Periodic Reporting for period 1 - FBXW7-InReg (Defining the role of FBXW7 in intestinal epithelium regeneration)
Reporting period: 2022-09-01 to 2024-08-31
Intestinal bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is a very severe disorder that affects the intestinal epithelium. The intestine gets irritated or injured often and does not heal properly, leading to a painful, sometimes infected area. In Europe, 2.5-3 million people are suffering from IBD, and unfortunately current therapies cannot restore fully the intestinal structure and function but only limit the inflammation. Over the years, it is consequently possible to identify in IBD patients the expansion of cells not behaving properly, that could also potentially cause colitis-assciated colorectal cancer (CA-CRC). It is therefore necessary and urgent to understand how we can repair completely the intestine and prevent the tumor formation.
The protein FBXW7 functions as a tumor suppressor, helping to prevent the development of cancer, but is often found mutated and misfunctioning in IBD patients, potentially increasing their risk of cancer. It is not fully clear yet how these FBXW7’s mutations affect the intestine, the goal of our project is to understand the role of FBXW7 and its mutation in the reparing intestine. This is likely to help us develop novel therapies for IBD patients.
When the intestinal epithelium is damaged and in need of repair, the remaing cells are able to re-acquire properties that were present in the fetal intestine. This transition is required for repairing the injured area. When the intestine is then healed, these cells modify their features again and return to the adult state as before injury.
FBXW7 controls the levels of specific proteins in the cell, making sure they are broken down and removed when no longer needed or in surplus, ultimately influencing the cell behaviour. Moreover, the regulation by FBXW7 is tightly linked to the activation of specific protein kinases also involved in tissue regeneration. We hypothesise that FBXW7 regulates the return of the cells into the adult state once the tissue repair is complete, and that when FBXW7 does not function correctly or is absent because of mutations, the transition back to homeostasis is dysfunctional elevating the risk of cancer.
To test this hypothesis we set the following objectives:
1. Study the effects of FBXW7 absence during intestinal repair
2. Understand the role of FBXW7 in intestinal repair
3. Identify proteins broken down by FBXW7 and modulate their abundance to rescue a non-functioning/absent FBXW7
The protein FBXW7 functions as a tumor suppressor, helping to prevent the development of cancer, but is often found mutated and misfunctioning in IBD patients, potentially increasing their risk of cancer. It is not fully clear yet how these FBXW7’s mutations affect the intestine, the goal of our project is to understand the role of FBXW7 and its mutation in the reparing intestine. This is likely to help us develop novel therapies for IBD patients.
When the intestinal epithelium is damaged and in need of repair, the remaing cells are able to re-acquire properties that were present in the fetal intestine. This transition is required for repairing the injured area. When the intestine is then healed, these cells modify their features again and return to the adult state as before injury.
FBXW7 controls the levels of specific proteins in the cell, making sure they are broken down and removed when no longer needed or in surplus, ultimately influencing the cell behaviour. Moreover, the regulation by FBXW7 is tightly linked to the activation of specific protein kinases also involved in tissue regeneration. We hypothesise that FBXW7 regulates the return of the cells into the adult state once the tissue repair is complete, and that when FBXW7 does not function correctly or is absent because of mutations, the transition back to homeostasis is dysfunctional elevating the risk of cancer.
To test this hypothesis we set the following objectives:
1. Study the effects of FBXW7 absence during intestinal repair
2. Understand the role of FBXW7 in intestinal repair
3. Identify proteins broken down by FBXW7 and modulate their abundance to rescue a non-functioning/absent FBXW7
In the laboratory we can explore the function of a protein by using mice or studying cells grown outside of the body under carefully controlled conditions. The two systems can also be used to study intestinal injury and repair. We made used of both of these systems to address our hypothesis.
We have developed a mouse model where FBXW7 can be mutated in the colon by specific compound administration. Similarly, we can trigger tissue damage by administrating another compound. We have tested and adjusted the amount of compounds needed to model properly tissue repair and FBXW7 absence, and started looking at what happens in the colon under such conditions.
At the same time, we have also grown normal human intestinal cells derived by healthy donors and mutated FBXW7. Several protocols have been tested and optimised to introduce FBXW7 mutatations, and one method in particular has been identified as the one providing mutations with highest efficiency. The mutant cells and the strategy used to obtain them have been shared among the lab members, so that they can be used again in the future. In parallel with studies in human cells, we have investigated FBXW7 function in mouse intestinal cells, in which FBXW7 can be removed by compound treatment. Several technologies have been used to study cells with or without FBXW7, among which RNA sequencing, allowing for identification of active genes that contain the recipe for producing proteins, and state-of-the-art microscopy, allowing to both record movies of cells growing in real time and to take snapshots of cells in a specific moment.
Our results showed that there are some clear phenotypic and behavioural differences in cells upon loss of FBXW7. This supports that FBXW7 plays a role in controlling cellular identity during tissues regeneration.
We have developed a mouse model where FBXW7 can be mutated in the colon by specific compound administration. Similarly, we can trigger tissue damage by administrating another compound. We have tested and adjusted the amount of compounds needed to model properly tissue repair and FBXW7 absence, and started looking at what happens in the colon under such conditions.
At the same time, we have also grown normal human intestinal cells derived by healthy donors and mutated FBXW7. Several protocols have been tested and optimised to introduce FBXW7 mutatations, and one method in particular has been identified as the one providing mutations with highest efficiency. The mutant cells and the strategy used to obtain them have been shared among the lab members, so that they can be used again in the future. In parallel with studies in human cells, we have investigated FBXW7 function in mouse intestinal cells, in which FBXW7 can be removed by compound treatment. Several technologies have been used to study cells with or without FBXW7, among which RNA sequencing, allowing for identification of active genes that contain the recipe for producing proteins, and state-of-the-art microscopy, allowing to both record movies of cells growing in real time and to take snapshots of cells in a specific moment.
Our results showed that there are some clear phenotypic and behavioural differences in cells upon loss of FBXW7. This supports that FBXW7 plays a role in controlling cellular identity during tissues regeneration.
This project has progressed our knowledge on FBXW7 function in the intestinal epithelium. We are currently looking more in depth into the differences found between cells with and without FBXW7 mutations, and identifying proteins broken down by FBXW7. We believe that this will help us understand how the temporary acquisition of fetal properties is regulated, and potentially uncover new strategies to overcome FBXW7 mutations during intestinal repair.