Noncanonical mTORC1-TFEB activation promotes hepatocyte plasticity and high-grade malignancy in hepatocellular carcinoma
Chen Zhang, Xiaojuan Chao, Sha Neisha Williams, Xiaoli Wei, Anthony M. DiGirolamo, Alisha Bajracharya, Lichun Ma, Ming Huang, Nicholas Dunn, Wanqing Liu, Kaito Ueda, Masayuki Sugimoto, Andrea Ballabio, Hong-Min Ni, Wen-Xing Ding
Chen Zhang, Xiaojuan Chao, Sha Neisha Williams, Xiaoli Wei, Anthony M. DiGirolamo, Alisha Bajracharya, Lichun Ma, Ming Huang, Nicholas Dunn, Wanqing Liu, Kaito Ueda, Masayuki Sugimoto, Andrea Ballabio, Hong-Min Ni, Wen-Xing Ding
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Research Article Cell biology Hepatology Oncology

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

Abstract

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.

Authors

Chen Zhang, Xiaojuan Chao, Sha Neisha Williams, Xiaoli Wei, Anthony M. DiGirolamo, Alisha Bajracharya, Lichun Ma, Ming Huang, Nicholas Dunn, Wanqing Liu, Kaito Ueda, Masayuki Sugimoto, Andrea Ballabio, Hong-Min Ni, Wen-Xing Ding

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Figure 8

TFEB-CLEAR network activity is elevated in human liver cancer and is associated with stage, survival, spatial tumor regions, and malignant cells.

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TFEB-CLEAR network activity is elevated in human liver cancer and is ass...
(A) TFEB-CLEAR network activity (mean expression/module score of curated CLEAR genes) in nontumor liver versus tumor across independent human cohorts, including the LCI dataset, the Mongolia dataset, and TCGA-LIHC (HCC). P values were determined by 2-sided Wilcoxon’s rank-sum test. (B) TFEB-CLEAR network score and TFEB expression stratified by clinical stage in TCGA-LIHC. ***P < 0.001, ****P < 0.0001. P values were determined by Kruskal-Wallis test with multiple-comparison adjustment. (C) TFEB expression in each TCGA-LIHC tumor sample was transformed as log2(TPM + 1). Patients were then dichotomized into TFEB-high and TFEB-low groups using the cohort median TFEB expression value as the cutoff, and Kaplan-Meier OS of TCGA-LIHC patients was stratified by TFEB expression (high versus low). (D) Log2(TPM + 1)–transformed expression values were standardized separately for each gene, and the CLEAR score was calculated as the mean of the genewise z scores across a curated gene panel related to lysosomal biogenesis, autophagy, and intracellular trafficking. Patients were subsequently classified into CLEAR-high and CLEAR-low groups using the cohort median CLEAR score as the cutoff. Kaplan-Meier overall survival of TCGA-LIHC patients stratified by ssGSEA-based TFEB-CLEAR module activity (high versus low). P value was determined by log-rank test. (E) Schematic of multiregional sampling in the single-cell dataset (GSE189903) study, including adjacent normal tissue (N), tumor border (B), and 3 tumor cores (T1, T2, and T3). (F) Heatmap showing regional expression of TFEB-CLEAR network genes across N, B, and individual tumor cores (T1–T3) in GSE189903. Values represent averaged (z-score–scaled) expression per region. (G) Same as F, with tumor cores T1–T3 merged into a single tumor group to summarize tumor versus border versus normal differences. TPM, transcripts per million; ssGSEA, single-sample gene set enrichment analysis; OS, overall survival.