HCC Model Research Articles

An open-label phase Ib/2 trial of TRC105 plus sorafenib in patients with advanced/metastatic hepatocellular carcinoma (HCC) (NCT01806064).

268 Background: TRC105, an endoglin antibody, potentiates the activity of sorafenib (S) in preclinical HCC models, and TRC105 + S demonstrated a 33% partial response rate (5/15 pts) by RECIST, at RP2D doses of TRC105 in HCC pts ( Clin Can Res 2017). Adverse events characteristic of each drug were not increased in frequency or severity when the two drugs were administered concurrently. Methods: P1: Compare wkly TRC105 dosing vs four wkly doses followed by every other wkly dosing + S 800 mg daily. P2: Four objective responses are required in 21 pts to reject the null hypothesis that the true response rate probability is < 5% with an alpha level of 0.1 and 80% power. Key inclusion criteria: disease not amendable to surgical or local therapies, ECOG ≤ 1; Child-Pugh A or B (7 points) classification. Results: Thirteen pts were enrolled in phase 1b at TRC105 10 mg/kg wkly for four doses and 15 mg/kg every other week thereafter + S. Mean serum levels of TRC105 exceeded the target conc. following 4 wkly doses of TRC105 at 10 mg/kg (mean = 34 µg/ml, range BLOQ-80). Mean trough conc. decreased following every other week dosing (mean = 13 µg/ml, range BLOQ-31), resulting in infusion reactions or a continued requirement for premedication. Therefore, wkly dosing of TRC105 at 10 mg/kg is the recommended Phase 2 dose. ADA were detected in 10 of 14 pts and correlated with lower than expected PK conc. Common TRC105 related AEs included ≤ G2 epistaxis, ≤ G2 fatigue and ≤ G2 headache. Common S related AEs included ≤ G3 fatigue, ≤ G3 hand foot syndrome and ≤G2 epistaxis. A total of 3 out of 14 evaluable patients (21%) enrolled in phase 1 and 2 achieved durable PR, 2 of these ongoing at week 45 and 17. Conclusions: TRC105 dosed at 10 mg/kg wkly was required to achieve target conc. due to higher clearance in HCC pts, which may have been influenced by a higher rate of ADA compared to studies of TRC105 in other tumor types. The combination of TRC105 + S demonstrated encouraging signs of activity, including durable PR in 2/9 evaluable pts in Phase 1b and 1/5 pts thus far in Phase 2. An additional 16 pts will be enrolled at the RP2D to assess the primary endpoint of ORR by RECIST. Clinical trial information: NCT01806064.

Ultrasmall hybrid protein-copper sulfide nanoparticles for targeted photoacoustic imaging of orthotopic hepatocellular carcinoma with a high signal-to-noise ratio.

Although photoacoustic imaging combined with second near infrared (NIR II) molecular probes for tumor diagnosis has drawn tremendous attention during the past few decades, the targeted photoacoustic imaging of orthotopic hepatocellular carcinoma (HCC) still remains a challenge due to high liver vascularization and non-specificity of probes in liver tumors. Herein, we report on cyclic arginine-glycine-aspartic acid (cRGD) peptide conjugated ultrasmall CuS nanoparticles (CuS@BSA-RGD NPs) which encapsulate bovine serum albumin (BSA) and possess high optical absorption at 1064 nm. The encapsulation of BSA results in great biocompatibility of CuS@BSA-RGD NPs along with excellent photostability and physiological stability. The cRGD conjugation enables the improvement of tumor uptake of CuS@BSA-RGD NPs by virtue of its positive tumor cell targeting capability. The efficient accumulation of CuS@BSA-RGD NPs in the tumor over time after intravenous administration to orthotopic HCC bearing mice was achieved, which resulted in highly sensitive photoacoustic visualization of the tumor region. Toxicity studies indicate that CuS@BSA-RGD NPs exhibited negligible systemic toxicity in vivo. The results demonstrate that the CuS@BSA-RGD NPs might hold great promise for future imaging and diagnosis of cancer.

The CCR2+ Macrophage Subset Promotes Pathogenic Angiogenesis for Tumor Vascularization in Fibrotic Livers.

Background & AimsHepatocellular carcinoma (HCC) typically arises in fibrotic or cirrhotic livers, which are characterized by pathogenic angiogenesis. Myeloid immune cells, specifically tumor-associated macrophages (TAMs), may represent potential novel therapeutic targets in HCC, complementing current ablative or immune therapies. However, the detailed functions of TAM subsets in hepatocarcinogenesis have remained obscure.MethodsTAM subsets were analyzed in-depth in human HCC samples and a combined fibrosis–HCC mouse model, established by i.p. injection with diethylnitrosamine after birth and repetitive carbon tetrachloride (CCl4) treatment for 16 weeks. Based on comprehensively phenotyping TAM subsets (fluorescence-activated cell sorter, transcriptomics) in mice, the function of CCR2+ TAM was assessed by a pharmacologic chemokine inhibitor. Angiogenesis was evaluated by contrast-enhanced micro–computed tomography and histology.ResultsWe show that human CCR2+ TAM accumulate at the highly vascularized HCC border and express the inflammatory marker S100A9, whereas CD163+ immune-suppressive TAM accrue in the HCC center. In the fibrosis–cancer mouse model, we identified 3 major hepatic myeloid cell populations with distinct messenger RNA profiles, of which CCR2+ TAM particularly showed activated inflammatory and angiogenic pathways. Inhibiting CCR2+ TAM infiltration using a pharmacologic chemokine CCL2 antagonist in the fibrosis–HCC model significantly reduced pathogenic vascularization and hepatic blood volume, alongside attenuated tumor volume.ConclusionsThe HCC microenvironment in human patients and mice is characterized by functionally distinct macrophage populations, of which the CCR2+ inflammatory TAM subset has pro-angiogenic properties. Understanding the functional differentiation of myeloid cell subsets in chronically inflamed liver may provide novel opportunities for modulating hepatic macrophages to inhibit tumor-promoting pathogenic angiogenesis.

Chemopreventive and antitumor effects of benzyl isothiocynate on HCC models: A possible role of HGF /pAkt/ STAT3 axis and VEGF.

Benzyl isothiocyanate (BITC) is a member of the isothiocyanate compounds that found in cruciferous vegetables. BITC has a potential anticancer effect in different types of tumors. Few studies referred to the antineoplastic effect of BITC against HCC. The mechanism of BITC concerning retardation of HCC progression is incompletely understood. This study evaluated the role of HGF, pAkt and STAT3 in BITC induced HCC growth retardation. HCC was induced in mice using diethylnitrosamine (DEN) 75 mg/kg once a week for 4 weeks. BITC 10 and 20 mg/kg was given to mice orally each day for 10 weeks. The HCC cell lines HepG2 and Huh-7 were also used to evaluate the effect of BITC on tumor cells behavior. Immunoassay was used to detect expressions of caspase-3 activity, VEGF, MMP-2, TNF-α, HGF and pAkt. STAT3 expression was detected in liver tissues using immunohistochemical staining. BITC has a potential role in suppressing hepatic precancerous lesion progression in mice. The drug increased caspase-3 activity in tumor cells and inhibited the angiogenic marker VEGF. It also decreased the metastatic marker MMP-2. This anticancer effect of BITC was observed in DEN treated mice as well as in hepatoma cell lines. The reported antineoplastic activity was correlated with downregulation of HGF and its downstream molecules pAkt and STAT3. The effect of BITC on HGF /pAkt/ STAT3 axis has a potential role in both chemopreventive and chemotherapeutic effects of BITC.

Abstract 162: UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common cancers worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and chemoresistant nature of this disease. Increasing evidence showed the critical role of cancer stem cells (CSCs) on tumor relapse and therapeutic resistance. Since normal stem cells and CSCs share high similarity, we would like to identify the molecules/pathways crucial in liver CSCs by determining what molecules involved in normal liver stem cells during liver regeneration. Comparison of expression profiles between early regenerating liver and intact liver revealed the upregulation of DNA Damage Response pathways in the self-renewing liver, in which Ubiquitin Conjugating Enzyme E2 T (UBE2T) was the most significantly upregulated. This, together with the publicly available dataset (GSE5975) shows upregulation of UBE2T in EpCAM-enriched liver CSC populations, suggest the potential role of UBE2T on regulation of cancer stemness. By qPCR analysis UBE2T mRNA level was overexpressed in 91% of the clinical HCC specimens (n=56) and associated with aggressive tumor behavior and poorer patients' survival. The overexpression of UBE2T was further confirmed by western blot and immunohistochemical analysis. By lentiviral based knock-down approach, we demonstrated the role of UBE2T in regulation of liver CSC properties, including self-renewal, tumorigenicity, drug resistance and expression of liver CSC markers. In orthotopic HCC model, UBE2T was found to play pivotal role in lung metastasis in vivo. Mechanistically, UBE2T is found to interact directly with E3 ligase Mule and regulate its protein expression via ubiquitination. Since Mule is found to directly degrade β-catenin protein, we are examining whether UBE2T mediates liver T-IC function through direct regulation of Mule-mediated β-catenin degradation. In conclusion, we have uncovered the novel role of UBE2T signaling cascade in regulation liver CSCs, which provides attractive therapeutic target for potential treatment of HCC. Citation Format: Pui Yu Ho, Kin Wah Lee. UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 162.

Abstract 4080: An inducible zebrafish model to dissect the role of beta catenin signaling in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third highest contributor to cancer-related mortality in the world. This disease is molecularly heterogeneous in nature, which obfuscates our understanding of the molecular etiology of the disease, giving rise to the essential and urgent need of delineating HCC mechanisms based on molecular subtypes. Various signaling pathways have been implicated in HCC development. The Wnt/β-catenin pathway remains of particular significance with more than a third of all HCC cases characterized by aberrant activation of the Wnt/β-catenin signaling axis. About 20% of HCC cases are characterized by activating mutations in the β-catenin gene (ctnnb1). These activating mutations prevent β-catenin degradation and allow it to enter the nucleus and activate transcription of various genes involved in cell proliferation and apoptosis. The need to elucidate the role of aberrant β-catenin gene activation in the development of liver malignancy is thwarted by the lack of relevant animal models. In mice, β-catenin mutation alone is not sufficient to induce HCC. To address this bottleneck, we have designed a vertebrate HCC model system of activated β-catenin-driven HCC in an inducible background using zebrafish (Danio rerio). In our previous work, we demonstrated that zebrafish expressing a mutated, phosphorylation-resistant, and constitutively-activated version of β-catenin develop HCC morphologically and transcriptionally similar to human HCC. Here, we describe a system in which spatially and temporally controlled expression of activated β-catenin is achieved using hepatocyte-specific, tamoxifen-induced expression of Cre recombinase. Using hepatocyte-specific constitutive/induced Cre-recombinase zebrafish lines together with floxed β-catenin and/or floxed fluorescent reporter lines, we show here that Cre recombinase is successfully able to switch on the expression of floxed transgenes in fishes at larval, juvenile and adult stages. Through histology and longitudinal studies, we are determining the ability of our inducible system to increase cellular β-catenin levels and Wnt signaling in the liver and to stimulate HCC when activated β-catenin is turned on at different developmental stages including adulthood. This inducible system will be useful for studying the impact of β-catenin activation on liver size and malignancy at different stages of liver development. The ability to induce the oncogene in adulthood will allow us to study HCC in a physiologically relevant setting. We will use this temporal and spatial control for further studies aimed at understanding the role of activated β-catenin in HCC initiation and developing treatment strategies. Citation Format: Srishti Kotiyal, Sharanya Maanasi Kalasekar, Kathryn Davis, Cindy Barba, Annika Young, Kimberley J. Evason. An inducible zebrafish model to dissect the role of beta catenin signaling in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4080.

Lenvatinib inhibits angiogenesis and tumor fibroblast growth factor signaling pathways in human hepatocellular carcinoma models.

Unresectable hepatocellular carcinoma (uHCC) is one of the most lethal and prevalent cancers worldwide, and current systemic therapeutic options for uHCC are limited. Lenvatinib, a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs), recently demonstrated a treatment effect on overall survival by statistical confirmation of noninferiority to sorafenib in a phase 3 study of uHCC. Here, we investigated mechanisms underlying the antitumor activity of lenvatinib in preclinical HCC models. In vitro proliferation assay of nine human HCC cell lines showed that lenvatinib selectively inhibited proliferation of FGF signal‐activated HCC cells including FGF19‐expressing Hep3B2.1‐7. Lenvatinib suppressed phosphorylation of FRS2, a substrate of FGFR1–4, in these cells in a concentration‐dependent manner. Lenvatinib inhibited in vivo tumor growth in Hep3B2.1‐7 and SNU‐398 xenografts and decreased phosphorylation of FRS2 and Erk1/2 within the tumor tissues. Lenvatinib also exerted antitumor activity and potently reduced tumor microvessel density in PLC/PRF/5 xenograft model and two HCC patient‐derived xenograft models. These results suggest that lenvatinib has antitumor activity consistently across diverse HCC models, and that targeting of tumor FGF signaling pathways and anti‐angiogenic activity underlies its antitumor activity against HCC tumors.

A novel function of platelets and CD40L in NAFLD-promoted HCC development

Abstract Hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer-related death worldwide. Chronic liver inflammation, such as non-alcoholic fatty liver disease (NAFLD), which is characterized by increased fat deposition in the liver, is one common cause of HCC. The prevalence of NAFLD is increasing rapidly with the growing epidemics of diabetes and obesity, but little is known about how NAFLD proceeds to HCC. Platelets, which are a critical component in hemostasis, have been shown to release CD40L after activation, and there is accumulating evidence for an important role of platelets in adaptive immunity. Platelet-derived CD40L, by interacting with its receptor CD40, is able to induce dendritic cell maturation, prime T cells, and stimulate B cell isotype switching. NAFLD patients have increased mean platelet volume, which is an indicator of platelet activation, and an increased level of CD40L in their serum. However, this potential role of platelets in NAFLD-promoted HCC is completely unknown. Here we studied the platelet function in a murine HCC model of NAFLD and HCC. We found that inhibition of platelet activation using Aspirin and Clopidogrel greatly increased HCC incidence. Similar results were found when mice were treated with a CD40L blocking antibody, while the combination treatment did not show an additive effect. However, Aspirin and Clopidogrel treatment had no effect on HCC in the absence of NAFLD, while using only a CD40 activating antibody significantly reduced HCC incidence without NAFLD. Finally, an increased CD40L level was found in the serum from mice with NAFLD using a bioassay. In summary, our results suggest an important role of platelets in NAFLD-promoted HCC, and will provide useful information for HCC treatment.

Abstract No. 495 Non-invasive liver tumor ablation using histotripsy in an in vivo subcutaneous murine hepatocellular carcinoma model

Histotripsy is a non-thermal, non-invasive ultrasound (US) ablation method that fractionates tissue through the precise control of acoustic cavitation guided by real-time US imaging. Histotripsy has the potential to improve treatment consistency and precision compared to thermal-based ablation methods. This study evaluates the feasibility and tumor volume reduction effects of histotripsy for liver cancer ablation in an in vivo murine HCC model. Subcutaneous xenograft tumors were generated by injecting human HCC Hep3B cells into 14 NSG mice (acute group A: treated n = 9, control n = 1 and chronic group B: treated n = 2, control n = 2) and 6 NOD-SCID mice (chronic group C: treated n = 6). Once tumors reached >5 mm, mice were treated by histotripsy using a custom built 1 MHz histotripsy transducer attached to a motorized positioner guided by US imaging system. 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa) were applied to the tumor volume. MRI was performed pre- and posttreatment to assess tumor ablation. Group A was sacrificed within 3 days post treatment. Groups B and C were monitored weekly using caliper measurements and MRI for 3 months or until tumors reached ∼1.8 cm. Tumor, brain and lung tissues were harvested for histology. Histotripsy-generated cavitation cloud and the treated region were visible on US imaging enabling real-time feedback. In group A, histopathology showed that the targeted region was completely fractionated into acellular debris with a sharp boundary. In groups B and C, MRI revealed effective tumor volume reduction post treatment as the homogenate and edema were resorbed within 3 weeks. However, as the subcutaneous tumor does not allow sufficient treatment margin, residual viable tumor cells developed into tumor regrowth at 3-9 weeks after treatment. Treated mice in group B survived 2-3 times longer than the control mice and showed no signs of metastasis in the lung and brain. At the time of submission, group C mice are being monitored until study endpoint. This study demonstrates the potential of histotripsy for non-invasive liver tumor ablation. Future work will study the biological response in immune competent orthotopic liver tumor models.

Region-based seed point cell segmentation and detection for biomedical image analysis

Salient region detection and segmentation from biological images is often a crucial step for image understanding. The initial contour selection during segmentation being a competent task and wrong differentiation between the foreground and background colours are compromised. In this paper, improved cell detection is introduced by using a region-based cell detection and segmentation method called Histogram Colour Contrast Seed Point Selection (HCC-SPS). In each pixel, the HCC model is able to group similar colour values, therefore addressing colour contrast in visual signal, resulting in accurate desired edge points. Second, considering the energy function, region-based seed point fine tunes the salient value and makes differentiation between salient and background points easier. Third, due to salient mapping function with pixel representation, the segmentation of biological images, done accurately. The results are compared with the existing system based on the parameters such as accuracy rate, segmentation time and mapping functions.

Alcoholic Liver Disease Accelerates Early Hepatocellular Cancer in aMouse Model.

HCC is a rapidly increasing cancer worldwide. Most HCC rises in the setting of chronic and advanced liver disease caused by viral hepatitis, alcohol use, non-alcoholic liver disease or their combination. We found that in the mouse model, alcohol alone does not induce HCC, however, it can promote HCC development after a carcinogen exposure. Multiple mechanisms are involved in carcinogenesis and alcohol affects many of those including epithelial-mesenchymal transition, cancer stem marker expression and inflammation as evidenced in our HCC model.

Region-based seed point cell segmentation and detection for biomedical image analysis

Salient region detection and segmentation from biological images is often a crucial step for image understanding. The initial contour selection during segmentation being a competent task and wrong differentiation between the foreground and background colours are compromised. In this paper, improved cell detection is introduced by using a region-based cell detection and segmentation method called Histogram Colour Contrast Seed Point Selection (HCC-SPS). In each pixel, the HCC model is able to group similar colour values, therefore addressing colour contrast in visual signal, resulting in accurate desired edge points. Second, considering the energy function, region-based seed point fine tunes the salient value and makes differentiation between salient and background points easier. Third, due to salient mapping function with pixel representation, the segmentation of biological images, done accurately. The results are compared with the existing system based on the parameters such as accuracy rate, segmentation time and mapping functions.

Pharmacologic Inhibition of the Menin-MLL Interaction Leads to Transcriptional Repression of PEG10 and Blocks Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for approximately 85% of malignant liver tumors and results in 600,000 deaths each year, emphasizing the need for new therapies. Upregulation of menin was reported in HCC patients and high levels of menin correlate with poor patient prognosis. The protein-protein interaction between menin and histone methyltransferase mixed lineage leukemia 1 (MLL1) plays an important role in the development of HCC, implying that pharmacologic inhibition of this interaction could lead to new therapeutic strategy for the HCC patients. Here, we demonstrate that the menin-MLL inhibitor MI-503 shows antitumor activity in in vitro and in vivo models of HCC and reveals the potential mechanism of menin contribution to HCC. Treatment with MI-503 selectively kills various HCC cell lines and this effect is significantly enhanced by a combination of MI-503 with sorafenib, the standard-of-care therapy for HCC. Furthermore, MI-503 reduces sphere formation and cell migration in in vitro HCC models. When applied in vivo, MI-503 gives a strong antitumor effect both as a single agent and in combination with sorafenib in mice xenograft models of HCC. Mechanistically, treatment with MI-503 downregulates expression of several genes known to play a critical role in proliferation and migration of HCC cells, including PEG10, and displaces the menin-MLL1 complex from the PEG10 promoter, resulting in reduced H3K4 methylation and transcriptional repression. Overall, our studies reveal a mechanistic link between menin and genes involved in HCC and demonstrate that pharmacologic inhibition of the menin-MLL interaction might represent a promising therapeutic approach for HCC. Mol Cancer Ther; 17(1); 26-38. ©2017 AACR.

Celastrol delays hepatic steatosis and carcinogenesis in a rapid AKT/c-Met-transfected hepatocellular carcinoma model via suppressing fatty acid synthase expression and AKT/ERK phosphorylation.

Although the suppressing effects of celastrol on hepatocellular carcinoma (HCC) have been demonstrated, evidence for the targeting of fatty acid synthetase (FASN) in the development of HCC by celastrol is still rare. In this study, the effect of celastrol on a rapid HCC model featuring co-activation of AKT/c-Met oncogenes in mice was studied. The effect of celastrol on the alpha-fetoprotein level in the liver and serum was also investigated. Protein expressions of PCNA, Ki67 and FASN in celastrol-treated AKT/c-Met HCC mice were observed. The molecular mechanism of celastrol on the AKT/c-Met signaling pathway was elucidated. The results revealed that celastrol significantly repressed the AKT/c-Met induced HCC development and down-regulated the mRNA expression of AFP in the liver and the AFP level in serum. Furthermore, the expression of proliferation-associated proteins in the HCC tissues was reduced by celastrol treatment. Moreover, the abundant steatosis that resulted from FASN accumulation in the liver in AKT/c-Met mice was also attenuated. Finally, the phosphorylation of AKT and ERK1/2 in HCC mice was repressed by celastrol treatment. Overall, our data demonstrate that celastrol exerts an antiproliferative and novel lipid-decreasing effect by targeting AKT/ERK and FASN in HCC development in vivo.

Gene expression, regulation of DEN and HBx induced HCC mice models and comparisons of tumor, para-tumor and normal tissues

BackgroundHepatocellular carcinoma (HCC) is the leading cause of cancer mortality. Chemical and virus induction are two major risk factors, however, the potential molecular mechanisms of their differences remain elusive. In this study, to identify the similarities and differences between chemical and virus induced HCC models, we compared the gene expression profiles between DEN and HBx mice models, as well as the differences among tumor, para-tumor and normal tissues.MethodsWe sequenced both gene and microRNA (miRNA) expression for HCC tumor tissues, para-tumor and normal liver tissues from DEN model mice (30-week-old) and downloaded the corresponding microarray expression data of HBx model from GEO database. Then differentially expressed genes (DEGs), miRNAs and transcription factors (TFs) were detected by R packages and performed functional enrichment analysis. To explore the gene regulatory network in HCC models, miRNA and TF regulatory networks were constructed by target prediction.ResultsFor model comparison, although DEGs between tumor and normal tissues in DEN and HBx models only had a small part of overlapping, they shared common pathways including lipid metabolism, oxidation-reduction process and immune process. For tissue comparisons in each model, genes in oxidation-reduction process were down-regulated in tumor tissues and genes in inflammatory response showed the highest expression level in para-tumor tissues. Genes highly expressed in both tumor and para-tumor tissues in two models mainly participated in immune and inflammatory response. Genes expressed in HBx model were also involved in cell proliferation and cell migration etc. Network analysis revealed that several miRNAs such as miR-381-3p, miR-142a-3p, miR-214-3p and TFs such as Egr1, Atf3 and Klf4 were the core regulators in HCC.ConclusionsThrough the comparative analyses, we found that para-tumor tissue is a highly inflammatory tissue while the tumor tissue is specific with both inflammatory and cancer signaling pathways. The DEN and HBx mice models have different gene expression pattern but shared pathways. This work will help to elucidate the molecular mechanisms underlying different HCC models.

Abstract LB-335: Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer

Abstract While ribosomal proteins are generally considered to be essential genes, the existence of ribosomopathies suggests that a therapeutic window for exploiting ribosomal proteins as targets for cancer therapy may exist. We report distinct stress responses of tumor cells and normal cells upon knockdown of Rpl15, a ribosomal protein which was identified in a direct in vivo shRNA screen for new therapeutic targets in liver cancer. While normal cells undergo a reversible cell cycle arrest, we found that in tumor cells, the knockdown of Rpl15 triggers a ribosomal stress response followed by induction of cellular senescence, designated ribosomal checkpoint induced senescence (RCIS). Importantly, Rpl15 suppression triggered RCIS independent of any reduction in global protein translation. Using well established therapy resistant murine HCC models, we show that shRNAs targeting Rpl15 potently suppress tumor development in genetically diverse murine HCCs. Using Rpl15 shRNA transgenic mice, allowing for ubiquitous shRNA mediated suppression of Rpl15, we show that systemic Rpl15 suppression can be tolerated for up to 5 days, thus revealing a therapeutic window for metronomic Rpl15 inhibitory therapies. However, molecular modeling analyses revealed that Rpl15 is not druggable by small molecule inhibitors and we thus set out to explore whether RCIS can be induced via interference with other ribosomal proteins or other factors involved in ribosome biogenesis. We generated and screened a focused shRNA library targeting 41 ribosomal proteins and 19 ribosome biogenesis factors and found that apart from Rpl15, only a small subset of shRNAs scored. From a druggability point of view it was interesting, that shRNAs targeting components of the RNA polymerase I complex had scored, as recently a pharmacological RNA polymerase I inhibitor became available. Both genetic and pharmacological inhibition of RNA polymerase I induced RCIS and mediated an excellent prolongation of survival (far superior to the clinically used standard therapy Sorafenib), however in contrast to targeting Rpl15, targeting RNA polymerase I failed to achieve full long term tumor suppression. Mechanistically we found a less efficient immune mediated clearance of senescent cells as a possible explanation. In line with the idea that distinct secretory profiles underlie differences in immune mediated clearance of RCIS cells, cytokine arrays revealed distinct secretory profiles of RCIS induced by Rpl15 suppression and RCIS induced via shRNA or pharmacological RNA polymerase I inhibition. Citation Format: Katharina Wolter, Marina Pesic, Sabrina Klotz, Nicolas Herranz, Torsten Wuestefeld, Tae-Won Kang, Marco Seehawer, Rishabh Chawla, Stefan Zwirner, Jonathan Cotton, Benyuan Zhou, Marcel Krüger, Frank Klawonn, Thomas Longerich, Bence Sipos, Bernd Pichler, Jesus Gil, Martin Eilers, Prem K. Premsrirut, Antti Poso, Lars Zender. Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer [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 LB-335. doi:10.1158/1538-7445.AM2017-LB-335

Abstract 1059: Enhanced anti-tumor efficacy of a checkpoint inhibitor in combination with the HDAC inhibitor belinostat in a murine hepato-cellular carcinoma preclinical model

Abstract Introduction Belinostat is an HDAC inhibitor currently marketed in the US for the treatment of PTCL. A new oral formulation is under development (positive PoC in preclinical PK study) and provides an increase potential to develop new indications of belinostat in combination with other drugs. It has been extensively demonstrated that some anti-tumor agents, besides their direct anti-tumor effect, may induce additional mechanisms involving activation of immune responses. Thus, combination of these drugs with other immunotherapeutic protocols may yield improved therapeutic benefits. The objective of the present study is the characterization of the therapeutic efficacy of a combination of checkpoint inhibitors (anti-CTLA-4 antibodies) with belinostat in a murine HCC model. In addition, immunoprofiling was performed in order to assess the associated immune response. Methods In vivo efficacy was performed in a Hepa 129 murine hepatocellular carcinoma model implanted subcutaneously in immune-competent C3H mice using anti-CTLA4 alone, belinostat alone or in combination. Treatments were optimized to be able to demonstrate positive effect of the combination. Tumor volume was the primary endpoint. Samples from the spleen were taken to analyze immune mechanism mediating the antitumor activity. Percent of CD4, CD8 T cells and regulatory T cells were determined by flow cytometry. Anti-tumor T cell response was measured by IFN-gamma ELISPOT assay. Results Belinostat improved anti-tumor therapeutic response induced by the anti-CTLA4 checkpoint inhibitor with a significant superior tumor growth inhibition compared to control groups. Treatment with the combination resulted in a complete cessation in tumor growth in all mice during the belinostat treatment period which continued for 1 week after the final dose. Mechanistic studies showed that the underlying immune response correlated with the observed therapeutic effect of the combination with enhancement of IFN-gamma production as antitumor T cell response and decrease in regulatory T cells in the spleens of treated animals. Conclusion These results provide a rational for using belinostat in combination with checkpoint inhibitors to reinforce therapeutic response. Currently only approx. 20% of patients respond to check point inhibitors alone. The oral formulation of belinostat will allow greater flexibility in dosing schedule and use in multiple clinical situations. Further studies are ongoing in order to fully characterize this finding and to facilitate its translation into patients. Citation Format: Diana Llopiz, Marta Ruiz, Perrine Pivette, Véronique Trochon-Joseph, Bérangère Vasseur, Caroline Lemarchand, Graham Dixon, Pablo Sarobe, Bruno Sangro. Enhanced anti-tumor efficacy of a checkpoint inhibitor in combination with the HDAC inhibitor belinostat in a murine hepato-cellular carcinoma preclinical model [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 1059. doi:10.1158/1538-7445.AM2017-1059

Chronic viral hepatitis and its association with liver cancer

Chronic infection with hepatitis viruses represents the major causative factor for end-stage liver diseases, including liver cirrhosis and primary liver cancer (hepatocellular carcinoma, HCC). In this review, we highlight the current understanding of the molecular mechanisms that drive the hepatocarcinogenesis associated with chronic hepatitis virus infections. While chronic inflammation (associated with a persistent, but impaired anti-viral immune response) plays a major role in HCC initiation and progression, hepatitis viruses can also directly drive liver cancer. The mechanisms by which hepatitis viruses induce HCC include: hepatitis B virus DNA integration into the host cell genome; metabolic reprogramming by virus infection; induction of the cellular stress response pathway by viral gene products; and interference with tumour suppressors. Finally, we summarise the limitations of hepatitis virus-associated HCC model systems and the development of new techniques to circumvent these shortcomings.

An anthracenecarboximide fluorescent probe for in vitro and in vivo ratiometric imaging of endogenous alpha-l-fucosidase for hepatocellular carcinoma diagnosis

The AFU level in the HCC model of zebrafish can be mapped by using a novel single-fluorophore based ratiometric fluorescent probe.

Abstract 4314: Using antioxidant to enhance liver cancer preventive effect of n-3 PUFAs

Abstract In various animal models, n-3 polyunsaturated fatty acids (PUFAs) have demonstrated cancer preventive effects. This protective effect is associated with reduced levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2). On the other hand, an endogenous DNA adduct formed from the peroxidation of ω-3 PUFAs, γ-hydroxy-1, N2-propanodeoxyguanosine (γ-OHPdG), is known to be mutagenic. In light of these observations, we hypothesized that the combination of n-3 PUFAs with an antioxidant to inhibit lipid peroxidation (e.g. α-lipoic acid) will result in an enhanced cancer preventive effect than n-3 PUFAs alone. The chemical carcinogen diethylnitrosamine (DEN) induced HCC model is employed in this study. The DEN-induced HCC model has a histology and genetic signature similar to that of human HCCs, such that it has poor prognosis and recapitulates a dependence on inflammatory signaling. It also reflects the same gender disparity seen in human HCCs. Fat-1 transgenic mice were used because they express a Caenorhabditis elegans desaturase converting n-6 to n-3 PUFAs endogenously without using dietary supplementation. This model underlies the importance of dietary control of PUFAs intake and ratios due to the inability of mammalian cells to generate anti-inflammatory n-3 PUFAs from pro-inflammatory n-6 PUFAs, which are a major component of the standard Western diets. The results showed that antioxidant can efficiently suppress the elevated level of γ-OHPdG in fat-1 mice. The tumor incidence, multiplicity, and size are monitored continuously. This work was supported by the NCI grant: RO1-CA-134892. Y.F. thanks the Prevent Cancer Foundation for his fellowship (Marcia and Frank Carlucci Charitable Foundation Award in Cancer Prevention and Early Detection) Citation Format: Ying Fu, Angela Y. Bai, Marcin Dyba, Jing X. Kang, Fung-Lung Chung. Using antioxidant to enhance liver cancer preventive effect of n-3 PUFAs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4314.