM, Euroclone), according to TATgCTTgCAgAgT-3 and reverse primer 5 -ggATCTg- ′ manufacturer’s instructions. Primary glioblastomas cell gAgTTA AAC TggTCCAg-3 . Te two transcripts of lines (WHO grade IV) were established from tumor mRNA DUSP6 were detected by PCR using the following ′ specimens of patients and cultured as described [1, primers: Forward primer 5 -CgAggACCgggACCgCTT ′ ′ 16]. Cells were plated at 80% of confuence on 100 mm CAC C-3 ; Reverse primer: 5 -AgTTAggggATATgTTg- ′ dishes and the day after infected with recombinant gATTTT-3 . Te expected size of the fragment was: adenovirus as previously described with the amounts 758 bp for the transcript variant 1 (NM_001946.4) and according to the scheme indicated in the fgures [16]. In 321 bp for the transcript variant 2 (NM_022652.4). Zuchegna et al. BMC Res Notes (2020) 13:374 Page 3 of 8 Cell invasion assay cascade activation was sustained in both primary and Trans-well
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