Noncanonical mTORC1-TFEB activation promotes hepatocyte plasticity and high-grade malignancy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is heterogeneous, and hepatocyte plasticity is linked to poorer patient outcomes. A subset of human HCC harboring tuberous sclerosis complex 1 (TSC1) mutations exhibits more aggressive behavior. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and cell fate. We analyzed human normal and HCC tissue arrays for TFEB and CK19 expression, as well as bulk and single-cell RNA-seq datasets from mouse and human HCC, to define TFEB-associated transcriptional programs. We performed biochemical, histological, metabolomic, and transcriptomic analyses in liver-specific Tsc1-knockout (L-Tsc1-KO) and L-Tsc1 Tfeb-double KO mice. Loss of hepatic Tsc1 led to increased phosphorylation of S6 and 4EBP1, with paradoxical increases in TFEB nuclear translocation and activation. L-Tsc1-KO mice showed increased hepatocyte plasticity, decreased HNF4α, increased YAP activation, and spontaneous HCC with increased SOX9- and CK19-positive biliary epithelial cell-like cells at 8-12 months. Deletion of Tfeb dampened hepatic metabolic reprogramming and hepatocyte fate changes and inhibited tumor progression in L-Tsc1-KO mice. Increased TFEB activity was associated with increased YAP and SOX9 gene expression and high-grade malignant HCC in humans. These findings indicate that loss of hepatic TSC1 leads to noncanonical TFEB activation, promoting hepatocyte plasticity and tumor heterogeneity associated with high-grade malignancy in both mouse and human HCC.