Periodic Reporting for period 4 - TSGPs-of-CFSs (Role of Tumour Suppressor Gene Products of Common Fragile Sites in Human Diseases)
Berichtszeitraum: 2020-11-01 bis 2021-10-31
Further investigation clearly suggest that gene products from CFSs play important roles in other human diseases. For example, we have evidence for the involvement of DNA damage and WWOX in neurological disease, particularly in childhood epilepsy.
Our project aims to investigate the role of tumor suppressor gene products (TSGPs) of CFSs in human diseases, particularly in cancer, metabolic and in neurological diseases. Three approaches were taken to tackle this question. First, molecular functions of TSGPs of CFSs in several cancer cells was determined using state-of-the-art genetic tools in vitro. Second, novel transgenic mouse tools were used to study CFSs and their associated TSGs in preneoplastic lesions and tumors in vivo, with confirmatory studies in human material. Third, discover potential involvement of CFSs and their TSGPs in metabolic diseases as well as in childhood epilepsy models and systems.
1. Generating engineered knockout (KO) cells of TSGs of CFSs; for example as a prototype for the WWOX gene. Several human cell types including MCF7 breast cancer epithelial cells, MCF10A breast epithelial cells, U2OS osteosarcoma mesenchymal cells, MC3T3 pre-osteoblast mesencymal cells, EndoC acinar pancreatic cells, and neonatal foreskin fibroblasts (FSE) were targeted and WWOX-KO clones were generated and validated. These KO cells together with their control cells were studied at cellular and on some at molecular levels. Tumorigenic traits of these cells were addressed in vitro and if successful their tumorigenic traits were/are examined in vivo (using immunocomprimised mice). Our studies revealed tumor suppressor function of TSGPs in several types of cancer cells.
2. Mapping for DNA double strand breaks (DSBs) at very high resolution and discovering new "hot spots" and CFSs in a cell-type specific manner. Using BLISS (Breaks Labeling In Situ and Sequencing), a method that maps DSBs at very high resolution, we were able to show novel mechanism of gene regulation in cancer cells that involves coupling between transcription and DNA repair.
3. Genetic engineered mouse models modelling loss of Wwox expression in different cell types further confirmed WWOX function as tumor suppressor. We mimicked somatic loss of WWOX in osteoblasts to show its importance in developing osteosarcomas. Somatic loss of WWOX in mammary epithelial cells revealed its icritical function in regulating p53 and Brca1 basal-like breast tumors. In addition, WWOX-specific knockout in hepatocytes revealed its association with hepatocellular carcinoma (HCC). Unpublished data also revealed WWOX function in developing pancreatic adenocarcinomas and squamous cell carcinomas.
4. Dissecting WWOX molecular function as a tumor suppressor was pursued using several Omics approaches and revealing importnbat functions in DNA damage response and cellular metabolism.
5. Mouse models mimicking WWOX loss of function in the brain revealed WWOX direct function in childhood epilepsy. Here we developed somatic loss of WWOX in neuronal stem cells and progenitors using Nestin-Cre mice and discovered that these mice resemble Wwox-null mice indicating the central WWOX function in central nervous system (CNS). We next went further to show that specific neuronal deletion of WWOX, but not in astrocytes or oligodendrocytes, phenocopies Wwox null mice. These mouse models displayed a remarkable phenotype of seizures and postnatal lethality, as documented in human WWOX-related epileptic encephalopathy (WOREE) patients.
6. The preceding findings (in 4) led us to propose possible therapeutic strategy using gene therapy of AAV9-WWOX as a possible treatment for WOREE syndrome.
7. Developing a human culture system to study brain development using induced-pluripotent stem cells (iPSCs) and brain organoids resembling WOREE syndrome phenotypes.
Our efforts contributed to address the question of whether TSGP of CFSs are playing direct roles not only in the tumorigenesis process but also in other maladies including neurological disorders.