MYC determines lineage commitment in KRAS-driven primary liver cancer development.

BACKGROUND & AIMS: Primary liver cancer (PLC) comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their tumour biology and responses to cancer therapies. Liver cells harbour a high degree of cellular plasticity and can give rise to either HCC or iCCA. However, little is known about the cell-intrinsic mechanisms directing an oncogenically transformed liver cell to either HCC or iCCA. The scope of this study was to identify cell-intrinsic factors determining lineage commitment in PLC. METHODS: Cross-species transcriptomic and epigenetic profiling was applied to murine HCCs and iCCAs and to two human PLC cohorts. Integrative data analysis comprised epigenetic Landscape In Silico deletion Analysis (LISA) of transcriptomic data and Hypergeometric Optimization of Motif EnRichment (HOMER) analysis of chromatin accessibility data. Identified candidate genes were subjected to functional genetic testing in non-germline genetically engineered PLC mouse models (shRNAmir knockdown or overexpression of full-length cDNAs). RESULTS: Integrative bioinformatic analyses of transcriptomic and epigenetic data pinpointed the Forkhead-family transcription factors FOXA1 and FOXA2 as MYC-dependent determination factors of the HCC lineage. Conversely, the ETS family transcription factor ETS1 was identified as a determinant of the iCCA lineage, which was found to be suppressed by MYC during HCC development. Strikingly, shRNA-mediated suppression of FOXA1 and FOXA2 with concomitant ETS1 expression fully switched HCC to iCCA development in PLC mouse models. CONCLUSIONS: The herein reported data establish MYC as a key determinant of lineage commitment in PLC and provide a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. IMPACT AND IMPLICATIONS: Liver cancer is a major health problem and comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their morphology, tumour biology, and responses to cancer therapies. We identified the transcription factor and oncogenic master regulator MYC as a switch between HCC and iCCA development. When MYC levels are high at the time point when a hepatocyte becomes a tumour cell, an HCC is growing out. Conversely, if MYC levels are low at this time point, the result is the outgrowth of an iCCA. Our study provides a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. Furthermore, our data harbour potential for the development of better PLC therapies.

LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer.

The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer.

Author Correction: Necroptosis microenvironment directs lineage commitment in liver cancer.

In this Article, the pCaMIN construct consisted of 'mouse MYC and mouse NrasG12V' instead of 'mouse Myc and human NRASG12V; and the pCAMIA construct consisted of 'mouse Myc and human AKT1' instead of 'mouse Myc and Akt1' this has been corrected online.

Necroptosis microenvironment directs lineage commitment in liver cancer.

Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.

TNFα sensitizes hepatocytes to FasL-induced apoptosis by NFκB-mediated Fas upregulation.

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.

microRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-Associated Protein 1 to Suppress Hepatocarcinogenesis.

BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. RESULTS: Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. CONCLUSIONS: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration.

Senescence is a form of cell cycle arrest induced by stress such as DNA damage and oncogenes. However, while arrested, senescent cells secrete a variety of proteins collectively known as the senescence-associated secretory phenotype (SASP), which can reinforce the arrest and induce senescence in a paracrine manner. However, the SASP has also been shown to favor embryonic development, wound healing, and even tumor growth, suggesting more complex physiological roles than currently understood. Here we uncover timely new functions of the SASP in promoting a proregenerative response through the induction of cell plasticity and stemness. We show that primary mouse keratinocytes transiently exposed to the SASP exhibit increased expression of stem cell markers and regenerative capacity in vivo. However, prolonged exposure to the SASP causes a subsequent cell-intrinsic senescence arrest to counter the continued regenerative stimuli. Finally, by inducing senescence in single cells in vivo in the liver, we demonstrate that this activates tissue-specific expression of stem cell markers. Together, this work uncovers a primary and beneficial role for the SASP in promoting cell plasticity and tissue regeneration and introduces the concept that transient therapeutic delivery of senescent cells could be harnessed to drive tissue regeneration.

Proteomic Analysis Reveals GMP Synthetase as p53 Repression Target in Liver Cancer.

Disruption of the tumor-suppressive p53 network is a key event in human malignancies, including primary liver cancer. In response to different types of stress, p53 mediates several antiproliferative cellular outcomes, such as cell cycle arrest, apoptosis, and senescence, by activation or repression of its target genes. Metabolic alterations initiating or being part of the p53 response have become an actively studied research area in the p53 field, with several aspects that still remain to be elucidated. Herein, we identified GMP synthetase (GMPS), a key enzyme of de novo purine biosynthesis, as an important p53 repression target using a large-scale proteomics approach. This p53-mediated repression of GMPS could be validated by immunoblotting in Sk-Hep1, HepG2, and HuH6 cells. Moreover, we found GMPS transcriptionally repressed in a p21-dependent manner and its repression maintained in the context of p53-mediated cellular senescence. More important, direct knockdown of GMPS by RNA interference resulted in reduced cell viability and was sufficient to trigger cellular senescence. Finally, by comparing murine hepatocellular carcinomas, which developed in p53 wild-type (+/+) versus p53 null (-/-) mice, we observed higher GMPS expression in the latter, supporting the in vivo relevance of our findings. We conclude that repression of GMPS by p53 through p21 is a functionally relevant part of the p53-mediated senescence program limiting tumor cell growth in liver cancer.

A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.