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MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

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Abstract

Glioblastoma is the most life-threatening tumor of the central nervous system. Despite recent therapeutic advancements, maximum survival of glioblastoma patients remains dismal. The mediator complex is a set of proteins, essential for eukaryotic gene expression. Abnormal expression/mutations of specific mediator genes have been associated with progression of various cancers, however, its role and status in glioblastoma remains largely unknown. Our work shows overexpression of a subunit of kinase assembly of mediator complex, MED12, in various glioblastoma patient cohorts including Indian glioblastoma patients and cell lines. Functional characterization of MED12 using both overexpression and knockdown approach revealed that it promotes glioblastoma cell proliferation, migration and inhibits apoptosis. Transcriptome analysis post MED12 knockdown revealed Vitamin D receptor (VDR) pathway to be one of the key pathways affected by MED12 in glioblastoma. We studied direct interaction of MED12 with VDR protein using docking studies and co-immunoprecipitation assay. We identify BCL6, a secondary regulator of VDR signaling, to be directly regulated by MED12 through a combination of chromatin immunoprecipitation, qRT-PCR and western analyses. We further show that MED12 brings about the inhibition of p53 levels and apoptosis partly through induction of BCL6 in glioblastoma. Overall, this stands as the first report of MED12 over-expression and involvement in glioblastoma pathogenesis and identifies MED12 as an important mediator of VDR signaling and an attractive molecule for development of new therapeutic interventions.

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Abbreviations

CDS:

Coding sequence

VDR:

Vitamin D receptor

qRT-PCR:

Quantitative real time polymerase chain reaction

T-ALL:

T-cell acute lymphoblastic leukemia

ESR1:

Estrogen receptor alpha

ESR2:

Estrogen receptor beta

THRA:

Thyroid hormone receptor alpha

siRNA:

Small interfering RNA

MED:

Mediator

MTT:

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

TCGA:

The Cancer Genome Atlas

CGGA:

Chinese Glioma Genome Atlas

GBM:

Glioblastoma

TGFBR2:

Transforming growth factor β receptor type 2

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Acknowledgements

SS thanks Ministry of Human Resource and Development (MHRD), Govt. of India for senior research fellowship. HM thanks Department of Science and Technology, Govt. of India for post-doctoral fellowship. VS thanks Department of Biotechnology, Govt. of India for the post-doctoral fellowship. CS thanks Science and Engineering Research Board (SERB), Govt. of India for the JC Bose fellowship.

Funding

RK and CS thank Department of Biotechnology (DBT), Government of India for financial support (BT/PR16851/MED/122/45/2016). CS also thanks the Science and Engineering Research Board, Govt. of India for the JC Bose fellowship.

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Authors and Affiliations

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

    Srishti Srivastava, Hima Makala, Vikas Sharma & Ritu Kulshreshtha

  2. Neuropathology Neurosciences Centre, All India Institute of Medical Sciences, New Delhi, 110029, India

    Vaishali Suri

  3. Department of Pathology, All India Institute of Medical Sciences, New Delhi, 110029, India

    Chitra Sarkar

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  1. Srishti Srivastava
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  2. Hima Makala
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  3. Vikas Sharma
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  4. Vaishali Suri
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  6. Ritu Kulshreshtha
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Contributions

RK conceptualized and coordinated the whole study. SS performed all the cell line experiments and data analyses of patient data. HM and SS performed co-immunoprecipitation experiments. HM performed docking studies.VS1, VS2 and CS performed analyses in the Indian GBM Patient samples. SS, HM and RK wrote the manuscript.

Corresponding author

Correspondence to Ritu Kulshreshtha.

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Conflict of interest

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

Ethics approval

The study was approved by the Ethics committee (Ref. No. IEC-130/07.04.2017, RP-24/2017) of All India Institute of Medical Sciences, New Delhi, and informed consent was obtained from the patients.

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Supplementary file1 Detailed methods employed to perform cellular assays (DOCX 14 KB)

18_2021_4056_MOESM2_ESM.xlsx

Supplementary file2 List of differentially regulated genes (p-value < 0.05, fold change > or =  + -1.5) obtained by gene expression profiling in A172 cells upon MED12 knockdown (XLSX 31 KB)

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Supplementary file3 Results of pathway analysis of differentially regulated genes (p-value < 0.05, fold change > or =  + -1.5) obtained by gene expression profiling in A172 cells upon MED12 knockdown (XLSX 15 KB)

18_2021_4056_MOESM4_ESM.docx

Supplementary file4 Supplementary tables with list of primers, list of amino acids in MED12 with VDR having hydrogen bond interactions and list of amino acids in MED12 with VDR having hydrophobic interactions. (DOCX 18 KB) 

18_2021_4056_MOESM5_ESM.tif

Supplementary file5 Supplementary Fig. 1: (1–10) IHC Images of glioma patients (antibody used: HPA003184) showing medium (n = 2) to high (n = 8) expression in MED12 protein. Images were retrieved from the Human Protein Atlas (TIF 21424 KB)

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Supplementary file6 Supplementary Fig. 2: MED12 transcript levels were measured 48 h post-transfection in GBM cells (a) qRT-PCR data showing knockdown of MED12 transcript levels in A172 cell line upon transfection with MED12 specific siRNA (Sigma). (b) qRT-PCR data show induction in MED12 transcript levels upon transfection with MED12 over-expression construct in A172 cell line. (c) qRT-PCR data showing knockdown of MED12 transcript levels in T98G cell line upon transfection with MED12 specific siRNA (Sigma). (d) qRT-PCR data shows induction in MED12 transcript levels upon transfection with MED12 over-expression construct in T98G cell line (TIF 21424 KB)

18_2021_4056_MOESM7_ESM.tif

Supplementary file7 Supplementary Fig. 3: (a, b) Graph showing fold change in expression of genes of VDR pathway analysed by qRT-PCR post MED12 over-expression in (a) A172 and (b) T98G cells (TIF 21424 KB)

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Supplementary file8 Supplementary Fig. 4: MED12 is enhanced in IDH mutant tumors. Data analysis from GlioVis showed enhanced expression of MED12 in IDH mutant tumors as compared to IDH wild type tumors across various datasets (a) analysis of CGGA patient dataset (b) analysis of TCGA_GBMLGG patient dataset (c) analysis of Gravendeel patient dataset (d) analysis of Bao patient dataset (TIF 21424 KB)

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Supplementary file9 Supplementary Fig. 5: BCL6 inhibits p53 expression in GBM cells. Western Blotting data to show inhibition in p53 levels post BCL6 overexpression in (a) A172 cells (b) T98G cells. The western blotting experiment was performed in duplicates. The actin blot was run on a different gel (TIF 7142 KB)

18_2021_4056_MOESM10_ESM.tif

Supplementary file10 Supplementary Fig. 6: Inhibition of apoptosis by MED12 is BCL6 mediated: Simultaneous over-expression of BCL6 and MED12 knockdown was performed in A172 and T98G cells. (a, b) Western blotting results showing effects of MED12 knockdown and BCL6 over-expression alone or in combination on cleaved PARP levels in (a) A172 cells (b) T98G cells. The western blotting experiment was performed in duplicates. (c) FACS based detection of phosphatidylserine externalisation was checked in A172 cells post simultaneous over-expression of BCL6 and MED12 (TIF 21424 KB)

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Srivastava, S., Makala, H., Sharma, V. et al. MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis. Cell. Mol. Life Sci. 79, 104 (2022). https://doi.org/10.1007/s00018-021-04056-6

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