HE-Färbung von Tumorgewebe
Überlebensrate nach Bestrahlung mittels Colony-Formation-Assay
Immunfluoreszenz für Rparaturfoci in Tumorzellen und Tumorgewebe nach Bestrahlung (ex-vivo-Assay)
Regulation of DNA Double Strand Break Repair in Tumors
Dr. Wael Mansour, Dipl.Biochem.
Dr. Sabrina Köcher, Dipl.Biotech.
Dr. Christoph Oing, Onkologie-Arzt
Stefanie Meien, BTA
Lena Nordquist, BTA
Alexandra Zielinski, BTA
Our goal is to make transformative discoveries that will provide important new insights into DSB repair processes that maintain genome and cellular integrity. We also explore how such insights can be translated to better understanding and treating cancer. To that end, we employ a variety of advanced techniques to analyze the regulation of DSB repair processes in tumors including: immunofluorescence analysis ex-vivo assay, Plasmid assay and in vitro assay (Fig. 1).
Fig. 1: Detection of DNA double-strand break repair. A) Detection of DSB repair in different cell cycle phases. Cells are pulsed-labeled for 30 min by EdU to mark S-phase cells (EdU+, red) or CENP-F to identify G2 cells (CENP-F+ / EdU-, green). B) Fine-needle punch biopsy taken from a prostate cancer, the inlet showing radiation-induced 53BP1 foci. C) DNA repair plasmids to detect efficiency and hierarchy. The plasmids were specifically established to determine the efficiency and hierarchy of I. NHEJ and HR, II.NHEJ and SSA, and III.NHEJ, SSA and HR. D) in vitro/vivo end joining assay. Linear plasmid is incubated with cell-free extract. After incubation, repair products are detected by transformation to E.Coli followed by PCR around DSB site.
Topic 1 — DSB repair hierarchy / DSB repair pathway choice
DNA double strand breaks (DSBs) represent one of the major threats to genome integrity. Eukaryotic cells possess at least three pathways for DSB repair: non-homologous end joining (NHEJ), single-strand annealing (SSA) and homologous recombination (HR).
Fig. 2: Representative functional hierarchy between DSB repair pathways. High fidelity NHEJ normally dominates over both HR and SSA. In the absence of Ku protein, the repair is switched to an error-prone PARP1-EJ, HR and SSA.
These pathways differ not only in the underlying mechanism but also in their repair product. While HR is basically error-free repair pathway, NHEJ can be mutagenic and associated with some errors that might eventually lead to mutations. SSA on the other hand are the most mutagenic repair pathway which results in large deletion. In our lab we have identified a functional hierarchy between these repair pathways which ensures fast and faithful repair of the DSBs. According to this hierarchy, the accurate NHEJ (Classical-NHEJ; C-NHEJ) predominates over the other repair pathways. Consequently, when C-NHEJ is not an option cells switch the repair to the other DSB repair pathways (HR and SSA, Fig.2). Importantly, we could identify an alternative end joining pathway which predominates when C-NHEJ is defect. This pathway is slower, associated with long deletion length and more important PARP1-dependent because either inhibition or knockdown of PARP1 abrogates this pathway.
In addition we aim to specifically determine how cells choose the appropriate DSB repair pathway. The initial processing of the DSB ends is a key determinant of DSB repair pathway choice and is tightly regulated during the cell cycle. Both end protection factors (such as 53BP1, RIF1, and RAP80) and end resection factors (such as BRCA1, MRE11 and CtIP) directly influence the choice between NHEJ and HR by regulating 5’ end resection but how this is achieved remains unclear. Interestingly, all these factors are regulated by ataxia-telangectasia mutated (ATM) gene which is the main coordinator of the DNA damage response after DSB. In the current project we aim to better understand the mechanism by which end protection and end resection factors cross-talk with each other to choose the appropriate repair pathway to be employed.
Selected recent papers
- Mansour WY, Schumacher S, Rosskopf R, Rhein T, Schmidt-Petersen F, Gatzemeier F, Haag F, Borgmann K, Willers H, Dahm-Daphi J (2008) Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks. Nucleic Acids Res 36:4088-4098
- Mansour WY, Rhein T, Dahm-Daphi J (2010) The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies. Nucleic Acids Res 38:6065-6077
- Mansour WY, Borgmann K, Petersen C, Dikomey E, Dahm-Daphi J (2013) The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining. DNA Repair (Amst) 12:1134-1142
- Kocher S, Rieckmann T, Rohaly G, Mansour WY, Dikomey E, Dornreiter I, Dahm-Daphi J (2012) Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase. Nucleic Acids Res 40:8336-8347
- Bakr A, Oing C, Köcher S, Borgmann K, Dornreiter I, Petersen C, Dikomey E and Mansour WY (2014), Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation, Nucleic Acids Res, Accepted
Topic 2 — Impact of DSB repair in development and targeting of prostate cancer
Prostate cancer (PCa) is the most frequent cancer for men. Generally, PCa grows slowly and initially responds to androgen ablation therapy (androgen-dependent, AD). However, eventually some cases become refractory to this therapy, and progress into androgen-independent prostate cancers (Hormone refractory, HRPC). So far, little information is available about the molecular mechanisms underlying the development and progression of PCa. It is believed that progression of PCa obviously occurs via two clearly separated mechanisms: one including the formation of translocations and the other leading to deletions. Both events are causally developed through DSB induction and repair. We study whether defects in specific DSB repair pathways or repair hierarchy may lead to defined subgroups of PCa. This will eventually enable us to establish biomarkers and also new targets for a specific treatment for each subgroup. Overall, our goal is to develop a new strategy for a personalized therapy of PCa.
Fig. 3: Concept of targeting Prostate cancer patient to enhance their radiosensitivity. DSB repair hierarchy is deregulated in PC patients either by deletion events like PTEN or CHD1 deletions leading to HR deficiency or through translocation which leads to overexpression of some oncogenes such as ERG or BCL2 which eventually leads to switch to PARP1-EJ. Both these deregulations render PC cells radiosensitized by PARP inhibitors.
Particularly, in PCa, we reported different deregulations in the DSB repair hierarchy which can be used to enhance the radiosensitization effect (Fig. 3). For instance, we reported a switch to the PARP1-dependent end joining (PARP1-EJ) pathway in PCa cell lines especially those with ERG or BCL2 overexpression. Another example for the disturbed DSB repair hierarchy in PCa is the deletion in either PTEN or CHD1 which leads to HR-deficiency. Importantly, we could show that either switch to PARP1-EJ or HR-deficiency can be used as a new strategy to specifically enhance the radiosensitization of PCa cells. Currently, we initiated an in vivo study to validate these findings.
Selected recent papers
- Kotter A, Cornils K, Borgmann K, Dahm-Daphi J, Petersen C, Dikomey E, Mansour WY (2014) Inhibition of PARP1-dependent end-joining contributes to Olaparib-mediated radiosensitization in tumor cells. Molecular oncology
- Kari V, Mansour WY, Raul SK, Baumgart S, Mund A, Sirma H, Simon R, Dikomey E, Will H, Johnsen SA (2014) ATP dependent chromatin remodeler CHD1 is required for the homologous recombination repair pathway. Oncogene in rev.
Topic 3 — miRNA-mediated targeting of DNA damage response as a tool to enhance Radiotherapy response in vivo
Basically, all potentially curative anti-cancer treatments including ionizing radiation (IR) work basically by causing DNA damage. ATM is the main coordinator of the DNA damage response (DDR) which eventually leads to (i) activation of cell cycle checkpoint, (ii) repair and (iii) apoptosis. Previously, we and others reported that ATM deficient cells show, so far, the most severe radiosensitity phenotype. Moreover, in our lab we identified a head and neck tumor patient who responded remarkably well to radiotherapy so as no further chemotherapy or surgery was described. We reported that this severe response was due to an up-regulation in the miR421 expression that caused ATM deficiency. With these detailed knowledge, we sought to be able to come up with more efficient radiotherapy by targeting ATM using specific miRNAs in vivo. To that end, in collaboration with the oncologists in UKE and Prof. M. Trepel who genetically modified adeno-associated virus (AVV) to carry specific peptide library (ESGLSQS) displayed on the capsid of AAV to allow a systemic delivery of different miRNAs selectively to the breast cancer tissue of the Polyoma middle-T transgenic (PymT) transgenic mice model (Fig. 4).
Fig. 4. In vivo bioluminescence imaging of transgene expression in FVB mice injected intravenously with rAAV-luciferase vectors harboring wild type capsid or capsids displaying the specificity of ESGLSQS peptide to the mammary glands of the mice (Michelfelder et al., PLoS One 2011)
Selected recent papers
- Kasten-Pisula U, Menegakis A, Brammer I, Borgmann K, Mansour WY, Degenhardt S, Krause M, Schreiber A, Dahm-Daphi J, Petersen C, Dikomey E, Baumann M (2009) The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair. Radiother Oncol 90:257-264
- Mansour WY, Bogdanova NV, Kasten-Pisula U, Rieckmann T, Kocher S, Borgmann K, Baumann M, Krause M, Petersen C, Hu H, Gatti RA, Dikomey E, Dork T, Dahm-Daphi J (2013) Aberrant overexpression of miR-421 downregulates ATM and leads to a pronounced DSB repair defect and clinical hypersensitivity in SKX squamous cell carcinoma. Radiother Oncol 106:147-154.