Hydrodynamic Tail Vein Injection Research Articles

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

Background STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Results Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR ( Nr1i3 ) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). Conclusions HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR ( Nr1i3 ) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ∼50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma

ObjectiveIntrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical...

Abstract 7102: The central dogma of hepatocellular carcinoma: Genomic, transcriptomic, and proteomic changes

Abstract Cancer is a disease condition characterized by remarkable heterogeneity, wherein numerous molecular features display considerable disparities even with the same patient cohorts, leading to varied treatment outcomes. The primary factor contributing to the substantial differences observed across cancers stems from unique combinations of genetic mutations. Epigenetic alterations further contribute to differential gene expression and splicing, thereby enhancing transcriptomic diversity. The protein products originating from the cancer transcriptome constitute the specific intracellular and extracellular environment, consequently exerting supplementary effects on epigenetic, transcriptomic, and proteomic changes. The complex interplay and mutual influence between genetic aberrations, transcriptomes, and proteomes significantly contribute to shaping the extensive spectrum of molecular characteristics observed in cancer. With label-free mass spectrometry, RNA-sequencing, and low-pass whole genome sequencing, the quantification of the proteome, transcriptome, and genetic abbreviation of the cancer can be evaluated. With the mouse hydrodynamic tail vein injection (HDVI) models of hepatocellular carcinoma (HCC), different subtypes of HCC derived from different genetic abbreviations can be generated. With the proteome, transcriptome, and genetic abbreviation data of the better-defined HCC from HDVI models and HCC patients, the patient-derived HCC can be classified according to the origin of the genetic abbreviation used in the generation of the HDVI-derived HCC. Furthermore, the proteome of patient-derived plasma will be analyzed, and the signature change unique to HCC classified by different origins of genetic abbreviation will provide an abstract of the proteome, transcriptome, and genetic abbreviation of the HCC tissue. The transcriptomics and proteomics data of HDVI mice produced from five distinct sets of genetic abbreviations have been measured. The proteome and transcriptome of tissue, as well as the proteome of plasma, from a small cohort of HCC patients were studied and classified using this resource to illuminate the fundamental landscape of HCC. The final results will be presented and discussed at the meeting. Citation Format: Hoi Tang MA, Chun Yin YU, Lau Yan NG. The central dogma of hepatocellular carcinoma: Genomic, transcriptomic, and proteomic changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7102.

Abstract 772: Investigation of gender disparity in liver tumor formation using a hydrodynamic tail vein injection mouse model

Abstract The incidence of hepatocellular carcinoma (HCC), the major form of liver cancer, is 4 times higher in men than in women. This gender disparity is difficult to study since it is believed to be multifactorial, due in part to differences in risk factors, behaviors, and hormones. The liver is a sexually dimorphic organ that is extremely susceptible to interactions with estrogens and androgens. Estrogen has been proven to have a protective effect against HCC with a demonstrable gender-related hormonal effect on the growth of HCC, though the underlying mechanisms are not fully understood. The hydrodynamic tail vein injection (HTVI) mouse model allows for the induction of liver tumors in the host. Eliminating the social and behavioral aspects of the gender disparity will allow us to determine what role estrogen plays in tumor development. In our study, a gender disparity in tumor development was observed between 6-week-old male and female FVB/NJ mice injected via HTVI of plasmids expressing c-MET, a mutated beta-catenin (b-Cat) and Sleeping Beauty transposon. HTVI of a Luciferase and Sleeping Beauty transposon expressing plasmid showed no significant difference in photon flux by bioluminescence imaging between male and female mice 24 hours after injection. When imaging was repeated one week after injection, the photon flux reduced by an order of magnitude in both male and female mice with the female mice showing more uniform reduction than the male mice. Similar luciferase gene copy number was detected in the liver tissue of male and female mice. These data suggest that there appears to be no gender difference in the transient expression and stable integration of the transfected plasmid. Plasma alpha-fetoprotein (AFP) was used to monitor tumor formation in the mice that were injected with c-MET and b-Cat without luciferase as luciferase is immunogenic. The male mice had significantly higher AFP levels at 3.5 weeks, 8 weeks, and 12 weeks post-injection than the female mice. At termination, the liver weight confirmed that female mice had much lower tumor burden than the males. We are currently using quantitative PCR to confirm similar copy number of exogenous c-MET and b-Cat inserted in the tumor cell genome and using RT-qPCR and Western blotting to determine whether the transgene expression is different in tumors from male and female mice. To determine whether estrogen played a role in preventing tumor development and progression in female mice, six-week-old male and ovariectomized female mice have been injected with the same oncogene plasmid combination plus the sleeping beauty. Interestingly, AFP levels between male and ovariectomized female mice were comparable when measured at 3 weeks. We will present our findings at the meeting. Citation Format: Araceli Bernal, Morgana McLaughlin, Ankur Tiwari, Francisco Cigarroa, LuZhe Sun. Investigation of gender disparity in liver tumor formation using a hydrodynamic tail vein injection mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 772.

Abstract 5335: Immune cell population variations in the livers of commonly used mouse strains

Abstract Previous studies have shown that the distribution of major immune cell populations differ across mouse strains in peripheral blood and hematopoietic organs at baseline. Additionally, variations in immune responses exist between mouse strains in Leishmania spp. and Melanoma models, but these studies in the context of liver diseases including Non Alcoholic Fatty Liver Disease (NAFLD) and Hepatocellular Carcinoma (HCC) have not been carried out. With the effort to state if these variations exist within the liver, we performed comprehensive spectral flow cytometry analysis on the liver and spleens of three female mouse strains commonly used in cancer research (BALB/c, C57BL/6, and FVBN). NAFLD was induced by providing mice with a methionine choline deficient diet. To promote HCC, a single genotype (myc/p53+/-) was induced by administering transposon-based vectors to overexpress Myc-Luc and CRISPR/Cas-9 vectors to knock down p53 via hydrodynamic tail-vein injection. To confirm vector integration and monitor tumor growth (luciferase activity), in vivo imaging was performed at different time points. As a result, we confirm throughout this study that differences exist within immune cell populations of the liver and spleen at baseline. Moreover, we depict differences in the shift of immune cell populations across mouse strains in the pathologic states included. In NAFLD, there is an increase of dendritic cells (DCs), macrophages, NK cells, ILC1s, T regulatory cells (Tregs), TH1, and TH2, and a decrease of CD4+ T cells, B cells, γδ-T cells, and NKT cells in the liver across all mouse strains, when compared to controls. However, the population of neutrophils in BALB/c decreases, and the population of CD8+ T cells increases in C57BL/6 mice, which differs from the patterns of change seen in the other mouse strains. Moreover, in spleens, NAFLD promotes an increase of TH1 and NK cells, and a decrease of TH2 and NKT Cells across the mouse strains included in the study, whereas the patterns of change differ between mouse strains among the remaining immune cell types. When evaluating liver immune cell population shifts on our HCC model, BALB/c mice showed a decrease in macrophages and ILC1s, C57BL/6 mice showed a decrease in γδ-T cells, prominent shifts across Neutrophils, TH1, and NKT cells, and null changes in TH2 and CD4+ T cell shifts, and FVBN mice had a less prominent decrease of B cells, which differed from the shifts observed in the mouse strains used for comparison. Immune cell populations in the spleens of our HCC mouse model also presented with differences across the mouse strains studied. In conclusion, we highlight that key differences in immune profiles exist across commonly used mouse strains for cancer research in the presence of liver pathology including HCC, suggesting that mouse strains could be possible confounders in the study of immune oncology of HCC. Citation Format: Francisco J. Rodriguez-Matos, Patrick Huang, Rajiv Trehan, Benjamin Ruf, Chi Ma, Tim F. Greten. Immune cell population variations in the livers of commonly used mouse strains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5335.

Abstract 2694: The role of innate lymphoid cells and innate like T cells in hepatocellular carcinoma

Abstract The major focus of cancer immunotherapy has been on conventional T cells due to their unique recognition of specific peptide antigens. Despite advances in the cancer immunotherapy field, including the use of atezolizumab and bevacizumab as first-line therapy for unresectable hepatocellular carcinoma (HCC), the overall mortality rate of HCC continues to rise. Innate lymphoid cells (ILC) and innate-like T cells (ILTC) are broadly categorized within the innate lymphoid population as playing an important role in inflammation, tissue repair and immune tolerance. Their ability to react rapidly to stimulants in their local tissue environment as well as to recruit and activate a variety of other immune cells makes these cells attractive targets for anti-tumor therapy. However, previous studies looking at ILCs and ILTCs in cancer have revealed both pro- and anti-tumor functions. Furthermore, the complex interplay between these cell populations in the tumor microenvironment is not well understood, particularly over the natural course of tumor development. In this study, we aim to better elucidate the role of ILC and ILTC populations in HCC progression in terms of both their kinetics and role as potential therapeutic targets. We performed hydrodynamic tail vein injections in mice with two different plasmid combinations to alter expression of commonly mutated oncogenes and tumor suppressors in human HCC (MYC;TP53 and MYC;CTNNB1). Histologic analysis of the livers was performed to determine appropriate time points to assess ILC and ILTC populations. We then designed a comprehensive 29-plex spectral flow panel to assess broad kinetic changes in the frequency of ILC, ILTC, and conventional T cell populations. We show that in early time points (4-7 days post-injection) when neoplastic transformation of hepatocytes has already occurred, mucosal-associated invariant T (MAIT) cells, group 1 ILCs, ILC2s, and ILC3s expand in frequency for both tumor models. Cytokine analysis shows a generalized decreased trend across timepoints in effector cytokine production in MAITs and group 1 ILCs, suggesting gradual dysfunction over time. Taken together, these preliminary findings show that ILC and ILTC populations expand early during tumor progression but gradually decrease in functionality with tumor progression. Citation Format: Patrick Huang, Benjamin Ruf, Rajiv Trehan, Dana Soika, Chi Ma, Tim F. Greten, Firouzeh Korangy. The role of innate lymphoid cells and innate like T cells in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2694.

Abstract 2703: Targeting STING pathway as a therapeutic strategy against β-catenin driven immune escape and resistance to anti-PD1 therapy in hepatocellular carcinoma

Abstract β-catenin mutation occurs in around 40% of hepatocellular carcinoma(HCC); yet no precision therapies are available to treat this tumor subset. Immune checkpoint inhibitors (ICIs) are now widely used for the treatment of patients with advanced HCC. Unfortunately, preliminary evidence showed that β-catenin mutation drives resistance to anti-PD1 immunotherapy in HCC. In this study, we studied the molecular mechanisms underlying β-catenin mutation during HCC pathogenesis to develop novel therapeutics targeting β-Catenin-driven HCC. Firstly, three HCC cohorts were examined, including 70 untreated, 20 treated patients with nivolumab, and patients from a public TCGA database. By single-cell RNA sequencing analysis, we identified that anti-PD-1 treatment displayed effectiveness with the increase of CD27+CD8+ T-cell infiltration, correlating to improved prognosis with longer progression-free survival (PFS) and OS. Importantly, multiplex IHC staining showed that β-catenin activation is associated with significantly reduced CD8+CD27+ T cell infiltration in tumors (n=70), demonstrating that β-catenin promoted immune escape and resistance to anti-PD-1 treatment involved defective cytotoxic T-cell activity. Subsequently, we found that the interaction of β-Catenin with sirtuin 1 (SIRT1) mediated suppression of IRF3/IRF7 activity, resulting in inhibition of Type I interferon (IFN-I) production. However, by CRISPRi screening, we found that disruption of β-catenin-SIRT1 interaction via STING caused persistent high-level IFN-I response which increased the transcription of T-cell costimulatory molecule CD27 via IL10/STAT3/BATF signaling on infiltrated CD8+ T cells. This effect was also confirmed in the STING null mice, emphasizing that STING signaling is a therapeutic target to overcome β-catenin driven immune escape. Further, using hydrodynamic tail-vein injection murine HCC models, we found that the STING agonist inhibited β-catenin-SIRT1 activity, restored IFN signaling to rescue T-cell cytotoxic capacities, and enhanced the anti-PD-1 therapy efficacy. In conclusion, we demonstrated that STING reprogrammed β-Catenin mutated the HCC tumor microenvironment and a combination of STING and anti-PD-1 treatment represents an effective novel therapeutic strategy for HCC patients with β-Catenin mutation. Citation Format: Qingmei Zhang, Jiacheng Bi, Oscar Wai-Ho Yeung, Tao Ding, Wing-Lim Chan, Yueqin Zhu, Jiang Liu, Kevin Tak-Pan Ng, Kwan Man. Targeting STING pathway as a therapeutic strategy against β-catenin driven immune escape and resistance to anti-PD1 therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2703.

CRKL dictates anti-PD-1 resistance by mediating tumor-associated neutrophil infiltration in hepatocellular carcinoma

The effectiveness of immune checkpoint inhibitor (ICI) therapy for hepatocellular carcinoma (HCC) is limited by treatment resistance. However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in resistance to anti-PD-1 therapy in HCC. Gene expression in HCC specimens from 10 patients receiving anti-PD-1 therapy was identified by RNA-sequencing. A total of 404 HCC samples from tissue microarrays were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53fl/fl) received hydrodynamic tail vein injections of a CRKL-overexpressing vector. Mass cytometry by time of flight was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues. CRKL was identified as a candidate anti-PD-1-resistance gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 treatment efficacy by mobilizing tumor-associated neutrophils (TANs), which block the infiltration and function of CD8+ T cells. PD-L1+ TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits APC (adenomatous polyposis coli)-mediated proteasomal degradation of β-catenin by competitively decreasing Axin1 binding, and thus promotes VEGFα and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/β-catenin/VEGFα and CXCL1. Targeting CRKL using CRISPR-Cas9 gene editing (CRKL knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid model. Activation of the CRKL/β-catenin/VEGFα and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 could be useful for the treatment of HCC. Here, we found that CRKL was overexpressed in anti-PD-1-resistant hepatocellular carcinoma (HCC) and that CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that upregulation of the CRKL/β-catenin/VEGFα and CXCL1 axis contributes to anti-PD-1 tolerance by promoting infiltration of tumor-associated neutrophils. These findings support the strategy of bevacizumab-based immune checkpoint inhibitor combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.

METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation

BackgroundWhile liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis.MethodsLiver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling.ResultsMETTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s).ConclusionsOur findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

STAT3 Decoy Oligodeoxynucleotides Suppress Liver Inflammation and Fibrosis in Liver Cancer Cells and a DDC-Induced Liver Injury Mouse Model.

Liver damage caused by various factors results in fibrosis and inflammation, leading to cirrhosis and cancer. Fibrosis results in the accumulation of extracellular matrix components. The role of STAT proteins in mediating liver inflammation and fibrosis has been well documented; however, approved therapies targeting STAT3 inhibition against liver disease are lacking. This study investigated the anti-fibrotic and anti-inflammatory effects of STAT3 decoy oligodeoxynucleotides (ODN) in hepatocytes and liver fibrosis mouse models. STAT3 decoy ODN were delivered into cells using liposomes and hydrodynamic tail vein injection into 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice in which liver injury was induced. STAT3 target gene expression changes were verified using qPCR and Western blotting. Liver tissue fibrosis and bile duct proliferation were assessed in animal experiments using staining techniques, and macrophage and inflammatory cytokine distribution was verified using immunohistochemistry. STAT3 decoy ODN reduced fibrosis and inflammatory factors in liver cancer cell lines and DDC-induced liver injury mouse model. These results suggest that STAT3 decoy ODN may effectively treat liver fibrosis and must be clinically investigated.

Hydrodynamic Transfection of Hepatocytes for the Study of Hepatocellular Carcinogenesis.

Hydrodynamic tail vein injection (HTVi), also called hydrodynamic gene transfer (HGT), is attracting increasing interest for modeling hepatic carcinogenesis. This highly versatile approach reproducibly provides efficient in vivo transfection of hepatocytes with naked DNA. Here, we give an in-depth description of the injection procedure, which is key for the success of the method. HTVi requires the injection of a large volume of a solution containing plasmids into the tail vein of the mouse. The transient right heart overload created by the injection forces the blood to flow back into the hepatic veins, enlarging the endothelial fenestrae and permeabilizing a fraction of hepatocytes for a few seconds. This results in the uptake of plasmids by the permeabilized hepatocytes, giving rise to their in vivo transfection. Including the Sleeping Beauty transposon system among the injected plasmids leads to the stable transfection of a subset of hepatocytes. HTVi is a powerful technique which enables numerous applications in liver cancer biology, such as a study of oncogene cooperation, of tumor heterogeneity, and interaction with the tumor microenvironment.

Pseudouridine synthase 1 promotes hepatocellular carcinoma through mRNA pseudouridylation to enhance the translation of oncogenic mRNAs.

Pseudouridine is a prevalent RNA modification and is highly present in the serum and urine of patients with HCC. However, the role of pseudouridylation and its modifiers in HCC remains unknown. We investigated the function and underlying mechanism of pseudouridine synthase 1 (PUS1) in HCC. By analyzing the TCGA data set, PUS1 was found to be significantly upregulated in human HCC specimens and positively correlated with tumor grade and poor prognosis of HCC. Knockdown of PUS1 inhibited cell proliferation and the growth of tumors in a subcutaneous xenograft mouse model. Accordingly, increased cell proliferation and tumor growth were observed in PUS1-overexpressing cells. Furthermore, overexpression of PUS1 significantly accelerates tumor formation in a mouse HCC model established by hydrodynamic tail vein injection, while knockout of PUS1 decreases it. Additionally, PUS1 catalytic activity is required for HCC tumorigenesis. Mechanistically, we profiled the mRNA targets of PUS1 by utilizing surveying targets by apolipoprotein B mRNA-editing enzyme 1 (APOBEC1)-mediated profiling and found that PUS1 incorporated pseudouridine into mRNAs of a set of oncogenes, thereby endowing them with greater translation capacity. Our study highlights the critical role of PUS1 and pseudouridylation in HCC development, and provides new insight that PUS1 enhances the protein levels of a set of oncogenes, including insulin receptor substrate 1 (IRS1) and c-MYC, by means of pseudouridylation-mediated mRNA translation.

Inhibition of Dickkopf-1 enhances the anti-tumor efficacy of sorafenib via inhibition of the PI3K/Akt and Wnt/β-catenin pathways in hepatocellular carcinoma

BackgroundSorafenib improves the overall survival in patients with advanced hepatocellular carcinoma (HCC). Dickkopf-1 (DKK1) is commonly overexpressed in HCC. In this study, we investigated whether the inhibition of DKK1 enhances the anti-tumor efficacy of sorafenib in HCC.MethodsHCC cells were treated with sorafenib and WAY-262611, which is an inhibitor of DKK1. Transgenic mouse models were also developed using hydrodynamic tail vein injection. Mice were orally administered with sorafenib (32 mg/kg), WAY-262611 (16 mg/kg), or sorafenib + WAY-262611 for 10 days. Mechanisms of sorafenib and WAY-262611 were explored via western blotting, immunostaining, and RNA sequencing.ResultsDKK1 was significantly overexpressed in patients with HCC than in the healthy controls and patients with liver diseases except HCC (all P < 0.05). Compared with sorafenib alone, sorafenib + WAY-262611 significantly inhibited the cell viability, invasion, migration, and colony formation by promoting apoptosis and altering the cell cycles in HCC cells (all P < 0.05). Moreover, sorafenib + WAY-262611 decreased the p110α, phospho-Akt (all P < 0.05), active β-catenin (all P < 0.05) and phospho-GSK-3β (Ser9) expression levels, while increasing the phospho-GSK-3β (Tyr216) expression levels compared with those in the sorafenib alone in vitro and in vivo. In addition, sorafenib + WAY-262611 inhibited tumor progression by regulating cell proliferation and apoptosis, significantly better than sorafenib alone in mouse models.ConclusionsOur results indicate that DKK1 inhibition significantly enhances the anti-tumor efficacy of sorafenib by inhibiting the PI3K/Akt and Wnt/β-catenin pathways via regulation of GSK3β activity, suggesting a novel therapeutic strategy for HCC.7yYqh-521_R5UtWAF2iJ8yVideo

Identification of DUSP4/6 overexpression as a potential rheostat to NRAS-induced hepatocarcinogenesis

BackgroundUpregulation of the mitogen-activated protein kinase (MAPK) cascade is common in hepatocellular carcinoma (HCC). Neuroblastoma RAS viral oncogene homolog (NRAS) is mutated in a small percentage of HCC and is hitherto considered insufficient for hepatocarcinogenesis. We aimed to characterize the process of N-Ras-dependent carcinogenesis in the liver and to identify potential therapeutic vulnerabilities.MethodsNRAS V12 plasmid was delivered into the mouse liver via hydrodynamic tail vein injection (HTVI). The resulting tumours, preneoplastic lesions, and normal tissue were characterized by NanoString® gene expression analysis, Western Blot, and Immunohistochemistry (IHC). The results were further confirmed by in vitro analyses of HCC cell lines.ResultsHTVI with NRAS V12 plasmid resulted in the gradual formation of preneoplastic and neoplastic lesions in the liver three months post-injection. These lesions mostly showed characteristics of HCC, with some exceptions of spindle cell/ cholangiocellular differentiation. Progressive upregulation of the RAS/RAF/MEK/ERK signalling was detectable in the lesions by Western Blot and IHC. NanoString® gene expression analysis of preneoplastic and tumorous tissue revealed a gradual overexpression of the cancer stem cell marker CD133 and Dual Specificity Phosphatases 4 and 6 (DUSP4/6). In vitro, transfection of HCC cell lines with NRAS V12 plasmid resulted in a coherent upregulation of DUSP4 and DUSP6. Paradoxically, this upregulation in PLC/PRF/5 cells was accompanied by a downregulation of phosphorylated extracellular-signal-regulated kinase (pERK), suggesting an overshooting compensation. Silencing of DUSP4 and DUSP6 increased proliferation in HCC cell lines.ConclusionsContrary to prior assumptions, the G12V NRAS mutant form is sufficient to elicit hepatocarcinogenesis in the mouse. Furthermore, the upregulation of the MAPK cascade was paralleled by the overexpression of DUSP4, DUSP6, and CD133 in vivo and in vitro. Therefore, DUSP4 and DUSP6 might fine-tune the excessive MAPK activation, a mechanism that can potentially be harnessed therapeutically.

Forced Hepatic Expression of NRF2 or NQO1 Impedes Hepatocyte Lipid Accumulation in a Lipodystrophy Mouse Model.

Lipodystrophy is a disorder featuring loss of normal adipose tissue depots due to impaired production of normal adipocytes. It leads to a gain of fat deposition in ectopic tissues such as liver and skeletal muscle that results in steatosis, dyslipidemia, and insulin resistance. Previously, we established a Rosa NIC/NIC::AdiCre lipodystrophy model mouse. The lipodystrophic phenotype that included hepatomegaly accompanied with hepatic damage due to higher lipid accumulation was attenuated substantially by amplified systemic NRF2 signaling in mice with hypomorphic expression of Keap1; whole-body Nrf2 deletion abrogated this protection. To determine whether hepatic-specific NRF2 signaling would be sufficient for protection against hepatomegaly and fatty liver development, direct, powerful, transient expression of Nrf2 or its target gene Nqo1 was achieved by administration through hydrodynamic tail vein injection of pCAG expression vectors of dominant-active Nrf2 and Nqo1 in Rosa NIC/NIC::AdiCre mice fed a 9% fat diet. Both vectors enabled protection from hepatic damage, with the pCAG-Nqo1 vector being the more effective as seen with a ~50% decrease in hepatic triglyceride levels. Therefore, activating NRF2 signaling or direct elevation of NQO1 in the liver provides new possibilities to partially reduce steatosis that accompanies lipodystrophy.

Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate.

Synthetic biology holds great promise to improve the safety and efficacy of future gene and engineered cell therapies by providing new means of endogenous or exogenous control of the embedded therapeutic programs. Here, we focused on gluconate as a clinically licensed small-molecule inducer and engineered gluconate-sensitive molecular switches to regulate transgene expression in human cell cultures and in mice. Several switch designs were assembled based on the gluconate-responsive transcriptional repressor GntR from Escherichia coli. Initially we assembled OFF- and ON-type switches by rewiring the native gluconate-dependent binding of GntR to target DNA sequences in mammalian cells. Then, we utilized the ability of GntR to dimerize in the presence of gluconate to activate gene expression from a split transcriptional activator. By means of random mutagenesis of GntR combined with phenotypic screening, we identified variants that significantly enhanced the functionality of the genetic devices, enabling the construction of robust two-input logic gates. We also demonstrated the potential utility of the synthetic switch in two in vivo settings, one employing implantation of alginate-encapsulated engineered cells and the other involving modification of host cells by DNA delivery. Then, as proof-of-concept, the gluconate-actuated genetic switch was connected to insulin secretion, and the components encoding gluconate-induced insulin production were introduced intotype-1 diabetic miceas naked DNA via hydrodynamic tail vein injection. Normoglycemia was restored, thereby showcasing the suitability of oral gluconate to regulate in situ production of a therapeutic protein.

Dyrk2 gene transfer suppresses hepatocarcinogenesis by promoting the degradation of Myc and Hras

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and has a poor prognosis. However, the molecular mechanisms underlying hepatocarcinogenesis and progression remain unknown. Invitro gain- and loss-of-function analyses in cell lines and xenografts revealed that dual-specificity tyrosine-regulated kinase 2 (DYRK2) influences tumour growth in HCC. To investigate the role of Dyrk2 during hepatocarcinogenesis, we developed liver-specific Dyrk2 conditional knockout mice and an invivo gene delivery system with a hydrodynamic tail vein injection and the Sleeping Beauty transposon. The antitumour effects of Dyrk2 gene transfer were investigated in a murine autologous carcinogenesis model. Dyrk2 expression was reduced in tumours, and that its downregulation was induced before hepatocarcinogenesis. Dyrk2 gene transfer significantly suppressed carcinogenesis. It also suppresses Myc-induced de-differentiation and metabolic reprogramming, which favours proliferative, and malignant potential by altering gene profiles. Dyrk2 overexpression caused Myc and Hras degradation at the protein level rather than at the mRNA level, and this degradation mechanism was regulated by the proteasome. Immunohistochemical analyses revealed a negative correlation between DYRK2 expression and MYC and longer survival in patients with HCC with high-DYRK2 and low-MYC expressions. Dyrk2 protects the liver from carcinogenesis by promoting Myc and Hras degradation. Our findings would pave the way for a novel therapeutic approach using DYRK2 gene transfer. Hepatocellular carcinoma (HCC) is one of the most common cancers, with a poor prognosis. Hence, identifying molecules that can become promising targets for therapies is essential to improve mortality. No studies have clarified the association between DYRK2 and carcinogenesis, although DYRK2 is involved in tumour growth in various cancer cells. This is the first study to show that Dyrk2 expression decreases during hepatocarcinogenesis and that Dyrk2 gene transfer is an attractive approach with tumour suppressive activity against HCC by suppressing Myc-mediated de-differentiation and metabolic reprogramming that favours proliferative and malignant potential via Myc and Hras degradation.

Abstract 1250: Aberrant polyunsaturated fatty acids desaturation impairs survival and anti-tumor immunity of tumor resident memory CD8+ T cell in hepatocellular carcinoma

Abstract Background &amp; Aim: Aberrant polyunsaturated fatty acids (PUFAs) metabolism is closely associated with the immunosuppressive tumor microenvironment (TME). Tissue-resident memory T cell (Trm) plays a central role against solid tumors, including HCC, depending on the uptake of exogenous fatty acids to maintain survival and anti-tumor immunity. Here we aimed to explore the potential interaction in TME between HCC-derived PUFAs and Trm function in a series of integrated clinical, basic and translational studies. Methods: Tissue interstitial fluids (TIFs) were isolated and analyzed from fresh paired adjacent tissues and HCC for detecting the PUFAs alterations in the HCC TME. Integrated analyses were performed in multiple public databases and local cohorts. Fatty acid desaturase 1 (FADS1) knockdown (KD) HCC cell line was generated to observe the tumor biology and metabolic function. The effects of FADS1 knockout (KO) or overexpression were investigated in multiple spontaneous HCC mouse models via hydrodynamic tail vein injection (HTVI) in vivo. Mutation landscape analysis was used to explore the mechanism of FADS1 transcription. Therapeutic interventions by downregulating FADS1 were also evaluated in vivo. Results: Clinically, we found that the arachidonic acid (ARA) level was enriched in HCC TIFs. A rate-limiting enzyme controlling PUFA biosynthesis, especially ARA production from dihomo-γ-linolenic acid (DGLA), FADS1, was overexpressed in HCC patients. High expression of FADS1 predicted poor survival and was negatively correlated with CD8+CD103+Trm infiltration. Moreover, in the local clinical trial, we noticed that the expression of FADS1 was upregulated in non-response patients with anti-PD-1 mAb treatment. Functionally, we demonstrated that the ARA level was decreased in the conditioned medium (CM) from FADS1 KD cells. Applying CM prolonged the survival of CD8+CD103+Trm ex vivo. FADS1 KO inhibited tumor development by increasing intratumor infiltration and function of CD8+CD103+Trm. In contrast, overexpression of FADS1 promoted HCC progression by restraining immune surveillance in vivo. Mechanistically, we found that the expression of FADS1 was upregulated in the TP53 mutation group by analyzing the mutation landscape of HCC. Mutant P53 protein could bind to the promoter region of FADS1. Therapeutically, recombinant adeno-associated 8 (rAAV8)-shRNAs delivery and anti-PD-1 mAb administration inhibited the tumor growth of anti-PD-1 mAb treatment-resistant HCC model driven by CTNNB1N90 and MYC. Conclusions: We demonstrated that mutant P53 protein directly promoted FADS1 transcription, increasing ARA accumulation in HCC TME. FADS1 remodeling the metabolic patterns of PUFAs impaired CD8+CD103+Trm anti-tumor immunity, possibly leading to HCC progression. Citation Format: Tao Ding, Li Pang, Runying Long, Shinuan Zeng, Kevin TP Ng, Qingmei Zhang, Zhiwei Chen, Kwan Man. Aberrant polyunsaturated fatty acids desaturation impairs survival and anti-tumor immunity of tumor resident memory CD8+ T cell in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1250.

Abstract 5799: SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma

Abstract Intratumor molecular heterogeneity of hepatocellular carcinoma (HCC) is partly attributed to the presence of cancer stem cells (CSCs), which we now know represents a critical root of tumor recurrence and chemotherapy resistance. CD133 is known to represent an important functional marker of liver CSCs. We have demonstrated CD133 to enrich following chemotherapy treatment, while CD133/Prom1-depletion would enhance sensitivity of HCC tumors to chemotherapy. Yet unfortunately, CD133 is not specific to HCC, but is also expressed in normal regenerating liver. Identifying critical factors expressed specifically in liver CD133+ CSCs, but not in liver normal CD133+ stem/progenitor cells may offer important therapeutic opportunities overcoming chemoresistance in HCC. RNA-seq profiling comparing sorted CD133+ and CD133- subsets of normal regenerating liver induced by DDC diet and HCC induced by either N-nitrosodiethylamine (DEN)/carbon tetrachloride (CCl4) or hydrodynamic tail vein injection of oncogenic plasmids AKT and NRAS identified Serine Peptidase Inhibitor Kazal Type I (SPINK1) to be distinctly expressed in the HCC CD133+ subpopulation but not in normal regenerating liver CD133+ subpopulation. SPINK1 overexpression in HCC clinical samples is correlated with a poor prognosis. Expression of SPINK1 increased during early liver progenitor development, peaked during the premature hepatocyte stage, decreased during hepatocyte maturation and increased progressively from well-differentiated to poorly differentiated HCCs. Enhanced transcriptional activity of SPINK1 was mediated by promoter binding of the epithelial cell-specific transcription factor ELF3, which like CD133, were both increased following chemotherapy treatment. SPINK1 inhibition by lentiviral-based knockdown or a specific monoclonal antibody (mAb) mitigated tumor initiation, self-renewal and chemoresistance. Mechanistically, secretory SPINK1 bound to epidermal growth factor receptor (EGFR) activating MEK/ERK signaling and consequently promoting the formation of CDK4/6-cyclin D1 complex to phosphorylate Rb and release E2F2, allowing the cells to overcome G1/S checkpoint promoting cell cycle progression as well as transcribing stemness, oncogenic dedifferentiation and chemoresistance-related genes. Collectively, our findings suggest SPINK1 to play a critical role in hepatocarcinogenesis and that SPINK1 mAb may represent a novel therapeutic option for the treatment of HCC, targeting at the CD133+ CSC tumor roots and overcoming chemoresistance. Citation Format: Ki-Fong Man, Lei Zhou, Ying-Tung Lam, Jun Yu, Jing-Ping Yun, Xin-Yuan Guan, Ming Liu, Stephanie Ma. SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5799.