Olfactomedin 4 orchestrates TGF-β/CEACAM6 axis, promoting cell-autonomous epithelial to mesenchymal transition during gallbladder epithelium carcinogenesis.

Gallbladder cancer (GBC) is a highly lethal disease which is usually diagnosed at advanced stage owing to unavailable screening tools and effective therapies. Persistent inflammation induced by various risk factors is the main cause of GBC, however, the molecular program remains elusive. Unveiling the molecular trajectory and events during gallbladder epithelium malignant transformation contribute to prevention and drug discovery for GBC. Single cell RNA sequencing was performed by using 4 gallbladder adenoma and cancer samples. Pseudotime trajectory analyses were employed to reconstruct epithelium transformation track in order to identify crucial genes which promoted GBC development. Functional and mechanism studies were performed to validate the regulatory network and cell behavior in vitro and in vivo. Three clusters of gallbladder epithelium were identified among GBC microenvironment which were characterized by distinct epithelial mesenchymal transition (EMT) and inflammation states. Cell adhesion molecular binding was the most significant GO term between EMT high and low states, in which OLFM4 was differentially expressed gene participated. Further studies implicated that OLFM4 could promote GBC metastasis and activate EMT through CEACAM6/AKT signaling cascade, and the CEACAM6 expression was regulated by TGF-β/Smad3 pathway. Interestingly, we disclosed that TGFβR1 was the functional receptor of OLFM4, through which the tumorigenic signaling of OLFM4 was transduced. These findings suggest the oncogenic role of OLFM4 during GBC carcinogenesis which can be a candidate biomarker of GBC, and OLFM4, TGFβR1 and downstream signaling elements are promising therapeutic targets for GBC.