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Benign prostatic hyperplasia (BPH) is considered an age-related disease of men with unknown etiopathophysiology. Chronic inflammation represents one of the most important pathophysiological mechanisms in this context, and there is also evidence for the involvement of autoimmune responses in an inflammatory environment in the prostate. Conventional therapeutic approaches for BPH are limited; however, immunosuppression by TNF (tumor necrosis factor)-𝛼 blockers seems to reduce symptoms in BPH patients due to the reduction of epithelial hyperplasia and macrophage-triggered inflammation. Therefore, this cumulative dissertation addresses biomarker-based BPH diagnosis by lens epithelium-derived growth factor splice variant 75 kDa (LEDGF/p75). LEDGF/p75 is an autoantigen that is overexpressed in solid tumors and functions as a stress-related transcriptional co-activator. It also plays an important role in cancer and DNA damage repair (DDR). The generation of CRISPR/Cas9-modified HEp-2 LEDGF deficient (KO) and HEp-2 LEDGF/p75 overexpressing (OE) cell lines provided new insights for the diagnosis and therapy of cancer and genetic disorders. Herein, IgG autoantibody reactivity to LEDGF/p75 was detected in patients with prostate cancer (PCa, n=89), bladder cancer (BCa, n=116), benign prostatic hyperplasia (BPH, n=103) and blood donors (BD, n=60) by indirect immunofluorescence assay (IFA) linear immunoassay (LIA) and Chritidia luciliae immunofluorescence test (CLIFT). In addition, proliferation and migration capacity as well as chemosensitivity of genome-edited cells were determined by optimized all-in-one approach, followed by investigation of DDR signaling pathways by Western blot and immunofluorescence. Surprisingly, no increased binding of autoantibodies (autoAbs) to LEDGF/p75 was detected in cancer patients, but autoAb reactivity to LEDGF/p75 O/E cells was unexpectedly increased in approximately 50% of patients with BPH. At the molecular level, LEDGF-deficient cells exhibit decreased proliferation and migration and increased sensitivity to the chemotherapeutic agent etoposide. A significantly Decreased DNA damage response could also be visualized. New diagnostic capabilities using HEp-2 cells overexpressing LEDGF/p75 or mitochondrial DNA as autoimmune targets could be used to identify BPH patients with autoimmune responses. Furthermore, this Thesis shows that LEDGF is not only involved in the recruitment of DNA damage response proteins, but also affects the proteosomal regulation of DDR signaling molecules.
Lens epithelium derived growth factor (LEDGF), is known to be overexpressed in different solid cancers and cancer cell lines but is also involved in acquired immunodeficiency syndrome (AIDS) and diverse inflammatory diseases. Due to its chromatin-binding ability, it acts as a transcriptional coactivator, promoting anti-apoptotic pathways leading to increased tumor aggressiveness and chemotherapy resistance. In this thesis, the role of LEDGF in laryngeal cancer cells was investigated. Thus, a knock-out of LEDGF in HEp-2 cells was successfully generated by CRISPR/Cas9 technology. Moreover, LEDGF-deficient HEp-2 cells exhibited a decreased proliferation and migration, as well as an increased chemotherapy sensitivity towards topoisomerase II inhibitor etoposide. The DNA damage response was in more detail investigated, where LEDGF depleted cells showed a drastic reduction in the recruitment of downstream damage-response related proteins, more precisely replication protein A subunit of 32kDa (RPA32), and an increased amount of DNA fragmentation, highlighting the role of LEDGF in homology-directed DNA repair.
In addition, a efficient method was established to allow genomic tagging of LEDGF at its C-terminus with EGFP which enables the monitoring of LEDGF in living cells. Therefore, an efficient Cas9-mediated DNA DSB induction was supplemented with a DNA template containing homology arms to the C-terminus as well as the EGFP sequence to allow homology-directed repair. Subsequently, EGFP-expressing cells were FACS sorted and single cells expanded. Almost all test clones showed a homozygous genotype for LEDGF-EGFP fusion which exhibited the typical nuclear pattern of LEDGF.