Identification of MIDORI: a novel microprotein that regulates epithelial-mesenchymal plasticity

Abstract

Recent advances in ribosome profiling, peptidomics and bioinformatics have revealed that many genomic regions previously misannotated as non-protein-coding actually code for evolutionarily conserved small proteins. These proteins, smaller that 100 amino acids in length, have been called microproteins, micropeptides or SEPs (small-ORF encoded polypeptides). To date, only few of them have been functionally characterised but they have been revealed as a new class of molecular regulators with crucial functions in many cellular processes, such as RNA splicing, DNA repair and mitochondrial metabolism. These surprising discoveries have opened a new field of study, with thousands of microproteins with clinical potential waiting to be characterised. _x000D_ Cellular plasticity is the ability of cells to change their original identity and transit between distinct cell states. It is known to be a fundamental property for embryonic development and tissue regeneration, but it has become clear that cellular plasticity is also crucial for cancer cells, which hijack this process to increase their adaptative capacity._x000D_ In this doctoral thesis, we aimed to identify and characterise novel microproteins as regulators of cancer cell plasticity. Using a bioinformatic pipeline, we have identified MIDORI, a microprotein conserved across evolution encoded by ZEB2-AS1, a gene misannotated as a long non-coding RNA (lncRNA). ZEB2-AS1 is known to be upregulated by TGFβ signalling and it is required for the epithelial-to-mesenchymal transition (EMT). Indeed, we have observed that MIDORI protein expression increases upon TGFβ as well as upon genotoxic and ER stress. Surprisingly, we demonstrate that MIDORI downregulates the mesenchymal transcriptional programme in many cell types and blocks the induction of EMT. _x000D_ Next, we analysed the role of MIDORI in several processes related with EMT. In breast cancer cells, MIDORI promotes the establishment of cell-to-cell junctions, impairs the secretion of proinflammatory cytokines and reduces cell migration and invasion in vitro. Moreover, MIDORI overexpression impairs metastatic colonization in vivo. Mechanistically, we have shown that MIDORI reduces the activation of the TGFβ effectors SMAD2, ERK1/2 and NF-kB. We have identified MYBBP1A, a NF-kB co-repressor, as a MIDORI interactor, suggesting that MIDORI could be blocking EMT by transcriptional regulation. _x000D_ In agreement with the role of MIDORI in cancer cells, our studies have also revealed that MIDORI enhances the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs), a process known to require a mesenchymal-to-epithelial transition (MET). Of note, the conditioned medium of MIDORI-overexpressing MEFs is enough to promote cellular reprogramming, suggesting the MIDORI enhances reprogramming in a cell extrinsic manner. Importantly, we have found that endogenous MIDORI is transiently expressed at the early stages of reprogramming, when MET takes place, and MIDORI deficiency drastically decreases reprogramming efficiency. Interestingly, we have observed that MIDORI-deficient MEFs display a dysregulated transcriptional dynamics of EMT-related genes and upregulate p16INK4A and p19ARF tumour suppressor genes during the reprogramming process. Indeed, MIDORI-deficient MEFs are prone to enter cellular senescence and MIDORI overexpression decrease the onset of senescence during reprogramming, suggesting that MIDORI regulates in vitro reprogramming -at least in part- by regulating the onset of cellular senescence. _x000D_ Altogether, in this work we have identified MIDORI, a novel microprotein that is expressed upon stress and acts as an effector of a negative feedback loop that shuts down the mesenchymal programme to revert EMT. Consistently, MIDORI reduces metastasis of breast cancer cells and improves cellular reprogramming to iPSCs. Our data shed light in the regulation of the epithelial-mesenchymal plasticity and uncover a potential new target for regenerative medicine and cancer therapy.

Date of Award	29 Nov 2022
Original language	English
Supervisor	Maria Abad Méndez (Director)