Mouse Hepatocellular Carcinoma Cell Research Articles

Abstract 1424: Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance in hepatocellular carcinoma

Abstract Background: Immune checkpoint blockade (ICB) therapies by antibodies have revolutionized the treatment paradigm for a variety of cancers. Although subsets of people exhibit durable responses, resistance and relapse are common in hepatocellular carcinoma (HCC). We therefore aim to delineate the mechanism underlying the failure of ICB therapy in HCC, and more importantly to identify ICB-sensitizing therapeutic approaches. Methods: ICB-resistant orthotopic-grafted murine models were established via a serial selection of HCC cells in ICB-treated mice. Integrated multi-omics analysis (single-cell RNA-seq, single-cell ATAC-seq and ChIP-seq) were applied to decode the mechanisms underlying the ICB resistance and an epigenetic-mediated ICB response. Results: We successfully established the ICB-resistant murine models of HCC, in which tumor intrinsic interferon-gamma (IFNγ) response was most significantly suppressed and characterized by a “cold” tumor microenvironment with decreased lymphocyte activation and intratumoral infiltration. On the other hand, single cell RNA-seq analysis of human HCC biopsies revealed that patients with HDAC1/2/3-high HCC exhibit poor survival upon ICB therapy. Notably, we found that a novel specific HDAC1/2/3 inhibitor CXD101 synergized with PD-(L)1 antibody to induce immunogenic cell death, eradicate tumor and prolong survival in our ICB-resistant mouse models, which was accompanied by enhanced intratumoral infiltration, activation and anti-tumor memory formation of cytotoxic lymphocytes. Mechanistically, CXD101 combined with PD-(L)1 inhibition synergistically reactivated tumor IFNγ pathway via enhancing chromatin accessibility of downstream genes associated with antigen presentation and lymphocytes recruitment. Moreover, the activated immune system induced tumor cell pyroptosis, further igniting antitumor immunity and ICB response. Conclusions: Our findings suggest that corruption of tumor intrinsic IFNγ signaling may confer ICB resistance upon ICB therapy, which can be rectified by class I HDAC inhibitor mediated IFNγ response and tumor cell pyroptosis. Based on these findings, we have commenced a Phase II clinical study of a new epigenetic immunotherapy (CXD101 plus anti-PD-1) for HCC patients resisting anti-PD-(L)1 alone. This project is supported by the CRF (C4045-18W), GRF (14115820), TRS (T11-706/18-N), Li Ka Shing Foundation, CUHK Strategic Seed Funding for Collaborative Research Scheme. We also acknowledge support (funding and study medications) by Celleron Therapeutics. Citation Format: Yalin Tu, Haoran Wu, Chengpeng Zhong, Zhewen Xiong, Jing Wang, Patrick Pak-Chun Wong, Weiqin Yang, Jiahuan Lu, Zhixian Liang, Shufen Chen, Lingyun Zhang, Siyun Chen, Jingying Zhou, Ka-Fai To, Joseph Jao-Yiu Sung, Stephen Lam Chan, David Kerr, Nick La Thangue, Alfred Sze-Lok Cheng. Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance 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 1424.

SCARB1-encoded circ _0029343 induces p73 splicing to promote growth and metastasis of hepatocellular carcinoma via miR-486-5p/SRSF3 axis.

Circular RNAs (circRNAs) exhibit essential regulation in the malignant development of hepatocellular carcinoma (HCC). This study aims to investigate the physiological mechanisms of circ_0029343 encoded by scavenger receptor class B member 1 (SCARB1) involved in the growth and metastasis of HCC. Differentially expressed mRNAs in HCC were obtained, followed by the prediction of target genes of differentially expressed miRNAs and gene ontology and kyoto encyclopedia of genes and genomes analysis on the differentially expressed mRNAs. Moreover, the regulatory relationship between circRNAs encoded by SCARB1 and differentially expressed miRNAs was predicted. In vitro cell experiments were performed to verify the effects of circ_0029343, miR-486-5p, and SRSF3 on the malignant features of HCC cells using the gain- or loss-of-function experiments. Finally, the effects of circ_0029343 on the growth and metastasis of HCC cells in xenograft mouse models were also explored. It was found that miR-486-5p might interact with seven circRNAs encoded by SCARB1, and its possible downstream target gene was SRSF3. Moreover, SRSF3 was associated with the splicing of various RNA. circ_0029343 could sponge miR-486-5p to up-regulate SRSF3 and activate PDGF-PDGFRB (platelet-derived growth factor and its receptor, receptor beta)signaling pathway by inducing p73 splicing, thus promoting the proliferation, migration, and invasion and inhibiting apoptosis of HCC cells. In vivo, animal experiments further confirmed that overexpression of circ_0029343 could promote the growth and metastasis of HCC cells in nude mice. circ_0029343 encoded by SCARB1 may induce p73 splicing and activate thePDGF-PDGFRB signaling pathway through the miR-486-5p/SRSF3 axis, thus promoting the growth and metastasis of HCC cells.

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice.

Constitutive activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) contributes to the malignancy of many cancers such as hepatocellular carcinoma (HCC) and is associated with poor prognosis. STAT3 activity is increased by the reversible palmitoylation of Cys108 by the palmitoyltransferase DHHC7 (encoded by ZDHHC7). Here, we investigated the consequences of S-palmitoylation of STAT3 in HCC. Increased ZDHHC7 abundance in HCC cases was associated with poor prognosis, as revealed by bioinformatics analysis of patient data. In HepG2 cells in vitro, DHHC7-mediated palmitoylation enhanced the expression of STAT3 target genes, including HIF1A, which encodes the hypoxia-inducible transcription factor HIF1α. Inhibiting DHHC7 decreased the S-palmitoylation of STAT3 and decreased HIF1α abundance. Furthermore, stabilization of HIF1α by cyclin-dependent kinase 5 (CDK5) enabled it to promote the expression of ZDHHC7, which generated a positive feedback loop between DHHC7, STAT3, and HIF1α. Perturbing this loop reduced the growth of HCC cells in vivo. Moreover, DHHC7, STAT3, and HIF1α were all abundant in human HCC tissues. Our study identifies a pathway connecting these proteins that is initiated by S-palmitoylation, which may be broadly applicable to understanding the role of this modification in cancer.

ZMIZ1 Upregulation of TET3-Mediated Hydroxymethylation Induces M2 Polarization of Kupffer Cells in Hepatocellular Carcinogenesis by Mediating Notch1/c-Myc Signaling

Hydroxymethylation of DNA, mediated by the ten-eleven translocation (TET) family of methylcytosine dioxygenases, represents a crucial epigenetic modification that manipulates gene expression in numerous biological processes. This study focuses on the effect of TET3 on the polarization of Kupffer cells (KCs) and its connection to the development of hepatocellular carcinoma (HCC). TET3 was found to be abundant in KCs, and its knockdown induced an M2–M1 phenotype shift, resulting in the suppression of viability, migration, and invasion of cocultured HCC cells. Additionally, the TET3 knockdown inhibited the tumorigenesis of HCC cells in nude mice. Downstream targets of TET3 were predicted using bioinformatics. TET3-mediated DNA hydroxymethylation of zinc finger MIZ-type containing 1 (ZMIZ1) promoter. The ZMIZ1 protein interacted with notch receptor 1 (Notch1) protein to activate the transcription of c-Myc. Silencing of ZMIZ1 in KCs similarly suppressed M2 polarization of KCs and malignant phenotype of cocultured HCC cells. However, these changes were counteracted by the overexpression of either Notch1 or c-Myc overexpression in KCs. In summary, this study demonstrates that TET3-mediated hydroxymethylation of ZMIZ1 enhances hepatocellular carcinogenesis by promoting M2 skewing of KCs through the Notch1/c-Myc axis.

Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis.

Lentinan (LNT) isolated from Lentinus edodes is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1‑6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using invitro and invivo approaches. Forinvitroapproaches, the effect of LNT on the proliferation of Hepa1‑6 cells was investigated by Cell Counting Kit‑8 assay. Annexin V‑FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p‑Akt), protein kinase B (Akt), B lymphocyte‑2 (Bcl‑2), Bcl2 family‑associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1‑6 cells. The association betweenEGR1andPTENwas explored by EGR1 overexpressionin cell lines. Forinvivomethods, a mouse model of diethylnitrosamine (DEN)‑induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin‑eosin staining and immunohistochemical assay. Invitro and invivo results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1‑6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti‑tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.

Salvigenin Suppresses Hepatocellular Carcinoma Glycolysis and Chemoresistance Through Inactivating the PI3K/AKT/GSK-3β Pathway

Salvigenin is a Trimethoxylated Flavone enriched in Scutellariae Barbatae Herba and Scutellariae Radix and is demonstrated to have anti-tumor properties in colon cancer. Notwithstanding, the function and mechanism of Salvigenin in hepatocellular carcinoma (HCC) are less well studied. Different doses of Salvigenin were taken to treat HCC cells. Cell viability, colony formation ability, cell migration, invasion, apoptosis, glucose uptake, and lactate production levels were detected. As shown by the data, Salvigenin concentration dependently dampened HCC cell proliferation, migration, and invasion, weakened glycolysis by abating glucose uptake and lactate generation, and suppressed the profiles of glycolytic enzymes. Moreover, Salvigenin strengthened HCC cells’ sensitivity to 5-fluorouracil (5-FU) and attenuated HCC 5-FU-resistant cells’ resistance to 5-FU. Through network pharmacological analysis, we found Salvigenin potentially regulates PI3K/AKT pathway. As shown by the data, Salvigenin repressed the phosphorylated levels of PI3K, AKT, and GSK-3β. The PI3K activator 740Y-P induced PI3K/AKT/GSK-3β pathway activation and promotive effects in HCC cells. However, Salvigenin substantially weakened 740Y-P-mediated effects. In-vivo assay revealed that Salvigenin hampered the growth and promoted apoptosis of HCC cells in nude mice. Collectively, Salvigenin impedes the aerobic glycolysis and 5-FU chemoresistance of HCC cells by dampening the PI3K/AKT/GSK-3β pathway.

Anticancer effect of Triphala extract on the hepatocellular carcinoma cells in mice

Context: Triphala is a traditional herbal formulation consisting of dried fruits from three plants namely, Terminalia bellirica, Terminalia chebula and Phyllanthus emblica. Its traditional used to treat many ailments, including various types of cancers, health promotion, and longevity. Aims: To evaluate the ability of an aqueous extract Triphala (TPL) to inhibit the growth of Hepatocellular carcinoma cells (HepG2) and in mice. Methods: The anticancer activity of TPL and its composite extracts was assessed in vitro by MTT assay with HepG2 cells. The mice were inoculated with HepG2 cells and then divided randomly into six groups (6 mice/group): control group, positive control group (intraperitoneal with 3 mg/kg body weight doxorubicin), and TPL treatment groups (oral administrated with 50, 100, and 200 mg/kg body weight TPL, respectively). Anticancer activity based on body weight, tumor growth volumes, survival time, relative organ weight, and hematological parameters was determined after administrating the TPL for 14 consecutive days. Results: The results demonstrated that the TPL extract exhibited antiproliferative activity against HepG2 cells in a time-dependent manner of treatment. The selectivity index (SI) showed that TPL is highly selective (SI>3) against the HepG2 cell line and has no activity against non-tumor cells. Oral administration with TPL extract at the dose of 200 mg/kg body weight inhibited tumor growth volume and increase the survival time. No abnormality of hematological parameters, relative organ weight, body weight, and morphology of organs were observed. Conclusions: The TPL extract at the test doses was a non-toxic drug. Our finding reveals the anticancer efficacy of TPL extract.

Facile synthesis of efficient red-emissive carbon quantum dots as a multifunctional platform for biosensing and bioimaging

The red-emissive carbon quantum dots (r-CQDs) have gained considerable interest in the field of fluorescence imaging as they can effectively overcome short-wave background interference. However, the development of efficient and simple synthetic pathways for the preparation of r-CQDs with high production yield and fluorescence quantum yield remains a great challenge. Herein, we prepared the intense red-emissive carbon quantum dots (r-CQDs) through one-pot hydrothermal treatment of methylene violet. The morphology and structure of the as-prepared r-CQDs were characterized through transmission electron microscopy, Raman spectra, Fourier transform infrared spectra, and X-ray photoelectron spectroscopy. r-CQDs were well dispersed in water with an average size of 2.39 nm. The surface of r-CQDs was full of oxygen-containing functional groups, which contributed to their high solubility in water. Under ultraviolet light (365 nm) irradiation, both the aqueous solution and powder of r-CQDs emitted intense red luminescence. r-CQDs exhibited the excitation-independent emission property with the maximum fluorescence peak at 596 nm. The fluorescence quantum yield of r-CQDs was calculated to be 26.46% in water using rhodamine B as a reference. Due to the significantly different fluorescence intensity in water and organic solvents, r-CQDs could be applied as the fluorescence sensor to quickly detect the trace amounts of water in various organic solvents. Moreover, the fluorescence of r-CQDs showed selective response to hypochlorite (ClO−) through an “on-off” sensing mechanism. The good biocompatibility of r-CQDs was verified by the cell counting kit-8 (CCK-8) method against mouse hepatocellular carcinoma (Hepa 1–6) cells. Finally, the r-CQDs were successfully used in cellular imaging and they were mainly located in the lysosomes of cancel cells. Thus, r-CQDs could be served as a powerful fluorescence nano-platform for biosensing and bioimaging.

Abstract 5985: Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression

Abstract Background: PD-1 blockade has been approved as treatment for advanced HCC. We previously demonstrated the efficacy of CDK9 inhibition in reducing the expression of inducible proteins and as a treatment for HCC (Shao et al. Oncology 2022). We thus examined the influence of CDK9 inhibition on the expression of PD-L1 in HCC and the potential of improving the efficacy of PD-1 blockade with the combination of CDK9 inhibitors. Methods: We first examined the influence of specific CDK9 inhibitors, AZD4573 and atuveciclib, on interferon-γ (IFN-γ) induced PD-L1 expression of human HCC cell lines HuH7 and Hep3B. Overexpression of CDK9 and siRNA to CDK9 were used to confirm the findings. Similar experiments were repeated in the mouse HCC cell line BNL. To test the in vivo efficacy, we orthotopically implanted BNL cells in the subcapsular area of BALB/c mice, which were then treated with vehicle, CDK9 inhibitors, anti-PD-L1 antibodies, or their combination. To compare with tumor sizes, all mice were sacrificed after 14 days of treatment. To compare survival, the mice were only sacrificed if they met the criteria of animal euthanasia. Results: Both HuH7 and Hep3B cells had minimal PD-L1 expression, which however could be induced using IFN-γ. AZD4573 and atuveciclib decreased the IFN-γ induced PD-L1 expression of HuH7 and Hep3B cells in a dose dependent manner. The flow cytometry results confirmed that membrane PD-L1 expression was also decreased by CDK9 inhibition. Overexpression of CDK9 decreased the influence of these CDK9 inhibitors on PD-L1 expression. CDK9 knockdown using siRNA reduced the IFN-γ induced PD-L1 expression. AZD4573 and atuveciclib also reduce the PD-L1 expression in BNL cell lines. In the orthotopic animal model, the tumors of the sacrificed mice were significantly smaller in mice treated with atuveciclib and an anti-PD-L1 antibody than mice treatment with either alone. The survival of mice treated with the combination was also longer than mice treated with each therapy alone. Conclusion: We demonstrated that CDK9 inhibition could reduce the IFN-γ induced PD-L1 expression of HCC cells. Combination of CDK9 inhibitors and anti-PD-L1 antibodies was more effective than either therapy alone. Citation Format: Ching-Tso Chen, Yu-Yun Shao, Hung-Wei Hsu, Rita R. Wo, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression. [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 5985.

NONHSAT021545/miR-330-3p/EREG: A Cooperative Axis in Breast Cancer Prognosis and Treatment

Lymphatic metastasis is the most common form in breast cancer (BC) progression. Previously, we observed that lnc045874, a most conservative homology of Homo Sapiens NONHSAT021545 (lnc021545), miR-330-3p, and EREG may have some effects in mouse hepatocarcinoma cell lines with different lymphatic metastasis potentials. Through data from TCGA and GEO database analysis, we speculated that miR-330-3p might be a tumor promoter, while EREG could be a tumor suppressor in BC. MiR-330-3p was upregulated, while lnc021545 and EREG were downregulated in 50 BC tissues. MiR-330-3p advanced the metastatic behaviors of BC cells, whereas lnc021545 and EREG resulted in the opposite effects. The three molecules' expressions were correlated respectively and showed that miR-330-3p targeted lnc021545 and EREG to affect their expressions. Lnc021545/miR-330-3p axis affected BC metastasis by regulating EREG in epithelial-to-mesenchymal transition. In 50 BC patients, these three molecules and their cooperation are associated with aggressive tumor phenotypes, patient outcomes, and trastuzumab therapy. We finally discovered that lnc021545, miR-330-3p, and EREG formed a multi-gene co-regulation system that affected the metastasis of BC and the cooperation reflects the synergistic effects of the three molecules, recommending that their cooperation may provide a more accurate index for anti-metastasis therapeutic and prognostic evaluation of BC.

SUMOylated IL-33 in the nucleus stabilizes the transcription factor IRF1 in hepatocellular carcinoma cells to promote immune escape.

Interleukin-33 (IL-33) functions both as a secreted cytokine and as a nuclear factor, with pleiotropic roles in cancer and immunity. Here, we explored its role in hepatocellular carcinoma (HCC) and identified that a posttranslational modification altered its nuclear activity and promoted immune escape for HCC. IL-33 abundance was overall decreased but more frequently localized to the nucleus in patient HCC tissues than in normal liver tissues. In human and mouse HCC cells in culture and in vivo, IL-33 overexpression inhibited proliferation and repressed the abundance of programmed death ligand 1 (PD-L1) at the transcriptional level by promoting the ubiquitin-dependent degradation of interferon regulatory factor 1 (IRF1). However, this interaction was disrupted by SUMOylation of IL-33 at Lys54 mediated by the E3 ligase RanBP2. IL-33 SUMOylation correlated with its nuclear localization in HCC cells and tumors. An increase in SUMOylated IL-33 in HCC cells in cocultures and in vivo stabilized IRF1 and increased PD-L1 abundance and chemokine IL-8 secretion, which prevented the activation of cytotoxic T cells and promoted the M2 polarization of macrophages, respectively. Mutating the SUMOylation site in IL-33 reversed these effects and suppressed tumor growth. These findings indicate that SUMOylation of nuclear IL-33 in HCC cells impairs antitumor immunity.

Hyperglycemia Alters O-GlcNAcylation Patterns of Hepatocytes in Mice Treated With Hepatoxic Carcinogen.

Diabetes mellitus (DM) is an established risk for hepatocellular carcinoma (HCC), with unclarified mechanisms. This study investigated the effects of hyperglycemia on O-GlcNacylation in hepatocytes and its associations with hepatocarcinogenesis. Mouse and human HCC cell lines were used in an in vitro model of hyperglycemia. Western blotting was used to determine the effects of high glucose on O-GlcNacylation in HCC cells. Twenty 4-week-old C3H/HeNJcl mice were randomized into four groups: non-DM control, non-DM plus diethylnitrosamine (DEN), DM, and DM plus DEN. DM was induced using intraperitoneal injection of a single high dose of streptozotocin. DEN was used to induce HCC. All mice were euthanized at week 16 after DM induction, and the liver tissues were histologically examined using hematoxylin and eosin, and immunohistochemistry. High glucose increased O-GlcNacylated proteins in mouse and human HCC cell lines compared with those cultured at normal glucose concentration. Mice with hyperglycemia or DEN treatment had increased O-GlcNacylated proteins in hepatocytes. No gross tumors were evident at the end of the experiment but hepatic morbidity was observed. Mice with hyperglycemia and DEN treatment showed greater histological morbidity in their livers, i.e. increased nuclear size, hepatocellular swelling and sinusoidal dilatation, compared with mice in the DM group or treated with DEN alone. Hyperglycemia increased O-GlcNAcylation in both in vitro and animal models. Increased O-GlcNAcylated proteins may be associated with hepatic histological morbidities which then promote HCC development in carcinogen-induced tumorigenesis.

Immunogenic Death of Hepatocellular Carcinoma Cells in Mice Expressing Caspase-Resistant ROCK1 Is Not Replicated by ROCK Inhibitors

The morphological changes during apoptosis help facilitate "immunologically silent" cell death. Caspase cleavage of the ROCK1 kinase results in its activation, which drives the forceful contraction of apoptotic cells. We previously showed that when ROCK1 was mutated to render it caspase-resistant, there was greater liver damage and neutrophil recruitment after treatment with the hepatotoxin diethylnitrosamine (DEN). We now show that acute DEN-induced liver damage induced higher levels of pro-inflammatory cytokines/chemokines, indicative of immunogenic cell death (ICD), in mice expressing non-cleavable ROCK1 (ROCK1nc). Hepatocellular carcinoma (HCC) tumours in ROCK1nc mice had more neutrophils and CD8+ T cells relative to mice expressing wild-type ROCK1, indicating that spontaneous tumour cell death also was more immunogenic. Since ICD induction has been proposed to be tumour-suppressive, the effects of two distinct ROCK inhibitors on HCC tumours was examined. Both fasudil and AT13148 significantly decreased tumour numbers, areas and volumes, but neither resulted in greater numbers of neutrophils or CD8+ T cells to be recruited. In the context of acute DEN-induced liver damage, AT13148 inhibited the recruitment of dendritic, natural killer and CD8+ T cells to livers. These observations indicate that there is an important role for ROCK1 cleavage to limit immunogenic cell death, which was not replicated by systemic ROCK inhibitor administration. As a result, concomitant administration of ROCK inhibitors with cancer therapeutics would be unlikely to result in therapeutic benefit by inducing ICD to increase anti-tumour immune responses.

Differential requirement of Hippo cascade during CTNNB1 or AXIN1 mutation-driven hepatocarcinogenesis.

Gain-of-function (GOF) mutations of CTNNB1 and loss-of-function (LOF) mutations of AXIN1 are recurrent genetic alterations in hepatocellular carcinoma (HCC). We aim to investigate the functional contribution of Hippo/YAP/TAZ in GOF CTNNB1 or LOF AXIN1 mutant HCCs. The requirement of YAP/TAZ in c-Met/β-Catenin and c-Met/sgAxin1-driven HCC was analyzed using conditional Yap , Taz , and Yap;Taz knockout (KO) mice. Mechanisms of AXIN1 in regulating YAP/TAZ were investigated using AXIN1 mutated HCC cells. Hepatocyte-specific inducible TTR-CreER T2KO system was applied to evaluate the role of Yap;Taz during tumor progression. Cabozantinib and G007-LK combinational treatment were tested in vitro and in vivo . Nuclear YAP/TAZ was strongly induced in c-Met/sgAxin1 mouse HCC cells. Activation of Hippo via overexpression of Lats2 or concomitant deletion of Yap and Taz significantly inhibited c-Met/sgAxin1 driven HCC development, whereas the same approaches had mild effects in c-Met/β-Catenin HCCs. YAP is the major Hippo effector in c-Met/β-Catenin HCCs, and both YAP and TAZ are required for c-Met/sgAxin1-dependent hepatocarcinogenesis. Mechanistically, AXIN1 binds to YAP/TAZ in human HCC cells and regulates YAP/TAZ stability. Genetic deletion of YAP/TAZ suppresses already formed c-Met/sgAxin1 liver tumors, supporting the requirement of YAP/TAZ during tumor progression. Importantly, tankyrase inhibitor G007-LK, which targets Hippo and Wnt pathways, synergizes with cabozantinib, a c-MET inhibitor, leading to tumor regression in the c-Met/sgAxin1 HCC model. Our studies demonstrate that YAP/TAZ are major signaling molecules downstream of LOF AXIN1 mutant HCCs, and targeting YAP/TAZ is an effective treatment against AXIN1 mutant human HCCs.

PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis

Metastasis indicates a grave prognosis in patients with hepatocellular carcinoma (HCC). Our previous studies showed that RNA binding motif protein Y-linked (RBMY) is potentially a biomarker for poor survival in HCC patients, but its role in metastasis is largely unclear. A total of 308 male patients with primary HCC were enrolled. RBMY expression was traced longitudinally by immunostaining from the manifestation of a primary HCC tumor to the formation of a distant metastasis, and its upstream regulators were screened with a protein microarray. Aseries of metastasis assays in mouse models and HCC cell lines were performed to explore new functional insights into RBMY. Cytoplasmic expression of RBMY was associated with rapid distant metastasis (approximately 1 year after resection) and had a predictive power of 82.4% for HCC metastasis. RBMY conferred high migratory and invasive potential upon phosphorylation by the provirus integration in Moloney 1 (PIM1) kinase. Binding of PIM1 to RBMY caused mutual stabilization and massive translocation of RBMY from nuclei to mitochondria, thereby preventing mitochondrial apoptosis and augmenting mitochondrial generation of adenosine triphosphate/reactive oxygen species to enhance cell motility. Depletion of RBMY suppressed Snail1/zinc finger E-box binding homeobox transcription factor 1-mediated epithelial-mesenchymal transition and dynamin-related protein 1-dependent mitochondrial fission. Inactivation and knockout of PIM1 down-regulated the expression of RBMY. In nude mice, cytoplasmic RBMY promoted liver-to-lung metastasis by increasing epithelial-mesenchymal transition, mitochondrial proliferation, and mitochondrial fission, whereas nuclear-restricted RBMY impeded the mitochondrial switch and failed to induce lung metastasis. This study showed the regulation of HCC metastasis by PIM1-driven cytoplasmic expression of RBMY and suggested a novel therapeutic target for attenuating metastasis.

Cyclin-Dependent Kinase 9 Inhibition as a Potential Treatment for Hepatocellular Carcinoma

Purpose: Composite cyclin-dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. Methods: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. Results: CDK9 inhibitors exhibited potent antiproliferative activities in HCC cells regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1 and survivin. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited significantly slower tumor growth than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving a CDK9 inhibitor also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Knockdown of CDK9 expression reduced the proliferative activities of HCC cells. Conclusion: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

Cyclin dependent kinase 9 inhibition as a potential treatment for hepatocellular carcinoma.

425 Background: Composite cyclin dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. Methods: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, on cell viability and apoptosis in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. Results: Both BAY1143572 and AZD4573 exhibited potent antiproliferative activities in HCC cell lines including HuH7, HLE, and HepG2, regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1, survivin, BimEL, and the X-linked inhibitor of apoptosis protein. Reduction of CDK9 expression through siRNA knockdown also reduced the phosphorylation of RNA polymerase II and the colony formations of HuH7 and HLE cells. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited a significantly lower tumor growth rate than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving BAY1143572 also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Conclusions: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

MiR-660-5p-loaded M2 macrophages-derived exosomes augment hepatocellular carcinoma development through regulating KLF3

ObjectiveM2 macrophages (M2) can affect tumor development by secreting various cytokines, including exosomes (Exo). Herein, we intended to explore how microRNA (miR)-660-5p-modified M2-Exo affected hepatocellular carcinoma (HCC) development through regulating Kruppel-like factor 3 (KLF3). MethodsmiR-660-5p and KLF3 levels were first measured in clinical HCC tissues. A miR-targeted relation was explored between miR-660-5p and KLF3. M2-Exo were modified by miR-660-5p-related oligonucleotides and co-cultured with HepG2 cells to determine their effects on cell proliferation, colony formation, invasion, migration, apoptosis and epithelial-mesenchymal transition (EMT). Xenografted tumors were collected from mice to further verify the in vitro results. ResultsHigher miR-660-5p and lower KLF3 levels were examined in HCC. KLF3 was targeted by miR-660-5p. Up-regulated miR-660-5p-modified M2-Exo boosted the grwoth and EMT of HepG2 cells, but this effect was impaired by overexpression of KLF3. miR-660-5p-loaded M2-Exo enhanced tumorigenic ability of HCC cells in mice. On the contrary, down-regulated miR-660-5p reduced M2-Exo-mediated promotion of growth of HCC cells in vitro and in vivo. ConclusionOur study summarizes that miR-660-5p-loaded M2-Exo augment HCC development through down-regulating KLF3.

Liver-specific over-expression of Cripto-1 in transgenic mice promotes hepatocyte proliferation and deregulated expression of hepatocarcinogenesis-related genes and signaling pathways.

In this study, we investigated the role of embryonic gene Cripto-1 (CR-1) in hepatocellular carcinoma (HCC) using hepatocyte-specific CR-1-overexpressing transgenic mice. The expression of truncated 1.7-kb CR-1 transcript (SF-CR-1) was significantly higher than the full-length 2.0-kb CR-1 transcript (FL-CR-1) in a majority of HCC tissues and cell lines. Moreover, CR-1 mRNA and protein levels were significantly higher in HCC tissues than adjacent normal liver tissues. Hepatocyte-specific over-expression of CR-1 in transgenic mice enhanced hepatocyte proliferation after 2/3 partial hepatectomy (2/3 PHx). CR-1 over-expression significantly increased in vivo xenograft tumor growth of HCC cells in nude mice and in vitro HCC cell proliferation, migration, and invasion. CR-1 over-expression in the transgenic mouse livers deregulated HCC-related signaling pathways such as AKT, Wnt/β-catenin, Stat3, MAPK/ERK, JNK, TGF-β and Notch, as well as expression of HCC-related genes such as CD5L, S100A8, S100A9, Timd4, Orm2, Orm3, PDK4, DMBT1, G0S2, Plk2, Plk3, Gsta1 and Gsta2. However, histological signs of precancerous lesions, hepatocyte dysplasia or HCC formation were not observed in the livers of 3-, 6- or 8-month-old hepatocyte-specific CR-1-overexpressing transgenic mice. These findings demonstrate that liver-specific CR-1 overexpression in transgenic mice deregulates signaling pathways and genes associated with HCC.

Caveolin-1 knockout mice have altered serum N-glycan profile and sialyltransferase tissue expression.

Caveolin-1 (Cav-1) is a constitutive protein within caveolar membranes. Previous studies from our group and others indicated that Cav-1 could mediate N-glycosylation, α2,6-sialylation, and fucosylation in mouse hepatocarcinoma cells in vitro. However, little is known about the effect of Cav-1 expression on glycosylation modifications in vivo. In this study, the N-glycan profiles in serum from Cav-1-/- mice were investigated by lectin microarray and mass spectrometric analysis approaches. The results showed that levels of multi-antennary branched, α2,6-sialylated, and galactosylated N-glycans increased, while high-mannose typed and fucosylated N-glycans decreased in the serum of Cav-1-/- mice, compared with that of wild-type mice. Furthermore, the real-time quantitative PCR analysis indicated that α2,6-sialyltransferase gene expression decreased significantly in Cav-1-/- mouse organ tissues, but α2,3- and α2,8-sialyltransferase did not. Of them, both mRNA and protein expression levels of the β-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) had dramatically reduced in Cav-1-/- mice organ tissues, which was consistent with the α2,6-sialyl Gal/GalNAc level reduced significantly in tissues instead of serum from Cav-1-/- mice. These results provide for the first time the N-glycans profile of Cav-1-/- mice serum, which will facilitate understanding the function of Cav-1 from the perspective of glycosylation.