Increased activity of alternative non-homologous end joining (A-NHEJ) as a possible mechanism of chromosomal aberration in pancreatic cancer.

• Applicant: Dr. Patryk Moskwa
• Subject Area: Gastroenterology ; Cell Biology
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269137599

A hallmark of all cancer roots in chromosomal aberrations but their causative mechanism remain unclear. The classical non-homologous end joining (cNHEJ) is one of the key mechanisms of DNA double-strand brake (DSB) repair and its dysfunction causes chromosomal aberrations, most likely through the compensatory alternative NHEJ (aNHEJ). aNHEJ generates microhomology at the DSB repair junction whereas its components remain elusive.Single miRNA has the ability to target multiple genes of a single or multiple pathways simultaneously. Aberrant expression of miRNA(s) may supress cNHEJ contributing thereby via the aNHEJ to chromosomal aberrations.Aim 1. Identification of miRNAs suppressing the cNHEJ pathway. In analogy to in Moskwa et al., we detected 6 miRNAs candidates. To eliminate those miRNAs which don't impact cNHEJ genes, all candidates will first be pretested with a functional genome-integrated cNHEJ reporter-assay and with luciferase assay. MiR-142 was already tested and showed promising results. Subsequently, we will measure the impact of these miRNAs on DNA repair capacity and kinetic upon gamma-ionizing radiation (gIR) and determine the cells' sensitivity overexpressing these miRNAs to gIR, PARP inhibitor and Bleomycin. To support in vivo significance of our miRNAs, we will analyze the DNA repair junction of chromosomal aberration in pancreatic cell lines and primary tumor tissue for microhomology as well as expression of miRNAs and their target proteins. This way, we will establish an inverse correlation of miRNAs and their protein targets as well as positive correlation with microhomology.Aim 2. Identification of new components of the aNHEJ pathway. We will induce an increased activity of aNHEJ employing specific expression of KrasG12D in pancreatic cells (mouse model) and shRNA-mediated downregulation of Ku70 (a core component of cNHEJ) ex vivo in isolated acinus cells. All upregulated genes on microarrays, common to both conditions, will be considered as candidates. Based on literature search, The Gene Ontology and prioritizing the top 20 genes, we will establish a pool of max. 50 genes. These genes will be knocked down using siRNA in a cell line caring the genomic aNHEJ reporter to eliminate all candidates with no affect on aNHEJ. We will also determine the impact of these proteins on DNA repair capacity and kinetic upon gIR to identify the top 3 candidates with strongest effect. Next, the expression of these candidates will be manipulated and the sensitivity toward gIR, Bleomycin and PARP inhibitors tested.For in vivo relevance, the expression of the candidates in KrasG12D/p48 murine pancreatic cancer model and in human pancreatic cancer will be analyzed. To determine the therapeutic value, candidategenes specific siRNA coupled to nanoparticles will be used to treat pancreatic cancer in the murine model and their impact alone or in combination with Bleomycin or PARP inhibitor on tumor growth/metastasis evaluated.

• DFG Programme: Research Grants