Mouse Model Of Hepatocellular Carcinoma Research Articles

Bimodal Treatment of Hepatocellular Carcinoma by Targeted Minimally Interventional Photodynamic/Chemotherapy Using Glyco-Covalent-Organic Frameworks-Guided Porphyrin/Sorafenib

Very limited treatment options are available to fight hepatocellular carcinoma (HCC), a serious global health concern with high morbidity and mortality. The integration of multiple therapies into one nanoplatform to exert synergistic therapeutic effects offers advantages over monotherapies. Here, we describe the construction of the nanoplatform Sor@GR-COF-366 for synergistic chemotherapy and photodynamic therapy (PDT) for HCC using a porphyrin-based covalent organic framework (COF-366) coated with N-acetyl-galactosamine (GalNAc) and rhodamine B (RhB), and loaded with the first-line agent, Sorafenib (Sor). The nanoplatform is targeted towards ASGPR-overexpressed HCC cells and liver tissues by GalNAc and observed by real-time imaging of RhB in vitro and in vivo. The nanoplatform Sor@GR-COF-366 exerts an enhanced synergistic tumor suppression effect in a subcutaneous HCC mouse model with a tumor inhibition rate (TGI) of 97% while significantly prolonging survival at very low toxicity. The potent synergistic therapeutic outcome is confirmed in an orthotopic mouse model of HCC with the TGI of 98% with a minimally invasive interventional PDT (IPDT). Sor@GR-COF-366 is a promising candidate to be combined with chemo-IPDT for the treatment of HCC. Statement of significanceThis work describes the construction of covalent-organic frameworks (COFs) modified with glyco-moieties to serve as hepato-targeted multitherapy delivery systems. They combine minimally invasive interventional photodynamic therapy (IPDT) triggered synergism with chemotherapy treatment for hepatocellular carcinoma (HCC). With the aid of minimally invasive intervention, PDT can elicit potent anti-cancer activity for deep solid tumors. This platform shows strong therapeutic outcomes in both subcutaneous and orthotopic mouse models, which can significantly prolong survival. This work showed an effective combination of a biomedical nano-formulation with the clinical operational means in cancer treatment, which is greatly promising in clinical translation.

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5′) pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.

Abstract No. 331 ▪ FEATURED ABSTRACT Immune modulation by molecularly targeted photothermal ablation in a mouse model of advanced hepatocellular carcinoma and cirrhosis

Immunotherapy is a promising new treatment approach for hepatocellular carcinoma (HCC), but there are numerous barriers to immunotherapy in HCC, including an immunosuppressive microenvironment and the “immunotolerance” of the liver. Hyperthermia treatment modalities are standard of care for early stage HCC, and hyperthermia is known to have immunomodulatory effects. We have developed a molecularly targeted photothermal ablation (MTPA) technology that provides thermally tunable, tumor-specific heat generation.

Novel Paired Cell Lines for the Study of Lipid Metabolism and Cancer Stemness of Hepatocellular Carcinoma.

There are few well-characterized syngeneic murine models for hepatocellular carcinoma (HCC), which limits immunological studies and the development of immunotherapies for HCC. We previously established an oncogene-induced spontaneous HCC mouse model based on transposon-mediated oncogene (AKT and NRASV12) insertion into the genome of hepatocytes to induce tumorigenesis. Two tumor clones with different levels of lipid droplets (LDs) showed similar in vitro growth but distinctive in vivo phenotypes, including divergent proliferative capability and varying induction of myeloid-derived suppressor cells (MDSCs). The two clones showed distinct gene expression related to lipid metabolism, glycolysis, and cancer stemness. Endogenous fatty acid (FA) synthesis and exogenous monounsaturated fatty acid (MUFA) consumption promoted both tumor proliferation and cancer stemness, and upregulated c-Myc in the HCC cell lines. Moreover, the LDhi HCC cell line expressed a higher level of type II IL-4 receptor, which promoted tumor proliferation through binding IL-4 or IL-13. The chromosomal DNA of two tumor clones, NHRI-8-B4 (LDhi) and NHRI-1-E4 (LDlo) showed five identical AKT insertion sites in chromosomes 9, 10, 13, 16 and 18 and two NRAS integration sites in chromosomes 2 and 3. Herein, we describe two novel HCC cell lines with distinct features of lipid metabolism related to cancer stemness and differential interplay with the immune system, and present this syngeneic HCC mouse model as a practical tool for the study of cancer stemness and discovery of new therapies targeting liver cancers.

Rejuvenating hepatic tumor microenvironment immunity with a phosphatidylserine-targeting small molecule drug conjugate

We report the design, synthesis and evaluation of a class of phosphatidylserine-targeting zinc (II) dipicolylamine drug conjugates and show that conjugate 16b elicits immune cell infiltration and remodels the “cold” hepatic tumor microenvironment to the inflamed “hot” tumor. Structure-property relationship study via linker modifications and subsequent pharmacokinetics profiling were carried out to improve the solubility and stability of the conjugates in vivo. In a spontaneous hepatocellular carcinoma mouse model, we showed that conjugate 16b exhibited better antitumor efficacy than sorafenib. In particular, significant increase of CD8+ T cell infiltration and granzyme B level was observed, providing insights in sensitizing tumors from intrinsic immune suppressive microenvironment. Evaluation of tumor inflammation-related mRNA expression profile revealed that conjugate 16b, through inductions of key gene expressions including STAT1, CXCL9, CCL5, and PD-L1, rejuvenated tumor microenvironment with enhancement in T cell-, macrophage-, NK cell-, chemokines and cytokines’- functions. Our study establishes that an apoptosis-targeting theranostic enables enrichment of multifaceted immune cells into the tumor mass, which provides potential therapeutic strategies in the combination with immune checkpoint blockade treatment.

Understanding Molecular Heterogeneity in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer‐related death in the United States with an estimated 32,000 annual deaths. Roughly 30% of HCC cases demonstrate mutations in the CTNNB1 oncogene that encodes a form of the ꞵ‐catenin protein that cannot be degraded. Recently, we developed a mouse model of HCC that genetically represents ~9‐12% of all human HCCs. Here, we combine hydrodynamic tail vein injections with the Sleeping Beauty Transposase – a non‐viral method of expressing genes of interest. Our model uses a clinically observed mutant of ꞵ‐catenin‐(T41A) that is co‐expressed with a mutant form of the nuclear factor erythroid 2–related factor 2 (Nrf2‐G31A) in the livers of wild‐type mice (B+N). This system results in microscopic HCC within 3 weeks and evident macroscopic disease by 7 weeks after induction.ꞵ‐catenin activation can be identified by the expression of its downstream target gene Glutamine Synthetase (GS) in the liver. Patient HCCs with ꞵ‐catenin mutations strongly correlate with GS expression. The evolution and function of GS in b‐catenin‐mutated HCC, remains unclear. Also, there is a gap in the knowledge whether elevated GS expression correlates with HCC biology, which were all subjects of the current study. B+N mouse model of HCC was examined temporally for development of HCC along with GS expression. B+N mice develop HCCs with heterogeneous GS expression in the form of GS‐high and GS‐low to GS‐negative foci. We next asked whether GS expression is associated with cell proliferation or death in the B+N model. We stained tissues for TUNEL and PCNA to mark cell apoptosis and proliferation, respectively in 7‐ and 9.5 weeks post‐injection livers, which were then correlated to GS staining of HCC nodules. While we found no correlation between apoptosis and GS, we found nodules with lower GS expression to have higher proliferation and vice versa. Finally, we asked if eliminating GS expression from B+N HCCs will impact tumor burden. Five weeks after B+N tumor induction in GS‐floxed mice, these animals were injected with AAV8‐TBG‐Cre virus to delete Glul,and mice were harvested after 2‐ or 4.5‐weeks. We found B+N mice with Glul deletion show increased liver weight to body weight ratio, a measurement of HCC burden, compared to controls. The livers of these mice were enriched for GS‐negative tumors. Together, our data suggests that HCC nodules with lower GS expression may outperform GS‐positive foci, and thus are more aggressive compared to the nodules with higher GS expression, which may be more differentiated. The mechanisms of GS heterogeneity and elucidation of its functional significance may help select the most effective targeted therapies for HCC patients with ꞵ‐catenin mutations.

Mitoribosomal defects aggravate liver cancer via aberrant glycolytic flux and T cell exhaustion

BackgroundMitochondria are involved in cancer energy metabolism, although the mechanisms underlying the involvement of mitoribosomal dysfunction in hepatocellular carcinoma (HCC) remain poorly understood. Here, we investigated the effects of mitoribosomal...

METTL3 Enhances Hepatocellular Carcinoma Progression by Regulating Polycomb Repressive Complex 1 (PRC1) Components BMI1 and RNF2

N6‐methyladenosine (m6A) modification is the most abundant mRNA modification which plays important roles in the regulation of mRNA stability, splicing, translocation, and translation. Methyltransferase‐like 3 (METTL3), a primary RNA methyltransferase that catalyzes RNA m6A modification, is implicated in several human cancers including hepatocellular carcinoma (HCC), although its mechanisms of actions remain to be further defined. In the current study, we aimed to further determine the effect and mechanism of METTL3‐derived m6A in HCC. We have analyzed the TCGA database and observed that METTL3 expression is significantly upregulated in HCC patients which is associated with lower survival rate (P < 0.001). In a mouse model of HCC induced by hydrodynamic tail vein injection of hyperactive form of YAP and β‐catenin in conjunction with the sleeping beauty (SB) transposon system, we observed that the expression of METTL3 was notably high in YAP/β‐catenin‐induced HCC. In cultured human HCC cell lines (Huh7 and PLC/PRF/5), we observed that stable knockdown of METTL3 by shRNA significantly decreased tumor cell proliferation, colony formation and migration, in vitro. When Huh and PLC/PRF/5 cells with shRNA knockdown of METTL3 were inoculated into the livers of SCID mice, we found that METTL3 knockdown significantly inhibited xenograft tumor growth, in vivo. We next delivered METTL3 and YAP expression plasmids to the livers of wild type mice via hydrodynamic tail vein injection and observed that co‐expression of METTL3 plus YAP induced the development of HCC which involves almost the entire livers (20 weeks after tail vein injection). These findings provide important evidence for a tumor‐promoting role of METTL3 in HCC development. Through N6‐methyladenosine‐sequencing (m6A‐Seq) and RNA sequencing (RNA‐Seq), we identified BMI1 and RNF2, two key components of the polycomb repressive complex 1 (PRC1), as direct downstream targets of METTL3 in HCC. Our further analyses revealed that both BMI1 and RNF2 were significantly elevated in HCC patients and were associated with lower survival rate and that the expression of BMI1 and RNF2 were positively correlated with METTL3 in human HCC tissues. Consistent with the above results, our further data showed that knockdown of METTL3 in Huh7 and PLC/PRF/5 significantly decreased the expression of BMI1 and RNF2. Accordingly, treatment of Huh7 and PLC/PRF/5 cells with the METTL3 inhibitor, STM2457, significantly reduced the expression of BMI1 and RNF2 and decreased tumor cell proliferation and colony formation. Moreover, our data showed that inhibition of the m6A reader YTHDF1 by siRNA significantly decreased the expression of BMI1 and RNF2 in human HCC cells. Collectively, our study provides important evidence that METTL3 promotes HCC development and progression through m6A modification of BMI1 and RNF2 mRNAs which involve a YTHDF1 dependent mechanism. It is conceivable that the METTL3‐m6A‐BMI1/RNF2 axis may represent a promising target for HCC treatment.

Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma

BackgroundImmunotherapy for hepatocellular carcinoma (HCC) exhibits limited clinical efficacy due to immunosuppressive tumor microenvironment (TME). Tumor-infiltrating macrophages (TIMs) account for the major component in the TME, and the dominance of...

Comprehensive Analysis of RAPGEF2 for Predicting Prognosis and Immunotherapy Response in Patients with Hepatocellular Carcinoma.

Background Hepatocellular carcinoma (HCC) is the sixth most common tumor worldwide. Additionally, deletion of RAPGEF2 plays a critical role in CNV and related to tumor immune microenvironment, whereas the prognostic potential of RAPGEF2 in HCC patient needs to be explored. Methods We looked for prognostic potential genes in HCC using a variety of R programs. Then, using the LASSO Cox regression, we thoroughly evaluated and integrated the RAPGEF2-related genes from TCGA database. Meanwhile, utilizing TCGA and ICGA databases, the link between RAPGEF2 and immunotherapy response in HCC was studied. In vivo, the effect of RAPGEF2 on tumor development and the capacity of natural killer (NK) cells to recruit were confirmed. To ascertain the connection between RAPGEF2-related genes and the prognosis of HCC, a prognostic model was created and validated. Result We demonstrated RAPGEF2 has a differential expression, and patients with deletion of RAPGEF2 gene get shorter survival in HCC. Additionally, the tissues without RAPGEF2 have a weaker ability to recruit the NK cells and response to immunotherapy. After that, we scoured the database for eight RAPGEF2-related genes linked with a better prognosis in HCC patients. Additionally, silencing RAPGEF2 accelerated tumor development in the HCC mouse model and decreased CD56+ NK cell recruitment in HCC tissues. TCGA database was used to classify patients into low- and high-risk categories based on the expression of related genes. Patients in the low-risk group had a significantly greater overall survival than those in the high-risk group (P < 0.001). Meanwhile, the low-risk group demonstrated connections with the NK cell and immunotherapy response. Finally, the prognostic nomogram showed a high sensitivity and specificity for predicting the survival of HCC patients at 1, 2, and 3 years. Conclusion The prognostic model based on RAPGEF2 and RAPGEF2-related genes showed an excellent predictive performance in terms of prognosis and immunotherapy response in HCC, therefore establishing a unique prognostic model for clinical assessment of HCC patients.

The combination of PD-1 blockade with interferon-α has a synergistic effect on hepatocellular carcinoma.

The efficacy of immune checkpoint inhibitors (ICIs), such as programmed cell death protein-1 (PD-1) or its ligand 1 (PD-L1) antibody, in hepatocellular carcinoma (HCC) is limited, and it is recommended that they be combined with other therapies. We evaluated the combination of pegylated interferon-α (Peg-IFNα) with PD-1 blockade in HCC mouse models. We analyzed the effects of Peg-IFNα on tumor-infiltrating immune cells and PD-1 expression in the HCC immune microenvironment and examined the underlying mechanism of its unique effect on the PD-1 pathway. The in vivo efficacy of anti-PD-1 and Peg-IFNα was evaluated in both subcutaneous and orthotopic mouse models of HCC. The combination of Peg-IFNα with PD-1 blockade dramatically enhanced T-cell infiltration, improved the efficacy of PD-1 antibody and prolonged mouse survival compared with PD-1 antibody monotherapy. Mechanistically, Peg-IFNα could recruit cytotoxic CD8+ T cells to infiltrate the HCC microenvironment by inducing tumor cells to secrete the chemokine CCL4. Nevertheless, the HCC microenvironment quickly overcame the immune responses by upregulating PD-1 expression in CD8+ T cells via the IFNα-IFNAR1-JAK1-STAT3 signaling pathway. The combination of PD-1 blockade with Peg-IFNα could restore the cytotoxic capacity of CD8+ T cells and exerted a significant synergistic effect on HCC. These results indicate that in addition to initiating the antitumor immune response itself, Peg-IFNα can also generate a microenvironment favoring PD-1 blockade. Thus, the combination of Peg-IFNα and PD-1 blockade can be a promising strategy for HCC.

Bi-specific T1 positive-contrast-enhanced magnetic resonance imaging molecular probe for hepatocellular carcinoma in an orthotopic mouse model

BACKGROUNDHepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. HCC-targeted magnetic resonance imaging (MRI) is an effective noninvasive diagnostic method that involves targeting clinically-related HCC biomarkers, such as alpha-fetoprotein (AFP) or glypican-3 (GPC3), with iron oxide nanoparticles. However, in vivo studies of HCC-targeted MRI utilize single-target iron oxide nanoprobes as negative (T2) contrast agents, which might weaken their future clinical applications due to tumor heterogeneity and negative MRI contrast. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles (approximately 5 nm) are potential optimal positive (T1) contrast agents. We previously verified the efficiency of AFP/GPC3-double-antibody-labeled iron oxide MR molecular probe in vitro. AIMTo validate the effectiveness of a bi-specific probe in vivo for enhancing T1-weighted positive contrast to diagnose the early-stage HCC.METHODSThe single- and double-antibody-conjugated 5-nm USPIO probes, including anti-AFP-USPIO (UA), anti-GPC3-USPIO (UG), and anti-AFP-USPIO-anti-GPC3 (UAG), were synthesized. T1- and T2-weighted MRI were performed on day 10 after establishment of the orthotopic HCC mouse model. Following intravenous injection of U, UA, UG, and UAG probes, T1- and T2-weighted images were obtained at 12, 12, and 32 h post-injection. At the end of scanning, mice were euthanized, and a histologic analysis was performed on tumor samples. RESULTST1- and T2-weighted MRI showed that absolute tumor-to-background ratios in UAG-treated HCC mice peaked at 24 h post-injection, with the T1- and T2-weighted signals increasing by 46.7% and decreasing by 11.1%, respectively, relative to pre-injection levels. Additionally, T1-weighted contrast in the UAG-treated group at 24 h post-injection was enhanced 1.52-, 2.64-, and 4.38-fold compared to those observed for single-targeted anti-GPC3-USPIO, anti-AFP-USPIO, and non-targeted USPIO probes, respectively. Comparison of U-, UA-, UG-, and UAG-treated tumor sections revealed that UAG-treated mice exhibited increased stained regions compared to those observed in UG- or UA-treated mice. CONCLUSIONThe bi-specific T1-positive contrast-enhanced MRI probe (UAG) for HCC demonstrated increased specificity and sensitivity to diagnose early-stage HCC irrespective of tumor size and/or heterogeneity.

PH-Responsive Liposomes Loaded with Targeting Procoagulant Proteins as Potential Embolic Agents for Solid Tumor-Targeted Therapy.

Selectively inducing tumor thrombosis and subsequent necrosis is a novel and promising antitumor strategy. We have previously designed a targeting procoagulant protein, called tTF-EG3287, which is a fusion of a truncated tissue factor (tTF) with EG3287, a short peptide against the neuropilin-1 (NRP1) binding site of vascular endothelial growth factor-A 165 (VEGF-A 165). However, off-target effects and high-dose requirements limit the further use of tTF-EG3287 in antitumor therapy. Therefore, we encapsulated tTF-EG3287 into poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine (PEOz-DSPE)-modified liposomes to construct pH-responsive liposomes as a novel vascular embolization agent, called tTF-EG3287@Liposomes. The liposomes had an average particle size of about 100 nm and showed considerable drug-loading capacity, encapsulation efficiency, and biocompatibility. Under the stimulation of acidic microenvironments (pH 6.5), the lipid membrane of tTF-EG3287@Liposomes collapsed, and the cumulative drug release rate within 72 h was 83 ± 1.26%. When administered to a mouse model of hepatocellular carcinoma (HCC), tTF-EG3287@Liposomes showed prolonged retention and enhanced accumulation in the tumor as well as a superior antitumor effec, compared with tTF-EG3287. This study demonstrates the potential of tTF-EG3287@Liposomes as a novel embolic agent for solid tumors and provides a new strategy for tumor-targeted infarction therapy.

Multi-omics joint analysis reveals the mechanism of action of the traditional Chinese medicine Marsdenia tenacissima (Roxb.) Moon in the treatment of hepatocellular carcinoma

Ethnopharmacological relevanceMarsdenia tenacissima (Roxb.) Moon, (M. tenacissima) a traditional herbal medicine, has been used for thousands of years. It is noted in Dian Nan Ben Cao that M. tenacissima is bitter in flavor and cold in property, and extracts possess diverse pharmacological effects, including immunomodulation and anti-tumor activities. Aim of the studyThe anti-tumor effects of M. tenacissima extracts (MTE) have been repeatedly confirmed, and this medicine has also been extensively applied in cancer treatment or prognostic adjuvant therapy, with significant curative effect. This study aims to comprehensively analyze the anti-tumor mechanism of M. tenacissima starting from the key features of traditional Chinese medicine and by studying the main active components individually to identify anti-tumor targets in the context of hepatocellular carcinoma. Materials and methodsMolecular network profiling and multi-omic joint analyses were conducted using an H22 mouse model of hepatocellular carcinoma to determine the main active ingredients in MTE and the underlying anti-tumor mechanisms. ResultsTenacissosides I, H, and G (TI,TH and TG) were found to be the likely active ingredients of MTE in the treatment of hepatocellular carcinoma. These compounds were shown to promote apoptosis, inhibit angiogenesis and improve immune function through targeting P53, JAK-1 and HIF1α, respectively. ConclusionsFor the first time, based on the theory that multiple components and multiple targets synergistically exert the beneficial effects of a traditional Chinese medicine, this paper comprehensively analyzes the mechanisms of action of M. tenacissima and provides a novel strategy for the subsequent development of anti-tumor therapies.

Pectin coated nanostructured lipid carriers for targeted piperine delivery to hepatocellular carcinoma

Piperine (PIP) is a herbal drug with well-known anticancer activity against different types of cancer including hepatocellular carcinoma. However, low aqueous solubility and extensive first-pass metabolism limit its clinical use. In this study, positively charged PIP-loaded nanostructured lipid carriers (PIP-NLCs) were prepared via melt-emulsification and ultra-sonication method followed by pectin coating to get novel pectin-coated NLCs (PIP-P-NLCs) targeting hepatocellular carcinoma. Complete in vitro characterization was performed. In addition, cytotoxicity and cellular uptake of nanosystems in HepG2 cells were evaluated. Finally, in vivo anticancer activity was tested in the diethylnitrosamine-induced hepatocellular carcinoma mice model. Successful pectin coating was confirmed by an increased particle size of PIP-NLCs from 150.28 ± 2.51 nm to 205.24 ± 5.13 nm and revered Zeta potential from 33.34 ± 3.52 mV to −27.63 ± 2.05 mV. Nanosystems had high entrapment efficiency, good stability, spherical shape, and sustained drug release over 24 h. Targeted P-NLCs enhanced the cytotoxicity and cellular uptake compared to untargeted NLCs. Furthermore, PIP-P-NLCs improved in vivo anticancer effect of PIP as proved by histological examination of liver tissues, suppression of liver enzymes and oxidative stress environment in the liver, and alteration of cell cycle regulators. To conclude, PIP-P-NLCs can act as a promising approach for targeted delivery of PIP to hepatocellular carcinoma.

TIA1 Loss Exacerbates Fatty Liver Disease but Exerts a Dual Role in Hepatocarcinogenesis

Simple SummaryHepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with few and poorly efficient therapeutic options. Adenylate–uridylate-rich-element-binding proteins (AUBPs) are potent post-transcriptional regulators of gene expression, whose alterations have been associated with the development of chronic liver diseases (e.g., fatty liver disease) and their progression toward HCC. Emerging evidence indicates that T-Cell-restricted intracellular antigen-1 (TIA1), an AUBP regulating mRNA translation, promotes HCC. Herein, we further characterize TIA1’s functions in hepatic cancer cells by identifying an entire set of oncogenes and tumor suppressors under TIA1’s influence, thus suggesting a dual role in hepatocarcinogenesis. In agreement, using in vivo models, we found that contrary to our observations in HCC cells, TIA1 can exert a tumor suppressive function and refrain from hepatic steatosis and fibrosis. Finally, TIA1 expression is reduced in human HCC. Our study provides new insights regarding the role for TIA1 in HCC and questions the current concept of TIA1 as a strict tumor promoter.Alterations in specific RNA-binding protein expression/activity importantly contribute to the development of fatty liver disease (FLD) and hepatocellular carcinoma (HCC). In particular, adenylate–uridylate-rich element binding proteins (AUBPs) were reported to control the post-transcriptional regulation of genes involved in both metabolic and cancerous processes. Herein, we investigated the pathophysiological functions of the AUBP, T-cell-restricted intracellular antigen-1 (TIA1) in the development of FLD and HCC. Analysis of TIA1 expression in mouse and human models of FLD and HCC indicated that TIA1 is downregulated in human HCC. In vivo silencing of TIA1 using AAV8-delivered shRNAs in mice worsens hepatic steatosis and fibrosis induced by a methionine and choline-deficient diet and increases the hepatic tumor burden in liver-specific PTEN knockout (LPTENKO) mice. In contrast, our in vitro data indicated that TIA1 expression promoted proliferation and migration in HCC cell lines, thus suggesting a dual and context-dependent role for TIA1 in tumor initiation versus progression. Consistent with a dual function of TIA1 in tumorigenesis, translatome analysis revealed that TIA1 appears to control the expression of both pro- and anti-tumorigenic factors in hepatic cancer cells. This duality of TIA1′s function in hepatocarcinogenesis calls for cautiousness when considering TIA1 as a therapeutic target or biomarker in HCC.

Targeted-detection and sequential-treatment of small hepatocellular carcinoma in the complex liver environment by GPC-3-targeted nanoparticles

Despite advancements in diagnostic methods and therapeutic strategies, the mortality rate of hepatocellular carcinoma (HCC) remains as high as its incidence rate. Most liver cancers are detected in the advanced stages, when treatment options are limited. Small HCC is difficult to diagnose and is often overlooked by current imaging methods because of the complexity of the liver environment, especially in cirrhotic livers. In the present study, we developed a tumor “cruise missile”, mesoporous Fe3O4-containing glucose oxidase-conjugated GPC3 peptide nanoparticles (FGP NPs). It was designed to enhance the accuracy of small HCC visualization to 85.7% using combined ultrasound/photoacoustic imaging in complex liver environment, which facilitated sequential catalytic targeted therapy for small HCC. In a carcinogen-induced mouse HCC model, FGP NPs could be used to accurately diagnose HCC in a liver cirrhosis background as well as distinguish HCC nodules from other abnormal liver nodules, such as cirrhosis nodules and necrotic nodules, by dynamic contrast-enhanced photoacoustic imaging. In a mouse xenograft HCC model, highly reactive oxygen species were formed by sequential catalytic reactions, which promoted HCC cell apoptosis, significantly increasing the survival of the model mice. The present study provides a basis for the precise detection and elimination of small HCCs in the complex liver environment.Graphical

Ratiometric co-delivery of hydroxychloroquine and calculated low-dose paclitaxel efficiently suppresses tumor growth in hepatocellular carcinoma mouse models in vivo

Utilizing a liposomal nanocarrier to co-encapsulate autophagy inhibitor hydroxychloroquine (HCQ) plus a calculated low-dose chemotherapeutic agent paclitaxel (PTX), we demonstrated a ratiometrically designed nanoformulation with synergistic anti-cancer efficacy in various hepatocellular carcinoma (HCC) mouse models. Using CompuSyn software to calculate drug synergy, we determined the synergistic drug ratios in cultured HCC cells, which became the reference to design ratiometric liposomes. Particle synthesis was accomplished by a film dispersion method for the calculated amount of PTX lipid incorporation and a remote loading process for HCQ import using ammonium sulfate. Ratiometric co-delivery led to synergistic killing effect and autophagy inhibition in cultured HCC cells and nude mice that were used to grow subcutaneous human HepG2 xenografts. Intravenous injection of ratiometric liposome yielded the most effective tumor shrinkage compared to various controls, including the free drug mix. A similar outcome was achieved in a HepG2 orthotopic model. We further explored the anti-cancer effect in a Hep1–6 murine HCC in immunocompetent mice, with or without co-administrated anti-PD1 antibody. Anti-PD1 antibody synergized with HCQ/PTX ratiometric liposome in the immunocompetent model.

Genetic tools for the stable overexpression of circular RNAs

ABSTRACT Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

The Transcription of ZIP9 Is Associated With the Macrophage Polarization and the Pathogenesis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common digestive system cancers (DSCs) with a poor prognosis. Zinc‐regulated transporter (ZRT)/iron‐regulated transporter (IRT) like protein transporters (ZIPs) encode membrane transport proteins, which are responsible for the absorption of zinc and play important roles in the pathogenesis of various human cancers. Tumor-associated macrophages (TAMs) are important participants in the regulation of tumor microenvironment and the development of HCC. Individual role of each ZIP involved in hepatocarcinogenesis remains elusive. In this study, the transcription patterns of ZIPs in the DSCs were screened firstly through GEPIA2 database. Interestingly, the analysis of the DSCs data showed the distinct mRNA levels of ZIPs between DSCs tissues and healthy controls. Notably, the transcription levels of ZIP2, ZIP5, ZIP8, ZIP9 and ZIP14 were decreased significantly in the tissues of human liver cancer compared to paracarcinoma liver tissues. To further confirm the mRNA transcriptional changes of Zips in HCC, N-Nitrosodiethylamine (DEN) combined with carbon tetrachloride (CCl4) inducing mouse model of HCC were established. Consistently, the mRNA levels of Zip2, Zip9, and Zip14 in liver tissues of HCC induced mice were also decreased compared with the healthy controls. In addition, mouse peritoneal elucidated macrophages (PEMs)-derived M1/M2 macrophages in vitro, as well as human patients of HCC-derived TAMs, were used to examine the transcription levels of ZIPs. Our results showed that both Zip2 and Zip9 were up-regulated in M2-polarized macrophages. Zip2 transcript was also up-regulated M1-polarized macrophages, but Zip9 was slightly down-regulated. TAMs generated from human liver cancer tissues also displayed a decrease in ZIP9 transcription compared to paracarcinoma tissues. To further explore the role of Zip9 in M1/M2 polarization, the siRNA knockdown results revealed that Zip9, but not Zip2, could promote M2 macrophage polarization and impair M1 macrophage polarization. Mechanistically, Zip9 enhances phosphorylated STAT6 to promote M2 macrophage polarization but suppresses the phosphorylation of IκBα/β to inhibit M1 macrophage polarization. Together, our results indicate that ZIP9 may involve in macrophages polarity in HCC development and may be a potent new biomarker for the diagnosis of HCC.