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Role of PP2A phosphatase and miR-19b in DNA damage response and temozolomide / radiotherapy resistance of glioblastoma tumours

English title Role of PP2A phosphatase and miR-19b in DNA damage response and temozolomide / radiotherapy resistance of glioblastoma tumours
Applicant Vassella Erik
Number 175656
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pathologie Medizinische Fakultät Universität Bern
Institution of higher education University of Berne - BE
Main discipline Experimental Cancer Research
Start/End 01.03.2018 - 28.02.2022
Approved amount 408'509.00
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All Disciplines (2)

Discipline
Experimental Cancer Research
Molecular Biology

Keywords (6)

DNA damage response; microRNA; glioblastoma; chemoresistance; serine/threonine phosphatase; signal transduction

Lay Summary (German)

Lead
Das Glioblastom ist der häufigste primäre Hirntumor bei Erwachsenen, der auf Grund seines infiltrativen Wachstums und seiner intrinsischen Resistenz gegenüber Radiochemotherapie zu den aggressivsten Neoplasien gehört. Die Erforschung der Chemoresistenzmechanismen dieser Tumore könnte in Zukunft wegweisend für die Entwicklung neuer Therapieformen sein. Es ist bekannt, dass DNA-Reparaturmechanismen eine wichtige Rolle in der Resistenzentwicklung spielen; die molekularen Mechanismen der Regulation dieser zellulären Prozesse sind jedoch weitgehend unbekannt. Wir stellen die Hypothese, dass microRNAs, kurze regulatorische RNAs, zur Resistenzentwicklung beitragen. In einem systematischen Screeningverfahren haben wir eine microRNA identifiziert, die Chemoresistenz gegenüber Temozolomid, einer gängigen chemotherapeutischen Substanz für die Behandlung des Glioblastoms, vermittelt. Serin/Threonin Phosphatase PP2A wurde als spezifisches Target dieser microRNA identifiziert.
Lay summary

Unsere präliminären Daten zeigen, dass die in diesem Screen identifizierte microRNA die Aktivität der DNA Reparatur beeinflusst. Dieses Resultat deckt sich weitgehend mit Befunden aus der Literatur, dass PP2A gewisse DNA Reparaturproteine dephosphoryliert. Wir möchten in einem ersten Teilprojekt die Rolle der microRNA und PP2A in der DNA Reparatur an Hand von Glioblastoma Zelllinien genauer untersuchen. Erkenntnisse aus diesem Teilbereich sollen auf Glioblastoma Stammzellen übertragen werden. Diese Tumorzell-Subpopulation ist für die Tumorentstehung, Tumorprogression und das Ansprechen auf eine Chemotherapie hauptverantwortlich. In einem orthotopischen Mausmodell planen wir, die in vivo Relevanz dieser microRNA sowie PP2A in der Tumorentwicklung und Therapieresponse zu untersuchen. In einem weiteren Teilprojekt planen wir einen translationellen Ansatz, um Tumore mit erworbener Resistenz gegenüber Temozolomid auf Mutationen bzw. Alterationen in der Genexpression von DNA Reparaturgenen näher zu untersuchen. Dieses Teilprojekt wird uns einen Einblick in Chemoresistenzmechanismen des Glioblastoms am Patientenmaterial gewähren.

Relevanz des Forschungsprojekts

Das Ziel dieses Forschungsprojekts ist, Einblick in neue Resistenzmechanismen am Beispiel des Glioblastoms zu erhalten. Erkenntnisse aus diesem Projekt könnten möglicherweise zur Entwicklung neuer Formen adjuvanter Therapie beitragen, mit dem Ziel, die Wirksamkeit von Temozolomid für die Behandlung des Glioblastoms zu verbessern.

Direct link to Lay Summary Last update: 15.12.2017

Responsible applicant and co-applicants

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Associated projects

Number Title Start Funding scheme
138129 Analysis of microRNAs implicated in the regulation of NF-kB and MGMT pathways for their role in conferring apoptosis and chemoresistance of glioma tumours 01.04.2012 Project funding (Div. I-III)

Abstract

Background: Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumour. Patients with GBM are normally treated by a combination of surgery, chemotherapy using temozolomide (TMZ) and radiation therapy (RT), but the tumour is virtually incurable due to its highly infiltrative growth and its intrinsic resistance to radio-chemotherapy. O6-methylguanine-DNA methyl-transferase (MGMT) repairs alkylated DNA, but many patients who show MGMT methylation in the tumour are resistant to TMZ/RT owing to replacement by the capacity of other repair mechanisms. Ser/Thr phosphatases are key regulators of checkpoint control responsible for initiating DNA repair. PP2A, which constitutes the major class of Ser/Thr phosphatases involved in DNA damage response, is downregulated in half of glioma cases, but its role in TMZ resistance is poorly understood. During the prior research period supported by the SNF, we performed a systematic screening of GBM cells using a lentiviral miRNA library. We showed using this unbiased approach that miR-19b/20b encoding lentiviral clones were selected reproducibly in TMZ resistance screens using two GBM cell lines. miR-19b is among the most abundant miRNAs in GBM tissues and is induced by ionizing radiation. Our preliminary results revealed that attenuation of miR-19b resulted in major changes in the phospho-proteome, enhanced phosphatase activity and reduced efficiency in TMZ-induced DNA damage response in a GBM cell line. miR-19b is potentially able to target PP2A subunits PPP2R2A, PPP2R1B, PPP2R5A and PPP2R5E. PPP2R5E targeting by miR-19b was confirmed experimentally. Consistent with these findings, miR-19b levels were inversely correlated with the expression of PP2A subunits in GBM tissues, suggesting that miR-19b controls the expression of PP2A. Specific aims: A Analysis of PP2A and DNA damage response genes in tissues of TMZ resistant recurrent GBM cases. B Mechanistic analysis of PP2A and miR-19b in patient-derived GBM stem cells (GSCs). C Assess PP2A regulation by miR-19b D Assess if targeting miR-19b can be exploited for anti-GBM adjuvant therapy in an orthotopic mouse model.Experimental design: Aim A: DNA repair capacity is not systematically analysed in recurrent GBM after TMZ treatment. We plan to perform an integrative analysis of matched tumour samples prior and post-therapy from an exceptionally large collection of 60 GBM cases from the Institute of Pathology of Bern by next generation sequencing using a designed panel of genes involved in DNA damage response and DNA repair and expression analysis using Nanostring technology to provide a network of pathways that are altered in resistant tumours. This tissue collective put us in a unique position to assess the role of PP2A in TMZ/RT resistance in patient material. Aim B: We plan to assess the role of PP2A and miR-19b in TMZ- and RT-induced DNA damage response of patient-derived primary GSCs. GSCs are the most relevant cells for addressing therapy response as tumour progression mainly depends on this subpopulation. To this end, a gene knockout of the catalytic subunit PPP2CA and a knockdown of miR-19b will be constructed in GSC cells. We will analyse changes in the phospho-proteome of genes implicated in DNA repair and will assess DNA repair capacity using multiplexed DNA repair assays for base excision repair, nucleotide excision repair, mismatch repair and MGMT. Aim C: We will assess the role of miR-19b in TMZ and RT response and will perform luciferase reporter assays to assess if PP2A subunits are targets of miR-19b. Aim D: An orthotopic mouse model of GBM will be used to assess TMZ-resistance elicited by miR-19b and PP2A. Relevance of the proposed work: (i) Identification of novel TMZ resistance mechanisms using a translational approach from a large cohort of matched cases prior and post-treatment with TMZ/RT. (ii) Providing information on the role of PP2A complexes in TMZ-induced DNA damage response. (iii) Assessing if miR-19b can be exploited as a target in anti-glioma adjuvant therapy.
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