INTEGRATED MOLECULAR INVESTIGATIONS OF MYRF AND SFRP2: UNVEILING REGULATORY MECHANISMS OF ER STRESS AND ECM REMODELLING IN PANCREATIC CANCER

Pancreatic ductal adenocarcinoma (PDAC) stands out as the most prevalent type of pancreatic tumors and the deadliest among common solid malignancies. One of the causes of this high lethality is the significant intratumor cell diversity, highlighting the need for enhanced molecular understanding. Indeed, PDACs are ensembles of three different subtypes with distinct morphology and gene expression programs that coexist in variable proportions in every tumor. Following the identification of transcriptional regulatory networks in the various biotypes, we decided to better characterize two pivotal proteins controlling specific hallmarks of pancreatic cancer biology, like evading growth suppression and activating invasion. First, we focused on the regulation of the myelin regulatory factor (MYRF), a poorly characterized transcription factor. Previous studies conducted in our laboratory demonstrated that MYRF, an endoplasmic reticulum (ER) transmembrane protein, maintains the homeostasis in the ER of pancreatic secretory glandular cells. After trimerization and self-cleavage, MYRF releases the N-terminal fragment which translocates into the nucleus to exert its transcriptional role by regulating the expression of genes encoding secretory proteins. However, the role of C-terminal fragment which remains inserted in the ER is unclear. Since the regulation of MYRF release from the ER membrane is still unknown, our hypothesis is that this fragment may interact with proteins in the ER lumen and regulate MYRF trimerization and cleavage. Using enzyme-catalyzed proximity labeling followed by biotin pull-down and mass spectrometry, we identified MYRF proximal interactors in the ER lumen. Currently, we were able to obtain a first validation of MYRF interaction with UGGT1, which could provide quality control for MYRF folding. Furthermore, in order to investigate the role of MYRF C-terminal fragment in the maintenance of ER functionality, we generated clonal cell lines lacking one of the two critical MYRF functional domains – DNA binding or transmembrane domains - by CRISPR/Cas9-mediated in-frame deletion. The analysis of the gene expression profile of MYRF domain-specific mutants revealed that MYRF has a structural functionality in the ER that is independent of its transcriptional activity. Additionally, we focused on the role of SFRP2 as a potential regulator influencing the characteristics of PDAC transitional cells. One of the key aspects of transitional cancer cells is the ability to contribute to the extracellular matrix (ECM) remodeling: we observed that SFRP2 led to a noteworthy augmentation in this process, along with increased migratory and invasive capabilities of PDAC cells. We also demonstrated that the abundance of ECM components contributes to different tissue density and rigidity, leading cells to mount a mechanoresponsive transcription program. This work could provide i) a mechanistic understanding of MYRF in the regulation of ER activity. Considering the impact of ER stress on cancer cell fitness and drug-induced toxicity, the possibility to influence ER regulation by modulating MYRF trimerization and cleavage by targeting UGGT1 may provide therapeutic opportunities; ii) a better characterization of SFRP2 and its functional effects on PDAC transitional cells, improving anti-cancer therapies.