Mouse Hepatoma Research Articles

Diarylheptanoids/sorafenib as a potential anticancer combination against hepatocellular carcinoma: the p53/MMP9 axis of action

Hepatocellular carcinoma (HCC) is a serious and potentially fatal form of cancer associated with liver damage. New anticancer drugs are increasingly needed due to the increasing number of cancer cases every year. In this study, diarylheptanoids (DAH) from Alpinia officinarum were examined for their antitumor activity against DAB-induced HCC in mice, as well as their ability to reduce liver damage. Assays for cytotoxicity were conducted using MTT. The DAB-induced HCC Swiss albino male mice were given DAH and sorafenib (SOR) either as single treatments or in combination, and the effects on tumour development and progression were monitored. Malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were evaluated along with biomarkers of liver enzymes (AST, ALT, and GGT). The apoptosis-related gene (CASP8), the apoptosis-related gene (p53), the anti-inflammatory genes (IL-6), the migration-related gene matrix metalloprotease-9 (MMP9), and the angiogenesis-related gene vascular endothelial growth factor (VEGF) were assessed using qRT-PCR in the hepatic tissue. As a final step, DAH and SOR were docked with CASP8 and MMP9 via molecular docking to propose potential mechanisms of action. Our results revealed that the combination of DAH and SOR has a potent inhibitory effect on the growth and viability of the HepG2 cell line. The outcomes demonstrated that DAH and SOR-treated HCC-bearing mice displayed a reduction in the tumour burden and liver damage as demonstrated by (1) parameters of repaired liver function; (2) low levels of hepatic MDA; (3) elevated levels of hepatic T-SOD; (4) p53, IL-6, CASP8, MMP9, and VEGF downregulation; and (5) enhanced hepatic structure. The best results were revealed in mice that were co-treated with DAH (given orally) and SOR (given intraperitoneally). The docking study also proposed that both DAH and SOR could inhibit CASP8 and MMP9’s oncogenic activities and had a high affinity for these enzymes. In conclusion, according to study findings, DAH enhances SOR antiproliferative and cytotoxic effects and identifies their molecular targets. Furthermore, the results revealed that DAH was able to boost the anticancer effects of the drug SOR and reduce liver damage caused by HCC in mice. This suggests that DAH could be a potential therapeutic agent against liver cancer.

Dysregulated sphingolipid metabolism contributes to NASH-HCC disease progression

Background: Nonalcoholic fatty liver disease (NAFLD) is the fastest-rising cause of end-stage liver disease for liver transplantation. The underlying mechanisms of NAFLD disease progression from nonalcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) remain unclear. It has been well-accepted that inflammation and dysregulation of sphingolipid metabolism are two important contributors to NASH progression. Recent advances in gene profiling technologies have significantly improved the current understanding of NASH-HCC pathogenesis. However, the key pathway changes underlying liver fibrosis and dysregulation of sphingolipid metabolism haven’t been fully elucidated and are the focus of this study. Methods: DIMOND NASH mouse model (C57BL/6J and 129S1/SvlmJ mixed background) was used in this study. Male mice (21 to 24-week-old) were fed with WDSW (Western diet, Harlan, TD88137, and a high fructose-glucose solution, 23.1g/L D-fructose plus 18.9g/L D-glucose) or standard chow diet ad libitum for six months or one year. At the end of the study, liver tissues were harvested for total RNA isolation using Trizol. Gene profiles were analyzed using the NanoString nCounter® Gene Expression Fibrosis V2 panel with sphingolipid metabolism pathways. The differentially expressed genes (DEGs) between groups were determined using Rosalind. QIAGEN Ingenuity Pathway Analysis (IPA) was used to analyze the DEGs. Human HCC patient data were downloaded from The Cancer Genome Atlas Program (TCGA). The sphingolipid profiles in the serum and liver were determined using LC-MS/MS. Results: All the mice developed NASH and HCC after feeding with WDSW for 6 months and 1 year, respectively. Bioinformatic analysis identified 191 DEGs in the NASH group and 250 DEGs in HCC group compared to the corresponding controls. The IPA analysis revealed that the major pathways related to inflammation and fibrosis were upregulated in both NASH and HCC mice. The expression levels of key genes related to ceramide synthesis and metabolism [serine palmitoyltransferase long chain base subunit 2 (Sptlc2), ceramide synthase 6 (Cers6), N-acylsphingosine amidohydrolase 2 (Asah2)] and sphingosine-1-phosphate receptor 2 (S1PR2) were significantly upregulated in NASH mice. In HCC group, Cers5, Cers6, sphingomyelin synthase 1 (Sgms1), Asah1, and S1PR3 were significantly increased. The expression levels of sphingosine kinase 2 and S1P lyase 1 were decreased both in NASH and HCC. Analysis of the TCGA data set showed that CERS5, CERS6, SPTLC2, ASAH1 and SGMS1 were highly expressed in HCC compared to healthy controls. The LC-MS/MS data also showed significant changes in sphingolipid profiles in serum and liver NASH and HCC. Conclusion: Our study suggests that dysregulated sphingolipid metabolism is associated with NASH and HCC disease progression. Further validation and research are needed for the potential application of sphingolipid profiles as diagnostic or prognostic markers in the clinic. This study was supported by VA Merit Award I01BX004033; Research Career Scientist Award (IK6BX004477); VA ShEEP grants (1IS1BX005517-01 and 1 IS1 BX004777-01) National Institutes of Health Grant R01 DK104893, R01DK-057543 and R21 AA026629-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tumor cell-macrophage crosstalk promotes immunotherapy resistance via lipid metabolism

Abstract Immune-checkpoint blockade (ICB) therapies have transformed the treatment landscapes of hepatocellular carcinoma (HCC). However, the immunosuppressive tumor microenvironment (TME) constructed by tumor cells restricts the responsiveness of ICB therapies to a minority of patients. Triggering receptor expressed on myeloid cells-2 (TREM2) counteracts inflammation and maintains metabolic fitness in myeloid cells. Single-cell RNA-sequencing analysis of our pembrolizumab study (NCT03419481) identified a subset of tumor-associated macrophages over-expressing TREM2 in non-responders. Consistently, our syngeneic ICB-resistant HCC mouse models verified a group of Trem2+ myeloid cells, which were accumulated in the lipid-rich TME of ICB-resistant tumors. Compared to the parental ICB-sensitive tumor cells, the conditional medium (CM) of ICB-resistant tumor cells significantly increased Trem2 expression in macrophages, which was abolished when lipids in the CM were depleted. Notably, lentivirus-mediated Trem2 ablation reduced lipid accumulation in TME and overcame anti-PD-1 resistance with increased cytotoxic CD8+ T cell infiltration. In conclusion, our study highlights TREM2 as an immuno-metabolic target to enhance HCC immunotherapy. This study is supported by Collaborative Research Fund (C4045-18W) and Li Ka Shing Foundation. Z.L. is supported by Hong Kong PhD Fellowship Scheme.

Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Pivotal role for the non-receptor tyrosine kinase SRMS in IRF3-dependent innate antiviral immune response in murine cells

Abstract Upon viral infections, RIG-I-like receptors (RLRs) sense cytosolic viral RNAs and initiate signaling pathways culminating in the activation of IRF3 and subsequent type I interferon (IFN) antiviral response. This intrinsic protective mechanism is orchestrated by a host of positive and negative regulators, which collectively govern the magnitude and duration of antiviral signaling. SRMS is an understudied non-receptor tyrosine kinase frequently overexpressed in breast cancers. SRMS restrains autophagy and promotes tumor growth, although its physiological function is unclear. We report here that SRMS is critical for IRF3-dependent innate antiviral response in murine cells. Depletion of SRMS impaired virus- or dsRNA-induced expression of type I and III IFNs and ISGs in mouse embryonic fibroblasts (MEFs), mouse hepatoma hepa1–6 cells, and primary mouse lung fibroblasts, concomitant with heightened viral replication. Mechanistically, SRMS loss led to diminished phosphorylations of TBK1 and IRF3, IRF3 dimerization and nuclear translocation. The kinase activity of SRMS, however, was dispensable. Reconstituting the expression of SRMS of mouse or human origin, be it wild-type or kinase-dead protein, in SRMS-null MEFs restored antiviral signaling. Autophagy regulation was not involved, either. Interestingly, antiviral gene expression via the RLRs in human U2OS and MDA-MB-231 cells was not impacted by SRMS deletion, suggesting signaling redundancy in human cells. Altogether, our data uncover a novel role of SRMS in controlling IRF3-dependent innate antiviral responses and describe a non-canonical, kinase- and autophagy-independent regulatory mechanism that operates differentially between mouse and human species. R21 AI142044

Neoantigen vaccination augments antitumor effects of anti-PD-1 on mouse hepatocellular carcinoma

Immune checkpoint inhibitors are groundbreaking resources for cancer therapy. However, only a few patients with hepatocellular carcinoma (HCC) have shown positive responses to anti-PD-1 therapy. Neoantigens are sequence-altered proteins resulting from somatic mutations in cancer. This study identified the neoantigens of Hep-55.1C and Dt81 Hepa1-6 HCCs by comparing their whole exome sequences with those of a normal C57BL/6 mouse liver. Immunogenic long peptides were pooled as peptide vaccines. The vaccination elicited tumor-reactive immune responses in C57BL/6 mice, as demonstrated by IFN-γ ELISPOT and an in vitro killing assay of splenocytes. In the treatment of three mouse HCC models, combined neoantigen vaccination and anti-PD-1 resulted in more significant tumor regression than monotherapies. Flow cytometry of the tumor-infiltrating lymphocytes showed decreased Treg cells and monocytic myeloid-derived suppressor cells, increased CD8+ T cells, enhanced granzyme B expression, and reduced exhaustion-related markers PD-1 and Lag-3 on CD8+ T cells in the combination group. These findings provide a strong rationale for conducting clinical studies of using neoantigen vaccination in combination with anti-PD-1 to treat patients with HCC.

The Combination of R848 with Sorafenib Enhances Antitumor Effects by Reprogramming the Tumor Immune Microenvironment and Facilitating Vascular Normalization in Hepatocellular Carcinoma.

Novel promising strategies for combination with sorafenib are urgently needed to enhance its clinical benefit and overcome toxicity in hepatocellular carcinoma (HCC). the molecular and immunomodulatory antitumor effects of sorafenib alone and in combination with the new immunotherapeutic agent R848 are presented. Syngeneic HCC mouse model is presented to explore the antitumor effect and safety of three sorafenib doses alone, R848 alone, or their combination in vivo. R848 significantly enhances the sorafenib antitumor activity at a low subclinical dose with no obvious toxic side effects. Furthermore, the combination therapy reprograms the tumor immune microenvironment by increasing antitumor macrophages and neutrophils and preventing immunosuppressive signaling. Combination treatment promotes classical M1 macrophage-to-FTH1high M1 macrophage transition. The close interaction between neutrophils/classical M1 macrophages and dendritic cells promotes tumor antigen presentation to T cells, inducing cytotoxic CD8+ T cell-mediated antitumor immunity. Additionally, low-dose sorafenib, alone or combined with R848, normalizes the tumor vasculature, generating a positive feedback loop to support the antitumor immune environment. Therefore, the combination therapy reprograms the HCC immune microenvironment and normalizes the vasculature, improving the therapeutic benefit of low-dose sorafenib and minimizing toxicity, suggesting a promising novel immunotherapy (R848) and targeted therapy (tyrosine kinase inhibitors) combination strategy for HCC treatment.

Thymidine kinase 1 drives hepatocellular carcinoma in enzyme-dependent and -independent manners

Metabolic reprogramming plays a crucial role in the development of hepatocellular carcinoma (HCC). However, the key drivers of metabolic reprogramming underlying HCC progression remain unclear. Using a large-scale transcriptomic database and survival correlation screening, we identify thymidine kinase 1 (TK1) as a key driver. The progression of HCC is robustly mitigated by TK1 knockdown and significantly aggravated by its overexpression. Furthermore, TK1 promotes the oncogenic phenotypes of HCC not only through its enzymatic activity and production of deoxythymidine monophosphate (dTMP) but also by promoting glycolysis via binding with protein arginine methyltransferase 1 (PRMT1). Mechanistically, TK1 directly binds PRMT1 and stabilizes it by interrupting its interactions with tripartite-motif-containing 48 (TRIM48), which inhibits its ubiquitination-mediated degradation. Subsequently, we validate the therapeutic capacity of hepatic TK1 knockdown in a chemically induced HCC mouse model. Therefore, targeting both the enzyme-dependent and -independent activity of TK1 may be therapeutically promising for HCC treatment.

Profiling of syngeneic mouse HCC tumor models as a framework to understand anti-PD-1 sensitive tumor microenvironments.

The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

Abstract LB208: A potent macrophage switching drug D-4559 reduces tumor GROWTH in a hepatocellular carcinoma mouse model

Abstract Tumor-associated macrophages (TAMs) play a role in cancer progression and are associated with Sorafenib resistance in hepatocellular carcinoma (HCC)1. D-4559 is a new potent macrophage switching nanomedicine technology that selectively inhibits VEGF receptor tyrosine kinases (VEGFR1, 2, 3) in TAMs leading to a functional reprogramming of TAMs toward a pro-inflammatory activated phenotype. Here, we evaluate the effect of D-4559 on the M1 and M2 polarization of TAMs and its anti-tumor efficacy in a murine HCC tumor model. In vivo efficacy of D-4559 was examined in the subcutaneous Hepa 1-6 liver tumor model in C57BL/6 mice. Animals (n=15/group) were treated with D-4559 (i.p., 200 mg/kg daily) and free drug Sorafenib (p.o., 40 mg/kg daily) as a positive control for 4 weeks. The treatment started when the mean tumor size reached approximately 100 mm3, then the animals were randomly allocated into study groups. The day of randomization and treatment was denoted as day 0. Tumor sizes were measured using a caliper for 27 days, M1/M2 macrophage polarization was examined by flow cytometry at day 16, and cytokine biomarkers were evaluated with MSD Cytokine Multiplex Assay at day 16 and 27.D-4559 significantly reduced Hepa 1-6 tumor growth (**p<0.01 vs. vehicle). D-4559 and Sorafenib efficacy were similar with 40% of mice with a tumor volume less than 500 mm3 after 27 days of treatment. At day 16, D-4559 significantly increased M1/M2 ratio by inducing M1 macrophage infiltration and reprogramming of TAMs into M1 macrophages compared to vehicle treatment groups (* p<0.05). In contrast, Sorafenib did not switch TAM polarization from M2 to M1 phenotype. Moreover, D-4559 significantly increased TNF-a and IL-8 cytokines in the tumor microenvironment at days 16 and 27, indicative of a M1 signature. Increased levels of TNF-a and IL-8 have also been correlated with better survival in HCC patients2,3. D-4559 significantly increased M1 infiltration, shifted TAM polarization from M2 to M1 phenotype and favored pro-inflammatory cytokines suggesting that D-4559 creates an immunopermissive tumor microenvironment leading to a reduction in tumor growth. This preclinical study supports the development of the potent macrophage switching drug D-4559 as a safe and effective agent that can be used systemically for the treatment of HCC or in combination therapies to improve anti-tumor immune response.

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.

Hepatocyte-specific deletion of Mettl3 promotes hepatocellular carcinoma in mice

Hepatocyte-specific deletion of Mettl3 promotes hepatocellular carcinoma in mice

Abstract 4854: PKM2 Modulates hepatic macrophage regulation of NASH, ferroptosis and HCC through TGF-β signaling

Abstract Background: Non-alcoholic steatohepatitis (NASH) and cirrhosis are major risk factors for hepatocellular carcinoma (HCC), which affects men 2-4 times more than women. In tumors Pyruvate kinase M2 (PKM2), induces aerobic glycolysis (Warburg effect) and proinflammatory and non-apoptotic cell death, ferroptosis. Raised levels of hepatic macrophage PKM2 expression are associated with poor prognosis HCC. We previously found that liver specific TGF-β/SMAD4 knockout mice develop iron deposition and hemochromatosis due to a dramatic loss of hepcidin. Moreover, Smad4+/−Sptbn1+/− mice develop gastrointestinal cancers but only in the presence of an altered microbiome. Recently, we found that liver-specific knockout of a TGF-β/SMAD3/4 adaptor protein, βII-spectrin (LSKO) blocks diet-induced NASH and HCC in mice, as well as in siRNA treated human NASH microfluidic cultures (Sci Transl Med. 2021;13(624): eabk2267). We hypothesized that βII-spectrin modulates PKM2 expression in NASH fatty liver, promoting proinflammatory cytokine release, inflammation and tumorigenesis. In addition, microbiome changes could alter hepcidin function and modulate ferroptosis. Our goal is to understand the molecular mechanism underlying PKM2 driven NASH and HCC. Methods: For our NASH mouse model, we fed SPTBN1Flox (control) and liver-specific βII-spectrin knockout (SPTBN1LSKO) mice a Western diet (WD). Body weight, total cholesterol, and triglyceride concentrations in serum were monitored, and PKM2 expression levels in the liver tissues was analyzed. SPTBN1Flox and SPTBN1LSKO mice were treated with both WD and Diethylnitrosamine (DEN) for HCC, and gut microbiome profiles were performed in these mice. To evaluate PKM2 expression in the NASH-associated HCC mouse model, immunohistochemical labeling was performed on liver tissues of these mice with PKM2-specific antibody. Inflammation, lipidosis and fibrosis were determined with H&E, Oil Red O and Sirius Red staining. Results: We found upregulation of PKM2 expression in NASH and HCC Kupffer cells (WD SPTBN1Flox mice). In contrast, PKM2 expression was markedly reduced in the liver in WD SPTBN1LSKO that blocked NASH and HCC. Interestingly, microbiome profiles were altered and TGF-β/SMAD3-regulated fibrosis as well as expression of inflammatory genes were significantly reduced in LSKO mice, compared to the NASH mice. Conclusions: Our findings suggest that the knockdown of the Smad3/4 adaptor βII-spectrin decreases PKM2 expression in Kupffer cells, thereby suppressing pro-inflammatory cytokine production, blocking NASH-associated HCC. Hepatic macrophages such as Kupffer cells and monocyte-derived macrophages could therefore play a critical role in the ferroptosis-mediated protumor immune microenvironment. In addition, our work provides new insight into potential mechanism for two disorders that affect males more than females, hemochromatosis and HCC. Citation Format: Kazufumi Ohshiro, Krishanu Bhowmick, Xiaochun Yang, Addison Klebanov, Sahara John, Dillon Voss, Adrian Krainer, Lopa Mishra. PKM2 Modulates hepatic macrophage regulation of NASH, ferroptosis and HCC through TGF-β signaling. [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 4854.

Abstract 4092: Development of nucleotide metabolic reprogrammed CAR T cell therapy suitable for solid tumors

Abstract CAR T cell therapy has represented an exciting breakthrough in the treatment of patients with hematologic malignancies. In contrast, the antitumor activity of CAR T cell therapy in solid tumors has been modest so far in clinical studies. The suboptimal clinical efficacy is a result of multiple factors including limited T cell trafficking, persistence, and the immunosuppressive tumor microenvironment (TME). While current strategies utilize costimulatory molecules and cytokines to activate CAR T cells, a key but largely overlooked problem of CAR T cell therapy is nutrient competition between tumor cells and T cells in the nutrient-poor TME. In our previous studies, a metabolic screen identified inosine as an alternative fuel for T cells which can support T cell growth and function in the absence of glucose. T cells could metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. Moreover, we demonstrated that T cells have advantage over cancer cells in utilizing inosine, and supplementation with inosine enhances the antitumor efficacy of PD-1 mAb and adoptive T cell transfer in mouse models. In this study, we therefore engineered CAR T cells to express CD26 on the cell surface and a secreted adenosine deaminase (ADA) fused with an anchor. These metabolic reprogrammed CAR (designated as MRCAR) T cells exerts their activities through different mechanisms. ADA irreversibly converts adenosine to inosine, overcoming adenosine-mediated immunosuppression and providing inosine for CAR T cell growth, while CD26 induces a rich chemokine receptor profile enabling CAR T cells to traffic to solid tumors. We demonstrated that CD26-overexpressed CAR T cells displayed superior migration capacity and resisted TGF-beta suppression, while the CD26 expression on unmodified CAR T cells was significantly down-regulated by TGF-beta. We further proved that ADA is conditionally secreted in stress condition (i.e., low pH, lack of nutrient and oxygen) based on ADA’s biological characteristics, suggesting that ADA activates CAR T cells as a trans-signaling to the CAR construct in a tumor specific manner. Remarkably, we observed that the MRCAR T cells inhibit tumor growth more effectively than unmodified CAR T cells in both Huh7 human hepatocellular carcinoma (GPC3-MRCAR) and A549 human non-small cell lung cancer (HER2-MRCAR) mouse models. Thus, MR-CAR T cell therapy represents a promising approach to improve CAR T cell therapy against solid tumors. Citation Format: Yue Hu, Abhijit Sarkar, Kevin Song, Magnus Hook, Andras Heczey, Xiaotong Song. Development of nucleotide metabolic reprogrammed CAR T cell therapy suitable for solid tumors. [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 4092.

Abstract 4452: FASN inhibition overcomes immune evasion by upregulating MHC-I in hepatocellular carcinoma

Abstract Immune evasion is one of the important factors leading to the progression of hepatocellular carcinoma(HCC). Restricted antigen presentation by decrease of MHC-I remains a huge barrier to triggering an immune attack. However, knowledge is limited on how to enhance antigen presentation of cancer cells. Here we identify that FASN inhibition by shRNA and inhibitors could boost MHC-I level on HCC cell surface. FASN deficiency also promotes antigen presentation and augments CD8+ T cell cytotoxicity. Mechanistically, FASN knock-down decreases the degradation of MHC-I in lysosomes. Moreover, ZDHHC3-mediated palmitoylation participates in this process. In vivo, orthotopic HCC mouse model shows that Fasn knock-down greatly improves the cytotoxicity of tumor-infiltrating CD8+ T cells and suppresses HCC tumor growth, indicating that inhibiting FASN is a promising way to enhance the efficiency of immunotherapy in HCC. Citation Format: Jiao Huang, Wai Ying Tsang, Xiaona Fang, Yu Zhang, Jie Luo, Lanqi Gong, Baifeng Zhang, Ching Ngar Wong, Beilei Liu, Jinlin Huang, Yuma Yang, Shan Liu, Zhihong Li, Xinyuan Guan. FASN inhibition overcomes immune evasion by upregulating MHC-I 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 4452.

Abstract 3718: FOXM1 switches protein conformations upon C-terminal domain-mediated-autoregulation dictating its transcriptional activity in cancer cell proliferation

Abstract Transcription factor Forkhead box M1 (FOXM1) participates in tumor initiation, tumor development and tumor metastasis through regulating transcriptional network of its target genes. Activated FOXM1 promotes G1/S and G2/M transition via manipulating Cdc25B, Cyclin B1, TOPOIIa, Aurora and Polo-like kinases, as well as cancer cell survival and invasion through upregulating Survivin, SNAIL and MMP-9. The depletion of Foxm1 repressed the oncogenic Kras (G12D) driven mice lung cancer, Rb/Trp53/Myc triple knockout mice small cell lung cancer and carcinogen induced mice hepatocellular carcinoma. Given the roles of FOXM1 in tumorigenesis and its relevance to clinical outcomes, deciphering the mechanism underlying the regulation of FOXM1 activity might provide new therapeutic target for cancer study. It is known that FOXM1 activity drops when the C-terminal transcription activation domain (TAD) is blocked by the N-terminal repression domain (NRD), which is called autorepression. Furthermore, the NRD-deleted FOXM1 displays extraordinarily high transactivity. Compared to the NRD, the TAD is relatively longer and consists of around 420 amino acid with most of them being intrinsically disordered region (IDR). Previous studies showed that the NRD binds to a relatively ordered small region on the TAD. However, it is poorly understood about whether the IDR of TAD partakes in the FOXM1 self-regulated activity and how FOXM1 protein structure switches in living cells. Our co-immunoprecipitation (co-IP) and dual-luciferase reporter assay (DLR) suggested that FOXM1 TAD self-interacts via its IDR and the C-terminal αβα motif, which promotes FOXM1 transactivation. The IDR-driven FOXM1 autoactivation was mimicked by the chemically-induced dimerization system (CID) that forces IDR-αβα proximity intermolecularly. Using fluorescence resonance energy transfer (FRET) microscopy, we observed that the IDR-αβα interaction was elevated when cell cycle progression from G1/S to G2/M in living cells. The IDR-αβα interaction and FOXM1 activity were both suppressed by PLK1 inhibition. Furthermore, the characteristics of IDR-αβα interaction did not resemble NRD-TAD interaction as revealed by FRET data where we showed the NRD-TAD interaction dropped after G1/S. The NRD-TAD interaction underlying FOXM1 autorepression was recapitulated by CID. Through co-IP and yeast-two hybrid assays (Y2H), we further identified the minimal yet biological functional unit ββαβ on NRD that suppressed FOXM1 activity and lung adenocarcinoma A549 cells in vitro and in vivo. Collectively, our work suggested that FOXM1 might be switched from the NRD-TAD mediated autorepression toward the IDR-αβα mediated activation by Plk1 during S-G2 phase. And ββαβ peptide treatment might be applied to lung cancer as a potential therapeutic strategy. Citation Format: Chia-Chan Hsu, Xiang Yao, I-Ching Wang. FOXM1 switches protein conformations upon C-terminal domain-mediated-autoregulation dictating its transcriptional activity in cancer cell proliferation. [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 3718.

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.

Abstract 5252: Salsolinol suppresses the STAT3-EMT axis in liver carcinogenesis

Abstract Liver cancer is one of the most common malignancies and a leading cause of death worldwide. However, it is still very difficult to treat and prevent liver cancer. Salsolinol (SAL) is an endogenous catechol isoquinoline generated by the condensation of dopamine with acetaldehyde, a major metabolite of ethanol. In the present study, we found that SAL inhibited the phosphorylation, dimerization, and subsequent nuclear translocation of STAT3 in a concentration-dependent manner in SK-Hep1 cells, a hepatic carcinoma cell line. SAL suppressed the expression of Cdk 4 and Cyclin D1, the major target protein of STAT3, and induced the cell cycle arrest at Go/G1 phase. However, SAL induced the expression of STAT1 in SK-Hep1 cells. In addition, SAL enhanced the expression of the cell cycle regulators such as p53 and p21 in SK-Hep1 cells. Epithelial-mesenchymal transition (EMT) promotes tumor invasion and metastasis in malignancies through STAT3 activation. SAL induced the expression of E-cadherin while suppressing the expression of Snail, N-cadherin, and Slug, which are related to EMT. SAL inhibited the migration of SK-Hep1 cells by suppressing the expression of MMP-9 and MMP-2. Furthermore, intraperitoneal injection of SAL reduced the number of tumors in the diethylnitrosamine (DEN)-induced liver carcinogenesis model without affecting the body weight change. Proliferative markers PCNA and KI-67 and p-STAT3 were down-regulated in the SAL-injected mice liver cancer tissue. The plasma level of alpha-fetoprotein, a tumor marker in the blood, was also reduced in the SAL-injected mice. Interestingly, SAL injection significantly reduced the anxiety-like behavior without affecting the locomotive activities in the DEN-induced hepatocellular carcinoma mouse model. Moreover, SAL injection recovered tyrosine hydroxylase, a key enzyme for producing dopamine from tyrosine in the substantia nigra compacta of DEN-treated mice. Together, SAL inhibits STAT3 signaling, thereby suppressing the cell cycle progression and EMT in SK-Hep1 cells, which may account for its anti-depressant and anti-carcinogenic activity in DEN-induced liver carcinogenesis. Citation Format: Hyeon Jeong Oh, Seah Park, Hong-Kyung Yang, Hoon Ryu, Young-Joon Surh, Dae-Yong Kim, Hye-Kyung Na. Salsolinol suppresses the STAT3-EMT axis in liver carcinogenesis. [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 5252.

Interactions between driver genes shape the signaling pathway landscape and direct hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, whose initiation and development are driven by alterations in driver genes. In this study, we identified four driver genes (TP53, PTEN, CTNNB1, and KRAS) that exhibit a high frequency of somatic mutations or copy number variations (CNVs) in patients with HCC. Four different spontaneous HCC mouse models were constructed to screen for changes in various kinase signaling pathways. The sgTrp53+sgPten tumor upregulated mTOR and noncanonical NF-κB signaling, which was shown to be strongly inhibited by rapamycin (an mTOR inhibitor) in vitro and in vivo. JAK-STAT signaling was activated in the Ctnnb1mut +sgPten tumor, the proliferation of which was strongly inhibited by napabucasin (a STAT3 inhibitor). MTOR, cytoskeleton, and AMPK signaling were upregulated while rapamycin and ezrin inhibitors exerted potent anti-proliferative effects in the sgPten+KrasG12D tumor. JAK-STAT, MAPK, and cytoskeleton signaling were activated in the sgTrp53+KrasG12D tumor and the combination of sorafenib and napabucasin led to the complete inhibition of tumor growth in vivo. In patients with HCC, who had the same molecular classification as our mouse models, the downstream signaling pathway landscapes associated with genomic alterations were identical. Our research provides novel targeted therapeutic options for the clinical treatment of HCC, based on the presence of specific genetic alterations within the tumor.

PPDPF suppresses the development of hepatocellular carcinoma through TRIM21-mediated ubiquitination of RIPK1

Pancreatic progenitor cell differentiation and proliferation factor (PPDPF) has been reported to play a role in tumorigenesis. However, its function in hepatocellular carcinoma (HCC) remains poorly understood. In this study, we report that PPDPF is significantly downregulated in HCC and the decreased PPDPF expression indicates poor prognosis. In the dimethylnitrosamine (DEN)-induced HCC mouse model, hepatocyte-specific depletion of Ppdpf promotes hepatocarcinogenesis, and reintroduction of PPDPF into liver-specific Ppdpf knockout (LKO) mice inhibits the accelerated HCC development. Mechanistic study shows that PPDPF regulates nuclear factor κB (NF-κB) signaling through modulation of RIPK1 ubiquitination. PPDPF interacts with RIPK1 and facilitates K63-linked ubiquitination of RIPK1 via recruiting the E3 ligase TRIM21, which catalyzes K63-linked ubiquitination of RIPK1 at K140. In addition, liver-specific overexpression of PPDPF activates NF-κB signaling and attenuates apoptosis and compensatory proliferation in mice, which significantly suppresses HCC development. This work identifies PPDPF as a regulator of NF-κB signaling and provides a potential therapeutic candidate for HCC.