TGF-β1 and TGF-β2 family members differentially modulate tumor initiation and invasiveness of primary liver cancer in a MMP14-dependent manner

Emerging evidence suggests that perturbation of TGF-β signaling in parenchymal cells drives malignant transformation and cancer progression in a subset of patients with primary liver cancer (PLC). TGF-β plays a crucial role in liver pathophysiology with diverse effects on various processes and cell types. In liver homeostasis, TGF-β signaling exerts tumor-suppressive functions that are often lost during carcinogenic transformation, when downstream signaling rebranches from tumor-suppressive to pro-tumorigenic, facilitating invasiveness and metastasis. In this study, primary patient-derived and established liver cancer cell lines were exposed to TGF-β1 and TGF-β2, and effects on tumor-initiating potential, invasion, and migration were assessed by in vitro analyses including colony/sphere formation and wound healing assays. RNA sequencing and reverse-phase protein array (RPPA) were employed to analyze differential gene and protein expression across treatments. Our findings demonstrate that TGF-β1 and TGF-β2 reduced proliferation, colony and spheroid formation in investigated cell lines. Notably, TGF-β1 increased migratory and invasive properties of both HCC and CCA cell lines, whereas TGF-β2 had no such effect. Transcriptome profiling revealed activation of gene sets associated with cell cycle regulation by both ligands. Pro-migratory effects of TGF-β1 were linked to epithelial-mesenchymal transition (EMT), including enrichment of matrix metalloproteinase (MMP) and actin cytoskeleton pathways. Specifically, TGF-β1 downregulated epithelial marker E-cadherin and upregulated mesenchymal markers Vimentin and SNAIL. RPPA indicated p21 induction by both ligands, causing cell cycle arrest, while TGF-β1 specifically upregulated MMP14, promoting EMT-related properties. In conclusion, targeting TGF-β1-MMP14-EMT pathway could complement TGF-β-based therapies in PLC management.