YAP1 LICENSES DIFFERENTIATED CELLS TO RETURN TO THE CELL CYCLE IN PALIGENOSIS VIA NON-CANONICAL HIPPO PATHWAY REGULATION

To unravel the origins of gastric cancer, we are investigating the initiation and progression of precursor metaplasias. Chief cells in the stomach are transformed into proliferative spasmolytic polypeptide expressing metaplasia (SPEM) cells through a recently described stepwise process known as paligenosis that involves dynamic mTORC1 regulation: Stage 1, chief cells upregulate autophagic activity, as decreasing mTORC1 activity; Stage 2, SPEM genes are expressed; and, Stage 3, the mTORC1 activity increases, prompting previously quiescent cells to re-enter the cell cycle. Dysregulated paligenosis enables mature, nondividing cells to re-enter the cell cycle despite genomic abnormalities, making paligenosis a potential initiator of gastric cancer. While the Hippo pathway is well-known for its role in regulating cell proliferation and differentiation via its main effectors YAP1/TAZ, its involvement in metaplasia and paligenosis remains ambiguous. Our study showed accumulation of nuclear YAP1 during Stages 1 and 2, resulting in upregulated expression of canonical transcriptional targets. The induction of mitosis in chief cells was observed upon overexpression of YAP1-S127A, which is a constitutively active mutant form of YAP1, via low-dose-tamoxifen-treated Mist1^CreERT2/+; ROSA26^{LSL-rtTA.IRES.EGFP/+}; tetO^Yap1S127A mice induced by doxycycline; or GIF^-rtTA ; tetO^Yap1S127A mice induced by doxycycline. In contrast, paligenosis was impeded upon YAP1 deletion in Mist1^CreERT2/+; ROSA26^LSL-Ai9/+; Yap1^flox/flox ; Taz^flox/flox mice models. The deletion of TAZ alone in Mist1^CreERT2/+; ROSA26^LSL-Ai9/+; Taz^flox/flox mice was not sufficient, leading to the failure of chief cells to go through paligenosis. There was no significant change in total YAP1 protein levels detected by proteomics or western blot, while single-cell RNA sequencing identified alterations in Yap1 downstream target gene mRNAs in acute SPEM cells, indicating the activation of YAP1 in paligenosis via a potential dephosphorylation. Our multi-omic data, corroborated by western blot and immunohistochemistry results, suggests a non-canonical Hippo pathway kinase-dependent modulation of YAP1 localization. This intricate regulation involves autophagy in Stage 1 and implicates the SMAD2/3 key effectors of TGF-β signaling as plausible downstream targets. Our findings emphasize the vital role of YAP1 activity in paligenosis and provide a comprehensive description of upstream kinase regulation and downstream targets associated with YAP1 regulation during the paligenosis process. These findings contribute to a deeper understanding of gastric cancer initiation and provide potential avenues for targeted therapeutic interventions.