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HIF1α and p53 Regulated MED30, a Mediator Complex Subunit, is Involved in Regulation of Glioblastoma Pathogenesis and Temozolomide Resistance

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Abstract

Glioblastoma (GBM) is the most common, malignant, and aggressive form of glial cell cancer with unfavorable clinical outcomes. It is believed that a better understanding of the mechanisms of gene deregulation may lead to novel therapeutic approaches for this yet incurable cancer. Mediator complex is a crucial component of enhancer-based gene expression and works as a transcriptional co-activator. Many of the mediator complex subunits are found to be deregulated/mutated in various malignancies; however, their status and role in GBM remains little studied. We report that MED30, a core subunit of the head module, is overexpressed in GBM tissues and cell lines. MED30 was found to be induced by conditions present in the tumor microenvironment such as hypoxia, serum, and glucose deprivation. MED30 harbors hypoxia response elements (HREs) and p53 binding site in its promoter and is induced in a HIF1α and p53 dependent manner. Further, MED30 levels also significantly positively correlated with p53 and HIF1α levels in GBM tissues. Using both MED30 overexpression and knockdown approach, we show that MED30 promotes cell proliferation while reduces the migration capabilities in GBM cell lines. Notably, MED30 was also found to confer sensitivity to chemodrug, temozolomide, in GBM cells and modulate the level of p53 in vitro. Overall, this is the first report showing MED30 overexpression in GBM and its involvement in GBM pathogenesis suggesting its diagnostic and therapeutic potential urging the need for further systematic exploration of MED30 interactome and target networks.

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Abbreviations

cDNA:

Complimentary DNA

DMEM:

Dulbecco’s Modified Eagle Medium

DMSO:

Dimethyl Sulfoxide

FBS:

Fetal Bovine Serum

MED:

Mediator

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

TCGA:

The Cancer Genome Atlas

THRAP/TRAP:

Thyroid Hormone Receptor-Associated Protein

TMZ:

Temozolomide

GBM:

Glioblastoma

siRNA:

Small Interfering Ribonucleic Acid

qPCR:

Quantitative Polymerase Chain Reaction

CGGA:

Chinese Glioma Genome Atlas

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Acknowledgements

AS, SS and JD thank Ministry of Human Resource and Development (MHRD), Govt. of India for fellowship.

Funding

This work was supported by internal IIT Delhi funds to RK.

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Author notes

  1. Anubha Shukla and Srishti Srivastava have contributed equally to this manuscript.

Authors and Affiliations

  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, 110016, India

    Anubha Shukla, Srishti Srivastava, Jayant Darokar & Ritu Kulshreshtha

Authors
  1. Anubha Shukla
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  2. Srishti Srivastava
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  3. Jayant Darokar
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  4. Ritu Kulshreshtha
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Contributions

RK conceptualized and coordinated the whole study. AS and SS performed the in vitro analysis of MED30 in GBM cell lines. AS performed functional assays with MED30 overexpression. AS performed MED30 regulation studies. SS, AS and JD performed functional assays with MED30 knockdown in GBM cell lines. AS, SS and RK wrote the manuscript.

Corresponding author

Correspondence to Ritu Kulshreshtha.

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The authors declare no competing financial or other interest in relation to this work.

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Electronic supplementary material

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10571_2020_920_MOESM1_ESM.tif

Supplementary file1 Prognostic Significance of MED30 in GBM and low grade glioma tissues: (a)Kaplan-Meier curves from GEPIA database displaying correlation of MED30 levels with the overall survival pattern of GBMLGG tissues (b) Kaplan-Meier curve from CGGA database showing correlation of MED30 expression with survival in Chinese patient cohort with WHO grade II primary glioma (TIF 1413 kb)

10571_2020_920_MOESM2_ESM.tif

Supplementary file2 MED30 levels measured 48hr post-transfection in GBM cells (a) qRT-PCR data shows induction in MED30 transcript levels upon transfection with MED30 over-expression construct in A172 cell line. β-actin was used as the normalization control. (b) qRT-PCR data showing significant knockdown in expression of MED30 transcript levels in A172 cell line upon transfection with different concentrations of MED30 specific siRNA (Sigma). β-actin was used as the normalization control. (c)Western Blotting data showing induction in MED30 protein expression in response to transfection with MED30-6X Histidine construct. Anti-His tag antibody was used for western blotting. The graphical data points represent mean ± S.D of at least three independent experiments (* represents p-value<0.05 and ** represents p-value<0.001). Error bars denote ± SD (TIF 629 kb)

10571_2020_920_MOESM3_ESM.tif

Supplementary file3 MED30 enhances TMZ sensitivity in GBM cells. Graph showing relative cell viability using MTT assay upon TMZ treatment post MED30 over-expression in (a) U87MG cells (b) A172 cells. Graph showing relative caspase 3/7 activity in TMZ treated GBM cell lines pre transfected pcDNA 3.1 (control vector) and MED30 over-expression plasmid in (c) U87MG cells and (d) A172 cells. qRT-PCR data showing p53 transcript levels upon TMZ treatment in GBM cell lines pre transfected with pcDNA 3.1(control vector) and MED30 over-expressing plasmid in(e) U87MG cells(f) A172 cells. β-actin was used as the normalization control The data shows that MED30 induces p53 transcript levels in TMZ treated GBM cells. The graphical data points represent mean ± S.D of at least two independent experiments (* represents p-value<0.05 and ** represents p-value<0.001). Error bars denote ± SD (TIF 750 kb)

Supplementary file4 List of Primers used for cloning and detection (DOCX 15 kb)

10571_2020_920_MOESM5_ESM.docx

Supplementary file5 Expression of MED30 in different cell types in human brain, data retrieved from Genevestigator tool (DOCX 14 kb)

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Shukla, A., Srivastava, S., Darokar, J. et al. HIF1α and p53 Regulated MED30, a Mediator Complex Subunit, is Involved in Regulation of Glioblastoma Pathogenesis and Temozolomide Resistance. Cell Mol Neurobiol 41, 1521–1535 (2021). https://doi.org/10.1007/s10571-020-00920-4

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