Nuclear envelope (NE) ruptures are doubly linked to cancer: they create chromatin herniations and genome instability and favor cancer cell diffusion in confined spaces. Endosomal sorting complexes required for transport (ESCRT) machinery is associated with membrane sealing at trafficking vesicles and at the midbody in cytokinesis and also controls NE integrity and completion of NE reorganization after mitosis. AKTIP (Ft1 in mouse) is a new factor enriched at the NE, controlling telomere function, genome stability and nuclear envelope integrity. AKTIP has multiple striking similarities with ESCRTs. It interacts with vesicle factors, it is enriched at the NE and at the midbody, and is structurally similar to the ESCRT-I TSG101. Along with this, Ft1 is a concausal element in the diffusion of lymphomas in mice and AKTIP reduction in human lymphoma has been reported.
We hypothesize that AKTIP/Ft1 depletion would impinge on NE and chromatin organization. Deriving fragile nuclei and DNA damage would contribute to diffusion of lymphomas.
On these premises, in this study we aim at dissecting the role of AKTIP/Ft1 in nuclear integrity and ESCRT activity, along with defining the contribution of Ft1-linked NE alterations to lymphoma diffusion.
We will use 3D SIM to better understand AKTIP NE localization. We will study in vivo AKTIP dynamics by imaging of CRISPR/Cas9 AKTIP-GFP edited cells. We will define whether, in the absence of AKTIP, ESCRT activity at the NE is impaired and we will monitor NE integrity. We will analyze DNA damage markers on cells with reduced AKTIP/Ft1 expression combined with modulation of ESCRTs or lamins. Finally, we will monitor nature of lymphomas in Ft1 mutant mice injected with lentivectors expressing hairpin RNAs directed to ESCRT factors.
We believe that this study will give relevant insights into the role of NE and NE sealing factors in genome stability, telomere function, which interrelate with cancer aggressiveness and metastatic behavior.
The rigidity of the nucleus creates a barrier to cancer cell passage through narrow spaces. Wounded nuclei are prone to genome instability that in turn generates further NE instability. All this taken together creates a vicious cancer-driving cycle. Recent works on the ESCRT complex have shed light on mechanisms controlling NE stability particularly relevant in this scenario. This area of research is recent, conceptually intriguing and has the potential to opening the road to new anticancer drugs. In this study, through the dissection of a factor with strong analogies with ESCRTs, we wish to give insights into the connections among NE,
ESCRTs and cancer diffusion, along with defining the properties of a new candidate ESCRT and putative cancer player.
Feasibility of the project
The proposed project is based on a robust collection of preliminary data and on our previous work. This guarantees a solid theoretical ground for successful development of the research along with useful technical tools, including the mouse model and the CRISPR/Cas9 edited cells that are instrumental for the experiments. We chose to design both high and low risk experiments to ensure the success of the study and potential breakthrough data on NE integrity, telomere function and ESCRT activity