Carcinogen-induced Hepatocellular Carcinoma Research Articles

MLK3 promotes prooncogenic signaling in hepatocellular carcinoma via TGFβ pathway.

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. Analysis of TCGA databases suggested elevated MAP3K11 (MLK3 gene) expression, and TMA studies showed higher MLK3 activation in human HCCs. To understand MLK3's role in HCC, we utlized carcinogen-induced HCC model and compared between wild-type and MLK3 knockout (MLK3-/-) mice. Our studies showed that MLK3 kinase activity is upregulated in HCC, and MLK3 deficiency alleviates HCC progression. MLK3 deficiency reduced proliferation in vivo and MLK3 inhibition reduced proliferation and colony formation in vitro. To obtain further insight into the mechanism and identify newer targets mediating MLK3-induced HCCs, RNA-sequencing analysis was performed. These showed that MLK3 deficiency modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promoted EMT via autocrine TGFβ signaling. Moreover, MLK3 deficiency attenuated activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes HSC activation via paracrine TGFβ signaling. These findings reveal TGFβ playing a key role at different steps of HCC, downstream of MLK3, implying MLK3-TGFβ axis to be an ideal drug target for advanced HCC management.

Peritumoral HLJ1-STAT3 pathway investigation in carcinogen-induced hepatocellular carcinoma.

e16251 Background: Hepatocellular carcinoma (HCC) is a major global health issue and its treatment options have been limited. Therefore, it is urgent to develop cancer biomarkers for defining cancer risk, diagnosis, prognosis, and even finding new therapeutic targets. Human Liver DnaJ-Like Protein (HLJ1), which belongs to DnaJ heat shock protein family (HSP40) member B4 (DNAJB4), has been identified as a tumor suppressor in various cancers including lung cancer and colorectal cancer. Though the biological function of HLJ1 in either cancer or the liver has emerged in recent years, the precise role of HLJ1 in HCC carcinogenesis is still unclear. Methods: Samples for gene expression cDNA microarray were from 6-8 weeks wild-type (WT) and HLJ1 knockout (HLJ1-/-) mice liver. For the long-term carcinogenesis study, both mice are injected with 20 mg/kgbw diethylnitrosamine (DEN) at postnatal day 15. After that, 50 mg/kgbw DEN was administrated weekly to mice between the ages of 4 to 12 weeks. Mice were sacrificed at ages 30 and 36 weeks to assess the tumor count and size. For the short-term DEN study, 6-8-week-old mice were injected with 100 mg/kg DEN, and 24 and 48 hours after administration the liver tissues were frozen and fixed for western blotting and IHC analysis. Cancer cell transplantation was performed by intrasplenic injection of HLJ1-wildtype LLC and B16F1 cells into both genotypes. Clinical information of 362 liver cancer patients from the TCGA database was analyzed for the correlation between HLJ1 expression and survival. Results: In cDNA microarray analysis, We demonstrated that HLJ1 deficiency led to HCC gene signatures associated with activated IL-6/STAT3 signaling pathways. We used the DEN as a carcinogen and found STAT3 and H2AX phosphorylation induced by short-term DEN treatment was amplified in HLJ1-deficient mice. In long-term DEN experiments, HLJ1 deletion promoted tumor initiation and proliferation at the early and late stages of HCC progression accompanied by pronounced STAT3 phosphorylation in the normal part instead of the tumor part. Furthermore, transplantation of HLJ1-wildtype B16F1 and LLC cell lines into HLJ1-deficient mice led to more tumorigenic phenotype than into wildtype mice. Clinical information of 362 liver cancer patients from the TCGA database exhibited that higher expression of HLJ1 in the tumor part is correlated to a lower disease-specific survival rate. Conclusions: In summary, HLJ1 protects against DEN-induced HCC hepatocarcinogenesis through the regulation of STAT3 signaling in the peritumoral normal cells. Hence, HLJ1 can be a prognostic biomarker for liver cancer and a promising therapeutic target for HCC in the future.

ATF4 suppresses hepatocarcinogenesis by inducing SLC7A11 (xCT) to block stress-related ferroptosis

Hepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown. Hepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development. Hepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH. Despite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset. Liver cancer or hepatocellular carcinoma (HCC) is associated with multiple aetiologies. Most HCC aetiologies cause hepatocyte stress and death, as well as subsequent inflammation, and compensatory proliferation, thereby accelerating HCCdevelopment. The contribution of individual stress effectors to HCC and their underlying mechanisms of action were heretofore unknown. This study shows that the stress-responsive transcription factor ATF4 blunts liver damage and cancer development by suppressing iron-dependent cell death (ferroptosis). Although ATF4 ablation prevents hepatic steatosis, it also increases susceptibility to ferroptosis, due to decreased expression of the cystine/glutamate antiporter SLC7A11, whose expression in human HCC and NASH correlates with ATF4. These findings reinforce the notion that benign steatosis may be protective and does not increase cancer risk unless accompanied by stress-induced liver damage. These results have important implications for prevention of liver damage and cancer.

Discovery of Novel Substrate-Competitive Lysine Methyltransferase G9a Inhibitors as Anticancer Agents.

Identification of structurally novel inhibitors of lysine methyltransferase G9a has been a subject of intense research in cancer epigenetics. Starting with the high-throughput screening (HTS) hit rac-10a obtained from the chemical library of the University of Tokyo Drug Discovery Initiative, the structure-activity relationship of the unique substrate-competitive inhibitors was established with the help of X-ray crystallography and fragment molecular orbital (FMO) calculations for the ligand-protein interaction. Further optimization of the in vitro characteristics and drug metabolism and pharmacokinetics (DMPK) properties led to the identification of 26j (RK-701), which is a structurally distinct potent inhibitor of G9a/GLP (IC50 = 27/53 nM). Compound 26j exhibited remarkable selectivity against other related methyltransferases, dose-dependent attenuation of cellular H3K9me2 levels, and tumor growth inhibition in MOLT-4 cells in vitro. Moreover, compound 26j showed inhibition of tumor initiation and growth in a carcinogen-induced hepatocellular carcinoma (HCC) in vivo mouse model without overt acute toxicity.

Discovery of an Orally Efficacious MYC Inhibitor for Liver Cancer Using a GNMT-Based High-Throughput Screening System and Structure-Activity Relationship Analysis.

Glycine-N-methyl transferase (GNMT) downregulation results in spontaneous hepatocellular carcinoma (HCC). Overexpression of GNMT inhibits the proliferation of liver cancer cell lines and prevents carcinogen-induced HCC, suggesting that GNMT induction is a potential approach for anti-HCC therapy. Herein, we used Huh7 GNMT promoter-driven screening to identify a GNMT inducer. Compound K78 was identified and validated for its induction of GNMT and inhibition of Huh7 cell growth. Subsequently, we employed structure-activity relationship analysis and found a potent GNMT inducer, K117. K117 inhibited Huh7 cell growth in vitro and xenograft in vivo. Oral administration of a dosage of K117 at 10 mpk (milligrams per kilogram) can inhibit Huh7 xenograft in a manner equivalent to the effect of sorafenib at a dosage of 25 mpk. A mechanistic study revealed that K117 is an MYC inhibitor. Ectopic expression of MYC using CMV promoter blocked K117-mediated MYC inhibition and GNMT induction. Overall, K117 is a potential lead compound for HCC- and MYC-dependent cancers.

From the Editor's Desk...

From the Editor's Desk...

Notch activity characterizes a common hepatocellular carcinoma subtype with unique molecular and clinicopathologic features

The hepatocyte Notch pathway is a pathogenic factor in non-alcoholic steatohepatitis (NASH)-associated fibrosis, but its role in hepatocellular carcinoma (HCC) is less well defined. Herein, we aimed to characterize the molecular and clinical features of Notch-active human HCC, and to investigate the mechanisms by which Notch affects NASH-driven HCC. Using a 14-gene Notch score, we stratified human HCCs from multiple comprehensively profiled datasets. We performed gene set enrichment analyses to compare Notch-active HCCs with published HCC subtype signatures. Next, we sorted Notch-active hepatocytes from Notch reporter mice for RNA sequencing and characterized Notch-active tumors in an HCC model combining a carcinogen and a NASH-inducing diet. We used genetic mouse models to manipulate hepatocyte Notch to investigate the sufficiency and necessity of Notch in NASH-driven tumorigenesis. Notch-active signatures were found in ~30% of human HCCs that transcriptionally resemble cholangiocarcinoma-like HCC, exhibiting a lack of activating CTNNB1 (β-catenin) mutations and a generally poor prognosis. Endogenous Notch activation in hepatocytes is associated with repressed β-catenin signaling and hepatic metabolic functions, in lieu of increased interactions with the extracellular matrix in NASH. Constitutive hepatocyte Notch activation is sufficient to induce β-catenin-inactive HCC in mice with NASH. Notch and β-catenin show a pattern of mutual exclusivity in carcinogen-induced HCC; in this mouse model, chronic blockade of Notch led to β-catenin-dependent tumor development. Notch activity characterizes a distinct HCC molecular subtype with unique histology and prognosis. Sustained Notch signaling in chronic liver diseases can drive tumor formation without acquiring specific genomic driver mutations. The Notch signaling pathway is known to be involved in the pathogenesis of liver fibrosis. However, its role in liver cancer has not been well defined. Herein, we show that Notch activity is increased in a subset of liver cancers and is associated with poor outcomes. We also used a mouse model to show that aberrant Notch activity can drive cancer progression in obese mice.

Early Exposure to Gut Microbiome Reduces Hepatocellular Carcinoma Risks in Mice

Aims. Liver cancer is a multietiological disease that has multiple factors contributing to the hepatocarcinogenic process, e.g., hepatitis viruses, carcinogens, male sex, or metabolic factors. Notably, emerging evidence reported that gut microbiota is crucial to the pathogenesis of hepatocellular carcinoma (HCC) via activation of innate immunity. However, the effect of time to gut microbiota exposure after birth is unknown. Using a germ-free animal housing environment, instead of antibiotics, we examined the effects of various time-to-exposure (TTE) to gut microbiota durations on HCC risk. Methods. HBV or carcinogen-mediated spontaneous HCC models were implemented in this study. The HCC incidence rates in mice either kept germ-free (GF; that is, with no exposure to gut microbiota) or exposed to gut microbiota after being moved to a specific pathogen-free (SPF) housing environment and with various time-to-exposure (TTE) durations, namely, 5 weeks after birth, 10 weeks after birth, or since conception (that is, 5-week TTE group, 10-week TTE group, and SPF group, respectively), were recorded. The mice were sacrificed at 30 or 40 weeks after birth, and macro-/microscopic observations and pathological diagnosis were performed. Results. The incidence of liver tumors among the male mice was higher than that among the female mice in the carcinogen-induced HCC mice sacrificed at 40 weeks after birth (with P=0.011, 0.035, 0.0003, and 0.012, respectively, in the GF group, 5-week TTE group, 10-week TTE group, and SPF group). Similarly, in the HBV-HCC model, the incidence of liver tumors among the male mice was significantly higher than that among the female mice (with P=0.013, 0.020, 0.012, and 0.002, respectively, in the GF group, 5-week TTE group, 10-week TTE group, and SPF group). These results suggest that gut microbiota exposure is irrelevant to the male sex preference of HCC. Surprisingly, when comparing carcinogen-induced HCC male mice in the 10-week TTE group (90%; n=10), 5-week TTE group (56%; n=9), and SPF group (30%; n=10) (P=0.020), we found that the incidence of liver tumors was higher in the mice with later exposure to gut microbiome. Similarly, when comparing HBV-HCC male mice in the 10-week TTE group (100%; n=11), 5-week TTE group (70%; n=10), and SPF group (33%; n=9) (P=0.080), we also found that the incidence of liver tumors was higher in the mice with later exposure to gut microbiome. Conclusions. Early (prepubertal) exposure to gut microbiome reduces the risk of HCC development, indicating a potentially important factor for cancer surveillance. Exploring the mechanisms by which such exposure affects HCC risk might lead to novel cancer vaccines.

Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

Sestrin 3 (Sesn3) belongs to a small protein family that has been implicated in multiple biological processes including anti-oxidative stress, anti-aging, cell signaling, and metabolic homeostasis. However, the role of Sesn3 in hepatocellular carcinoma (HCC) remains unclear. Here we generated a Sesn3 knockout mouse model and induced HCC development by a combination of a single dose of diethylnitrosamine and chronic feeding of a choline deficient-high fat diet. After 6 months of the dietary treatment, Sesn3 knockout mice developed more severe HCC with higher levels of alpha-fetoprotein, arginase 1, and cytokeratin 19, but also higher metastatic rates than wild-type mice. Histological analysis revealed elevated extracellular matrix and cancer stem cell markers including Acta2, Cd44, and Cd133. Signaling analysis showed activated IL6-Stat3 and Akt pathways. Biochemical and microscopic analyses uncovered a novel inhibitory regulation of Gli2, a downstream transcription factor of the hedgehog signaling, by Sesn3. Two of the Gli2-regulated genes – Pdgfrb and Cd44 were upregulated in the Sesn3-deficient liver tissue. In conclusion, our data suggest that Sesn3 plays a critical tumor suppressor role in the liver partly through the inhibition of the hedgehog signaling.

Eicosapentaenoic acid attenuates obesity-related hepatocellular carcinogenesis.

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of obesity, is an emerging risk factor for hepatocellular carcinoma (HCC). Accumulating evidence has shown that chronic inflammation represents a plausible link between obesity and HCC and that the pro-inflammatory cytokine interleukin (IL)-6 contributes to the development of obesity-related HCC. In the present study, we aimed to examine the therapeutic potential of the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which exerts anti-inflammatory effects. The results showed that the development of carcinogen-induced HCC was significantly less in mice fed a high-fat diet (HFD) supplemented with EPA than in those fed HFD only, suggesting that EPA attenuates the development of obesity-related HCC. Although EPA did not appear to affect obesity-linked inflammation, it suppressed the activation of the pro-tumorigenic IL-6 effector STAT3, contributing to the inhibition of tumor growth. These findings suggest a clinical implication of EPA as a treatment for obesity-related HCC.

Abstract 3056: Genetic deletion of insulin-like growth factor binding protein-7 (IGFBP7) promotes hepatocellular carcinoma (HCC): a novel role of IGFBP7 in regulating antitumor immune surveillance

Abstract In the US, the incidence and mortality rates of hepatocellular carcinoma (HCC) are alarmingly increasing since no effective therapy is available for the advanced disease. Activation of IGF signaling is a major oncogenic event in diverse cancers, including HCC. Insulin-like growth factor binding protein-7 (IGFBP7) inhibits IGF signaling by binding to IGF-1 receptor (IGF-1R) and functions as a potential tumor suppressor for hepatocellular carcinoma (HCC). IGFBP7 abrogates tumors by inducing cancer-specific senescence and apoptosis and inhibiting angiogenesis. We now document that Igfbp7 knockout (Igfbp7-/-) mouse shows constitutive activation of IGF signaling, presents with pro-inflammatory and immunosuppressive microenvironment, and develops spontaneous tumors in lungs and liver and markedly accelerated carcinogen-induced HCC. Loss of Igfbp7 resulted in increased proliferation and decreased senescence in hepatocytes and mouse embryonic fibroblasts that could be blocked by an IGF-1 receptor inhibitor. A significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- livers which was associated with marked inhibition in antigen cross-presentation by Igfbp7-/- dendritic cells. IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice which could be abolished by depletion of CD4+ or CD8+ T lymphocytes. Our studies unravel modulation of immune response as a novel component of pleiotropic mechanisms by which IGFBP7 suppresses HCC. Even though HCC has an immunosuppressive milieu, immune targeted therapies are beginning to demonstrate significant objective responses in clinical trials. IGFBP7 might be an effective anti-HCC therapeutic by directly inhibiting cancer cells and stimulating an anti-tumor immune response. Citation Format: Maaged A. Akiel, Chunqing Guo, Xia Li, Devaraja Rajasekaran, Rachel G. Mendoza, Nidhi H. Jariwala, Chadia L. Robertson, Mark A. Subler, Jolene J. Windle, Paul B. Fisher, Devanand Sarkar. Genetic deletion of insulin-like growth factor binding protein-7 (IGFBP7) promotes hepatocellular carcinoma (HCC): a novel role of IGFBP7 in regulating antitumor immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3056. doi:10.1158/1538-7445.AM2017-3056

Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models

Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC), a life-threatening malignancy with limited treatment options in the clinic that responds poorly to immunotherapy, remains to be investigated. Exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) were investigated in three different HCC mouse models systemically. Tumor growth and microenvironment were monitored. DEXAFP elicited strong antigen-specific immune responses and resulted in significant tumor growth retardation and prolonged survival rates in mice with ectopic, orthotopic and carcinogen-induced HCC tumors that displayed antigenic and pathological heterogeneity. The tumor microenvironment was improved in DEXAFP-treated HCC mice, demonstrated by significantly more γ-interferon (IFN-γ)-expressing CD8+ T lymphocytes, elevated levels of IFN-γ and interleukin-2, and fewer CD25+Foxp3+ regulatory T (Treg) cells and decreased levels of interleukin-10 and transforming growth factor-β in tumor sites. Lack of efficacy in athymic nude mice and CD8+ T cell-depleted mice showed that T cells contribute to DEXAFP-mediated antitumor function. Dynamic examination of the antitumor efficacy and the immune microenvironment in DEXAFP-treated orthotopic HCC mice at different time-points revealed a positive correlation between tumor suppression and immune microenvironment. Our findings provide evidence that AFP-enriched DEXs can trigger potent antigen-specific antitumor immune responses and reshape the tumor microenvironment in HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy. Lay summary: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC) remains unknown. Here, we investigated exosomes from HCC antigen-expressing DCs in three different HCC mouse models and proved their feasibility and capability of treating HCC, and thus provide a cell-free vaccine for HCC immunotherapy.

A pro-tumorigenic function of S100A8/A9 in carcinogen-induced hepatocellular carcinoma

Human hepatocellular carcinoma (HCC) is a heterogeneous disease, driven by different risk factors and presenting diverse clinicopathological features and outcomes. Epidemiological and experimental data indicate that the damage-associated molecular pattern molecules S100A8 and S100A9, forming a heterodimer called calprotectin, might be critically involved in HCC development. However, deletion of S100a9 in an inflammation- and cirrhosis-driven mouse model did not show any impairment in liver tumorigenesis, most likely due to functional compensation by other inflammatory cytokines. Here, we investigated the effect of calprotectin ablation in mice treated with diethylnitrosamine, a carcinogen-driven HCC model mimicking cancer development caused by acute liver damage in the absence of prominent chronic inflammation and tissue damage. We found that tumor cell proliferation was diminished in the absence of S100A8/A9, leading to significant reduction of tumor size.Our results demonstrate that calprotectin is required for the progression of non-inflammation driven liver tumor and might represent a therapeutic target for the treatment of HCC formed in non-cirrhotic liver.

The camKK2/camKIV relay is an essential regulator of hepatic cancer.

Hepatic cancer is one of the most lethal cancers worldwide. Here, we report that the expression of Ca(2+) /calmodulin-dependent protein kinase kinase 2 (CaMKK2) is significantly up-regulated in hepatocellular carcinoma (HCC) and negatively correlated with HCC patient survival. The CaMKK2 protein is highly expressed in all eight hepatic cancer cell lines evaluated and is markedly up-regulated relative to normal primary hepatocytes. Loss of CaMKK2 function is sufficient to inhibit liver cancer cell growth, and the growth defect resulting from loss of CaMKK2 can be rescued by ectopic expression of wild-type CaMKK2 but not by kinase-inactive mutants. Cellular ablation of CaMKK2 using RNA interference yields a gene signature that correlates with improvement in HCC patient survival, and ablation or pharmacological inhibition of CaMKK2 with STO-609 impairs tumorigenicity of liver cancer cells in vivo. Moreover, CaMKK2 expression is up-regulated in a time-dependent manner in a carcinogen-induced HCC mouse model, and STO-609 treatment regresses hepatic tumor burden in this model. Mechanistically, CaMKK2 signals through Ca(2+) /calmodulin-dependent protein kinase 4 (CaMKIV) to control liver cancer cell growth. Further analysis revealed that CaMKK2 serves as a scaffold to assemble CaMKIV with key components of the mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway and thereby stimulate protein synthesis through protein phosphorylation. The CaMKK2/CaMKIV relay is an upstream regulator of the oncogenic mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway, and the importance of this CaMKK2/CaMKIV axis in HCC growth is confirmed by the potent growth inhibitory effects of genetically or pharmacologically decreasing CaMKK2 activity; collectively, these findings suggest that CaMKK2 and CaMKIV may represent potential targets for hepatic cancer.

P0243 : Role of S100A8/A9 in carcinogen-induced hepatocellular carcinoma onset and development

P0243 : Role of S100A8/A9 in carcinogen-induced hepatocellular carcinoma onset and development

TIMP3 controls cell fate to confer hepatocellular carcinoma resistance.

Inflammation enables human cancers and is a critical promoter of hepatocellular carcinoma (HCC). TIMP3 (Tissue inhibitor of metalloproteinase 3), a natural metalloproteinase inhibitor, controls cytokine and growth factor bioavailability to keep inflammation in check and regulate cell survival in the liver. TIMP3 is also found silenced in human cancers. We therefore tested whether Timp3 affects HCC predisposition. Remarkably, genetic loss of Timp3 protected from carcinogen-induced HCC through the immediate engagement of several tumor suppressor pathways, while tumor necrosis factor (TNF) signaling was dispensable for this protection. All wild-type mice developed HCC by 12 months, whereas HCC incidence was reduced to 33% at 12 months and 57% at 15 months in Timp3 null mice. Upon acute carcinogen treatment the deficient livers exhibited greater cytokine expression, but lower cell death and higher hepatocyte senescence. We found that precocious activation of p53, p38 and Notch preceded senescence and hepatic cell differentiation, and these events were conserved throughout tumorigenesis. Timp3-deficient mouse embryo fibroblasts also responded to carcinogen by favoring senescence over apoptosis. We conclude that Timp3 status determines p53, p38 and Notch coactivation to instruct hepatic cell fate and transformation and uncover mechanisms that are protective even within a pro-inflammatory microenvironment.

Androgen receptor enhances cell adhesion and decreases cell migration via modulating β1-integrin-AKT signaling in hepatocellular carcinoma cells

The androgen receptor (AR) has been shown to promote the initiation and development of hepatocellular carcinoma (HCC) during the early stage of the disease process and to suppress HCC cell invasion during the later stages of the disease. The mechanisms governing these dual yet opposite roles have yet to be elucidated. Using carcinogen-induced HCC in vivo mouse models and the in vitro human HCC cell line SKhep1, we found that knockout of AR in primary HCC cells led to a decrease in HCC cell focal adhesion capacity compared to cells from wildtype mice. Similar results were obtained after adding functional AR into human HCC SKhep1 cells. Further analysis revealed that the role AR plays in adhesion of HCC cells is governed, at least in part, by its ability to up-regulate β1-integrin and activate the PI3K/AKT pathway. We also found that AR-β1-integrin-mediated cell adhesion suppresses cell migration. Those findings indicate that the AR-β1-integrin-PI3K/AKT signaling pathway might play a role in the bimodal function of AR on cell adhesion and migration at the cellular level.

Engineering AFP and GPC3 to create highly immunogenic gene vaccines to prevent carcinogen-induced murine autochthonous hepatocellular carcinoma (VAC12P.1015)

Abstract In this study, we investigated whether alpha-fetoprotein (AFP) and glypican 3 (GPC3) could be engineered to create effective vaccines to potently activate CD8 T cells to prevent clinically relevant carcinogen-induced autochthonous hepatocellular carcinoma (HCC) in mice. We found that the approach of epitope-optimization created a highly immunogenic AFP and GPC3 and that immunization with lentivector expressing the epitope-optimized AFP and GPC3, but not wild-type AFP and GPC3, potently activated CD8 T cells. Critically, the activated CD8 T cells not only cross-recognized native AFP peptides, but also recognized and killed tumor cells expressing native AFP and GPC3 protein. Immunization with lentivector expressing optimized AFP, but not native AFP, completely protected mice from tumor challenge and reduced the incidence of carcinogen-induced autochthonous HCC. In addition, prime-boost immunization with the optimized AFP significantly increased the frequency of AFP-specific memory CD8 T cells in the liver that were highly effective against emerging HCC tumor cells, further enhancing the tumor prevention of carcinogen-induced autochthonous HCC. Our data demonstrate that epitope-optimization is required to potently activate AFP- and GPC3- specific CD8 T cells, generating effective antitumor effect to prevent carcinogen-induced HCC in mice. Our study provides a roadmap to develop effective human HCC vaccines that may result in an improved antitumor effect.

CCAAT/Enhancer-Binding Protein Homologous (CHOP) Protein Promotes Carcinogenesis in the DEN-Induced Hepatocellular Carcinoma Model

Background and AimsC/EBP homologous protein (CHOP) plays pro-apoptotic roles in the integrated stress response. Recently, a tumor suppressive role for CHOP was demonstrated in lung cancer via regulation of tumor metabolism. To explore the role of CHOP in hepatocarcinogenesis, we induced hepatocellular carcinoma (HCC) in wild type (wt) and CHOP knockout (KO) mice using the carcinogen N-diethylnitrosamine (DEN).ResultsAnalysis of tumor development showed reduced tumor load, with markedly smaller tumor nodules in the CHOP KO animals, suggesting oncogenic roles of CHOP in carcinogen-induced HCC. In wt tumors, CHOP was exclusively expressed in tumor tissue, with minimal expression in normal parenchyma. Analysis of human adenocarcinomas of various origins demonstrated scattered expression of CHOP in the tumors, pointing to relevance in human pathology. Characterization of pathways that may contribute to preferential expression of CHOP in the tumor identified ATF6 as a potential candidate. ATF6, a key member of the endoplasmic reticulum stress signaling machinery, exhibited a similar pattern of expression as CHOP and strong activation in wt but not CHOP KO tumors. Because HCC is induced by chronic inflammation, we assessed whether CHOP deficiency affects tumor-immune system crosstalk. We found that the number of macrophages and levels of IFNγ and CCL4 mRNA were markedly reduced in tumors from CHOP KO relative to wt mice, suggesting a role for CHOP in modulating tumor microenvironment and macrophage recruitment to the tumor.ConclusionOur data highlights a role for CHOP as a positive regulator of carcinogen-induced HCC progression through a complex mechanism that involves the immune system and modulation of stress signaling pathways.

BEZ235 in combination with everolimus for advanced solid malignancies: Preliminary results of a phase Ib dose-escalation study.

e13518 Background: The mammalian target of rapamycin (mTOR) is a critical signaling pathway in many tumors including cancers of the breast and colon, glioblastoma multiforme, and hepatocellular carcinoma (HCC). Preclinical studies demonstrated the combination of BEZ235, a competitive dual phosphatidylinositide 3-kinase (PI3K)/mTOR inhibitor, and the mTOR inhibitor, everolimus led to greater regression of a carcinogen-induced HCC than treatment with higher doses of either drug alone. Based on this, we initiated a study of BEZ235 combined with everolimus in patients with advanced solid tumors. Methods: A single institution phase Ib dose-escalation study. Patients with advanced solid malignancies, no available standard of care treatment option, and ECOG performance status 0-2 were eligible. Prior treatment with PI3K inhibitors was not allowed. Sequential cohorts of 3-6 patients were treated. The starting dose was everolimus 2.5 mg and BEZ235 200 mg daily in an oral sachet formulation. Cohort 2 received everolimus 2.5 mg and BEZ235 400 mg daily. Pharmacokinetic and pharmacodynamic studies were performed during the first cycle. The phosphorylation of specific downstream effectors of the mTOR pathway was assessed in peripheral blood mononuclear cells (PBMC). Results: Eleven patients, median age 58 (36-73 years) were treated. Tumors included non-small cell lung cancer, colon cancer, and glioblastoma, hepatocellular carcinoma, pancreatic cancer, esophageal cancer, adenoid cystic carcinoma of the larynx, and appendiceal carcinoma. Four patients were treated on cohort 1. None experienced dose-limiting toxicity. Seven patients were treated on the second dose cohort. One patient withdrew necessitating replacement and another developed grade 3 stomatitis from herpes virus requiring cohort expansion. The most common adverse events were thrombocytopenia, lymphopenia, transaminitis, diarrhea, nausea and fatigue. No tumor responses were noted. PBMC showed a decrease in 4E-BP1S65 phosphorylation on day 28 in 2 of 3 patients in cohort 1. Conclusions: The combination of BEZ235 and everolimus was well tolerated at these doses. The trial remains open to accrual. Clinical trial information: NCT01508104.