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Role of Tumour Suppressor Gene Products of Common Fragile Sites in Human Diseases

Periodic Reporting for period 3 - TSGPs-of-CFSs (Role of Tumour Suppressor Gene Products of Common Fragile Sites in Human Diseases)

Reporting period: 2019-05-01 to 2020-10-31

Common fragile sites (CFSs) are large chromosomal regions identified by conventional cytogenetics as sequences prone to breakage in cells subjected to replication stress. The interest in CFSs stems from their key role in DNA damage, resulting in chromosomal rearrangements. The instability of CFSs was correlated with genome instability in precancerous lesions and during tumor progression. Two opposing views dominate the discussion regarding the role of CFSs. One school of thought suggested that genomic instability during cancer progression causes collateral damage to genes residing within CFSs, such as WWOX and FHIT. These genes are proposed to be unselected ‘‘passenger’’ mutations. The counter argument is that deletions and other genomic alterations in CFSs occur early in cancer development. Cancer cells with deletions in genes that span CFSs are then selectively expanded due to loss of tumor suppressor functions such as protection of genome stability, coordination of cell cycle or apoptosis.
We have recently proposed another model by which these two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities (Hazan et. al. Plos Genetics, 2016).
Recent observations from my lab clearly suggest that gene products from CFSs play driver roles in cancer transformation. Moreover, we have evidence for the involvement of DNA damage and Wwox in pancreatic β-cells in the context of diabetes. Here, we propose to investigate the role of tumor suppressor gene products (TSGPs) of CFSs in human diseases. Three approaches will be taken to tackle this question. First, molecular functions of TSGPs of CFSs will be determined using state-of-the-art genetic tools in vitro. Second, novel transgenic mouse tools will be used to study CFSs and their associated TSGs in preneoplastic lesions and tumors in vivo, with confirmatory studies in human material. Third, we will examine the potential involvement of CFSs and their TSGPs in type-2 diabetes (T2D).
The expected outcome is a detailed molecular understanding of CFSs and their associated TSGPs in genomic instability as well as their roles in cancer and metabolic diseases.
"During the first 30-months of our project, our work included 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 levels. Tumorigenic traits of these cells were addressed in vitro and if successful their tumorigenic traits were/are examined in vivo (using immunocomprimised mice).
To map for DNA double strand breaks (DSBs) at very high resolution and discover new ""hot spots"" and CFSs in a cell-type specific manner, we are using BLISS (Breaks Labeling In Situ and Sequencing). BLISS is a method that can map DSBs at very high resolution. Using BLISS, we were able to show novel mechanism of gene regulation in cancer cells that involves coupling between transcription and DNA repair.
The effect of WWOX loss on replication stress was also addressed using DNA combing, a method that can detect the progression of DNA replication fork (speed and symmetry). Our preliminary data suggest that WWOX KO cells display increased DNA replication stress.
In parallel to our work on cell culture, we also generated mouse models to mimic WWOX loss in cancer. Mouse models of Wwox-KO in mammary gland epithelium, pancreatic acing cells and osteoblasts were generated using Cre-Loxp technology. Our findings of mouse models of Wwox loss of function show that alteration in WWOX synergises with other cancer driver genes to accelerate tumorigenesis. For example, loss of WWOX together with RAS activation in acinar pancreatic cells results in accelerated pancreatic lesions and tumors. Moreover, combined loss of WWOX and p53 results in increased incidence in Keratin-14 epithelial tumors. These tumor models are currently being dissected in-depth to reveal role of TSGPs-of-CFSs in their development.
To study the role of TSGPs of CFSs in T2D, we are using BLISS to profile DSBs in the EndoC β-cell. Preliminary results suggest increased DSBs in high glucose-treated β-cells. We have also generated specific KO of WWOX in -cells using Insulin-Cre transgenic line, however these mice didn't show any phenotype related to T2D. In contrast, targeted deletion of WWOX in skeletal muscles was associated with hyperglycaemia and obesity. Mechanistically, we reported that WWOX modulates the levels of HIF1a and AMPK, two central proteins in regulating the metabolic status of the cells.
The merging role of genes spanning CFS was recently expanded to include neurological disorders. Recent evidence associated WWOX, for example, with childhood epilepsy. In fact, WWOX gremlin mutations are linked today with WWOX-related epileptic encephalopathy (WOREE) syndrome. To address the precise role of WWOX in WOREE syndrome we developed and utilised specific mouse model systems. Mouse models mimicking WWOX loss of function in the brain: 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 WOREE patients. At present, we are dissecting the molecular changes in these mouse models. This development also led us to propose possible therapeutics using gene therapy of AAV-WWOX (PoC-ERC), that is currently under development.
This later observation of WWOX involvement in neurological disorders also led us to look for other gene products of CFSs that might play a role in CNS. We are currently modelling those to uncover their direct contribution to human diseases."
We have generated new tools (cells and mouse models) and used state-of-the-art technologies to test our hypothesis. This included using of CRISPR/CAS9 technology to generate KO cells and optimization of BLISS (Breaks Labeling In Situ and Sequencing) to profile DNA double strand breaks (DSBs) in vitro. We are also employing RNA sequencing and mass spectrometry to study molecular changes of manipulated and sorted cells.
Expected results. Our efforts continue 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 diabetes and neurological disorders. Our preliminary data support this assumption and will continue in developing resources to address this hypothesis.
Modelling somatic loss of WWOX expression in several tissues revealed its importance in tissue/organ homeostasis and tumor suppressor function. Furthermore, our results are revealing WWOX as a key regulator of brain homeostasis and that its loss results in severe neurological disorders.
Role of common fragile sites and their gene products in cancer development