Isolation of the Hepatic Ubiquitome/NEDDylome by Streptavidin Pull-Down Assay in the Biotinylated Ubiquitin (bioUb)/Biotinylated NEDD8 (bioNEDD8) Transgenic Mice.

In the last years, our group and others have uncovered the role of ubiquitin (Ub) and ubiquitin-like proteins such as the neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-mediated modifications in several types of liver disease, including nonalcoholic fatty liver disease, liver fibrosis, and hepatocellular carcinoma. For this purpose, we have taken advantage of biotinylated ubiquitin (bioUb) and biotinylated NEDD8 (bioNEDD8) mice, transgenic mouse models in which ubiquitin and NEDD8, respectively, are biotinylated in vivo. Using these genetic tools and pull-down assays that exploit the strong biotin-streptavidin interaction, denaturing lysis conditions, and stringent washing procedures, only proteins modified by Ub or NEDD8 are isolated from mammalian tissues in vivo. Here, we report a protocol of streptavidin pull-down of ubiquitinated and NEDDylated liver proteins using the bioUb and bioNEDD8 mice that can potentially be used to characterize both the hepatic ubiquitome and NEDDylome in different models of liver injury.

Hu Antigen R (HuR) Protein Structure, Function and Regulation in Hepatobiliary Tumors.

Hu antigen R (HuR) is a 36-kDa ubiquitous member of the ELAV/Hu family of RNA-binding proteins (RBPs), which plays an important role as a post-transcriptional regulator of specific RNAs under physiological and pathological conditions, including cancer. Herein, we review HuR protein structure, function, and its regulation, as well as its implications in the pathogenesis, progression, and treatment of hepatobiliary cancers. In particular, we focus on hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), tumors where the increased cytoplasmic localization of HuR and activity are proposed, as valuable diagnostic and prognostic markers. An overview of the main regulatory axes involving HuR, which are associated with cell proliferation, invasion, metastasis, apoptosis, and autophagy in HCC, is provided. These include the transcriptional, post-transcriptional, and post-translational modulators of HuR function, in addition to HuR target transcripts. Finally, whereas studies addressing the relevance of targeting HuR in CCA are limited, in the past few years, HuR has emerged as a potential therapeutic target in HCC. In fact, the therapeutic efficacy of some pharmacological inhibitors of HuR has been evaluated, in early experimental models of HCC. We, further, discuss the major findings and future perspectives of therapeutic approaches that specifically block HuR interactions, either with post-translational modifiers or cognate transcripts in hepatobiliary cancers.

Methionine Cycle Rewiring by Targeting miR-873-5p Modulates Ammonia Metabolism to Protect the Liver from Acetaminophen.

Drug-induced liver injury (DILI) development is commonly associated with acetaminophen (APAP) overdose, where glutathione scavenging leads to mitochondrial dysfunction and hepatocyte death. DILI is a severe disorder without effective late-stage treatment, since N-acetyl cysteine must be administered 8 h after overdose to be efficient. Ammonia homeostasis is altered during liver diseases and, during DILI, it is accompanied by decreased glycine N-methyltransferase (GNMT) expression and S-adenosylmethionine (AdoMet) levels that suggest a reduced methionine cycle. Anti-miR-873-5p treatment prevents cell death in primary hepatocytes and the appearance of necrotic areas in liver from APAP-administered mice. In our study, we demonstrate a GNMT and methionine cycle activity restoration by the anti-miR-873-5p that reduces mitochondrial dysfunction and oxidative stress. The lack of hyperammoniemia caused by the therapy results in a decreased urea cycle, enhancing the synthesis of polyamines from ornithine and AdoMet and thus impacting the observed recovery of mitochondria and hepatocyte proliferation for regeneration. In summary, anti-miR-873-5p appears to be an effective therapy against APAP-induced liver injury, where the restoration of GNMT and the methionine cycle may prevent mitochondrial dysfunction while activating hepatocyte proliferative response.

Sphingolipids in Non-Alcoholic Fatty Liver Disease and Hepatocellular Carcinoma: Ceramide Turnover.

Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the main causes of chronic liver disease worldwide. NAFLD comprises a group of conditions characterized by the accumulation of hepatic lipids that can eventually lead to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), the fifth most common cancer type with a poor survival rate. In this context, several works have pointed out perturbations in lipid metabolism and, particularly, changes in bioactive sphingolipids, as a hallmark of NAFLD and derived HCC. In the present work, we have reviewed existing literature about sphingolipids and the development of NAFLD and NAFLD-derived HCC. During metabolic syndrome, considered a risk factor for steatosis development, an increase in ceramide and sphigosine-1-phosphate (S1P) have been reported. Likewise, other reports have highlighted that increased sphingomyelin and ceramide content is observed during steatosis and NASH. Ceramide also plays a role in liver fibrosis and cirrhosis, acting synergistically with S1P. Finally, during HCC, metabolic fluxes are redirected to reduce cellular ceramide levels whilst increasing S1P to support tumor growth.

Post-translational modifiers of liver kinase B1/serine/threonine kinase 11 in hepatocellular carcinoma.

Liver kinase B1 (LKB1) also referred to as serine/threonine kinase 11 (STK11) encodes a 50 kDa evolutionary conserved serine/threonine kinase that is ubiquitously expressed in adult and fetal tissues. LKB1 is a master kinase known to phosphorylate and activate several kinases including AMP-activated protein kinase, a crucial cellular energy sensor. LKB1 shows pleiotropic activity playing diverse roles in multiple processes, including cell polarity and other processes relevant in cancer pathology, such as energy metabolism, proliferation and apoptosis. In spite of the fact that LKB1 is often considered a tumor suppressor in a wide variety of organs, in the last years, several studies have shown that LKB1 is unexpectedly high in hepatocellular carcinoma (HCC), the most common type of primary liver cancer. Post-translational modifications of LKB1 are potentially relevant in HCC. Herein, we provide a comprehensive revision of post-translational modifications of LKB1 in HCC and how they modulate LKB1 function by different mechanisms such as regulation of its activity, localization or stability. Overall, the signature post-translational modifications of LKB1 in HCC appear to play an important role in the rather unique role of LKB1 as an oncogenic driver in liver cancer and may provide an alternative valuable therapeutic approach to regulate LKB1 expression and/or activity in HCC.

Neddylation, a novel paradigm in liver cancer.

Liver cancer is the sixth most prevailing cancer worldwide. Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, has a rather heterogeneous pathogenesis making it highly refractive to current therapeutic approaches. Hence, HCC patients have a poor and gloomy prognosis making liver cancer the second leading cause of global cancer-related deaths. On this basis, a more global mechanism, such as post-translational modifications (PTMs) of proteins, may provide a valuable therapeutic approach for HCC clinical management by simultaneously regulating multiple disrupted signaling pathways. In the last years, the ubiquitin-like molecule NEDD8 (Neural precursor cell-expressed developmentally downregulated-8) conjugation pathway, neddylation, was shown to be aberrant in HCC patients with a significant positive correlation found among global levels of neddylation and poorer prognosis. Even though the best-established role for NEDD8 is the activation of ubiquitin E3 ligase family of cullin-RING ligases, the putative role for other NEDD8 substrates has been explored in recent years leading to the identification of novel neddylation targets in HCC. Importantly, treatment with the small pharmacological inhibitor Pevonedistat (MLN4924) (Millennium Pharmaceuticals, Takeda Pharmaceutical), currently in clinical trials for the treatment of some types of leukemias and other advanced solid tumors, was shown to suppress the outgrowth of hepatoma cells and liver cancer in pre-clinical mouse models. Overall, considering that the neddylation inhibitor Pevonedistat was well-tolerated and displayed a significant antitumor effect in pre-clinical models, combinatory pharmacological treatment based on Pevonedistat are highly recommended to enter clinical trials targeting advanced HCC.