Cooperation Between the NRF2 Pathway and Oncogenic ß-catenin During HCC Tumorigenesis.

CTNNB1 (catenin beta 1)-mutated hepatocellular carcinomas (HCCs) account for a large proportion of human HCCs. They display high levels of respiratory chain activity. As metabolism and redox balance are closely linked, tumor cells must maintain their redox status during these metabolic alterations. We investigated the redox balance of these HCCs and the feasibility of targeting this balance as an avenue for targeted therapy. We assessed the expression of the nuclear erythroid 2 p45-related factor 2 (NRF2) detoxification pathway in an annotated human HCC data set and reported an enrichment of the NRF2 program in human HCCs with CTNNB1 mutations, largely independent of NFE2L2 (nuclear factor, erythroid 2 like 2) or KEAP1 (Kelch-like ECH-associated protein 1) mutations. We then used mice with hepatocyte-specific oncogenic ß-catenin activation to evaluate the redox status associated with ß-catenin activation in preneoplastic livers and tumors. We challenged them with various oxidative stressors and observed that the ß-catenin pathway activation increased transcription of Nfe2l2, which protects ß-catenin-activated hepatocytes from oxidative damage and supports tumor development. Moreover, outside of its effects on reactive oxygen species scavenging, we found out that Nrf2 itself contributes to the metabolic activity of ß-catenin-activated cells. We then challenged ß-catenin activated tumors pharmacologically to create a redox imbalance and found that pharmacological inactivation of Nrf2 was sufficient to considerably decrease the progression of ß-catenin-dependent HCC development. Conclusion: These results demonstrate cooperation between oncogenic ß-catenin signaling and the NRF2 pathway in CTNNB1-mediated HCC tumorigenesis, and we provide evidence for the relevance of redox balance targeting as a therapeutic strategy in CTNNB1-mutated HCC.

Lkb1 suppresses amino acid-driven gluconeogenesis in the liver.

Excessive glucose production by the liver is a key factor in the hyperglycemia observed in type 2 diabetes mellitus (T2DM). Here, we highlight a novel role of liver kinase B1 (Lkb1) in this regulation. We show that mice with a hepatocyte-specific deletion of Lkb1 have higher levels of hepatic amino acid catabolism, driving gluconeogenesis. This effect is observed during both fasting and the postprandial period, identifying Lkb1 as a critical suppressor of postprandial hepatic gluconeogenesis. Hepatic Lkb1 deletion is associated with major changes in whole-body metabolism, leading to a lower lean body mass and, in the longer term, sarcopenia and cachexia, as a consequence of the diversion of amino acids to liver metabolism at the expense of muscle. Using genetic, proteomic and pharmacological approaches, we identify the aminotransferases and specifically Agxt as effectors of the suppressor function of Lkb1 in amino acid-driven gluconeogenesis.

ß-catenin-activated hepatocellular carcinomas are addicted to fatty acids.

OBJECTIVES: CTNNB1-mutated hepatocellular carcinomas (HCCs) constitute a major part of human HCC and are largely inaccessible to target therapy. Yet, little is known about the metabolic reprogramming induced by ß-catenin oncogenic activation in the liver. We aimed to decipher such reprogramming and assess whether it may represent a new avenue for targeted therapy of CTNNB1-mutated HCC. DESIGN: We used mice with hepatocyte-specific oncogenic activation of ß-catenin to evaluate metabolic reprogramming using metabolic fluxes on tumourous explants and primary hepatocytes. We assess the role of Pparα in knock-out mice and analysed the consequences of fatty acid oxidation (FAO) using etomoxir. We explored the expression of the FAO pathway in an annotated human HCC dataset. RESULTS: ß-catenin-activated HCC were not glycolytic but intensively oxidised fatty acids. We found that Pparα is a ß-catenin target involved in FAO metabolic reprograming. Deletion of Pparα was sufficient to block the initiation and progression of ß-catenin-dependent HCC development. FAO was also enriched in human CTNNB1-mutated HCC, under the control of the transcription factor PPARα. CONCLUSIONS: FAO induced by ß-catenin oncogenic activation in the liver is the driving force of the ß-catenin-induced HCC. Inhibiting FAO by genetic and pharmacological approaches blocks HCC development, showing that inhibition of FAO is a suitable therapeutic approach for CTNNB1-mutated HCC.

LKB1 signaling is activated in CTNNB1-mutated HCC and positively regulates ß-catenin-dependent CTNNB1-mutated HCC.

Hepatocellular carcinomas (HCCs) are known to be highly heterogenous. Within the extensive histopathological and molecular heterogeneity of HCC, tumors with mutations in CTNNB1, encoding ß-catenin (CTNNB1-mutated HCC), constitute a very homogeneous group. We previously characterized a distinctive metabolic and histological phenotype for CTNNB1-mutated HCC. They were found to be well-differentiated, almost never steatotic, and often cholestatic, with a microtrabecular or acinar growth pattern. Here, we investigated whether LKB1, which controls energy metabolism, cell polarity, and cell growth, mediates the specific phenotype of CTNNB1-mutated HCC. The LKB1 protein was overexpressed in CTNNB1-mutated HCC and oncogenic activation of ß-catenin in human HCC cells induced the post-transcriptional accumulation of the LKB1 protein encoded by the LKB1 (STK11) gene. Hierarchical clustering, based on the expression of a murine hepatic liver Lkb1 (Stk11) signature in a human public dataset, identified a HCC cluster, composed of almost all the CTNNB1-mutated HCC, that expresses a hepatic liver LKB1 program. This was confirmed by RT-qPCR of an independent cohort of CTNNB1-mutated HCC and the suppression of the LKB1-related profile upon ß-catenin silencing of CTNNB1-mutated human hepatoma cell lines. Previous studies described an epistatic relationship between LKB1 and CTNNB1 in which LKB1 acts upstream of CTNNB1. Thus, we also analyzed the consequences of Lkb1 deletion on the zonation of hepatic metabolism, known to be the hallmark of ß-catenin signaling in the liver. Lkb1 was required for the establishment of metabolic zonation in the mouse liver by positively modulating ß-catenin signaling. We identified positive reciprocal cross talk between the canonical Wnt pathway and LKB1, both in normal liver physiology and during tumorigenesis that likely participates in the amplification of the ß-catenin signaling by LKB1 and the distinctive phenotype of the CTNNB1-mutated HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

AXIN deficiency in human and mouse hepatocytes induces hepatocellular carcinoma in the absence of ß-catenin activation.

BACKGROUND & AIMS: The Wnt/ß-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Inactivating mutations of the gene encoding AXIN1, a known negative regulator of the Wnt/ß-catenin signaling pathway, are observed in about 10% of HCCs. Whole-genome studies usually place HCC with AXIN1 mutations and CTNNB1 mutations in the group of tumors with Wnt/ß-catenin activated program. However, it has been shown that HCCs with activating CTNNB1 mutations form a group of HCCs, with a different histology, prognosis and genomic signature to those with inactivating biallelic AXIN1 mutations. We aimed to elucidate the relationship between CTNNB1 mutations, AXIN1 mutations and the activation level of the Wnt/ß-catenin program. METHODS: We evaluated two independent human HCC datasets for the expression of a 23-ß-catenin target genes program. We modeled Axin1 loss of function tumorigenesis in two engineered mouse models and performed gene expression profiling. RESULTS: Based on gene expression, we defined three levels of ß-catenin program activation: strong, weak or no activation. While more than 80% CTNNB1-mutated tumors were found in the strong or in the weak activation program, most of the AXIN1-mutated tumors (>70%) were found in the subgroup with no activation. We validated this result by demonstrating that mice with a hepatocyte specific AXIN1 deletion developed HCC in the absence of ß-catenin induction. We defined a 329-gene signature common in human and mouse AXIN1 mutated HCC that is highly enriched in Notch and YAP oncogenic signatures. CONCLUSIONS: AXIN1-mutated HCCs occur independently of the Wnt/ß-catenin pathway and involve Notch and YAP pathways. These pathways constitute potentially interesting targets for the treatment of HCC caused by AXIN1 mutations. LAY SUMMARY: Liver cancer has a poor prognosis. Defining the molecular pathways involved is important for developing new therapeutic approaches. The Wnt/ß-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Mutations of AXIN1, a member of this pathway, represent about 10% of HCC mutations. Using both human HCC collections and engineered mouse models of liver cancers with AXIN1 mutation or deletion, we defined a common signature of liver tumors mutated for AXIN1 and demonstrate that these tumors occur independently of the activation of the Wnt/ß-catenin pathway.

RSK Regulates PFK-2 Activity to Promote Metabolic Rewiring in Melanoma.

Metabolic reprogramming is a hallmark of cancer that includes increased glucose uptake and accelerated aerobic glycolysis. This phenotype is required to fulfill anabolic demands associated with aberrant cell proliferation and is often mediated by oncogenic drivers such as activated BRAF. In this study, we show that the MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to maintain glycolytic metabolism in BRAF-mutated melanoma cells. RSK directly phosphorylated the regulatory domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate during glycolysis. Inhibition of RSK reduced PFKFB2 activity and glycolytic flux in melanoma cells, suggesting an important role for RSK in BRAF-mediated metabolic rewiring. Consistent with this, expression of a phosphorylation-deficient mutant of PFKFB2 decreased aerobic glycolysis and reduced the growth of melanoma in mice. Together, these results indicate that RSK-mediated phosphorylation of PFKFB2 plays a key role in the metabolism and growth of BRAF-mutated melanomas.Significance: RSK promotes glycolytic metabolism and the growth of BRAF-mutated melanoma by driving phosphorylation of an important glycolytic enzyme. Cancer Res; 78(9); 2191-204. ©2018 AACR.