Proteomic analysis of DEN and CCl4-induced hepatocellular carcinoma mouse model.

Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced HCC mouse model almost recapitulates the characteristic of HCC with fibrosis and inflammation, it is taken as an essential tool to investigate the pathogenesis of HCC. However, a comprehensive understanding of the protein expression profile of this model is little. In this study, we performed proteomic analysis of this model to elucidate its proteomic characteristics. Compared with normal liver tissues, 432 differentially expressed proteins (DEPs) were identified in tumor tissues, among which 365 were up-regulated and 67 were down-regulated. Through Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), protein-protein interaction networks (PPI) analysis and Gene-set enrichment analysis (GSEA) analysis of DEPs, we identified two distinguishing features of DEN and CCl4-induced HCC mouse model in protein expression, the upregulation of actin cytoskeleton and branched-chain amino acids metabolic reprogramming. In addition, matching DEPs from the mouse model to homologous proteins in the human HCC cohort revealed that the DEN and CCl4-induced HCC mouse model was relatively similar to the subtype of HCC with poor prognosis. Finally, combining clinical information from the HCC cohort, we screened seven proteins with prognostic significance, SMAD2, PTPN1, PCNA, MTHFD1L, MBOAT7, FABP5, and AGRN. Overall, we provided proteomic data of the DEN and CCl4-induced HCC mouse model and highlighted the important proteins and pathways in it, contributing to the rational application of this model in HCC research.

Chronic Administration of Diethylnitrosamine and 2-Acetylaminofluorene Induces Hepatocellular Carcinoma in Wistar Rats.

This study aimed to analyze the biochemical, histological, and gene expression alterations produced in a hepatocarcinogenesis model induced by the chronic administration of diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) in Wistar rats. Thirteen rats weighing 180 to 200 g were divided into two groups: control and treated. Rats in the treated group were administered an intraperitoneal (i.p.) injection of DEN (50 mg/kg/week) and an intragastric (i.g.) dose of 2-AAF (25 mg/kg/week) for 18 weeks. The treated group had significant increases in their total cholesterol, HDL-C, AST, ALT, ALKP, and GGT levels. Furthermore, a histological analysis showed the loss of normal liver architecture with nuclear pleomorphism in the hepatocytes, atypical mitosis, and fibrous septa that were distributed between the portal triads and collagen fibers through the hepatic sinusoids. The gene expressions of 24 genes related to fibrosis, inflammation, apoptosis, cell growth, angiogenesis, lipid metabolism, and alpha-fetoprotein (AFP) were analyzed; only TGFß, COL1α1, CYP2E1, CAT, SOD, IL6, TNF-α, and ALB showed significant differences when both groups were compared. Additionally, lung histopathological alterations were found in the treated group, suggesting metastasis. In this model, the chronic administration of DEN+2-AAF induces characteristic alterations of hepatocellular carcinoma in Wistar rats without AFP gene expression changes, highlighting different signatures in hepatocellular carcinoma heterogeneity.

Dietary folate drives methionine metabolism to promote cancer development by stabilizing MAT IIA.

Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.

hnRNPC induces isoform shifts in miR-21-5p leading to cancer development.

MicroRNA (miRNA) processing is a critical step in mature miRNA production. Its dysregulation leads to an increase in miRNA isoforms with heterogenous 5'-ends (isomiRs), which can recognize distinct target sites because of their shifted seed sequence. Although some miRNA genes display productive expression of their 5'-isomiRs in cancers, how their production is controlled and how 5'-isomiRs affect tumor progression have yet to be explored. In this study, based on integrative analyses of high-throughput sequencing data produced by our group and publicly available data, we demonstrate that primary miR-21 (pri-miR-21) is processed into the cancer-specific isomiR isomiR-21-5p | ±1, which suppresses growth hormone receptor (GHR) in liver cancer. Treatment with antagomirs against isomiR-21-5p | ±1 inhibited the in vitro tumorigenesis of liver cancer cells and allowed the recovery of GHR, whereas the introduction of isomiR-21-5p | ±1 mimics attenuated these effects. These effects were validated in a mouse model of spontaneous liver cancer. Heterogeneous nuclear ribonucleoprotein C and U2 small nuclear RNA auxiliary factor 2 were predicted to bind upstream of pre-miR-21 via a poly-(U) motif and influence Drosha processing to induce the production of isomiR-21-5p | ±1. Our findings suggest an oncogenic function for the non-canonical isomiR-21-5p | ±1 in liver cancer, and its production was shown to be regulated by hnRNPC.

MicroRNA-15a/16-1 Prevents Hepatocellular Carcinoma by Disrupting the Communication Between Kupffer Cells and Regulatory T Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is characterized by intratumoral accumulation of regulatory T cells (Tregs), which suppresses antitumor immunity. This study was designed to investigate how microRNAs regulate immunosuppression in HCC. METHODS: FVB/NJ mice were hydrodynamically injected with AKT/Ras or c-Myc and Sleeping Beauty transposon to induce HCC. The Sleeping Beauty system was used to deliver microRNA-15a/16-1 into livers of mice. Flow cytometry and immunostaining were used to determine changes in the immune system. RESULTS: Hydrodynamic injection of AKT/Ras or c-Myc into mice resulted in hepatic enrichment of Tregs and reduced cytotoxic T cells (CTLs) and HCC development. HCC impaired microRNA-15a/16-1 biogenesis in Kupffer cells (KCs) of AKT/Ras and c-Myc mice. Hydrodynamic injection of microRNA-15a/16-1 fully prevented HCC in AKT/Ras and c-Myc mice, while 100% of control mice died of HCC. Therapeutically, microRNA-15a/16-1 promoted a regression of HCC in both mouse models, impaired hepatic enrichment of Tregs, and increased hepatic CTLs. Mechanistically, a significant increase was observed in serum C-C motif chemokine 22 (CCL22) and transcription of Ccl22 in KCs of AKT/Ras and c-Myc mice. MicroRNA-15a/16-1 prevented KCs from overproducing CCL22 by inhibiting nuclear factor-κB that activates transcription of Ccl22. By reducing CCL22 binding to C-C chemokine receptor type 4 on Tregs, microRNA-15a/16-1 impaired Treg chemotaxis. Disrupting the interaction between microRNA-15a/16-1 and nuclear factor-κB impaired the ability of microRNA-15a/16-1 to prevent hepatic Treg accumulation and HCC. Depletion of cluster of differentiation 8+ T cells and additional treatment of CCL22 recovered growth of HCC that was fully prevented by microRNA-15a/16. CONCLUSIONS: MicroRNA-15a/16-1 attenuates immunosuppression by disrupting CCL22-mediated communication between KCs and Tregs. MicroRNA-15a/16-1 represents a potential immunotherapy against HCC.

An RNA-RNA crosstalk network involving HMGB1 and RICTOR facilitates hepatocellular carcinoma tumorigenesis by promoting glutamine metabolism and impedes immunotherapy by PD-L1+ exosomes activity.

Hepatocellular carcinoma (HCC) is the global leading cause of cancer-related deaths due to the deficiency of targets for precision therapy. A new modality of epigenetic regulation has emerged involving RNA-RNA crosstalk networks where two or more competing endogenous RNAs (ceRNAs) bind to the same microRNAs. However, the contribution of such mechanisms in HCC has not been well studied. Herein, potential HMGB1-driven RNA-RNA crosstalk networks were evaluated at different HCC stages, identifying the mTORC2 component RICTOR as a potential HMGB1 ceRNA in HBV+ early stage HCC. Indeed, elevated HMGB1 mRNA was found to promote the expression of RICTOR mRNA through competitively binding with the miR-200 family, especially miR-429. Functional assays employing overexpression or interference strategies demonstrated that the HMGB1 and RICTOR 3'untranslated regions (UTR) epigenetically promoted the malignant proliferation, self-renewal, and tumorigenesis in HCC cells. Intriguingly, interference against HMGB1 and RICTOR in HCC cells promoted a stronger anti-PD-L1 immunotherapy response, which appeared to associate with the production of PD-L1+ exosomes. Mechanistically, the HMGB1-driven RNA-RNA crosstalk network facilitated HCC cell glutamine metabolism via dual mechanisms, activating a positive feedback loop involving mTORC2-AKT-C-MYC to upregulate glutamine synthetase (GS) expression, and inducing mTORC1 signaling to derepress SIRT4 on glutamate dehydrogenase (GDH). Meanwhile, this crosstalk network could impede the efficacy of immunotherapy through mTORC1-P70S6K dependent PD-L1 production and PD-L1+ exosomes activity. In conclusion, our study highlights the non-coding regulatory role of HMGB1 with implications for RNA-based therapeutic targeting together with a prediction of anti-PD-L1 immunotherapy in HCC.

GOLM1 exacerbates CD8+ T cell suppression in hepatocellular carcinoma by promoting exosomal PD-L1 transport into tumor-associated macrophages.

The immunosuppressive microenvironment plays an important role in tumor progression and immunotherapy responses. Golgi membrane protein 1 (GOLM1) is correlated to hepatocellular carcinoma (HCC) progression and metastasis. However, little is known about the role of GOLM1 in regulating the immunosuppressive environment and its impact on immunotherapeutic efficacy in HCC. In this study, GOLM1 was positively correlated with infiltrating tumor-associated macrophages (TAMs) expressed high levels of programmed death-ligand 1 (PD-L1) and CD8+ T cell suppression in HCC tissues. Both gain- and loss-of-function studies determined a close correlation between GOLM1 and immunosuppression. In the mechanism, GOLM1 promoted COP9 signalosome 5-mediated PD-L1 deubiquitination in HCC cells and increased the transport of PD-L1 into exosomes via suppression of Rab27b expression. Furthermore, co-culture with exosomes derived from HCC cells upregulated the expression of PD-L1 on macrophages. Zoledronic acid in combination with anti-PD-L1 therapy reduced PD-L1+ TAMs infiltration and alleviated CD8+ T cell suppression, resulting in tumor growth inhibition in the mouse HCC model. Together, our study unveils a mechanism by which GOLM1 induces CD8+ T cells suppression through promoting PD-L1 stabilization and transporting PD-L1 into TAMs with exosome dependent. Targeting PD-L1+ TAM could be a novel strategy to enhance the efficacy of anti-PD-L1 therapy in HCC.

Regulation of Tumorigenesis in Hepatocellular Carcinoma via the AKT3 Pathway in Cell Lines.

BACKGROUND: The estimation of hepatocellular carcinoma (HCC) is tremendously inferior because of the formation of chemoresistance, integrated with essentially increased stemness belongings. At present, the relevance between miR-122 and cancer development was mostly undisclosed with single study reflecting its importance in glioblastoma. Material and Methods. The research here was focused to investigate the task of miR-122 to modulate tumorigenesis in hepatocellular carcinoma by aiming AKT3 in the management of hepatocellular carcinoma stemness and chemosensitivity. The method of QRT-PCR was performed to investigate the aspect of miR-122 and AKT3 in tissue sample and cell lines. The evaluation was done using gain- or loss-of-function in order to retrieve the function of miR-122 in the hepatocellular carcinoma cells, including cell multiplication and stemness effects. The properties of hepatocellular carcinoma were discovered by the development of sphere development, cell feasibility, and the emergence of colony. RNA immunoprecipitation (RIP), luciferase reporter, and the RNA pull down evaluation were conducted to investigate the communication between the miR-122 and AKT3. Therefore, a naked mouse xenograft specimen was set up for the in vivo analysis. RESULTS: Here, we determined the new role of AKT3 and unmediated target of miR-122. The reimposition of miR-122 appearance in the hepatocellular carcinoma cell lines reduces the levels of AKT3 and also hinders the relocation and expansion of cells by prompting apoptosis. These phenotypes are antitumor in nature and can be retrieved by the reorganization of the AKT3 expression which signals the crucial role of AKT3 in the miR-122 arbitrated HCC modification. The in vivo analysis demonstrated the reimposition of miR-122 entirely obstructing the xenograft expansion to manage tumorigenesis in hepatocellular carcinoma and a prospective remedial entrant for the liver cancer. CONCLUSION: In this study, it was observed that miR-122 encourages the stemness role of hepatocellular carcinoma and diminishes the chemosensitivity by cleaning the miR-122 in order to initiate the AKT3. The in vivo study reflected the restoration of miR-122 which completely hinders the xenograft growth to regulate the tumorigenesis in the HCC.

c-Myc-driven Hepatocarcinogenesis.

BACKGROUND/AIM: Dysregulation of the c-Myc gene is frequently found in human hepatocellular carcinoma (HCC), often accompanied by genetic and epigenetic alterations in other cancer-related genes. Here, we investigated the tumorigenic potential of c-Myc in diverse genetic environments in which the Ras, Wnt/ß-catenin, Sonic hedgehog, or P53 pathways were either activated or inactivated. MATERIALS AND METHODS: Hydrodynamic tail vein injection was employed to administer expression transposons and generate transgenic livers expressing c-Myc together with a constitutively active form of RAS (HRASG12V), ß-catenin (ß-cateninS33Y), Smo (SmoM2), or short hairpin RNA targeting P53 (shp53). RESULTS: c-Myc was most tumorigenic when the RAS signaling pathway was activated, whereas no tumors were found in mice when either ß-cateninS33Y or SmoM2 was co-expressed with c-Myc. Approximately 40% of mice had HCC when c-Myc was over-expressed under P53 inactivation. Furthermore, we investigated the effect of mutation in c-Myc on hepatocarcinogenesis. CONCLUSION: No significant differences in tumorigenic potential were found between wild type c-Myc and c-MycT58A, minimizing the role of the mutation in hepatocarcinogenesis.

MicroRNA-26a systemic administration attenuates tumor formation in hepatocellular carcinoma mouse model.

MicroRNA (miRNA)-26a is one of the tumor suppressor genes that has been down regulated during the development of hepatocellular carcinoma (HCC). This work was conducted to evaluate the possible preventive effect of exogenous miRNA-26a administration on diethylnitrosamine (DEN)-mediated HCC. Balb/C mice were intraperitoneally injected with saline (Normal group), DEN (HCC group) or miRNA-26a (HCC+miRNA-26a group). On week 8, 12, 16 and 20, the concentrations of alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), the levels of helper T cells-associated cytokines, and the vascular endothelial growth factor (VEGF), were measured. Flow cytometry determined the frequencies of regulatory T (Treg) cells. The concentrations of AFP, DCP and VEGF, as well as the frequency of Treg cells showed significantly lower values following miRNA-26a administration than in HCC group. miRNA-26a administration has reduced the levels of IL (interleukin)-2 and TNF (tumor necrosis factor)-α, in contrast, IL-10 level was markedly elevated in comparison to HCC model at all experimental time points. The restore of miRNA-26a function significantly (P<0.001) down regulated the expression levels of survivin & caspase-3 compared to HCC group. The obtained data introduce an evidence for the suppressive impact of miRNA-26a on liver tumor formation and its possible manipulation as a therapeutic design for HCC.

Identification of potential targets with high centrality indicated by diethylnitrosamine + thioacetamide-induced hepatocellular carcinoma model.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC), a primary liver malignancy, represents a continuous challenge to clinicians as it is a leading cause of death due to cancer widely. Early detection is the only hope to cure patients from this deadly disease or possibly increase life expectancy. Mouse models are most acceptable studies as they have ability to manipulate their genome and transcriptome to evaluate mechanistic changes. In addition, system biology can improvise the understanding of molecular mechanism of HCC and also can reveal the protein hub involved in every stage of HCC. MATERIALS AND METHODS: Herein, diethylnitrosamine and thioacetamide (TAA) were used to develop stage-specific HCC in Wistar rats. Histopathological changes, biochemical parameters, and the oxidative stress were measured in hepatocytes. We have reanalyzed the microarray dataset to identify the complex signaling pathways involved in hepatocarcinogenesis induced by TAA. GSE45050 dataset was downloaded from Gene Expression Omnibus database, and the gene expression profile of nontumor, cirrhosis, and HCC was compared. RESULTS: The study reveals stage-specific development of chronic HCC rat model and promising stage-specific targets (EHMT2, GMPS, and SPRY2) of HCC. CONCLUSIONS: EHMT2, GMPS, and SPRY found as high centrality nodes in protein-protein interaction studies using high-throughput microarray data which tend to be present in signaling pathways and co-occur in a biological state of HCC. These genes can be targeted to understand the possible pathology, molecular changes, and target strategy under cirrhosis and HCC condition.

Multi-Modal Imaging Probe for Glypican-3 Overexpressed in Orthotopic Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is rising steadily in incidence, and more effective methods are needed for early detection and image-guided surgery. Glypican-3 (GPC3) is a cell surface biomarker that is overexpressed in early-stage cancer but not in cirrhosis. An IRDye800-labeled 12-mer amino acid sequence was identified, and specific binding to GPC3 was validated in vitro and in orthotopically implanted HCC tumors in vivo. Over 4-fold greater binding affinity and 2-fold faster kinetics were measured by comparison with previous GPC3 peptides. Photoacoustic images showed peak tumor uptake at 1.5 h post-injection and clearance within ∼24 h. Laparoscopic and whole-body fluorescence images showed strong intensity from tumor versus adjacent liver with about a 2-fold increase. Immunofluorescence staining of human liver specimens demonstrated specific binding to HCC versus cirrhosis with 79% sensitivity and 79% specificity, and normal liver with 81% sensitivity and 84% specificity. The near-infrared peptide is promising for early HCC detection in clinical trials.

Ablating putative Ku70 phosphorylation sites results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma.

Multiple pathways mediate the repair of DNA double-strand breaks (DSBs), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that mutating five putative phosphorylation sites on the non-homologous end joining (NHEJ) factor, Ku70, results in sustained retention of human Ku70/80 at DSB ends and attenuation of DSB repair via homologous recombination (HR). In this study, we generated a knock-in mouse, in which the three conserved putative phosphorylation sites of Ku70 were mutated to alanine to ablate potential phosphorylation (Ku703A/3A), in order to examine if disrupting DSB repair pathway choice by modulating Ku70/80 dynamics at DSB ends results in enhanced genomic instability and tumorigenesis. The Ku703A/3A mice developed spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting decreased DNA repair. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that mutating the putative Ku70 phosphorylation sites results in defective DNA damage repair and disruption of this process drives genomic instability and accelerated development of HCC.

Fructose-1,6-Bisphosphate Aldolase B Depletion Promotes Hepatocellular Carcinogenesis Through Activating Insulin Receptor Signaling and Lipogenesis.

BACKGROUND AND AIMS: Insulin receptor (IR) transduces cell surface signal through phosphoinositide 3-kinase (PI3K)-AKT pathways or translocates to the nucleus and binds to the promoters to regulate genes associated with insulin actions, including de novo lipogenesis (DNL). Chronic activation of IR signaling drives malignant transformation, but the underlying mechanisms remain poorly defined. Down-regulation of fructose-1,6-bisphosphate aldolase (ALDO) B in hepatocellular carcinoma (HCC) is correlated with poor prognosis. We aim to study whether and how ALDOB is involved in IR signaling in HCC. APPROACH AND RESULTS: Global or liver-specific ALDOB knockout (L-ALDOB-/- ) mice were used in N-diethylnitrosamine (DEN)-induced HCC models, whereas restoration of ALDOB expression was achieved in L-ALDOB-/- mice by adeno-associated virus (AAV). 13 C6 -glucose was employed in metabolic flux analysis to track the de novo fatty acid synthesis from glucose, and nontargeted lipidomics and targeted fatty acid analysis using mass spectrometry were performed. We found that ALDOB physically interacts with IR and attenuates IR signaling through down-regulating PI3K-AKT pathways and suppressing IR nuclear translocation. ALDOB depletion or disruption of IR/ALDOB interaction in ALDOB mutants promotes DNL and tumorigenesis, which is significantly attenuated with ALDOB restoration in L-ALDOB-/- mice. Notably, attenuated IR/ALDOB interaction in ALDOB-R46A mutant exhibits more significant tumorigenesis than releasing ALDOB/AKT interaction in ALDOB-R43A, whereas knockdown IR sufficiently diminishes tumor-promoting effects in both mutants. Furthermore, inhibiting phosphorylated AKT or fatty acid synthase significantly attenuates HCC in L-ALDOB-/- mice. Consistently, ALDOB down-regulation is correlated with up-regulation of IR signaling and DNL in human HCC tumor tissues. CONCLUSIONS: Our study reports a mechanism by which loss of ALDOB activates IR signaling primarily through releasing IR/ALDOB interaction to promote DNL and HCC, highlighting a potential therapeutic strategy in HCC.

Treatment of Retinoblastoma 1-Intact Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4/6 Inhibitor Combination Therapy.

BACKGROUND AND AIMS: Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy. APPROACH AND RESULTS: Loss of all Rb family members in transformation related protein 53 (Trp53)-/- mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an I kappa B kinase (IKK)ß inhibitor Bay 11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/ß phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay 11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact KRAS Kirsten rat sarcoma viral oncogene homolog mutated lung and colon cancers. CONCLUSIONS: In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.

Butyrate-containing structured lipids act on HDAC4, HDAC6, DNA damage and telomerase activity during promotion of experimental hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) presents with a high treatment resistance and poor prognosis. Early diagnosis and preventive approaches such as chemoprevention are essential for the HCC control. Therefore, we evaluated the chemopreventive effects of butyrate-containing structured lipids (STLs) administered during the promotion stage of hepatocarcinogenesis in rats submitted to the 'resistant hepatocyte' (RH) model. Administration of butyrate-containing STLs inhibited the incidence and mean number of visible hepatic nodules per rat and reduced the number and area of glutathione S-transferase placental form-positive (GST-P+) preneoplastic focal lesions in the livers. This was accompanied by the induction of apoptosis and an increased level of hepatic butyric acid. Treatment with butyrate-containing STLs resulted in increased histone H3 lysine 9 (H3K9) acetylation, reduction of total histone deacetylase (HDAC) activity, and lower levels of HDAC4 and HDAC6 proteins. The chemopreventive effect of butyrate-containing STLs was also associated with the increased nuclear compartmentalization of p53 protein and reduced expression of the Bcl-2 protein. In addition, rats treated with butyrate-containing STLs showed decreased DNA damage and telomerase activity in the livers. These results demonstrate that the suppressive activity of butyrate-containing STLs is associated with inhibition of elevated during hepatocarcinogenesis chromatin-modifying proteins HDAC4 and HDAC6, subcellular redistribution of the p53 protein, and decreased DNA damage and telomerase activity.

Glucose deprivation-induced aberrant FUT1-mediated fucosylation drives cancer stemness in hepatocellular carcinoma.

Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK/eIF2α/ATF4 signaling axis to drive fucosyltransferase 1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal, and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR, and EPHA2 are glycoprotein targets of FUT1, in which such fucosylation would consequently converge on deregulated AKT/mTOR/4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecularly targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR, and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.

Myelocytomatosis-Protein Arginine N-Methyltransferase 5 Axis Defines the Tumorigenesis and Immune Response in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: HCC is a leading cause of cancer-related deaths globally with poor outcome and limited therapeutic options. Although the myelocytomatosis (MYC) oncogene is frequently dysregulated in HCC, it is thought to be undruggable. Thus, the current study aimed to identify the critical downstream metabolic network of MYC and develop therapies for MYC-driven HCC. APPROACH AND RESULTS: Liver cancer was induced in mice with hepatocyte-specific disruption of Myc and control mice by administration of diethylnitrosamine. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses revealed that urinary dimethylarginine, especially symmetric dimethylarginine (SDMA), was increased in the HCC mouse model in an MYC-dependent manner. Analyses of human samples demonstrated a similar induction of SDMA in the urines from patients with HCC. Mechanistically, Prmt5, encoding protein arginine N-methyltransferase 5, which catalyzes SDMA formation from arginine, was highly induced in HCC and identified as a direct MYC target gene. Moreover, GSK3326595, a PRMT5 inhibitor, suppressed the growth of liver tumors in human MYC-overexpressing transgenic mice that spontaneously develop HCC. Inhibition of PRMT5 exhibited antiproliferative activity through up-regulation of the tumor suppressor gene Cdkn1b/p27, encoding cyclin-dependent kinase inhibitor 1B. In addition, GSK3326595 induced lymphocyte infiltration and major histocompatibility complex class II expression, which might contribute to the enhanced antitumor immune response. Combination of GSK3326595 with anti-programed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) improved therapeutic efficacy in HCC. CONCLUSIONS: This study reveals that PRMT5 is an epigenetic executer of MYC, leading to repression of the transcriptional regulation of downstream genes that promote hepatocellular carcinogenesis, highlights a mechanism-based therapeutic strategy for MYC-driven HCC by PRMT5 inhibition through synergistically suppressed proliferation and enhanced antitumor immunity, and finally provides an opportunity to mitigate the resistance of "immune-cold" tumor to ICT.

Tumor restriction by type I collagen opposes tumor-promoting effects of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAF) may exert tumor-promoting and tumor-suppressive functions, but the mechanisms underlying these opposing effects remain elusive. Here, we sought to understand these potentially opposing functions by interrogating functional relationships among CAF subtypes, their mediators, desmoplasia, and tumor growth in a wide range of tumor types metastasizing to the liver, the most common organ site for metastasis. Depletion of hepatic stellate cells (HSC), which represented the main source of CAF in mice and patients in our study, or depletion of all CAF decreased tumor growth and mortality in desmoplastic colorectal and pancreatic metastasis but not in nondesmoplastic metastatic tumors. Single-cell RNA-Seq in conjunction with CellPhoneDB ligand-receptor analysis, as well as studies in immune cell-depleted and HSC-selective knockout mice, uncovered direct CAF-tumor interactions as a tumor-promoting mechanism, mediated by myofibroblastic CAF-secreted (myCAF-secreted) hyaluronan and inflammatory CAF-secreted (iCAF-secreted) HGF. These effects were opposed by myCAF-expressed type I collagen, which suppressed tumor growth by mechanically restraining tumor spread, overriding its own stiffness-induced mechanosignals. In summary, mechanical restriction by type I collagen opposes the overall tumor-promoting effects of CAF, thus providing a mechanistic explanation for their dual functions in cancer. Therapeutic targeting of tumor-promoting CAF mediators while preserving type I collagen may convert CAF from tumor promoting to tumor restricting.