Model Of Human Hepatocellular Carcinoma Research Articles

Radiosensitization by combining an aurora kinase inhibitor with radiotherapy in hepatocellular carcinoma through cell cycle interruption

Radiotherapy has been integrated into the multimodal treatment of hepatocellular carcinoma (HCC), especially of localized hepatic tumor(s) refractory to conventional treatment. However, tumor control remains unsatisfactory mainly because of insufficient dose, and sublethally irradiated tumor may associate with metastasis. Our aim was to assess the effect of combining a molecularly targeted Aurora kinase inhibitor, VE-465, with radiotherapy in in vitro and in vivo models of human HCC. Human HCC cell lines (Huh7 and PLC-5) were used to evaluate the in vitro synergism of combining VE-465 with irradiation. Flow cytometry analyzed the cell cycle changes, while western blot investigated the protein expressions after the combined treatment. Severe combined immunodeficient (SCID) mice bearing ectopic and orthotopic HCC xenografts were treated with VE-465 and/or radiotherapy for the in vivo response. VE-465 significantly enhanced radiation-induced death in HCC cells by a mechanism involving the enhanced inhibition of histone H3 phosphorylation and interruption of cell cycle change. In SCID, mice bearing ectopic HCC xenografts, pretreatment with VE-465 (20 mg/kg/day × 9 days) significantly enhanced the tumor-suppressive effect of radiotherapy (5 Gy/day × 5 days) by 54.0%. A similar combinatorial effect of VE-465 and radiotherapy was observed in an orthotopic model of Huh7 tumor growth by 17.2%. In the orthotopic Huh7 xenografts, VE-465 significantly enhanced radiation-induced tumor growth suppression by a mechanism involving the increased apoptosis. VE-465 is a potent inhibitor of Aurora kinase with therapeutic value as a radiosensitizer of HCC.

Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

Increasing evidence suggests that the renin-angiotensin system (RAS) plays an important role in tumorigenesis. The interaction between Angiotensin II (AngII) and angiotensin type 1 receptor (AT1R) may have a pivotal role in hepatocellular carcinoma (HCC) and therefore, AT1R blocker and angiotensin I-converting enzyme (ACE) inhibitors may have therapeutic potential in the treatment of hepatic cancer. Although the involvement of AT1R has been well explored, the role of the angiotensin II Type 2 receptor (AT2R) in HCC progression remains poorly understood. Thus, the aim of this study was to explore the effects of AT2R overexpression on HCC cells in vitro and in mouse models of human HCC. An AT2R recombinant adenoviral vector (Ad-G-AT2R-EGFP) was transduced into HCC cell lines and orthotopic tumor grafts. The results indicate that the high dose of Ad-G-AT2R-EGFP–induced overexpression of AT2R in transduced HCC cell lines produced apoptosis. AT2R overexpression in SMMC7721 cells inhibited cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells through changing expression of CDK4 and cyclinD1. The data also indicate that overexpression of AT2R led to apoptosis via cell death signaling pathway that is dependent on activation of p38 MAPK, pJNK, caspase-8 and caspase-3 and inactivation of pp42/44 MAPK (Erk1/2). Finally, we demonstrated that moderately increasing AT2R expression could increase the growth of HCC tumors and the proliferation of HCC cells in vivo. Our findings suggest that AT2R overexpression regulates proliferation of hepatocellular carcinoma cells in vitro and in vivo, and the precise mechanisms of this phenomenon are yet to be fully determined.

Mn2+-doped silica nanoparticles for hepatocyte-targeted detection of liver cancer in T1-weighted MRI

With an aim to examine the possibility of developing a liver-specific MRI contrast agent that takes advantages of brightly enhanced MR images by Mn2+ whilst making up the limitations of the pre-developed contrast agent, the Mn2+-doped SiO2 nanoparticles (Mn–SiO2) were synthesized and their characteristics as MR contrast agents were investigated. The in vitro and in vivo investigations showed that Mn–SiO2 has unique MR contrast-enhancing characteristics that activate positive contrast enhancement in T1-weighted MR images only under low pH conditions by liberating Mn2+ ions from MR inactive nanoparticles. The administration of Mn–SiO2 to an orthotopic xenograft model of human hepatocellular carcinoma (HCC) resulted in a differentiation of enhancement periods between HCC and normal parenchyma tissues on T1-weighted MR images and consequently presented the duplicates of the highly contrast-enhanced liver image with an equal liver-to-HCC contrast ratio but opposite contrast. The Mn–SiO2-enhanced MR imaging therefore allowed for the repetitive detection of the HCC within a single MR imaging session, which can help us to achieve more reliable diagnosis and characterization of liver lesions than is possible with any currently used Mn2+-based contrast agent. In addition, the in vivo biodistribution study also supported the effectiveness of Mn–SiO2 nanoparticles as a liver-specific MRI contrast agent, which efficiently delivers and releases the T1-contrasting Mn2+ ions to targeted hepatocytes.

Targeting therapy of hepatocellular carcinoma with doxorubicin prodrug PDOX increases anti-metastatic effect and reduces toxicity: a preclinical study

BackgroundThis study was to investigate the effects and safety of cathepsin B-cleavable doxorubicin (DOX)-prodrug (PDOX) for targeting therapy of metastatic human hepatocellular carcinoma (HCC) using DOX as a positive control drug.MethodsThe orthotopic nude mice model of highly metastatic HCC was established and the animals were randomized and treated with PDOX, DOX and saline, respectively. Hematology, biochemistry and tumor markers were studied. At autopsy, liver tumor weight and size, ascites, abdominal lymph nodes metastases, experimental peritoneal carcinomatosis index (ePCI), and tumor-host body weight ratio were investigated. Immunohistochemical studies and western blotting were done to investigate key molecules involved in the mechanism of action.ResultsCompared with Control, both PDOX and DOX could similarly and significantly reduce liver tumor weight and tumor volume by over 40%, ePCI values, retroperitoneal lymph node metastases and lung metastases and serum AFP levels (P < 0.05). The PDOX group had significantly higher WBC than the DOX group (P < 0.05), and higher PLT than Control (P < 0.05). Serum BUN and Cr levels were lower in the PDOX group than DOX and Control groups (P < 0.05). Compared with Control, DOX increased CK and CK-MB; while PDOX decreased CK compared with DOX (P < 0.05). Multiple spotty degenerative changes of the myocardium were observed in DOX-treated mice, but not in the Control and PDOX groups. PDOX could significantly reduce the Ki-67 positive rate of tumor cells, compared with DOX and Control groups. PDOX produced the effects at least via the ERK pathway.ConclusionCompared with DOX, PDOX may have better anti-metastatic efficacy and reduced side effects especially cardio-toxicities in this HCC model.

Inflammation-Induced Hepatocellular Carcinoma Is Dependent on Ccr5 in Mice

Human hepatocellular carcinoma (HCC) is an inflammation-induced cancer, which is the third-leading cause of cancer mortality worldwide. We investigated the role of the chemokine receptors, CCR5 and CCR1, in regulating inflammation and tumorigenesis in an inflammation-induced HCC model in mice. Multidrug resistance 2 gene (Mdr2)-knockout (Mdr2-KO) mice spontaneously develop chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC. We generated two new strains from the Mdr2-KO mouse, the Mdr2:CCR5 and the Mdr2:CCR1 double knockouts (DKOs), and set out to compare inflammation and tumorigenesis among these strains. We found that in Mdr2-KO mice lacking the chemokine receptor, CCR5 (Mdr2:CCR5 DKO mice), but not CCR1 (Mdr2:CCR1 DKO), macrophage recruitment and trafficking to the liver was significantly reduced. Furthermore, in the absence of CCR5, reduced inflammation was also associated with reduced periductal accumulation of CD24(+) oval cells and abrogation of fibrosis. DKO mice for Mdr2 and CCR5 exhibited a significant decrease in tumor incidence and size. Our results indicate that CCR5 has a critical role in both the development and progression of liver cancer. Therefore, we propose that a CCR5 antagonist can serve for HCC cancer prevention and treatment.

Mitogenic Insulin Receptor-A Is Overexpressed in Human Hepatocellular Carcinoma due to EGFR-Mediated Dysregulation of RNA Splicing Factors

Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.

Establishment of NOD/SCID mouse models of human hepatocellular carcinoma via subcutaneous transplantation of histologically intact tumor tissue.

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers, but it is very difficult to establish an animal model by using surgical specimens. In the present experiment, histologically intact fresh surgical specimens of HCC were subcutaneously transplanted in non-obese diabetic/severe combined immunodeficienccy (NOD/SCID) mice. The biological characteristics of the original and the corresponding transplanted tumors and cell lines were investigated. The results showed that 5 new animal models and 2 primary cell lines were successfully established from surgical specimens. Hematoxylin-eosin staining showed that xenografts retained major histological features of the original surgical specimens. The two new cell lines had been cultivated for 3 years and successively passaged for more than 100 passages in vitro. The morphological characteristics and biologic features of the two cell lines were genetically similar to the original tumor. The subcutaneous transplant animal models with histologically intact tumor tissue and primary cell lines could be useful for in vivo and in vitro testing of anti-cancer drugs and be ideal models to study various biologic features of HCC.

An armed oncolytic measles vaccine virus eliminates human hepatoma cells independently of apoptosis

Due to late diagnosis and a pronounced chemoresistance, most patients with hepatocellular carcinoma (HCC) have an overall poor prognosis. Measles vaccine viruses (MeV) have been shown to possess anti-tumor properties and their efficacy has been enhanced by arming with suicide genes. To test armed MeV for the treatment of HCC, we equipped it with the suicide gene Super-cytosine deaminase (SCD) and tested the efficacy in cell culture and in a mouse xenograft model of human HCC. Prodrug conversion was investigated in cell culture and quantified by high-performance liquid chromatography. We observed a strong oncolytic activity of MeV-SCD against human HCC in vitro and in vivo. The prodrug was efficiently converted in infected cells leading to a significant enhancement of the cytotoxic effect. Treatment of HCC xenografts with MeV caused long-term virus replication in tumor tissue. We show that the suicide gene therapy induces an apoptosis-like cell death but is not dependent on intact apoptosis pathways. These results demonstrate that MeV-based suicide gene therapy is a promising novel therapy regimen for HCC overcoming resistance towards conventional therapy. The independence from apoptosis raises hopes for the treatment of patients whose tumor cells exert defects in this cell death mechanism.

Sprengerinin C exerts anti-tumorigenic effects in hepatocellular carcinoma via inhibition of proliferation and angiogenesis and induction of apoptosis

The multi-targeted therapy for liver cancer has been considered as a novel strategy to fight hepatocellular carcinoma. In this study, we first found that sprengerinin C, a naturally derived compound strongly suppressed tumor angiogenesis in human umbilical vein endothelial cells. A mechanism study revealed that sprengerinin C blocked vascular endothelial growth factor receptor 2-dependent phosphoinositide 3-kinase/Akt/mTOR/matrix metalloproteinase and p38 MAPK/matrix metalloproteinase pathways, two major pathways for tumor angiogenesis. Moreover, sprengerinin C inhibited vascular endothelial growth factor release, a vital event for early angiogenesis response, from hypoxic HepG-2/BEL7402 cells by suppressing hypoxia-inducible factor-1α transcriptional activity. Furthermore, sprengerinin C induced HepG-2/BEL7402 cell apoptosis by activating NADPH oxidase/reactive oxygen species-dependent caspase apoptosis pathway and suppressed HepG-2/BEL7402 cell growth through p53-mediated G2/M-phase arrest. Sprengerinin C also showed a significant anti-tumor effect in the nude mouse xenograft model of human hepatocellular carcinoma. These results provide new insights into development of potent candidate compounds for liver cancer through affecting multiple tumor progression steps of angiogenesis, apoptosis and proliferation.

Abstract 5661: Nanoparticle based combinatorial siRNA therapy against human hepatocellular carcinoma (HCC) .

Abstract Background: We have previously demonstrated the therapeutic effect of lipid nanoparticles (LNP) loaded with single siRNA targeting CSN5 and WEE1 against human HCC in mouse models. Aim: To test the benefit of a combinatorial versus single siRNA therapy in mouse models of human HCC and to identify molecular mechanism(s) involved in therapeutic response by extensive microarray analyses. Materials and Methods: LNP formulations of chemically modified siRNAs targeting CSN5 and WEE1 were produced by Tekmira® Pharmaceuticals. SCID-beige mice were used for subcutaneous (Hep3B) and intra-hepatic (Huh7-luciferase) tumor transplantation. Mice with established tumors were treated intravenously with 4 mg/kg single agent siRNA or 2 mg/kg + 2 mg/kg siCSN5:siWEE1 siRNA co-encapsulated in the same LNP. Tumors were assayed following 1 to 9 injected doses. Tumor progression in the Huh7 orthotopic model was monitored by bioluminescence imaging and metastases were evaluated at endpoint. Results: Significant target silencing was observed in tumors after single or repeat administration with no antagonism between siRNAs occurring in the CSN5:WEE1 combination. We observed significant inhibition of tumor growth and metastases in mice treated with active siRNAs compared to LNP containing a non-targeting control sequence. Potency was not lost with siCSN5:siWEE1 LNP, where the concentration of each siRNA is halved in combination, relative to the most efficacious single agent. Data from preliminary microarray analyses demonstrate a strong transcriptome difference between each treatment group. Conclusion: We demonstrate a clear efficacy of a LNP based combinatorial siRNA therapy in human mouse models of HCC. Overall this therapy led to a significant decrease of tumor size induced by mRNA inhibition Citation Format: Thomas Decaens, Valentina M. Factor, Iva Kulic, Jesper B. Andersen, Daekwan Seo, Yun-Han Lee, Adam D. Judge, Elizabeth A. Conner, Ian MacLachlan, Snorri S. Thorgeirsson. Nanoparticle based combinatorial siRNA therapy against human hepatocellular carcinoma (HCC) . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5661. doi:10.1158/1538-7445.AM2013-5661

Insulin-like growth factor-binding protein-7 (IGFBP7): a promising gene therapeutic for hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, targeted therapies to elicit prolonged survival benefit to the patients. Insulin-like growth factor-binding protein-7 (IGFBP7), a secreted protein belonging to the IGFBP family, functions as a potential tumor suppressor for HCC. In the present study, we evaluated the therapeutic efficacy of a replication-incompetent adenovirus expressing IGFBP7 (Ad.IGFBP7) in human HCC. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing reactive oxygen species (ROS) and activating a DNA damage response (DDR) and p38 MAPK. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intrahepatic metastasis. In a nude mice subcutaneous model, xenografts from human HCC cells were established in both flanks and only left-sided tumors received intratumoral injection of Ad.IGFBP7. Growth of both left-sided injected tumors and right-sided uninjected tumors were markedly inhibited by Ad.IGFBP7 with profound suppression of angiogenesis. These findings indicate that Ad.IGFBP7 might be a potent therapeutic eradicating both primary HCC and distant metastasis and might be an effective treatment option for terminal HCC patients.

Emodin suppresses migration and invasion through the modulation of CXCR4 expression in an orthotopic model of human hepatocellular carcinoma.

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Fe3O4/MnO hybrid nanocrystals as a dual contrast agent for both T1- and T2-weighted liver MRI

To investigate whether it is possible to develop a dual magnetic resonance (MR) contrast agent, Fe3O4/MnO hybrid nanocrystals were modified to integrate the T1 and T2 contrast-enhancing abilities of each compound, and their characteristics as MR contrast agents were investigated. In vitro and in vivo investigations revealed that the Fe3O4/MnO dumbbell-shaped nanocrystal exerted a negative T2 contrast effect in its intact form and also gave rise to a positive contrast effect in T1-weighted MR imaging by releasing Mn2+ ions in a low pH environment. This induced organ-specific contrast enhancement for both T1- and T2-weighted in vivo MR imaging. The usefulness of the Fe3O4/MnO hybrid nanocrystals as dual contrast agents was evaluated by in vivo MR imaging of an orthotopic xenograft model of human hepatocellular carcinoma (HCC). After injection of the Fe3O4/MnO hybrid nanocrystals, dual contrast-enhanced MR images that synergistically combined the T2 and T1 contrast effects from the Fe3O4 grain and released Mn2+ ions were obtained by a single acquisition of MR imaging. This facilitated the detection of HCC with a high degree of conspicuity that could not be achieved with any single contrast agent.

A nude mouse model of hepatocellular carcinoma single cell-derived organ site-specific metastasis

To establish a single cell-derived organ site-specific metastatic model of human hepatocellular carcinoma (HCC) in the nude mouse. Using the limited dilution method, HCCLM3-R-LM1 and HCCLM3-R-LnM1 cell lines were used to generate eight (LM1-S2, -S3, -S4, -S5, -S11, -S15, -S21, and -S23) and five (LnM1-S7, -S11, -S13, -S17, and -S20) single cell-derived monoclonal cell lines, respectively. The monoclonal cell lines were seeded into 4-week-old nude mice, and three weeks later the resultant subcutaneous tumor tissues were orthotopically transplanted into the livers of nude mice. At six weeks after implantation, lung and lymph node were extracted for analysis of the metastatic foci fluorescence area and pathology to assess the number of metastatic foci. Among the 13 mice implanted with the established monoclonal cell lines, six grew subcutaneous tumors. When orthotopically transplanted, the six tumors showed remarkably different metastatic potential and organ site-specific tropism. The fluorescence areas of lung metastatic foci were: LM1-S3, 80 923+/-10 162; LM1-S4, 1506 000+/-297 064; LM1-S5, 36 140+/-8 210; and LM1-S11, 508 448+/-134 272 (P less than 0.01); no lymph node metastases were found for these lines. For LnM1-S11, the fluorescence areas of lung and lymph node metastatic foci were 435 062+/-206 620 and 1 254 000+/-225 171, respectively. We successfully established several monoclonal cell lines and nude mouse models of HCC with different metastatic potential and organ tropism. Among them, LM1-S3, LM1-S4, LM1-S5, and LM1-S11 have metastasis organotropism to lung. The LnM1-S11 line exhibits dual metastasis organotropism to lung and lymph node. These monoclonal cell lines and nude mouse models may represent useful tools for study of HCC metastasis organotropism.

Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines

Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate.

Assessment and comparison of magnetic nanoparticles as MRI contrast agents in a rodent model of human hepatocellular carcinoma

The purpose of this study was to synthesize, characterize and tailor the surface properties of magnetic nanoparticles with biocompatible copolymer coatings and to evaluate the efficiency of the resulting nanoconjugates as magnetic resonance imaging (MRI) contrast agents for liver imaging. Magnetic nanoparticles with core diameters of 10 and 30 nm were synthesized by pyrolysis and were subsequently coated with a copolymer containing either carboxyl (SHP) or methoxy groups as termini. All four formulas, and ferumoxides (Feridex I.V.(®)), were individually injected intravenously into separate, normal Balb/C mice (at 2.5, 1.0 and 0.56 mg Fe kg(-1)), and the animals underwent T(2)-weighted MRI at multiple time points post injection (p.i.) to evaluate the hepatic uptake and clearance. Furthermore, we compared the abilities of the new formulas and Feridex to detect tumors in an orthotropic Huh7 tumor model. Transmission electron microscopy (TEM) revealed a narrow size distribution of both the 10 and 30 nm nanoparticles, in contrast to a wide size distribution of Feridex. MTT, apoptosis and cyclin/DNA flow cytometry assays showed that the polymer coated nanoparticles had no adverse effect on cell growth. Among all the tested formulas, including Feridex, SHP-30 showed the highest macrophage uptake at the in vitro level. In vivo MRI studies on normal mice confirmed the superiority of SHP-30 in inducing hypointensities in the liver tissue, especially at clinical dose (0.56 mg Fe kg(-1)) and 3 T field. SHP-30 showed better contrast-to-noise ratio than Feridex on the orthotropic Huh7 tumor model. SHP-30 was found to be an efficient contrast agent for liver MR imaging. The success of this study suggests that, by improving the synthetic approach and by tuning the surface properties of IONPs, one can arrive at better formulas than Feridex for clinical practice.

Integrative genomics: Liver regeneration and hepatocellular carcinoma

Numerous genome wide profiles of gene expression changes in human hepatocellular carcinoma (HCC), compared to normal liver tissue, have been reported. Hierarchical clustering of these data reveal distinct patterns, which underscore conservation between human disease and mouse models of HCC, as well as suggest specific classification of subtypes within the heterogeneous disease of HCC. Global profiling of gene expression in mouse liver, challenged by partial hepatectomy to regenerate, reveals alterations in gene expression that occur in response to acute injury, inflammation, and re-entry into cell cycle. When we integrated datasets of gene expression changes in mouse models of HCC and those that are altered at specific times of liver regeneration, we saw shared, conserved alterations in gene expression within specific biological pathways, both up-regulated, for example, cell cycle, cell death, and cellular development, or down-regulated, for example, vitamin and mineral metabolism, lipid metabolism, and molecular transport. Additional molecular mechanisms shared by liver regeneration and HCC, as yet undiscovered, may have important implications in tumor development and recurrence. These comparisons may offer a way to judge how liver resection, in the treatment of HCC, introduces challenges to care of the disease. Further, uncovering the pathways conserved in inflammatory response, hypertrophy, proliferation, and architectural remodeling of the liver, which are shared in liver regeneration and HCC, versus those specific to tumor development and progression in HCC, may reveal new biomarkers or potential therapeutic targets in HCC.

Abstract 4373: Targeting FGFR system in a model of HCC

Abstract Activation of the fibroblast growth factor receptor (FGF/FGFR) system has been reported in a large number of human cancers including human hepatocellular carcinoma (HCC). Furthermore, expression of FGFR2 has been associated with aggressive tumor growth and poor prognosis in this tumor entity. Moreover, HCC is known to be a hypervascularized tumor and activation of the FGF/FGFR system is a crucial event in tumor angiogenesis. Hence, we addressed the issue of targeting the FGFR system in HCC (Huh-7, HepG2) and stromal cells (endothelial cells (EC), vascular smooth muscle cells (VSMC), hepatic stellate cells (HSC)). The FGFR inhibitor BGJ398 (Novartis Oncology, Basel, Switzerland) was used to inhibit FGFR activation. Effects of targeting FGFR on growth of cells were determined by MTT assays. Inhibition of constitutive and growth factor induced cell motility was investigated in modified Boyden Chamber assays. Activation of signaling pathways upon treatment with BGJ398 was assessed by Western Blot analyses whereas effects on mediators of tumor angiogenesis (VEGF-A, PDGF-B) where measured by RT-PCR and ELISA, respectively. Results show that inhibition of the FGF/FGFR system with BGJ398 led to dose-dependent inhibition of growth in tumor cells and ECs whereas only minor effects were observed on VSMC and HSCs. Moreover, motility of tumor and stromal cells (ECs, VSMCs, HSCs) was significantly impaired upon constitutive conditions (P&amp;lt;0.05 for all). In addition, FGFR inhibition led to decrease in HSC motility even when cell were incubated with conditioned media from cancer cells (P&amp;lt;0.05). With regards to activation of signaling intermediates, FGFR inhibition impaired constitutive and growth factor induced phosphorylation of Akt and ERK in HCC cell lines, ECs and HSCs whereas only minor effect was observed in VSMCs. Moreover, treatment with BGJ398 led to inhibition of c-Myc expression and significantly reduced VEGF-A production from cancer cells, as determined by Western blotting and ELISA, respectively. Evaluation of effectiveness of BGJ398 in animal models is currently ongoing. However, results so far indicate that targeting FGFR impairs angiogenic signaling and motility in HCC cell lines and stromal cells. Therefore, inhibition of FGFR warrants further evaluation and might be an interesting novel approach for anti-neoplastic treatment protocols in human HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4373. doi:1538-7445.AM2012-4373

Abstract 1387: The importance of FGF pathway in hepatocellular carcinoma and its role in acquired resistance to anti-angiogenic therapies

Abstract INTRODUCTION: Fibroblast Growth Factor (FGF) signaling has an important role in different biological functions and human cancers, being some of its members described as driver oncogenes and components of evasive resistance to anti-angiogenic therapies. AIMS: 1) To identify molecular alterations of FGF signaling in human hepatocellular carcinoma (HCC), 2) to study the mechanism of acquired resistance to anti-angiogenic therapy in HCC to identify novel targets, and 3) to evaluate the role of brivanib in overcoming acquired resistance in animal models of HCC. METHODS: FGF pathway was analyzed in 104 early HCCs by whole-genome expression, genome-wide methylation status, microRNA expression and chromosomal aberrations. Subcutaneous tumors in nude mice (n=26) were treated with sorafenib (30 mg/kg/day) until development of acquired resistance, afterwards mice were randomized into either brivanib (BMS, 100 mg/kg/day) or sorafenib treatment. Anti-tumoral effect and survival were assessed. The mechanisms involved in acquired resistance were studied by gene expression profiling and protein evaluation. RESULTS: 31% of the HCC samples presented overexpression of oncogenic ligands (i.e. FGF19, FGF 21 and FGF2), and 19% overexpression of FGFR3 and FGFR4 -feature clustered in the proliferation HCC subclass (Chiang, Cancer Res, 2008). Promoter hypermethylation of FGFR2 (FDR=0.015) and regulation of FGFR3 expression by miR-483-5p (6 fold-change, FDR&amp;lt;0.05) were also observed, as well as high level amplification in FGF19 locus (8% of samples), and allelic losses of FGFR2 and Spry2 (p&amp;lt;0.05). FGF2 overexpression was associated with WNT-beta-catenin HCC subclass and correlated with expression of WNT-liver specific targets (i.e. GLUL, LGR5, TBX3, SPARCL1, p&amp;lt;0.05) suggesting a crosstalk among FGF and WNT pathways in HCC. Regarding the in vivo model, 65% mice acquired resistance after sorafenib therapy [median: 42 days; tumor size: 855 mm3 resistant group vs 316 mm3 sorafenib-responder group (p=0.002)]. This acquired resistance phenotype has been evaluated by gene profiling and GSEA revealing an enrichment of gene signatures of poor prognosis and resistance to cancer therapies (FDR&amp;lt;0.05), and identifying IGF-1R and FGFR1 overexpression as the potential drivers of the development of this resistance. Brivanib delayed tumor growth (p=0.03), increased survival after development of acquired resistance (median survival: 32 days vs 15 days on sorafenib, p=0.07) and inhibited FGF downstream signaling, reverting the resistant phenotype. CONCLUSIONS: FGF pathway is activated in a subclass of early HCC due to deregulation of ligands and receptors by different mechanisms. Acquired resistance to sorafenib in experimental models seems to be driven by FGF and IGF signaling pathways. Brivanib significantly delayed tumor growth and increased survival in the sorafenib-resistant in vivo model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1387. doi:1538-7445.AM2012-1387