Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - TSGPs-of-CFSs (Role of Tumour Suppressor Gene Products of Common Fragile Sites in Human Diseases)

Teaser

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...

Summary

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.

Work performed

\"During the first 30-months of our project, our work included generating engineered knockout (KO) cells of TSGs of CFSs; for example 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. Characterization of these mice reveals that WWOX is important for normal physiology and for tumorigenesis. At the molecular level, we performed RNA profiling to study the molecular changes taking place in these tumors relative to normal tissues. Our results reveal that WWOX loss renders the genome less stable leading to loss of key guardians of the genome, such as p53, contributing to chromosomal instability and tumor cells’ expansion. Future work will also include studies in human specimens.
We have also generated mouse models of WWOX deletion in central nervous system (CNS) and different cell types of the brain. Our initial findings imply that WWOX regulates homeostasis of the CNS and its deletion is associated with neuropathy, including early-onset epilepsy. A large accumulating evidence links gremline mutations of WWOX with epilepsy and ataxia.
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 WWOXIn skeletal muscles was associated with hyperglycaemia and obesity.
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Final results

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 tutor suppressor function.

Website & more info

More info: https://lautenbergcenter.org/people/faculty/prof-rami-aqeilan/.