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Differential gene expression in peritumoral brain zone of glioblastoma: role of SERPINA3 in promoting invasion, stemness and radioresistance of glioma cells and association with poor patient prognosis and recurrence

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Abstract

Purpose

Glioblastoma (GBM) is a highly invasive tumor. Despite advances in treatment modalities, tumor recurrence is common, seen mainly in the peritumoral brain zone (PBZ). We aimed to molecularly characterize PBZ, to understand the pathobiology of tumor recurrence.

Methods/patients

We selected eight differentially regulated genes from our previous transcriptome profiling study on tumor core and PBZ. Expression of selected genes were validated in GBM (tumor core and PBZ, n = 37) and control (n = 22) samples by real time quantitative polymerase chain reaction (qPCR). Serine protease inhibitor clade A, member 3 (SERPINA3) was selected for further functional characterization in vitro by gene knockdown approach in glioma cells. Its protein expression by immunohistochemistry (IHC) was correlated with other clinically relevant GBM markers, patient prognosis and tumor recurrence.

Results

The mRNA expression of selected genes from the microarray data validated in tumor core and PBZ and was similar to publicly available databases. SERPINA3 knock down in vitro showed decreased tumor cell proliferation, invasion, migration, transition to mesenchymal phenotype, stemness and radioresistance.

SERPINA3 protein expression was higher in PBZ compared to tumor core and also was higher in older patients, IDH wild type and recurrent tumors. Finally, its expression showed positive correlation with poor patient prognosis.

Conclusions

SERPINA3 expression contributes to aggressive GBM phenotype by regulating pro-tumorigenic actions in vitro and is associated with adverse clinical outcome.

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Data availability

All data generated or analyzed during this study are included in this article and its Supplementary Information files.

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Acknowledgements

Indian Council of Medical Research (ICMR) is acknowledged for Fellowship to VPN. The results presented in this study are in part based on the data generated by Ivy GAP database, The Cancer Genome Atlas established by NCI and NHGRI. Information about Ivy GAP and TCGA database is available at http://glioblastoma.alleninstitute.org/ and http://www.cancergenome.nih.gov/ respectively. The use of dataset from REMBRANDT is acknowledged. We thank Professor G. Subba Rao, Department of Microbiology and Cell Biology, Indian Institute of Science, for providing shSERPINA3 constructs as a kind gift. We acknowledge Dr. Ruchi Jain and Ms. Nandaki NK for helping with gene expression data analysis and collection of patient data respectively. We acknowledge the Faculty of Department of Neurosurgery, NIMHANS for helping in clinical data collection. We acknowledge Mr. Prasad Nimbalkar for the preparation of figure montages. We also thank Mr. Chandrashekar, Mr. Suresh for all the technical support. All the project investigators and project assistants of DBT-COE are acknowledged.

Funding

This study was funded by the Department of Biotechnology (DBT), Government of India as a part of the project under the umbrella of Centre of Excellence (COE) in Neuro-Oncology (Grant No. BT/COE/34/Sp15885/2016); PK is supported by Indian National Science Academy (INSA) Fellowship and Department of Biotechnology- Indian Institute of Science (DBT-IISc) Partnership Program (Grant No. BT/PR27952/INF/22/212/2018).

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Authors and Affiliations

  1. Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, 560029, India

    Vidya P. Nimbalkar, Banavathy S. Kruthika, Palavalasa Sravya, Shilpa Rao, Harsha S. Sugur, Yasha T. Chickabasaviah & Vani Santosh

  2. Department of Neurosurgery, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, 560029, India

    Arimappamagan Arivazhagan

  3. Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    Brijesh Kumar Verma & Paturu Kondaiah

Authors
  1. Vidya P. Nimbalkar
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  2. Banavathy S. Kruthika
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  3. Palavalasa Sravya
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  4. Shilpa Rao
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  5. Harsha S. Sugur
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  8. Arimappamagan Arivazhagan
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Contributions

Conceptualization: VPN, PK, VS; Methodology: VPN, BSK, SP, SR, HS, BK; Formal analysis and investigation: VPN, BSK, SP, SR, HS, BK; Writing—original draft preparation: VPN; Writing—review and editing: AA, YTC, PK, VS; Funding acquisition: PK, VS; Resources: AA, YTC, VS, PK; Supervision: VS, PK.

Corresponding author

Correspondence to Vani Santosh.

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The authors declare that they have no conflict of interest.

Ethical approval

This study has been approved by the Institutional Ethics Committee (NIMHANS/DO/ETHICS SUB-COMMITTEE 30TH MEETING/2016) and have been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

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Informed consent was obtained from all individual participants included in the study.

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11060_2020_3685_MOESM1_ESM.png

(PNG 35 kb) Fig. S1 Expression of SERPINA3 in five different glioma cell lines namely; A172, LN229, U251, U343 and U373 show uniformly increased expression of SERPINA3. GAPDH was used as a normalizing gene

11060_2020_3685_MOESM2_ESM.png

(PNG 104 kb) Fig. S2 Cell cycle analysis representing increased number of cells in G0/G1 phase of cell cycle and decreased number of cells in G2/M phase on SERPINA3-KD in both LN229 and U251 cells at 24 h time point resulting in reduction in proliferation in SERPINA3-KD cells compared to control cells

11060_2020_3685_MOESM3_ESM.png

(PNG 101 kb) Fig. S3 mRNA expression of epithelial to mesenchymal transition (EMT) markers in SERPINA3 knockdown (KD) cells compared to control cells in LN229 and U251 cell lines. Bar graphs showing significant reduction in the expression of N-cadherin, SNAIL and TWIST in SERPINA3-KD cells compared to control LN229 cells. However in U251 cell line we observed reduction in the expression of SNAIL in the SERPINA3-KD cells. Each experiment was performed in duplicate. Analysis was performed using one-way ANOVA with repeated measures followed by Bonferroni post hoc test (n = 3, p ≤ 0.05). All the data is represented as mean ± SD of three independent experiments. *, ** and *** represents p ≤ 0.05, 0.001 and 0.0001 respectively

11060_2020_3685_MOESM4_ESM.png

(PNG 738 kb) Fig S4 mRNA expression of stem cell markers in SERPINA3 knockdown (KD) cells compared to control cells in LN229 and U251 cell lines. Bar graphs showing significant reduction in the expression of SOX2, OCT4 and NANOG in SERPINA3-KD cells compared to control cells in both the cell lines. Each experiment was performed in duplicate. Analysis was performed using one-way ANOVA with repeated measures followed by Bonferroni post hoc test (n = 3, p ≤ 0.05). All the data is represented as mean ± SD of three independent experiments. *, ** and *** represents p ≤ 0.05, 0.001 and 0.0001 respectively

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Nimbalkar, V.P., Kruthika, B.S., Sravya, P. et al. Differential gene expression in peritumoral brain zone of glioblastoma: role of SERPINA3 in promoting invasion, stemness and radioresistance of glioma cells and association with poor patient prognosis and recurrence. J Neurooncol 152, 55–65 (2021). https://doi.org/10.1007/s11060-020-03685-4

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