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Loss of SPINT2 expression frequently occurs in glioma, leading to increased growth and invasion via MMP2

  • Márcia Santos Pereira
  • Sónia Pires Celeiro
  • Ângela Margarida Costa
  • Filipe Pinto
  • Sergey Popov
  • Gisele Caravina de Almeida
  • Júlia Amorim
  • Manuel Melo Pires
  • Célia Pinheiro
  • José Manuel Lopes
  • Mrinalini Honavar
  • Paulo Costa
  • José Pimentel
  • Chris Jones
  • Rui Manuel ReisEmail author
  • Marta Viana-PereiraEmail author
Original paper

Abstract

Purpose

High-grade gliomas (HGG) remain one of the most aggressive tumors, which is primarily due to its diffuse infiltrative nature. Serine proteases and metalloproteases are known to play key roles in cellular migration and invasion mechanisms. SPINT2, also known as HAI-2, is an important serine protease inhibitor that can affect MET signaling. SPINT2 has been found to be frequently downregulated in various tumors, whereby hypermethylation of its promoter appears to serve as a common mechanism. Here, we assessed the clinical relevance of SPINT2 expression and promoter hypermethylation in pediatric and adult HGG and explored its functional role.

Methods

A series of 371 adult and 77 pediatric primary HGG samples was assessed for SPINT2 protein expression (immunohistochemistry) and promoter methylation (methylation-specific PCR) patterns. After SPINT2 knockdown and knock-in in adult and pediatric HGG cell lines, a variety of in vitro assays was carried out to determine the role of SPINT2 in glioma cell viability and invasion, as well as their mechanistic associations with metalloprotease activities.

Results

We found that SPINT2 protein expression was frequently absent in adult (85.3%) and pediatric (100%) HGG samples. The SPINT2 gene promoter was found to be hypermethylated in approximately half of both adult and pediatric gliomas. Through functional assays we revealed a suppressor activity of SPINT2 in glioma cell proliferation and viability, as well as in their migration and invasion. These functions appear to be mediated in part by MMP2 expression and activity.

Conclusions

We conclude that dysregulation of SPINT2 is a common event in both pediatric and adult HGG, in which SPINT2 may act as a tumor suppressor.

Keywords

Glioma SPINT2 Hypermethylation Metalloproteases 

Notes

Acknowledgements

The authors thank Dr. Scholm for providing the 4/T0 vector.

Funding information

This work was funded by the Fundação para a Ciência e Tecnologia (FCT), Portugal (www.fct.pt) (grants PTDC/SAU-ONC/115513/2009_FCOMP-01-0124-FEDER-015949 and PTDC/SAU-TOX/114549/2009 to RMR). This article has been developed under the scope of project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through FCT, under the scope of project UID/Multi/50026. MVP was recipient of a FCT Post-Doctoral fellowship (SFRH/BPD/104290/2014). SPC was recipient of a BOLSA LIGA PORTUGUESA CONTRA CANCRO (LPCC)/Fundação PT 2018 grant. AMC was recipient of fellow DL 57/2016/CP1360/CT0009. FP was recipient of a FCT Post-Doctoral fellowship (SFRH/BPD/115730/2016); CJ acknowledges NHS funding to the NIHR Biomedical Research Centre at The Royal Marsden and the ICR.

Compliance with ethical standards

Conflict of interests

The authors disclose no potential conflicts of interest.

Supplementary material

13402_2019_475_MOESM1_ESM.jpg (112 kb)
ESM 1 Fig. S1. Median expression intensity for SPINT2 mRNA levels in in silico analysis. a) Analysis of SPINT2 mRNA expression in Rembrandt database. SPINT2 is down-regulated compared with non-tumor tissues. b) Analysis of SPINT2 mRNA expression in TCGA database. The mRNA levels are showed in Log2 median values and normalized to normal brain. Each point corresponds to one patient. Only two patients present higher levels of SPINT2 than normal brain. (JPG 111 kb)
13402_2019_475_MOESM2_ESM.jpg (177 kb)
ESM 2 Fig. S2.SPINT2 mRNA, protein and methylation characterization of adult glioma cell lines. a)SPINT2 promoter methylation analysis by MSP (M-methylated and UM-unmethylated product) showed SPINT2 methylation in all adult glioma cell lines. b)SPINT2 mRNA expression analysis by RT-PCR, using as a reference gene β-Actin, showed a decrease in SPINT2 mRNA levels from normal brain (adult and fetal brain tissues and normal cell line, NHAi) to glioma cell lines. c) Immunocytochemistry and d) immunofluorescence for SPINT2 confirmed the low expression levels observed in RNA analysis. (JPG 177 kb)
13402_2019_475_MOESM3_ESM.jpg (98 kb)
ESM 3 Fig. S3. Modulation of SPINT2 expression in SPINT2-negative U87 adult glioma cell lines. a-c) U87 cell line transfected with empty vector (4/T0) or SPINT2 transcript (4/T0-SPINT2). mRNA and protein expression analysis confirmed the over-expression of SPINT2. DAPI stained nucleus in blue and SPINT2 was stained in green. DAPI/SPINT2 is the merge of both staining. (JPG 97 kb)
13402_2019_475_MOESM4_ESM.jpg (124 kb)
ESM 4 Fig. S4. Functional assays for U87 adult glioma cell line under over-expression of SPINT2. a) MTS, (b) BrdU, (c) Colonigenic assay, (d) Matrigel invasion assay, (e) quantitative PCR for MMP2 and (f) zymogram showed that over-expression of SPINT2 decreased viability, proliferation, colonies formation, invasion capacity and MMP2 activity and expression. *p < 0.05, **p < 0.01, *p < 0.001 (JPG 123 kb)
13402_2019_475_MOESM5_ESM.jpg (43 kb)
ESM 5 Figure S5_ The effect of SPINT2 on MMP2 activity through HGF-MET pathway. MMP2 activity was detected by zymogram using conditioned medium from a) SF188 and b) U87 glioma cells, supplemented or not with 100 ng of HGF for 24 h. The stimulation of MMP2 activity was only observed in SPINT2-negative cells. Zymograms are representative of 3 independent experiences. Cells with empty vector - 4/T0; Cells with SPINT2 transcript (4/T0-SPINT2); Pro-MMP2 – total form of MMP2; Cleaved-MMP2 – activated form of MMP2. (JPG 42 kb)
13402_2019_475_MOESM6_ESM.docx (14 kb)
ESM 6 (DOCX 14 kb)

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Copyright information

© International Society for Cellular Oncology 2019

Authors and Affiliations

  • Márcia Santos Pereira
    • 1
    • 2
  • Sónia Pires Celeiro
    • 1
    • 2
  • Ângela Margarida Costa
    • 3
    • 4
  • Filipe Pinto
    • 1
    • 2
    • 3
    • 5
  • Sergey Popov
    • 6
  • Gisele Caravina de Almeida
    • 7
  • Júlia Amorim
    • 8
  • Manuel Melo Pires
    • 9
  • Célia Pinheiro
    • 10
  • José Manuel Lopes
    • 5
    • 11
  • Mrinalini Honavar
    • 12
  • Paulo Costa
    • 13
  • José Pimentel
    • 14
  • Chris Jones
    • 15
  • Rui Manuel Reis
    • 1
    • 2
    • 16
    Email author
  • Marta Viana-Pereira
    • 1
    • 2
    Email author
  1. 1.Life and Health Sciences Research Institute (ICVS), School of MedicineUniversity of MinhoBragaPortugal
  2. 2.ICVS/3B’s - PT Government Associate LaboratoryBraga/GuimarãesPortugal
  3. 3.I3S - Instituto de Investigação e Inovação em SaúdePortoPortugal
  4. 4.INEB - Institute of Biomedical EngineeringPortoPortugal
  5. 5.IPATIMUP – Institute of Molecular Pathology and ImmunologyUniversity of PortoPortoPortugal
  6. 6.Department of Cellular PathologyUniversity Hospital of WalesCardiffUnited Kingdom
  7. 7.Department of PathologyBarretos Cancer HospitalS. PauloBrazil
  8. 8.Department of OncologyHospital de BragaBragaPortugal
  9. 9.Unity of NeuropathologyCentro Hospitalar Universitário PortoPortoPortugal
  10. 10.Department of NeurosurgeryCentro Hospitalar Universitário PortoPortoPortugal
  11. 11.Department of PathologyHospital São JoãoPortoPortugal
  12. 12.Department of PathologyHospital Pedro HispanoMatosinhosPortugal
  13. 13.Department of RadiotherapyHospital de BragaBragaPortugal
  14. 14.Laboratory of NeuropathologyHospital de Santa MariaLisbonPortugal
  15. 15.Divisions of Molecular Pathology and Cancer TherapeuticsInstitute of Cancer ResearchLondonUnited Kingdom
  16. 16.Molecular Oncology Research Center, Barretos Cancer HospitalBarretos, São PauloBrazil

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