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MRTF/SRF signalling in regulation of cell senescence and melanoma progression

Periodic Reporting for period 1 - MRTFSen (MRTF/SRF signalling in regulation of cell senescence and melanoma progression)

Reporting period: 2020-04-01 to 2022-03-31

Mammalian cells contain a skeleton named cytoskeleton that is responsible for shaping them and giving them mechanical resistance. The cytoskeleton is formed by a network of different proteins, actin being one of them. The actin cytoskeleton is organized by actin filaments which can grow (polymerize) and shorten (depolymerize) by adding or removing molecules of actin. Hence, the actin cytoskeleton is highly dynamic and allowing cell shape changes to adapt to its environment, gives continuous structural support and is involved in many essential cellular processes such as migration and contraction. These processes need the controlled interplay of different proteins with actin. Regulation of actin dynamics can happen at different levels, from actin gene expression to actin polymerization or degradation. We have found that one of the pathways controlling actin dynamics is also involved in preventing cells from replication, causing what is known as cellular senescence.
Cells self-replicate to generate an increased number of cells. To do so they undergo a process known as the cell cycle which involves the duplication of all cell components, followed by the physical separation of the original cell into two identical daughter cells. The cell cycle is a tightly regulated multistep process which has distinct checkpoints to ensure no errors are inherited to the daughter cells. If one of these checkpoints is not passed, cells will arrest their proliferation and no-longer self-replicate. As a consequence, cells will die (apoptosis) or enter a permanent state of cell cycle arrest named cell senescence. Cell senescence was first observed in laboratory cells which could not continuously replicate and have stopped further division. Later it has been shown that damaged cells can also enter cell cycle arrest or senescence. Therefore, cell senescence can actually be beneficial during tissue remodelling, cancer, fibrosis or aging to prevent proliferation of damaged cells. There are also physiological senescence states, such as experienced by melanocytes when forming nevi. Melanocytes are pigmented cells in the skin that, when certain genetic alterations or mutations are present, can first proliferate and then enter senescence. Accumulation of senescent melanocytes in the skin is observed as nevus. These senescent melanocytes can subsequently reactivate proliferation and generate tumours, known as melanomas.
In this project we aim to unravel how decrease dysregulation of the actin machinery and dynamics causes cells to senesce, and how this could influence melanoma development and progression. Cell senescence is important in certain diseases such as fibrosis or cancer. Therefore, the study of this process is of clinical interest. This proposal wants to study a new mechanism for cell senescence and the results of this project are potentially interesting to investigate new drugs or therapies to modulate the senescence process in these diseases.
The overall objectives of this project have been:
To find the mediators and components involved in cell senescence when actin machinery is not dysfunctional.
To study whether this form of senescence affects different cell types equally
To investigate how this new senescence mechanism affects melanoma progression
The goal of this proposal was to investigate the role of cytoskeletal regulators in controlling cell cycle arrest. For that purpose, we established different objectives which we have addressed during the project.
To find the mediators and components involved in senescence when the actin machinery is dysfunctional, we have taken a high-throughput approach: a CRISPR-KO screen. By doing this screen, we have deleted the expression of many single genes in our senescent cells using the CRISPR/Cas9 system. As a result, we have generated individual cells which lack the expression of one gene. From these, only the ones proliferating are the ones which have avoided senescence. These cells were labelled so we can track which gene has been deleted causing cells to continue proliferating rather than senesce. Subsequently, we can relate which components are involved in cell senescence when the actin cytoskeleton is not fully functional. These results are still under analysis.
We also examined whether this mechanism of senescence happens to different cell types. Specifically, we wanted to study it in stem cells. For that purpose, we generated stem cells with the same mutation affecting the actin cytoskeleton. We have analysed and confirmed that this mutation also causes senescence in these stem cells.
To investigate how this type of senescence influences cancer development, we have used a transgenic mouse model which develops melanoma. We have introduced the same mutation affecting the actin cytoskeleton in the transgenic mice and observed when they develop tumours and analysed their characteristics. We can confirm that the mutation delays melanoma onset and reduces tumour size. These tumours have less proliferative cells and less fibrotic components. In addition, lung metastasis in the mice with defective actin cytoskeletal function seem to occur later. The details and mechanisms affecting tumour development and metastasis are still under study.

We plan on continuing analysing the mechanism that causes senescence when the regulators of the actin cytoskeleton are mutated. Once the project has concluded we will submit the results to a high impact scientific journal. We also plan on presenting the data at international meetings now that they have resumed. To communicate our findings to scientists and non-scientific audiences we will employ the Crick’s public engagement department which disseminates results through press releases, social media (twitter or facebook) or annual reports.

We anticipate that the results could have a potential clinical interest. Thus, by the end of the project we will discuss their potential exploitation with the Scientific Translation Team at the Crick.
As mentioned above, part of the project is still ongoing. We expect from further developing the project to find the mediators of senescence and unravel the mechanism. In addition, we want to prove the new mechanism in other cell types and further decipher how it regulates tumour onset and progression.
The outcome of this project will benefit the scientific community as it will provide new insight into different fields of biology, such as cell cycle regulation, aging, actin cytoskeleton and cancer.
Considering the medical interest in modulating senescence programs for new therapies, the results from this project would be valuable for translating into clinics. Deciphering a new senescence mechanism and revealing the modulators will have the potential to influence the design of new anti-cancer or anti-fibrosis therapies. This altogether will contribute to the public health and welfare.
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