Liver Tumor Growth Research Articles

High Serum Levels of Cholesterol Increase Antitumor Functions of Nature Killer Cells and Reduce Growth of Liver Tumors in Mice

The relationship between serum cholesterol level and development of hepatocellular carcinoma (HCC) remains unclear. We investigated the effects of serum cholesterol level on development of liver tumors in mice. We performed studies with C57BL/6J mice, mice with disruption of the low-density lipoprotein receptor gene (Ldlr-/-mice), and mice with conditional deletion of nature killer (NK) cells (NKdele mice). Some C57BL/6J and NKdele mice were given injections of diethylinitrosamine to induce liver tumor formation. Mice were placed on a normal diet (ND) or high-cholesterol diet (HCD) to induce high serum levels of cholesterol. We also studied mice with homozygous disruption of ApoE (ApoE-/- mice), which spontaneously develop high serum cholesterol. C57BL/6J and NKdele mice on the ND or HCD were implanted with Hep1-6 (mouse hepatoma) cells and growth of xenograft tumors and lung metastases were monitored. Blood samples were collected from mice and analyzed by biochemistry and flow cytometry; liver and tumor tissues were collected and analyzed by histology, immunohistochemistry, and RNA-sequencing analysis. NK cells were isolated from mice and analyzed for cholesterol content, lipid raft formation, immune signaling, and changes in functions. We obtained matched tumor tissues and blood samples from 30 patients with HCC and blood samples from 40 healthy volunteers; levels of cholesterol and cytotoxicity of NK cells were measured. C57BL/6J mice on HCD and ApoE-/- mice with high serum levels of cholesterol developed fewer and smaller liver tumors and lung metastases after diethylinitrosamine injection or implantation of Hep1-6 cells than mice on ND. Liver tumors from HCD-fed mice and ApoE-/- mice had increased numbers of NK cells compared to tumors from ND-fed mice. NKdele mice or mice with antibody-based depletion for NK cells showed similar tumor number and size in ND and HCD groups after diethylinitrosamine injection or implantation of Hep1-6 cells. NK cells isolated from C57BL/6J mice fed with HCD had increased expression of NK cell-activating receptors (natural cytotoxicity triggering receptor 1 and natural killer group 2, member D), markers of effector function (granzyme B and perforin), and cytokines and chemokines compared with NK cells from mice on ND; these NK cells also had enhanced cytotoxic activity against mouse hepatoma cells, accumulated cholesterol, increased lipid raft formation, and immune signaling activation. NK cells isolated from HCD-fed Ldlr-/- mice did not have increased cholesterol content or cytotoxic activity against mouse hepatoma cells compared with ND-fed Ldlr-/- mice. Serum levels of cholesterol correlated with number and activity of NK cells isolated from human HCCs. Mice with increased serum levels of cholesterol due to an HCD or genetic disruption of ApoE develop fewer and smaller tumors after injection of hepatoma cells or a chemical carcinogen. We found cholesterol to accumulate in NK cells and activate their effector functions against hepatoma cells. Strategies to increase cholesterol uptake by NK cells can be developed for treatment of HCC.

Mitochondrial Fusion Via OPA1 and MFN1 Supports Liver Tumor Cell Metabolism and Growth.

Metabolic reprogramming universally occurs in cancer. Mitochondria act as the hubs of bioenergetics and metabolism. The morphodynamics of mitochondria, comprised of fusion and fission processes, are closely associated with mitochondrial functions and are often dysregulated in cancer. In this study, we aim to investigate the mitochondrial morphodynamics and its functional consequences in human liver cancer. We observed excessive activation of mitochondrial fusion in tumor tissues from hepatocellular carcinoma (HCC) patients and in vitro cultured tumor organoids from cholangiocarcinoma (CCA). The knockdown of the fusion regulator genes, OPA1 (Optic atrophy 1) or MFN1 (Mitofusin 1), inhibited the fusion process in HCC cell lines and CCA tumor organoids. This resulted in inhibition of cell growth in vitro and tumor formation in vivo, after tumor cell engraftment in mice. This inhibitory effect is associated with the induction of cell apoptosis, but not related to cell cycle arrest. Genome-wide transcriptomic profiling revealed that the inhibition of fusion predominately affected cellular metabolic pathways. This was further confirmed by the blocking of mitochondrial fusion which attenuated oxygen consumption and cellular ATP production of tumor cells. In conclusion, increased mitochondrial fusion in liver cancer alters metabolism and fuels tumor cell growth.

Polycyclic polyprenylated acylphloroglucinols from Garcinia xanthochymus fruits exhibit antitumor effects through inhibition of the STAT3 signaling pathway.

The fruits of Garcinia xanthochymus can be eaten raw or processed into jams, preserves and vinegar. They provide not only vitamin and protein nutrients, but also pharmacologically active compounds, among which polycyclic polyprenylated acylphloroglucinols (PPAPs) are a major class. According to the literature, PPAPs exhibited good anti-cancer effects. This study investigated the antitumor effects and the underlying mechanism of S1 (the regioisomeric mixture of xanthochymol and guttiferone E) and S2 (the regioisomeric mixture of isoxanthochymol and cycloxanthochymol) isolated from the fruits of G. xanthochymus. In an H22 allograft mouse model, S1 and S2 could suppress the liver tumor growth and phosphorylation of STAT3. Computational modeling showed that S1 and S2 could form hydrogen bonds with the SH2 domain of STAT3. In HepG2 and MCF-7 cell lines, S1 and S2 downregulated the expression of p-STAT3Tyr705. Moreover, S1 and S2 inhibited the phosphorylation of JAK2 and Src, which are the upstream kinases of STAT3, and the expression of various STAT3-regulated genes, including anti-apoptotic (Bcl-XL, Mcl-1 and survivin), proliferative (cyclin D1) and angiogenic (VEGF) genes. As a result, S1 and S2 arrested the cell cycle and induced cell apoptosis, which were proved by the activation of cleaved caspase-3 and caspase-8. These results demonstrated that S1 and S2 from G. xanthochymus exhibited antitumor effects through the inactivation of STAT3, and could be promising candidates for cancer treatment.

MicroRNA-19a-3p regulates cell growth through modulation of the PIK3IP1-AKT pathway in hepatocellular carcinoma.

There are some controversies about the involvement of microRNA (miR)-19a-3p in hepatocellular carcinoma (HCC) biology, even though many studies have shown that it plays an important role in cancer. In this study, we found that miR-19a-3p is usually overexpressed in HCC tissues compared with corresponding peritumorous tissues, and its expression was associated with tumor size and poor overall survival. MiR-19a-3p promoted cell proliferation significantly, and more cells were found in the S phase. In vivo, miR-19a-3p promoted liver tumor growth, and more HCC cells were found in the active cell cycle. Sequencing and bioinformatics analysis predicted that PIK3IP1 is a likely target gene of miR-19a-3p, and we next confirmed it by luciferase and rescue assays. Altogether, our data showed an important role of PIK3IP1 downregulation by miR-19a-3p in HCC progression, and the miR-19a-3p-PIK3IP1-AKT pathway may be a potential therapeutic target.

Peritumoral YAP and TAZ Expression Suppresses Tumor Growth in Mice

Peritumoral expression of Hippo-pathway members YAP and TAZ restricts liver-tumor growth in mice.

Ultrasound-Targeted Microbubble Destruction Enhances the Antitumor Efficacy of Doxorubicin in a Mouse Hepatocellular Carcinoma Model

The aim of the study described here was to investigate whether ultrasound-mediated microbubble destruction (UTMD) of targeted microbubbles conjugated with an anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) antibody can enhance the therapeutic effect of doxorubicin (DOX) on a mouse hepatocellular carcinoma (HCC) model bearing HEP-G2-RFP tumors. The growth of liver tumors in mice was inhibited by using Visistar VEGFR2 plus ultrasound irradiation and by DOX alone. DOX plus UTMD had an inhibitory effect on tumor growth beginning on the seventh day of treatment, while Visistar VEGFR2 alone and DOX alone had inhibitory effects beginning on the 11th day. DOX + UTMD significantly decreased tumor volume and tumor weight compared with DOX alone (p < 0.05) and Visistar VEGFR2 alone (p < 0.05). Compared with DOX alone and Visistar VEGFR2 alone, DOX + UTMD had the highest inhibitory effect on tumor angiogenesis and the highest apoptosis index. UTMD-targeted microbubbles can significantly enhance the antitumor effect of DOX on a mouse HCC model, inhibit angiogenesis and induce apoptosis in tumor cells.

An Ultrasound Activated Vesicle of Janus Au‐MnO Nanoparticles for Promoted Tumor Penetration and Sono‐Chemodynamic Therapy of Orthotopic Liver Cancer

AbstractSonodynamic therapy (SDT) has the advantages of high penetration, non‐invasiveness, and controllability, and it is suitable for deep‐seated tumors. However, there is still a lack of effective sonosensitizers with high sensitivity, safety, and penetration. Now, ultrasound (US) and glutathione (GSH) dual responsive vesicles of Janus Au‐MnO nanoparticles (JNPs) were coated with PEG and a ROS‐sensitive polymer. Upon US irradiation, the vesicles were disassembled into small Janus Au‐MnO nanoparticles (NPs) with promoted penetration ability. Subsequently, GSH‐triggered MnO degradation simultaneously released smaller Au NPs as numerous cavitation nucleation sites and Mn2+ for chemodynamic therapy (CDT), resulting in enhanced reactive oxygen species (ROS) generation. This also allowed dual‐modality photoacoustic imaging in the second near‐infrared (NIR) window and T1‐MR imaging due to the released Mn2+, and inhibited orthotopic liver tumor growth via synergistic SDT/CDT.

An Ultrasound Activated Vesicle of Janus Au-MnO Nanoparticles for Promoted Tumor Penetration and Sono-Chemodynamic Therapy of Orthotopic Liver Cancer.

Sonodynamic therapy (SDT) has the advantages of high penetration, non-invasiveness, and controllability, and it is suitable for deep-seated tumors. However, there is still a lack of effective sonosensitizers with high sensitivity, safety, and penetration. Now, ultrasound (US) and glutathione (GSH) dual responsive vesicles of Janus Au-MnO nanoparticles (JNPs) were coated with PEG and a ROS-sensitive polymer. Upon US irradiation, the vesicles were disassembled into small Janus Au-MnO nanoparticles (NPs) with promoted penetration ability. Subsequently, GSH-triggered MnO degradation simultaneously released smaller Au NPs as numerous cavitation nucleation sites and Mn2+ for chemodynamic therapy (CDT), resulting in enhanced reactive oxygen species (ROS) generation. This also allowed dual-modality photoacoustic imaging in the second near-infrared (NIR) window and T1 -MR imaging due to the released Mn2+ , and inhibited orthotopic liver tumor growth via synergistic SDT/CDT.

Computational/experimental evaluation of liver metastasis post hepatic injury: interactions with macrophages and transitional ECM

The complex interactions between subclinical changes to hepatic extracellular matrix (ECM) in response to injury and tumor-associated macrophage microenvironmental cues facilitating metastatic cell seeding remain poorly understood. This study implements a combined computational modeling and experimental approach to evaluate tumor growth following hepatic injury, focusing on ECM remodeling and interactions with local macrophages. Experiments were performed to determine ECM density and macrophage-associated cytokine levels. Effects of ECM remodeling along with macrophage polarization on tumor growth were evaluated via computational modeling. For primary or metastatic cells in co-culture with macrophages, TNF-α levels were 5× higher with M1 vs. M2 macrophages. Metastatic cell co-culture exhibited 10× higher TNF-α induction than with primary tumor cells. Although TGFβ1 induction was similar between both co-cultures, levels were slightly higher with primary cells in the presence of M1. Simulated metastatic tumors exhibited decreased growth compared to primary tumors, due to high local M1-induced cytotoxicity, even in a highly vascularized microenvironment. Experimental analysis combined with computational modeling may provide insight into interactions between ECM remodeling, macrophage polarization, and liver tumor growth.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Disrupts Control of Cell Proliferation and Apoptosis in a Human Model of Adult Liver Progenitors.

The aryl hydrocarbon receptor (AhR) activation has been shown to alter proliferation, apoptosis, or differentiation of adult rat liver progenitors. Here, we investigated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated AhR activation on a human model of bipotent liver progenitors, undifferentiated HepaRG cells. We used both intact undifferentiated HepaRG cells, and the cells with silenced Hippo pathway effectors, yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which play key role(s) in tissue-specific progenitor cell self-renewal and expansion, such as in liver, cardiac, or respiratory progenitors. TCDD induced cell proliferation in confluent undifferentiated HepaRG cells; however, following YAP, and, in particular, double YAP/TAZ knockdown, TCDD promoted induction of apoptosis. These results suggested that, unlike in mature hepatocytes, or hepatocyte-like cells, activation of the AhR may sensitize undifferentiated HepaRG cells to apoptotic stimuli. Induction of apoptosis in cells with silenced YAP/TAZ was associated with upregulation of death ligand TRAIL, and seemed to involve both extrinsic and mitochondrial apoptosis pathways. Global gene expression analysis further suggested that TCDD significantly altered expression of constituents and/or transcriptional targets of signaling pathways participating in control of expansion or differentiation of liver progenitors, including EGFR, Wnt/β-catenin, or tumor growth factor-β signaling pathways. TCDD significantly upregulated cytosolic proapoptotic protein BMF (Bcl-2 modifying factor) in HepaRG cells, which could be linked with an enhanced sensitivity of TCDD-treated cells to apoptosis. Our results suggest that, in addition to promotion of cell proliferation and alteration of signaling pathways controlling expansion of human adult liver progenitors, AhR ligands may also sensitize human liver progenitor cells to apoptosis.

Selective Killing of Cancer Cells by Nonplanar Aromatic Hydrocarbon-Induced DNA Damage.

A large number of current chemotherapeutic agents prevent the growth of tumors by inhibiting DNA synthesis of cancer cells. It has been found recently that many planar polycyclic aromatic hydrocarbons (PAHs) derivatives, previously known as carcinogenic, display anticancer activity through DNA cross‐linking. However, the practical use of these PAHs is substantially limited by their low therapeutic efficiency and selectivity toward most tumors. Herein, the anticancer property of a nonplanar PAH named [4]helicenium, which exhibits highly selective cytotoxicity toward liver, lung cancer, and leukemia cells compared with normal cells, is reported. Moreover, [4]helicenium effectively inhibits tumor growth in liver cancer‐bearing mice and shows little side effects in normal mice. RNA sequencing and confirmatory results demonstrate that [4]helicenium induces more DNA damage in tumor cells than in normal cells, resulting in tumor cell cycle arrest and apoptosis increment. This study reveals an unexpected role and molecular mechanism for PAHs in selectively killing tumor cells and provides an effective strategy for precision cancer therapies.

Abstract 87: Recapitulating the orthotopic tumor microenvironment using bioluminescent syngeneic models in immune competent mice for checkpoint inhibitor interrogation

Abstract BACKGROUND: Checkpoint inhibitors have shown promising therapeutic benefit in various cancer types, but more advanced preclinical models are required to combat resistance and evaluate opportunities for combination treatments. Subcutaneous syngeneic models are routinely used to evaluate the impact of immunotherapies on tumour growth as well as tumour invading leucocytes (TILs). However, the tumour microenvironment of orthotopic models is more comparable to the patient due to the organ-specific location of the tumour which facilitates metastatic spread and also recapitulates the immune and stromal component interactions with the tumour. Bioluminescent imaging (BLI) enables non-invasive longitudinal monitoring of orthotopic tumour burden and end stage quantification of tumour spread. Here we report the generation of a panel of bioluminescent syngeneic cell lines for orthotopic and metastatic modeling with an aim to assess the impact of standard of care agents, immune checkpoint therapies and combinations in a more clinically relevant environment. METHODS: Bioluminescent variants of syngeneic cell lines were established by lentiviral transduction. Orthotopic models were established via direct implantation into various organs e.g. liver, breast, pancreas and bladder and tumour growth assessed by BLI in-life as well as end stage (Spectrum CT; PerkinElmer). Response to standard of care agents such as sorafenib, gemcitabine, docetaxel and immune checkpoint therapy (anti-CTLA-4, anti-PD-1) was also evaluated and TIL infiltration was assessed by flow cytometry analysis and IHC. RESULTS: The success rate of tumour transplantation into different organs ranged from 60-100% as confirmed by both in-life imaging and ex vivo imaging at termination. For example, Hepa 1-6 appeared to grow within the parenchyma of the liver whereas 4T1 metastasized to the lungs and bone from the mammary fat pad, which was easily quantified by BLI. Real-time quantification of tumour size could be correlated with biomarkers and end stage assessment, for example, treatment with Sorafenib and check point inhibitors was correlated with alpha-fetoprotein (AFP) and end stage tumour burden, showing significant response in the orthotopic setting (p&amp;lt;0.001, Two way ANOVA). Different responses to check point inhibitors were observed between tumors inoculated at subcutaneous sites and orthotopic sites. CONCLUSIONS: Bioluminescent syngeneic models enables clinically relevant interrogation of standard of care agents, immunotherapies and combination which is distinct from the subcutaneous setting. The orthotopic microenvironment influences the tumour growth and response to immuno therapy. These models also enable the assessment of disease progression and modelling the metastatic environment. Citation Format: Joyce Afrakoma Obeng, Maya Jetha, Jane Wrigley, Simon Jiang, Jason King, Rajendra Kumari, Yinfei Yin. Recapitulating the orthotopic tumor microenvironment using bioluminescent syngeneic models in immune competent mice for checkpoint inhibitor interrogation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 87.

Aspirin targets P4HA2 through inhibiting NF-κB and LMCD1-AS1/let-7g to inhibit tumour growth and collagen deposition in hepatocellular carcinoma.

BackgroundAbnormal construction of the extracellular matrix (ECM) is intimately linked with carcinogenesis and the development of solid tumours, especially hepatocellular carcinoma (HCC). As the major component of the ECM, collagen plays a pivotal role in carcinogenesis. P4HA2, the essential enzyme during collagen formation, becomes an important target in HCC treatment. Here, we tried to decipher whether aspirin (ASA), a classic anti-inflammatory drug, could improve the prognosis of HCC through targeting P4HA2.MethodsWestern blotting, qRT-PCR assay, immunofluorescence staining, luciferase reporter gene assay, and ChIP assay were applied to demonstrate the molecular mechanism of the regulation of P4HA2 expression by aspirin. A mouse xenograft model, cell viability assay, colony formation assay, and immunohistochemistry analysis were used to evaluate the anti-fibrosis effect of aspirin through targeting the NF-κB/P4HA2 axis and LMCD1-AS1/let-7g/P4HA2 axis in vitro and in vivo. The TCGA database was used to evaluate the correlation among P4HA2, let-7g, LMCD1-AS1 and overall survival of HCC patients.FindingsIn xenograft mice, aspirin was capable of targeting P4HA2 to decrease collagen deposition, resulting in the inhibition of liver tumour growth. TCGA database analysis revealed the close association between a higher P4HA2 concentration in HCC patients and shorter overall survival or a higher cancer stage and the pathological grade. Mechanistically, NF-κB can bind to the promoter of P4HA2 to activate its transcription. Moreover, lncRNA LMCD1-AS1 functions as a molecular sponge of let-7g to post-transcriptionally induce the target gene of let-7g, namely, P4HA2.InterpretationOur findings disclose the novel role and regulatory mechanism of aspirin in the suppression of HCC by disrupting abnormal collagen deposition.Funds973 Program, National Natural Scientific Foundation of China, Fundamental Research Funds for the Central Universities, Project of Prevention and Control of Key Chronic Non-Infectious Diseases.

NOD-like receptor C4 Inflammasome Regulates the Growth of Colon Cancer Liver Metastasis in NAFLD.

Nonalcoholic fatty liver disease (NAFLD) enhances the growth and recurrence of colorectal cancer (CRC) liver metastasis. With the rising prevalence of NAFLD, a better understanding of the molecular mechanism underlying NAFLD-associated liver metastasis is crucial. Tumor-associated macrophages (TAMs) constitute a large portion of the tumor microenvironment that promotes tumor growth. NOD-like receptor C4 (NLRC4), a component of an inflammasome complex, plays a role in macrophage activation and interleukin (IL)-1β processing. We aimed to investigate whether NLRC4-mediated TAM polarization contributes to metastatic liver tumor growth in NAFLD. Wild-type and NLRC4-/- mice were fed low-fat or high-fat diet for 6 weeks followed by splenic injection of mouse CRC MC38 cells. The tumors were analyzed 2 weeks after CRC cell injection. High-fat diet-induced NAFLD significantly increased the number and size of CRC liver metastasis. TAMs and CD206-expressing M2 macrophages accumulated markedly in tumors in the presence of NAFLD. NAFLD up-regulated the expression of IL-1β, NLRC4, and M2 markers in tumors. In NAFLD, but not normal livers, deletion of NLRC4 decreased liver tumor growth accompanied by decreased M2 TAMs and IL-1β expression in tumors. Wild-type mice showed increased vascularity and vascular endothelial growth factor (VEGF) expression in tumors with NAFLD, but these were reduced in NLRC4-/- mice. When IL-1 signaling was blocked by recombinant IL-1 receptor antagonist, liver tumor formation and M2-type macrophages were reduced, suggesting that IL-1 signaling contributes to M2 polarization and tumor growth in NAFLD. Finally, we found that TAMs, but not liver macrophages, produced more IL-1β and VEGF following palmitate challenge. Conclusion: In NAFLD, NLRC4 contributes to M2 polarization, IL-1β, and VEGF production in TAMs, which promote metastatic liver tumor growth.

RA-XII Suppresses the Development and Growth of Liver Cancer by Inhibition of Lipogenesis via SCAP-dependent SREBP Supression.

Lipogenesis plays a critical role in the growth and metastasis of tumors, which is becoming an attractive target for anti-tumor drugs. RA-XII, one of the cyclopeptide glycosides isolated from Rubia yunnanensis, exerts anti-tumor effects on liver cancer. However, the underlying mechanisms are not clear. In the present study, the effects of RA-XII on lipogenesis were evaluated and the underlying mechanisms were investigated. The results indicated that RA-XII strongly inhibited tumor growth and lipogenesis (triglycerides and lipid droplets) in HepG2 cells, and the expression of key factors involved in lipogenesis (SREBP, SCD, FASN) was also obviously downregulated. Further investigation showed that the anti-tumor effects of RA-XII were attenuated by SREBP knockdown. Moreover, RA-XII downregulated the expression of SREBP cleavage-activating protein (SCAP), an upstream regulator of SREBP, and siRNA of SCAP prevented its restrained effects on tumor growth and lipogenesis. In addition, the in vivo experiment showed that RA-XII strongly restrained the lipogenesis and growth of liver tumor in nude mice xenograft model. Taken together, these results indicate that RA-XII suppresses the liver cancer growth by inhibition of lipogenesis via SCAP-dependent SREBP suppression. The findings reveal the potentials of RA-XII to be used in a novel therapeutic approach for treating liver cancer.

Decrease of MLK4 prevents hepatocellular carcinoma (HCC) through reducing metastasis and inducing apoptosis regulated by ROS/MAPKs signaling

Hepatocellular carcinoma (HCC) results in large amounts of deaths each year worldwide. To develop more effective treatments for HCC, it is very necessary to define the molecular mechanisms in hepatocarcinogenesis. Mixed lineage kinase (MLK)-4 is a member of the MLK family of mitogen-activated protein kinase kinase kinases, and modulates different cellular responses. However, its role in the meditation of HCC progression remains unclear. In the study, we found that MLK4 was over-expressed in tumor samples of HCC patients. High MLK4 expression was significantly associated with shorter overall survival in HCC. Knockdown of MLK4 inhibited HCC cell proliferation and metastasis, which was partly through reducing matrix metalloproteinase (MMP)-13, MMP2, enhancer of zeste homolog 2 (EZH2) and Vimentin expressions. Apoptosis was significantly induced by MLK4 knockdown in HCC cells via decreasing Bcl-2 and increasing cleaved poly (ADP-ribose) polymerase (PARP), Caspase-7 and -3 expression levels. In addition, MLK4 silence led to a significant reactive oxygen species (ROS) production in liver cancer cells, accompanied with elevated expression of phosphorylated p38, c-Jun N-terminal kinase (JNK) and ERK1/2. Notably, reducing ROS generation and blocking MAPKs (p38/JNK/ERK1/2) signaling markedly abrogated MLK4 knockdown-induced apoptosis in HCC cells. Moreover, MLK4 silence-prevented metastasis was also rescued by scavenging ROS generation and repressing MAPKs pathway. In vivo, injection of MLK4 siRNA markedly inhibited liver tumor growth in xenograft models, and MLK4 knockdown reduced HCC lung metastasis. Together, our study indicated the essential function of MLK4 in HCC progression, providing crucial therapeutic hypothesis for the prevention of hepatocellular carcinoma.

The GalNAc-T Activation (GALA) Pathway: Drivers and markers

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway.

Polymeric Drug Delivery System with Actively Targeted Cell Penetration and Nuclear Targeting for Cancer Therapy.

Targeted tumor cell killing using polymeric micelles with active targeting strategies has been demonstrated to be effectively therapeutic for liver cancers. To implement this strategy, enhancing the cellular uptake of the drug delivery system with targeted anticancer drugs, such as doxorubicin toward nuclear targeting, is of vital importance for increasing drug efficiency and reducing the systemic side effects of encapsulated drugs. In this study, a multifunctional polymeric drug delivery system was designed with actively targeted cell penetration and nuclear targeting for efficient cancer therapy. The nanocarriers were self-assembled from poly(ethylene glycol)-block-poly(ε-caprolactone), decorated with folic acid (FA-PECL) for active targeting via amide reaction for selective delivery of drugs to tumors. A cell penetration peptide (CPP) was decorated with doxorubicin (DOX), and the conjugate (CPP-DOX) was encapsulated in the carrier system for efficient cell penetration and nuclear targeting of drugs. An in vitro study showed an enhanced in vitro cytotoxicity and showed that the tumor volume decreased more than 5 times compared with the nontargeted system, by utilizing the drug-loaded system (FA-PECL/CPP-DOX) with active tumor cell targeting and subsequent nuclear targeting. The FA-PECL/CPP-DOX drug-loading system was well-targeted and enriched on tumor sites, resulting in significant suppression of the liver tumor growth.

PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis

BackgroundAltered glucose metabolism endows tumor cells with metabolic flexibility for biosynthesis requirements. Phosphoenolpyruvate carboxykinase 1 (PCK1), a key enzyme in the gluconeogenesis pathway, is downregulated in hepatocellular carcinoma (HCC) and predicts poor prognosis. Overexpression of PCK1 has been shown to suppress liver tumor growth, but the underlying mechanism remains unclear.MethodsmRNA and protein expression patterns of PCK1, AMPK, pAMPK, and the CDK/Rb/E2F pathway were determined using qRT-PCR and western blotting. Cell proliferation ability and cell cycle were assessed by MTS assay and flow cytometric analysis. The effect of PCK1 on tumor growth was examined in xenograft implantation models.ResultsBoth gain and loss-of-function experiments demonstrated that PCK1 deficiency promotes hepatoma cell proliferation through inactivation of AMPK, suppression of p27Kip1 expression, and stimulation of the CDK/Rb/E2F pathway, thereby accelerating cell cycle transition from the G1 to S phase under glucose-starved conditions. Overexpression of PCK1 reduced cellular ATP levels and enhanced AMPK phosphorylation and p27Kip1 expression but decreased Rb phosphorylation, leading to cell cycle arrest at G1. AMPK knockdown significantly reversed G1-phase arrest and growth inhibition of PCK1-expressing SK-Hep1 cells. In addition, the AMPK activator metformin remarkably suppressed the growth of PCK1-knockout PLC/PRF/5 cells and inhibited tumor growth in an orthotropic HCC mouse model.ConclusionThis study revealed that PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis and supports a potential therapeutic and protective effect of metformin on HCC.

Abstract B095: Mapping the genetic features of immune checkpoint responsiveness using AAV-CRISPR mediated in vivo screen

Abstract Immune checkpoint blockade has achieved tremendous clinical success across many tumor types, but fails to induce clinical responses in many patients. The mechanisms underlying checkpoint resistance remain poorly characterized. Recent studies have applied next generation sequencing techniques to catalog the mutational burden of patient tumors, which provides a wealth of data to determine common mutations. To map the genetic features of response to checkpoint blockade immunotherapy as well as correlating the clinical efficacy with certain mutations, we developed a novel direct in vivo CRISPR screening approach for high-throughput profiling of functional cancer drivers in an autochthonous manner by injecting AAVs carrying an sgRNA library targeting the top 50 TCGA pan-cancer recurrently mutated tumor suppressor genes (mTSG) into the immunocompetent Cas9 transgenic mice. All mice that received the AAV-mTSG library developed liver cancer and died within four months. We then utilized MIP sequencing of sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis as well as identifying specific gene pairs that were co-occurring across mice. Using this approach, we also mapped the mutation landscape changes under the pressures of immune checkpoint inhibitors, anti-PD1 or anti-CTLA4. We monitored liver tumor growth in AAV-mTSG injected LSL-Cas9;LSL-Fluc mice by using intravital bioluminescent imaging system (IVIS) in combination with dissection check before drug administration. Using IVIS data, we grouped them into 3 size-matched cohorts to receive anti-PD1 or anti-CTLA4 treatments or PBS control. According to the survival data, the mice with mTSG-induced liver tumor benefit from anti-PD1 or anti-CLTA4 treatment. By comparing the mutation frequencies of liver tumors in the mice receiving either checkpoint inhibitors or PBS treatment, we mapped the mutation landscape changes associated with anti-PD1 or anti-CTLA4 treatment. We are performing validation studies on top targets such as Arid1a, Stk11, and B2M. Using this approach, we systematically mapped the correlation of these top 50 driver mutations with cancer immune evasion and immunotherapy responsiveness, providing a valuable reference for patient stratification when considering immunotherapy as well as novel targets for synergistic interventions. Citation Format: Guangchuan Wang, Ryan Chow, Zhigang Bai, Lupeng Ye, Sidi Chen. Mapping the genetic features of immune checkpoint responsiveness using AAV-CRISPR mediated in vivo screen [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B095.