Mouse Model Of Hepatocellular Carcinoma Research Articles

METTL3-m6A-EGFR-axis drives lenvatinib resistance in hepatocellular carcinoma

Lenvatinib is emerging as the first-line therapeutic option for advanced hepatocellular carcinoma (HCC), but drug resistance remains a major hurdle for its long-term therapy efficiency in clinic. N6-methyladenosine (m6A) is the most abundant mRNA modification. Here, we aimed to investigate the modulatory effects and underlying mechanisms of m6A in lenvatinib resistance in HCC. Our data revealed that m6A mRNA modification was significantly upregulated in the HCC lenvatinib resistance (HCC-LR) cells compared to parental cells. Methyltransferase-like 3 (METTL3) was the most significantly upregulated protein among the m6A regulators. Either genetic or pharmacological inhibition of m6A methylation through METTL3 deactivation in primary resistant cell line MHCC97H and acquired resistant Huh7-LR cells decreased cell proliferation and increased cell apoptosis upon lenvatinib treatment in vitro and in vivo. In addition, the specific METTL3 inhibitor STM2457 improved tumor response to lenvatinib in multiple mouse HCC models, including subcutaneous, orthotopic and hydrodynamic models. The MeRIP-seq results showed that epidermal growth factor receptor (EGFR) was a downstream target of METTL3. EGFR overexpression abrogated the METTL3 knocked down-induced cell growth arrest upon lenvatinib treatment in HCC-LR cells. Thus, we concluded that targeting METTL3 using specific inhibitor STM2457 improved the sensitivity to lenvatinib in vitro and in vivo, indicating that METTL3 may be a potential therapeutic target to overcome lenvatinib resistance in HCC.

Disruption of SLFN11 Deficiency–Induced CCL2 Signaling and Macrophage M2 Polarization Potentiates Anti–PD-1 Therapy Efficacy in Hepatocellular Carcinoma

The therapeutic effect of immune checkpoint inhibitors (ICIs) is poor in hepatocellular carcinoma (HCC) and varies greatly among individuals. Schlafen (SLFN) family members have important functions in immunity and oncology, but their roles in cancer immunobiology remain unclear. We aimed to investigate the role of the SLFN family in immune responses against HCC. Transcriptome analysis was performed in human HCC tissues with or without response to ICIs. A humanized orthotopic HCC mouse model and a co-culture system were constructed, and cytometry by time-of-flight technology was used to explore the function andmechanism of SLFN11 in the immune context of HCC. SLFN11 was significantly up-regulated in tumors that responded to ICIs. Tumor-specific SLFN11 deficiency increased the infiltration of immunosuppressive macrophages and aggravated HCC progression. HCC cells with SLFN11 knockdown promoted macrophage migration and M2-like polarization in a C-C motif chemokine ligand 2-dependent manner, which in turn elevated their own PD-L1 expression by activating the nuclear factor-κB pathway. Mechanistically, SLFN11 suppressed the Notch pathway and C-C motif chemokine ligand 2 transcription by binding competitively with tripartite motif containing 21 to the RNA recognition motif 2 domain of RBM10, thereby inhibiting tripartite motif containing 21-mediated RBM10 degradation to stabilize RBM10 and promote NUMB exon 9 skipping. Pharmacologic antagonism of C-C motif chemokine receptor 2 potentiated the antitumor effect of anti-PD-1 in humanized mice bearing SLFN11 knockdown tumors. ICIs were more effective in patients with HCC with high serum SLFN11 levels. SLFN11 serves as a critical regulator of microenvironmental immune properties and an effective predictive biomarker of ICIs response in HCC. Blockade of C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 signaling sensitized SLFN11low HCC patients to ICI treatment.

Combinatorial treatment with Silybum marianum essential oil enhances the therapeutic efficacy of a 5-fluorouracil base therapy for hepatocellular carcinoma.

Silybum marianum seeds contain a family of flavonolignans which can regulate cancer cell proliferation and apoptosis. However, research has rarely focused on the effect of S. marianum essential oil in combination with another anticancer drug. Here, we evaluated the antitumor effect of a combination of 5-fluorouracil (5-FU) and S. marianum essential oils on some pathways in hepatocellular carcinoma (HCC) in vitro and in vivo. Gas chromatography-mass spectrography results indicated there was no significant difference between the components of essential oils isolated from two geographical areas (Khuzestan or Isfahan, Sm-K or Sm-I). Each preparation decreased the viability of H22 cells compared to the control group. S. marianum essential oils alone, and combined with 5-FU, reduced the migration and invasion of H22 cells. Angiogenesis-related proteins were significantly reduced both in vivo and in vitro. Apoptosis and autophagy-related proteins were modulated both in vivo and in vitro. Each treatment decreased phospho-NF-κB (p65) and NF-κB (p65) protein levels. Expression levels of Wnt pathway-related genes were also decreased both in vivo and in vitro. Results revealed that the combination of S. marianum and 5-FU prolonged survival in a mouse model of HCC compared to either treatment alone.

Fe3+-binding transferrin nanovesicles encapsulating sorafenib induce ferroptosis in hepatocellular carcinoma

BackgroundFerroptosis, iron-dependent cell death, is an established mechanism for cancer suppression, particularly in hepatocellular carcinoma (HCC). Sorafenib (SOR), a frontline drug for the treatment of HCC, induces ferroptosis by inhibiting the Solute Carrier family 7 member 11 (SLC7A11), with inadequate ferroptosis notably contributing to SOR resistance in tumor cells.MethodsTo further verify the biological targets associated with ferroptosis in HCC, an analysis of the Cancer Genome Atlas (TCGA) database was performed to find a significant co-upregulation of SLC7A11 and transferrin receptor (TFRC), Herein, cell membrane-derived transferrin nanovesicles (TF NVs) coupled with Fe3+ and encapsulated SOR (SOR@TF-Fe3+ NVs) were established to synergistically promote ferroptosis, which promoted the iron transport metabolism by TFRC/TF-Fe3+ and enhanced SOR efficacy by inhibiting the SLC7A11.ResultsIn vivo and in vitro experiments revealed that SOR@TF-Fe3+ NVs predominantly accumulate in the liver, and specifically targeted HCC cells overexpressing TFRC. Various tests demonstrated SOR@TF-Fe3+ NVs accelerated Fe3+ absorption and transformation in HCC cells. Importantly, SOR@TF-Fe3+ NVs were more effective in promoting the accumulation of lipid peroxides (LPO), inhibiting tumor proliferation, and prolonging survival rates in HCC mouse model than SOR and TF- Fe3+ NVs alone.ConclusionsThe present work provides a promising therapeutic strategy for the targeted treatment of HCC.Graphical abstract

Rational combination of SHP2 and mTOR inhibition for the treatment of hepatocellular carcinoma.

Liver cancer is the fourth most common cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the main primary malignancy affecting the liver. Unfortunately, there are still limited therapeutic options for HCC, and even the latest advances have only increased the overall survival modestly. Thus, new treatment strategies and rational drug combinations are urgently needed. Reactivation of receptor tyrosine kinases (RTK) has been described as a mechanism of intrinsic resistance to targeted therapies in a variety of cancers, including inhibitors of mTOR. The design of rational combination therapies to overcome this type of resistance is complicated by the notion that multiple RTK can be upregulated during the acquisition of resistance. SHP2, encoded by the gene PTPN11, acts downstream of virtually all RTK, and has proven to be a good target for small molecule inhibitors. Here, we report activation of multiple RTK upon mTOR inhibition in HCC which, through SHP2, leads to reactivation of the mTOR pathway. We show that co-inhibition of both mTOR and SHP2 is highly synergistic in vitro by triggering apoptosis. More importantly, the combination is well-tolerated and outperforms the monotherapies in impairing tumor growth in multiple HCC mouse models. Our findings suggest a novel rational combination therapy for the treatment of HCC.

Human Epidermal Growth Factor Receptor 2 (HER2) Expression by Immunohistochemistry and Its Clinical Significance in Hepatocellular Carcinoma: A Single-Center Analysis.

Human epidermal growth factor receptor 2 (HER2) is a member of the tyrosine kinase receptor family. It has been identified as an oncogene and is associated with poor outcomes in multiple tumortypes. In hepatocellular carcinoma (HCC), there are contradictory data regarding the HER2 expression and its role in tumor development and progression. Some studies have identified HER2 expression as an early event during tumorigenesis, which decreases with progression and metastasis. Additional data provided evidence that treatment with anti-HER2 therapy resulted in local response and reduction in the metastasis rate in HCC micemodels. Patients with histological diagnoses of HCC between 2010 and 2020 were included. HER2 staining was performed by immunohistochemistry (IHC), and scoring was done in accordance with the gastric cancer guidelines as 0, 1+, 2+, and 3+. Clinicopathological features were accessed by medical records. This study aims to evaluate HER2 expression by IHC in HCCand to correlate this expression with some clinicopathological features such as Barcelona Clinic Liver Cancer (BCLC) staging,number of hepatic lesions,alpha-fetoprotein level, underlying liver disease, presence of livercirrhosis, Child-Pugh score, and tumor recurrence. A total of 57 specimens from 54 patients were included. Of the patients, 85% were men, and the median age at diagnosis was 71 years (interquartile range: 59-75 years). Regarding stage, 61% were at stage 0-A of BCLC. Of the patients,57% had a solitary HCC nodule. Concerning treatment, surgery was performed in 50% of the patients.HER2 expression was identified in seven patients: five in the membrane and two in the cytoplasm. Concerning the membrane staining, HER2 expression was scored as 1+/2+ in 7.4% (n = 4 patients). Of the patients with HER2 expression, fourhad a BCLC stage of 0-A and a single HCC nodule; alpha-fetoprotein was <400 ng/mL in all cases. There was no correlation to clinicopathological features. In one patient with HER2 2+ expressionat diagnosis, this expression was not identified at tumor progression. Median disease-free survival in HER2 with IHC scores 1+/2+ and cytoplasmatic was 38 months versus 22 months in HER2 with a score of 0 (p = 0.604). HER2 expression is a rare event in HCC. It was not possible to identify any relation to clinicopathological features. However, when we relate our data to previous trials, HER2 appears to be an early event in the course of HCC.

Androgen receptor variant 7 exacerbates hepatocarcinogenesis in a c-MYC-driven mouse HCC model

Androgen receptor variant 7 (AR-V7), an AR isoform with a truncated ligand-binding domain, functions as a transcription factor in an androgen-independent manner. AR-V7 is expressed in a subpopulation of hepatocellular carcinoma (HCC), however, its role(s) in this cancer is undefined. In this study, we investigated the potential roles of AR-V7 in hepatocarcinogenesis in vivo in a c-MYC-driven mouse HCC model generated by the hydrodynamic tail-vein injection system. The impacts of AR-V7 on gene expression in mouse HCC were elucidated by RNA-seq transcriptome and ontology analyses. The results showed that AR-V7 significantly exacerbated the c-MYC-mediated oncogenesis in the livers of both sexes. The transcriptome and bioinformatics analyses revealed that AR-V7 and c-MYC synergistically altered the gene sets involved in various cancer-related biological processes, particularly in lipid and steroid/sterol metabolisms. Importantly, AR-V7 suppressed a tumor suppressor Claudin 7 expression, upregulated by c-MYC overexpression via the p53 signaling pathway. Claudin 7 overexpression significantly suppressed the c-MYC-driven HCC development under p53-deficient conditions. Our results suggest that the AR-V7 exacerbates the c-MYC-driven hepatocarcinogenesis by potentiating the oncogenic roles and minimizing the anti-oncogenic functions of c-MYC. Since AR-V7 is expressed in a subpopulation of HCC cases, it could contribute to the inter- and intra-heterogeneity of HCC.

Synergistic effects of concurrent photodynamic therapy with indocyanine green and chemotherapy in hepatocellular carcinoma cell lines and mouse models.

Photodynamic therapy (PDT) using an 808nm laser irradiation with indocyanine green (ICG) has shown tumoricidal effects in a hepatocellular (HCC) orthotopic xenograft model. Recently, combining PDT with concurrent chemotherapy has shown synergistic outcomes and a better therapeutic effect for cancer treatment. In the present study, we utilized a combination of chemotherapy drugs and PDT using ICG in vitro and in vivo in a patient-derived orthotopic xenograft (PDoX) model. We independently performed PDT and chemotherapy with sorafenib or doxorubicin in the Huh-7 and Hep3b cell lines by increasing the sorafenib or doxorubicin concentration and increasing the total energy of 808nm light. Subsequently, we combined the two treatments to confirm the effects on cell viability. The combination index (CI) was evaluated in vitro, and thereafter, in the HCC PDoX mouse model, 808nm laser irradiation with intravenously injected ICG and chemotherapy using doxorubicin were performed for twelve days. The viability of the Huh-7 and Hep3B cell lines decreased rapidly as the concentration of sorafenib or doxorubicin increased and as the total energy of 808nm light increased. The cell viability of the Huh-7 and Hep3b cell lines with combined PDT and chemotherapy was less than that with PDT or chemotherapy alone. The CI was <1 in the sorafenib- or doxorubicin-treated Huh-7 and Hep3b cell lines. In the HCC PDoX mouse model, tumor size was markedly decreased, and complete remission achieved compared to that of the single chemotherapy or PDT and control groups. The synergistic effect of concurrent PDT and chemotherapy in the HCC cell line and PDoX model was confirmed with no definite adverse effect. Concurrent PDT and chemotherapy could be applied in further preclinical studies.

HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

BackgroundHepatic inflammation is a common initiator of liver diseases and considered as the primary driver of hepatocellular carcinoma (HCC). However, the precise mechanism of inflammation-induced HCC development and immune evasion remains elusive and requires extensive investigation. This study sought to identify the new target that is involved in inflammation-related liver tumorigenesis.MethodsRNA-sequencing (RNA-seq) analysis was performed to identify the differential gene expression signature in primary human hepatocytes treated with or without inflammatory stimulus. A giant E3 ubiquitin protein ligase, HECT domain and RCC1-like domain 2 (HERC2), was identified in the analysis. Prognostic performance in the TCGA validation dataset was illustrated by Kaplan–Meier plot. The functional role of HERC2 in HCC progression was determined by knocking out and over-expressing HERC2 in various HCC cells. The precise molecular mechanism and signaling pathway networks associated with HERC2 in HCC stemness and immune evasion were determined by quantitative real-time PCR, immunofluorescence, western blot, and transcriptomic profiling analyses. To investigate the role of HERC2 in the etiology of HCC in vivo, we applied the chemical carcinogen diethylnitrosamine (DEN) to hepatocyte-specific HERC2-knockout mice. Additionally, the orthotopic transplantation mouse model of HCC was established to determine the effect of HERC2 during HCC development.ResultsWe found that increased HERC2 expression was correlated with poor prognosis in HCC patients. HERC2 enhanced the stemness and PD-L1-mediated immune evasion of HCC cells, which is associated with the activation of signal transducer and activator of transcription 3 (STAT3) pathway during the inflammation-cancer transition. Mechanically, HERC2 coupled with the endoplasmic reticulum (ER)-resident protein tyrosine phosphatase 1B (PTP1B) and limited PTP1B translocation from ER to ER-plasma membrane junction, which ameliorated the inhibitory role of PTP1B in Janus kinase 2 (JAK2) phosphorylation. Furthermore, HERC2 knockout in hepatocytes limited hepatic PD-L1 expression and ameliorated HCC progression in DEN-induced mouse liver carcinogenesis. In contrast, HERC2 overexpression promoted tumor development and progression in the orthotopic transplantation HCC model.ConclusionOur data identified HERC2 functions as a previously unknown modulator of the JAK2/STAT3 pathway, thereby promoting inflammation-induced stemness and immune evasion in HCC.

ULK1 Depletion Protects Mice from Diethylnitrosamine-Induced Hepatocarcinogenesis by Promoting Apoptosis and Inhibiting Autophagy.

The uncoordinated-51 like kinase 1 (ULK1) is an important serine/threonine protein kinase involved in autophagy, especially for the initiation stage. Previous studies have shown that ULK1 could be used as a prognostic marker in predicting poor progression-free survival and a therapeutic target for hepatocellular carcinoma (HCC) when treated with sorafenib; however, its role during hepatocarcinogenesis remains to be elucidated. CCK8 and colony formation assay were used to detect cell growth ability. Western blotting was performed to determine expression level of protein. Data from public database were downloaded to analyze expression of ULK1 at mRNA level and predict survival time. RNA-seq was conducted to reveal disturbed gene profile orchestrated by ULK1 depletion. A diethylnitrosamine (DEN)-induced HCC mice model was used to uncover the role of ULK1 in hepatocarcinogenesis. ULK1 was up-regulated in liver cancer tissues and cell lines, and knockdown of ULK1 promoted apoptosis and suppressed proliferation of liver cancer cells. In in vivo experiments, Ulk1 depletion attenuated starvation-induced autophagy in mice liver, reduced diethylnitrosamine (DEN)-induced hepatic tumor number and size, and prevented tumor progression. Further, RNA-seq analysis revealed a close relationship between Ulk1 and immunity with significant changes in gene sets enriched in the interleukin and interferon pathways. ULK1 deficiency prevented hepatocarcinogenesis and inhibited hepatic tumor growth, and might be a molecular target for the prevention and treatment of HCC.

Semaphorin 7A interacts with nuclear factor NF-kappa-B p105 via integrin β1 and mediates inflammation

Semaphorin7a (SEMA7A), a membrane-anchored member of the semaphorin protein family, could be involved in a diverse range of immune responses via its receptor integrin β1. Recently, we reported that the SEMA7AR148W mutation (a gain-of-function mutation, Sema7aR145W in mice) is a risk factor for progressive familial intrahepatic cholestasis and nonalcoholic fatty liver disease via upregulated membrane localization. In this study, we demonstrated that integrin β1 is a membrane receptor for nuclear factor NF-kappa-B p105 (NF-κB p105) and a critical mediator of inflammation. Integrin β1 could interact with the C-terminal domain of NF-κB p105 to promote p50 generation and stimulate the NF-κB p50/p65 signalling pathway, upregulate TNF-α and IL-1β levels, and subsequently render hepatocytes more susceptible to inflammation. The induction of integrin β1 depends on elevated Sema7a membrane localization. Moreover, we revealed elevated levels of Sema7aWT (SEMA7AWT) in hepatocellular carcinoma (HCC) patients and an HCC mouse model. In line with our findings, the NF-κB p50/p65 pathway could also be activated by high Sema7a expression and repressed by integrin β1 silencing. In conclusion, our findings suggest that the Sema7aR145W (SEMA7AR148W) mutation and high Sema7aWT (SEMA7AWT) expression both activate the NF-κB p50/p65 pathway via integrin β1 and play a crucial role in inflammatory responses.CuX12wCaSN9kKvLmEwshrPVideo

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of hepatocellular carcinoma.

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

SERPINA12 promotes the tumorigenic capacity of HCC stem cells through hyperactivation of AKT/β-catenin signaling.

HCC is an aggressive disease with poor clinical outcome. Understanding the mechanisms that drive cancer stemness, which we now know is the root cause of therapy failure and tumor recurrence, is fundamental for designing improved therapeutic strategies. This study aims to identify molecular players specific to CD133 + HCC to better design drugs that can precisely interfere with cancer stem cells but not normal stem cell function. Transcriptome profiling comparison of epithelial-specific "normal" CD133 + cells isolated from fetal and regenerating liver against "HCC" CD133 + cells isolated from proto-oncogene-driven and inflammation-associated HCC revealed preferential overexpression of SERPINA12 in HCC but not fetal and regenerating liver CD133 + cells. SERPINA12 upregulation in HCC is tightly associated with aggressive clinical and stemness features, including survival, tumor stage, cirrhosis, and stemness signatures. Enrichment of SERPINA12 in HCC is mediated by promoter binding of the well-recognized β-catenin effector TCF7L2 to drive SERPINA12 transcriptional activity. Functional characterization identified a unique and novel role of endogenous SERPINA12 in promoting self-renewal, therapy resistance, and metastatic abilities. Mechanistically, SERPINA12 functioned through binding to GRP78, resulting in a hyperactivated AKT/GSK3β/β-catenin signaling cascade, forming a positive feed-forward loop. Intravenous administration of rAAV8-shSERPINA12 sensitized HCC cells to sorafenib and impeded the cancer stem cell subset in an immunocompetent HCC mouse model. Collectively, our findings revealed that SERPINA12 is preferentially overexpressed in epithelial HCC CD133 + cells and is a key contributor to HCC initiation and progression by driving an AKT/β-catenin feed-forward loop.

A novel cuproptosis-related prognostic gene signature and validation of differential expression in hepatocellular carcinoma.

Background: Cuproptosis is a newly discovered form of programmed cell death, which is characterized by accumulation of intra-cellular copper ion leading to the aggregation of lipoproteins and destabilization of Fe-S cluster proteins in mitochondrial metabolism, thereby affecting the prognosis of patients with cancer. However, the role of cuproptosis-related genes (CRGs) in hepatocellular carcinoma (HCC) remains elusive. Methods: Mutation signature, copy number variation and the expression of 10 CRGs were assessed in HCC from TCGA-LIHC dataset. ICGC-LIRI-JP dataset was used as further validation cohort. The least absolute shrinkage and selection operator (LASSO) was used to construct the prognostic model. Kaplan Meier curves, time-ROC curves, nomogram, univariate and multivariate Cox regression were utilized to evaluate the predictive efficacy of CRGs-score. Immune infiltration was analyzed by CIBERSOFT, ssGSEA algorithm, and TIMER database. The expression of prognostic CRGs was validated by qPCR both in-vitro and in-vivo. Drug sensitivity analysis was performed by pRRophetic. Results: All of the CRGs were differentially expressed in HCC and 5 out of them (CDKN2A, DLAT, GLS, LIPT1, MTF1) correlated with patient survival. These signature genes were selected by LASSO analysis to establish a prognosis model to stratify HCC patients into high and low CRGs-score subgroups. High CRGs-score was associated with a worse prognosis. Subsequently, univariate and multivariate Cox regression verified that CRGs-score was an independent cancer risk factor that correlated with clinical factors including stage and grade. Nomogram integrating the CRGs-score and clinical risk factors performed well to predict patient survival. Immune infiltration analysis further revealed that the expression of immune checkpoint genes was significantly enhanced in high CRGs-score group, especially PD-1 and PD-L1. An independent validation cohort (ICGC) confirmed that CRGs-score as a stable and universally applicable indicator in predicting HCC patient survival. Concordantly, the expression of five confirmed signature genes were also differentially expressed in human HCC cell lines and mouse HCC model. In addition, we also analyzed the sensitivity of 10 clinical targeted therapies between high and low CRGs-score groups. Conclusion: This study elucidated the role of dysregulated CRGs in HCC cohort, with validation with in-vitro and in-vivo models. The CRGs-score might be applied as a novel prognostic factor in HCC.

In situ vaccination followed by intramuscular poly-ICLC injections for the treatment of hepatocellular carcinoma in mouse models

The efficacy of treatment for advanced hepatocellular carcinoma (HCC) has remained limited. Polyinosinic–polycytidylic acid–poly-L-lysine carboxymethylcellulose (poly-ICLC) is a synthetic double-stranded RNA that serves as a viral mimic and induces an immune response. Intratumoral (IT) poly-ICLC injections can induce an autovaccination effect and prime the immune system, whereas intramuscular (IM) injection of poly-ICLC can attract and maintain tumor-specific cytotoxic T lymphocytes in tumors. We found that IT injection of poly-ICLC upregulated the expression of CD83 and CD86 on conventional type 1 dendritic cells in tumors. Combination therapy with IT followed by IM injections of poly-ICLC significantly inhibited tumor growth and increased the tumor-infiltrating CD8+ T cells in two syngeneic mouse models of HCC. Depletion of CD8+ T cells attenuated the antitumor effect. An IFN-γ enzyme-linked immunospot of purified tumoral CD8+ T cells revealed a significant proportion of tumor-specific T cells. Finally, the sequential poly-ICLC therapy induced abscopal effects in two dual-tumor models. This study provides evidence that the sequential poly-ICLC therapy significantly increased infiltration of tumor-specific CD8+ T cells in the tumors and induced CD8+ T cell–dependent inhibition of tumor growth, as well as abscopal effects.

MicroRNA-223 attenuates hepatocarcinogenesis by blocking hypoxia-driven angiogenesis and immunosuppression

ObjectiveThe current treatment for hepatocellular carcinoma (HCC) to block angiogenesis and immunosuppression provides some benefits only for a subset of patients with HCC, thus optimised therapeutic regimens are unmet needs,...

CDCBLD2 mediates sorafenib resistance in hepatocellular carcinoma by sponging miR-345-5p binding to the TOP2A coding sequence.

Sorafenib is a first-line chemotherapy drug for treating advanced hepatocellular carcinoma (HCC). However, its therapeutic effect has been seriously affected by the emergence of sorafenib resistance in HCC patients. The underlying mechanism of sorafenib resistance is unclear. Here, we report a circular RNA, cDCBLD2, which plays an important role in sorafenib resistance in HCC. We found that cDCBLD2 was upregulated in sorafenib-resistant (SR) HCC cells, and knocking down cDCBLD2 expression could significantly increase sorafenib-related cytotoxicity. Further evidence showed that cDCBLD2 can bind to microRNA (miR)-345-5p through a competing endogenous RNA mechanism, increase type IIA topoisomerase (TOP2A) mRNA stability through a miRNA sponge mechanism, and reduce the effects of sorafenib treatment on HCC by inhibiting apoptosis. Our findings also suggest that miR-345-5p can negatively regulate TOP2A levels by binding to the coding sequence region of its mRNA. Additionally, targeting cDCBLD2 by injecting a specific small interfering RNA (siRNA) could significantly overcome sorafenib resistance in a patient-derived xenograft (PDX) mouse model of HCC. Taken together, our study provides a proof-of-concept for a potential strategy to overcome sorafenib resistance in HCC patients by targeting cDCBLD2 or TOP2A.

Targeting β-catenin and PD-L1 simultaneously by a racemic supramolecular peptide for the potent immunotherapy of hepatocellular carcinoma.

Objective: The low clinical utility of immune checkpoint inhibitors (ICIs) against PD-1 or PD-L1 has recently been associated with the activation of the Wnt/β-catenin signaling pathway in hepatocellular carcinoma (HCC), which promotes tumor immune escape and resistance to anti-PD-1/PD-L1 therapy. Hence, we aimed to fabricate a supramolecular peptide which could target the Wnt/β-catenin signaling pathway coupled with ICIs blockage therapy for optimizing HCC immunotherapy. Methods: A racemic spherical supramolecular peptide termed sBBI&PDP nanoparticle was constructed by hierarchical self-assembly, comprising an L-enantiomeric peptide as an inhibitor of BCL9 and β-catenin (sBBI) and a D-enantiomeric peptide as an inhibitor of PD-1/PD-L1 (PDP). Results: sBBI&PDP nanoparticle potently suppressed the hyperactivated Wnt/β-catenin signaling pathway in vitro and in vivo, while blocking endogenous PD-L1 effectively. Furthermore, sBBI&PDP increased the infiltration and action of CD8+ T cells at tumor sites. Notably, compared with the original sBBI and commercial Anti-PD-L1 inhibitors, the designed sBBI&PDP showed stronger antitumor efficacy in an orthotopic homograft mice model of HCC and a PDX HCC model in Hu-PBMC-NSG mice. Moreover, sBBI&PDP possessed a favorable biosafety profile. Conclusion: The successful implementation of this strategy could revitalize ICIs blockage therapy and promote the discovery of artificial peptides for HCC immunotherapy.

Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression.

Rationale: CRISPR-Cas13a is an efficient tool for robust RNA knockdown with lower off-target effect, which may be a potentially powerful and safe tool for cancer gene therapy. However, therapeutic effect of current cancer gene therapy that targeting monogene was compromised by the multi-mutational signal pathway alterations of tumorigenesis. Methods: Here, hierarchically tumor-activated nanoCRISPR-Cas13a (CHAIN) is fabricated for multi-pathway-mediated tumor suppression by efficient microRNA disruption in vivo. A fluorinated polyetherimide (PEI; Mw=1.8KD) with graft rate of 33% (PF33) was utilized to compact the CRISPR-Cas13a megaplasmid targeting microRNA-21 (miR-21) (pCas13a-crRNA) via self-assemble to constitute a nanoscale 'core' (PF33/pCas13a-crRNA), which was further wrapped by modified hyaluronan (HA) derivatives (galactopyranoside-PEG2000-HA, GPH) to form CHAIN. Results: The dual-tumor-targeting and tumor-activated CHAIN not only manifested long-term circulation, but augmented tumor cellular uptake and endo/lysosomal escape, thus achieving efficient transfection of CRISPR-Cas13a megaplasmid (~ 13 kb) in tumor cells with minimal toxity. Efficient knockdown of miR-21 by CHAIN restored programmed cell death protein 4 (PDCD4) and reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) and further crippled downstream matrix metalloproteinases-2 (MMP-2), which undermined cancer proliferation, migration and invasion. Meanwhile, the miR-21-PDCD4-AP-1 positive feedback loop further functioned as an enhanced force for anti-tumor activity. Conclusion: Treatment with CHAIN in hepatocellular carcinoma mouse model achieved significant inhibition of miR-21 expression and rescued multi-pathway, which triggered substantial tumor growth suppression. By efficient CRISPR-Cas13a induced interference of one oncogenic microRNA, the CHAIN platform exerted promising capabilities in cancer treatment.

Hepatocellular Carcinoma Mouse Model Induced by SV40 T Antigen Expression: Whole Liver Transcriptome Analysis with Time- and Diet-varying RNA Expression

Hepatocellular Carcinoma Mouse Model Induced by SV40 T Antigen Expression: Whole Liver Transcriptome Analysis with Time- and Diet-varying RNA Expression