Hepatocellular Carcinoma Patient-derived Xenograft Research Articles

Varlitinib to demonstrate anti-tumour efficacy in patient-derived hepatocellular carcinoma xenograft models.

e15598Background: Varlitinib (ASLAN001) is a potent, reversible, small molecule pan-HER inhibitor that selectively binds to HER1 (IC50 = 7 nM), HER2 (IC50 = 2 nM) and HER4 (IC50 = 4 nM). Hepatocell...

Activating JAK1 mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.

Anti-CD47 antibody suppresses tumour growth and augments the effect of chemotherapy treatment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is often associated with metastasis and recurrence leading to a poor prognosis. Therefore, development of novel treatment regimens is urgently needed to improve the survival of HCC patients. In this study, we aimed to investigate the in vitro and in vivo effects of anti-CD47 antibody alone and in combination with chemotherapy in HCC. In this study, we examined the functional effects of anti-CD47 antibody (B6H12) on cell proliferation, sphere formation, migration and invasion, chemosensitivity, macrophage-mediated phagocytosis and tumourigenicity both in vitro and in vivo. The therapeutic efficacy of anti-CD47 antibody alone or in combination with doxorubicin was examined in patient-derived HCC xenograft. Blocking CD47 with anti-CD47 monoclonal antibody (B6H12) at 10 μg/ml could suppress self-renewal, tumourigenicity and migration and invasion abilities of MHCC-97L and Huh-7 cells. Interestingly, anti-CD47 antibody synergized the effect of HCC cells to chemotherapeutic drugs including doxorubicin and cisplatin. Blockade of CD47 by anti-CD47 antibody induced macrophage-mediated phagocytosis. Using a patient-derived HCC xenograft mouse model, we found that anti-CD47 antibody (400 μg/mouse) in combination with doxorubicin (2 mg/kg) exerted maximal effects on tumour suppression, as compared with doxorubicin and anti-CD47 antibody alone. Anti-CD47 antibody treatment could complement chemotherapy which may be a promising therapeutic strategy for the treatment of HCC patients.

Abstract 688: Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models

Abstract Hepatocellular carcinoma (HCC) is the fifth most common type of cancer worldwide. However, current therapeutic approaches for this epidemic disease are limited and its 5-year survival rate has not been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited point mutations in JAK1 (Janus Kinase) gene, which were confirmed by Sanger sequencing. To explore the transforming capability, these mutations were then introduced into 293T and Ba/F3 cells by transient transfection and retroviral infection, respectively. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, a modest sensitivity to the treatment of a JAK1/2 inhibitor, Ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly inhibited. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo. JAK-STAT pathway might represent a new therapeutic opportunity for HCC. Monotherapy using a more potent inhibitor and combinatory therapy may be further explored in JAK1S703I mutant PDX model. Citation Format: Shuqun Yang, Chonglin Luo, Qingyang Gu, Qiang Xu, Hongye Sun, Ziliang Qian, Yexiong Tan, Hao Wu, Yuxin Qin, Yuhong Shen, Xiaowei Xu, Shu-Hui Chen, Chi-Chung Chan, Hongyang Wang, Mao Mao, Douglas D. Fang. Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 688. doi:10.1158/1538-7445.AM2015-688

Nuclear factor kappa B-mediated CD47 up-regulation promotes sorafenib resistance and its blockade synergizes the effect of sorafenib in hepatocellular carcinoma in mice.

Sorafenib is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, the survival benefit of this treatment is modest, partly owing to drug resistance. Recent evidence has demonstrated the existence of tumor-initiating cells (T-ICs) as the culprit for treatment resistance. To examine whether sorafenib resistance was a result of the presence of liver T-ICs, we developed sorafenib-resistant HCC cells both in vitro and in vivo through continuous exposure to sorafenib. Using these models, we found that sorafenib-resistant clones demonstrated enhanced T-IC properties, including tumorigenicity, self-renewal, and invasiveness. In addition, several T-IC markers were found to be up-regulated, among which CD47 was found to be most significant. Using chromatin immunoprecipitation assays and expression analyses, CD47 expression was found to be regulated by nuclear factor kappa B (NF-κB) through a specific response element in the promoter of CD47, and the site occupancy and expression were increased and decreased upon stimulation and inhibition of NF-κB, respectively. Consistently, NF-κB was activated in sorafenib-resistant HCC cells, and this finding was confirmed in clinical HCC samples, which showed a positive correlation between NF-κB and CD47 expression. Functional characterization of CD47 in sorafenib-resistant HCC cells was evaluated using a lentivirus-based knockdown approach and showed increased sensitization to sorafenib upon CD47 knockdown. Furthermore, blockade of CD47 using anti-CD47 antibody (Ab) showed a similar effect. Using a patient-derived HCC xenograft mouse model, we found that anti-CD47 Ab (500 μg/mouse) in combination with sorafenib (100 mg/kg, orally) exerted synergistic effects on tumor suppression, as compared with sorafenib and anti-CD47 Ab alone. NF-κB-mediated CD47 up-regulation promotes sorafenib resistance, and targeting CD47 in combination with sorafenib is an attractive therapeutic regimen for the treatment of HCC patients.

Genomic characterization of a large panel of patient-derived hepatocellular carcinoma xenograft tumor models for preclinical development.

Lack of clinically relevant tumor models dramatically hampers development of effective therapies for hepatocellular carcinoma (HCC). Establishment of patient-derived xenograft (PDX) models that faithfully recapitulate the genetic and phenotypic features of HCC becomes important. In this study, we first established a cohort of 65 stable PDX models of HCC from corresponding Chinese patients. Then we showed that the histology and gene expression patterns of PDX models were highly consistent between xenografts and case-matched original tumors. Genetic alterations, including mutations and DNA copy number alterations (CNAs), of the xenografts correlated well with the published data of HCC patient specimens. Furthermore, differential responses to sorafenib, the standard-of-care agent, in randomly chosen xenografts were unveiled. Finally, in the models expressing high levels of FGFR1 gene according to the genomic data, FGFR1 inhibitor lenvatinib showed greater efficacy than sorafenib. Taken together, our data indicate that PDX models resemble histopathological and genomic characteristics of clinical HCC tumors, as well as recapitulate the differential responses of HCC patients to the standard-of-care treatment. Overall, this large collection of PDX models becomes a clinically relevant platform for drug screening, biomarker discovery and translational research in preclinical setting.

The over-expression of survivin enhances the chemotherapeutic efficacy of YM155 in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. The inability of chemotherapeutic drugs to selectively target HCC tumor cells because of their predominant resistant phenotype to most conventional anticancer agents bestows a major obstacle for the clinical management of HCC. In this report, we have examined and demonstrated the remarkable heterogeneity of expression of survivin and its phosphorylated active form (p-survivin) in HCC patients' tissues and cell lines. Furthermore, the expression of survivin and p-survivin in HCC cell lines was found to be associated with response to the small-molecule survivin suppressant YM155. Therefore, in the HCC cell lines that express elevated level of survivin and p-survivin, YM155 efficiently inhibited their proliferation, induced cell cycle arrest and apoptosis resulting in DNA damage through the dysregulation of cell-cycle checkpoint-related regulatory genes. Importantly, YM155 yielded significantly better therapeutic effect than sorafenib when tested in an orthotopic mouse model using patient-derived HCC xenografts with elevated survivin and p-survivin expression. Our results clearly demonstrated that the level of survivin and p-survivin expression could serve as molecular predictive biomarkers to select potential YM155-responsive patients, in a move towards delivering precision medicine for HCC patients.

Targeted Hsp70 expression combined with CIK-activated immune reconstruction synergistically exerts antitumor efficacy in patient-derived hepatocellular carcinoma xenograft mouse models.

The patient-derived tumor xenograft (PDTX) models can reproduce a similar natural genetic background and similar biological behaviors to tumor cells in patients, which is conducive to the assessment of personalized cancer treatment. In this study, to verify the targeting and effectiveness of the therapeutic strategy using a Survivin promoter-regulated oncolytic adenovirus expressing Hsp70, the PDTX models of hepatocellular carcinoma (HCC) were established in nude mice and the cytokine-induced killer (CIK) cells were intravenously infused into mice to partially reconstruct the mouse immune function. The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited. However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells. Oncolytic adenovirus mediated the specific expression of Hsp70 in cancer tissues allowed the CIK chemotaxis, and induce the infiltration of CD3+ T cells in tumor stroma, thereby exhibiting anti-tumor activity. The anti-tumor effect was more effective for the highly malignant tumor xenografts with highly Survivin expression. This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts.

Abstract 2839: Antitumor activities of FGFR inhibitors in FGFR1-overexpressing hepatocellular carcinoma patient-derived xenograft tumor models

Abstract Hepatocellular carcinoma (HCC) is intrinsically resistant to conventional chemotherapies. Developing effective therapeutics becomes critical to tackle the unmet medical need. The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling axis plays an important role in liver development and homeostasis in adults. Deregulation of the FGF/FGFR signaling pathway through genetic modification or overexpression of the receptors (or their ligands) has been increasingly recognized in HCC. Accordingly, therapeutic intervention of the pathway has been extensively explored. In this study, analysis of a panel of patient-derived xenograft (PDX) HCC models by Affymetrix human gene expression arrays revealed overexpression of FGFR1 in 13 out of 43 (30%) models. Antitumor activities of FGFR inhibitors, including lenvatinib, dovitinib, ENMD-2076, and LY2874455, were then tested in five models. Lenvatinib exhibited the most potent activity in all models, whereas LY2874455 failed to show the effect in one of models. IC50s for FGFR1 inhibition of ENMD-2076, lenvatinib, dovitinib and LY2874455 were 145 nM, 134.5 nM, 53.5 nM and 0.6 nM, respectively. In SNU-16 and NCI-H520 cell lines, IC50s of dovitinib, lenvatinib, ENMD-2076, and LY2874455 were 76/1355 nM, 94/4770 nM, 306/2280 nM, and 0.3/664 nM, respectively. Furthermore, analyses of PDX tissues by Affymetrix SNP Array 6.0 arrays revealed no amplification of FGFR1/2 genes in four models except for amplifications of FGFR3 gene in two models. Whole exome sequencing did not detect nonsynonymous genetic alterations, including SNPs and indels, in FGFR1/2 genes in these xenografts except for a few genetic variations in FGFR3/4 genes. Overall, due to the multi-targeting mechanisms of these inhibitors, in vitro inhibitory effects of the compounds on FGFR1 enzyme and cell proliferation may not correlate with the in vivo anti-tumor activities. Overexpression of FGFR1 gene may serve as a predictive biomarker for therapeutic intervention of the FGF/FGFR pathway in HCC by using lenvatinib, dovitinib and ENMD-2076. Further studies are required to define additional biomarkers for LY2874455. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in a subset of HCC patients expressing a high level of FGFR1 gene. Citation Format: Douglas D. Fang, Bin Zhang, Weiguo Xu, Kang Yan, Qingyang Gu, Qiang Xu, Yexiong Tan, Hongye Sun, Qin Luo, Weifeng Mao, Chiho Li, Jiangpeng Liao, Guibai Liang, Shu-Hui Chen, Chi-Chung Chan. Antitumor activities of FGFR inhibitors in FGFR1-overexpressing hepatocellular carcinoma patient-derived xenograft tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2839. doi:10.1158/1538-7445.AM2014-2839

Novel celastrol derivatives inhibit the growth of hepatocellular carcinoma patient-derived xenografts.

The molecular co-chaperone CDC37 is over-expressed in hepatocellular carcinoma (HCC) cells, where it functions with HSP90 to regulate the activity of protein kinases in multiple oncogenic signaling pathways that contribute towards hepatocarcinogenesis. Disruption of these signaling pathways via inhibition of HSP90/CDC37 interaction is therefore a rational therapeutic approach. We evaluated the anti-tumor effects of celastrol, pristimerin, and two novel derivatives (cel-D2, and cel-D7) on HCC cell lines in vitro and on orthotopic HCC patient-derived xenografts in vivo. All four compounds preferentially inhibited viability of HCC cells in vitro,and significantly inhibited the growth of three orthotopic HCC patient-derived xenografts in vivo; with the novel derivatives cel-D2 and cel-D7 exhibiting lower toxicity. All four compounds also induced cell apoptosis; and promoted degradation and inhibited phosphorylation of protein kinases in the Raf/MEK/ERK and PI3K/AKT/mTOR signaling pathways. We demonstrated that HSP90/CDC37 antagonists are potentially broad spectrum agents that might be beneficial for treating the heterogeneous subtypes of HCC, either as monotherapy, or in combination with other chemotherapeutic agents.

Imaging of hepatocellular carcinoma patient-derived xenografts using 89Zr-labeled anti-glypican-3 monoclonal antibody

Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in >50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe 89Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with 89Zr, and evaluated its tumor-targeting capacity. In vitro, 89Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, 89Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, 89Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, 89Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention.

MicroRNA-148a dysregulation discriminates poor prognosis of hepatocellular carcinoma in association with USP4 overexpression.

Hepatocellular carcinoma (HCC) is classified as a poor prognostic tumor, and becomes frequently aggressive. MicroRNAs emerge as key contributors to tumor progression. This study investigated whether miR-148a dysregulation differentiates poor prognosis of HCC, exploring new targets of miR-148a. miR-148a dysregulation discriminated not only the overall survival and recurrence free survival rates of HCC, but the microvascular invasion. In the human HCC samples, ubiquitin specific protease 4 (USP4) and sphingosine 1-phosphate receptor 1 (S1P1) were up-regulated as the new targets of miR-148a. USP4 and S1P1 were up-regulated in mesenchymal-type liver-tumor cells with miR-148a dysregulation, facilitating migration and proliferation of tumor cells. The inverse relationship between miR-148a and the identified targets was verified in a tumor xenograft model. In the analysis of human samples, the expression of USP4, but not S1P1, correlated with the decrease of miR-148a. In a heterotropic patient-derived HCC xenograft model, USP4 was also overexpressed in G1 and G2 tumors when miR-148a was dysregulated, reflecting the closer link between miR-148a and USP4 for a shift in the expansion phase of tumorgraft. In conclusion, miR-148a dysregulation affects the poor prognosis of HCC. Of the identified targets of miR-148a, USP4 overexpression may contribute to HCC progression towards more aggressive feature.

Transcriptional dissection of pancreatic tumors engrafted in mice.

BackgroundEngraftment of primary pancreas ductal adenocarcinomas (PDAC) in mice to generate patient-derived xenograft (PDX) models is a promising platform for biological and therapeutic studies in this disease. However, these models are still incompletely characterized. Here, we measured the impact of the murine tumor environment on the gene expression of the engrafted human tumoral cells.MethodsWe have analyzed gene expression profiles from 35 new PDX models and compared them with previously published microarray data of 18 PDX models, 53 primary tumors and 41 cell lines from PDAC. The results obtained in the PDAC system were further compared with public available microarray data from 42 PDX models, 108 primary tumors and 32 cell lines from hepatocellular carcinoma (HCC). We developed a robust analysis protocol to explore the gene expression space. In addition, we completed the analysis with a functional characterization of PDX models, including if changes were caused by murine environment or by serial passing.ResultsOur results showed that PDX models derived from PDAC, or HCC, were clearly different to the cell lines derived from the same cancer tissues. Indeed, PDAC- and HCC-derived cell lines are indistinguishable from each other based on their gene expression profiles. In contrast, the transcriptomes of PDAC and HCC PDX models can be separated into two different groups that share some partial similarity with their corresponding original primary tumors. Our results point to the lack of human stromal involvement in PDXs as a major factor contributing to their differences from the original primary tumors. The main functional differences between pancreatic PDX models and human PDAC are the lower expression of genes involved in pathways related to extracellular matrix and hemostasis and the up- regulation of cell cycle genes. Importantly, most of these differences are detected in the first passages after the tumor engraftment.ConclusionsOur results suggest that PDX models of PDAC and HCC retain, to some extent, a gene expression memory of the original primary tumors, while this pattern is not detected in conventional cancer cell lines. Expression changes in PDXs are mainly related to pathways reflecting the lack of human infiltrating cells and the adaptation to a new environment. We also provide evidence of the stability of gene expression patterns over subsequent passages, indicating early phases of the adaptation process.

Preclinical Evaluation of Transcriptional Targeting Strategy for Human Hepatocellular Carcinoma in an Orthotopic Xenograft Mouse Model

Gene regulation of many key cell-cycle players in S-, G(2) phase, and mitosis results from transcriptional repression in their respective promoter regions during the G(0) and G(1) phases of cell cycle. Within these promoter regions are phylogenetically conserved sequences known as the cell-cycle-dependent element (CDE) and cell-cycle genes homology regions (CHR) sites. Thus, we hypothesize that transcriptional regulation of cell-cycle regulation via the CDE/CHR region together with liver-specific apolipoprotein E (apoE)-hAAT promoter could bring about a selective transgene expression in proliferating human hepatocellular carcinoma. We show that the newly generated vector AH-6CC-L2C could mediate hepatocyte-targeted luciferase gene expression in tumor cells and freshly isolated short-term hepatocellular carcinoma cultures from patient biopsy. In contrast, normal murine and human hepatocytes infected with AH-6CC-L2C expressed minimal or low luciferase activities. In the presence of prodrug 5-fluorocytosine (5-FC), AH-6CC-L2C effectively suppressed the growth of orthotopic hepatocellular carcinoma patient-derived xenograft mouse model via the expression of yeast cytosine deaminase (yCD) that converts 5-FC to anticancer metabolite 5-fluoruracil. More importantly, we show that combination treatment of AH-6CC-L2C with an EZH2 inhibitor, DZNep, that targets EpCAM-positive hepatocellular carcinoma, can bring about a greater therapeutic efficacy compared with a single treatment of virus or inhibitor. Our study showed that targeting proliferating human hepatocellular carcinoma cells through the transcriptional control of therapeutic gene could represent a feasible approach against hepatocellular carcinoma.

Abstract 2779: Establishment and molecular characterization of a panel of Asian patient-derived tumor xenograft models .

Abstract Rodent tumor models with histological and molecular resemblance of human tumors and improved predictive value for clinical drug response are highly desired for oncology drug discovery and development. Patient-derived xenograft (PDX) tumor models are believed to better preserve features of human malignancy than cancer cell line-derived xenograft models. We have established more than 170 PDX models from Asian-prevalent human tumors in SCID or nude mice. Here we report molecular and pharmacological profiling of a panel of pancreatic adenocarcinoma (PAC) and hepatocellular carcinoma (HCC) models. Among 13 PAC models, Sequenom analysis showed KRAS mutation in all models, p53 mutation in 3, p16/CDKN2A deletion in 9 and SMAD4 deletion in 4 models. Comparison of transcriptomes by Affymetrix U133Plus2 between the original tumor and derived xenografts at different passages (P1-P6) in one PAC model revealed a high degree of similarity (R2 =0.92-0.97). Also, the gene mutational status and histological characters remained unchanged across the parental tumor and different passages of the PDX model. Evaluation of response to Gemzar treatment (60 mpk, Q4D x 3) showed significant tumor regression in 6 PAC models and partial tumor growth inhibition in 4 PAC models. Notably, the regression cohort and the partial response cohort displayed differential gene expression patterns. In addition, primary tumor cells derived from PAC models via tissue digestion were tested in vitro with Gemzar. The in vitro sensitivity to Gemzar of derived cells correlated with in vivo response of the parental PAC models. Gene expression analysis in 11 HCC models found aberrant gene expression involving several signaling pathways such as WNT, EGF/IGF and TGF-β, which recapitulate the alteration of these pathways in human HCC. These HCC PDX models exhibited major features of three HCC subclasses defined by the study in human HCC: S1 with activation of WNT and TGF-β pathways, S2 with enriched AKT and MYC activation and S3 with β-catenin activation. The response of these models to Sorafenib was also examined and varying degrees of sensitivity to Sorafenib were observed. Together, we have successfully generated PDX tumor models that preserve the histological and biological properties of the original human tumor and represent a heterogeneous patient population with varying degree of response to current stand of care. These models are a relevant and powerful tool for evaluation of anticancer therapeutics. Whole genome sequencing and gene expression profiling by RNAseq on PDX models are being preformed. Citation Format: Xiaoran Qin, Zhonghua Tang, Gang Hu, Kedong Ouyang, Ke Wang, Fu Li, Fubo Xie, Qiuming Pan, Min Shi, Gang Zhao, Yixin Zhang, Chunchao Zhu, Danyi Wen, Weikang Tao. Establishment and molecular characterization of a panel of Asian patient-derived tumor xenograft models . [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 2779. doi:10.1158/1538-7445.AM2013-2779

Foretinib demonstrates anti-tumor activity and improves overall survival in preclinical models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. The purpose of this study is to assess the anti-tumor and anti-angiogenic activities of foretinib, a vascular endothelial growth factor receptor 2 (VEGFR-2) and c-Met inhibitor using mouse models of human HCC. SK-HEP1 and 21-0208 HCC cells as well as patient-derived HCC models were employed to study the anti-tumor and antiangiogenic activities of foretinib. Changes of biomarkers relevant to hepatocyte growth factor (HGF) signaling pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Treatment of SK-HEP1 cells with foretinib resulted in growth inhibition, G2/M cell cycle arrest, reduced colony formation and blockade of HGF-induced cell migration. In both orthotopic and ectopic models of HCC, foretinib potently inhibited tumor growth in a dose-dependent manner. Inhibition of angiogenesis correlated with inactivation of VEGFR-2/c-Met signaling pathways. Foretinib also caused elevation of p27 and Bim but reduced cyclin B1 expression and p-c-Myc, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, foretinib potently inhibited primary tumor growth and significantly prolonged mouse survival. Foretinib demonstrated significant antitumor activities in patient-derived HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.

Abstract B3: Allosteric MEK inhibitor BAY 86-9766 acts synergistically with sorafenib to inhibit tumor growth, angiogenesis, and tumor cell survival in ectopic patient-derived xenograft models of human hepatocellular carcinoma.

Abstract Background: Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor and the Raf/MEK/ERK pathway is activated in HCC. Sorafenib has been shown to prolong survival of patients with advanced HCC. BAY 86-9766 (RDEA119) is an orally available, potent, allosteric MEK inhibitor currently in Phase I/II trials. A combination therapy of MEK inhibitor BAY 86-9766 with sorafenib might enhance the therapeutic efficacy of sorafenib in HCC. This study aims to investigate the potential of such a combined therapeutic approach in preclinical models of HCC. Methods: We treated 15 patient-derived HCC xenograft models (Asian origin) with i) sorafenib, ii) BAY 86-9766, and iii) sorafenib plus BAY 86-9766. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry. Results: We report here that sorafenib significantly suppressed tumor growth with the T/C ratios ranging from 0.25 to 0.6, angiogenesis and cell proliferation with modest elevation of pERK Thr202/Tyr204. BAY 86-9766 treatment caused a modest tumor growth inhibition (ranging from 30 to 60%) and dephosphorylation of ERK, Bim and p90RSK. The addition of BAY 86-9766 to sorafenib significantly augmented the antitumor activity of sorafenib in 15 models of HCC. The T/C ratios for the sorafenib/BAY 86-9766 ranged from 0.03 to 0.2. Such inhibition led to further increase in Bad, Bim and apoptosis. Profound inhibition of cell proliferation, angiogenesis and inactivation of p90RSK and mTOR targets were also observed in the combined therapy. Conclusion: We show that sorafenib/BAY 86-9766 treatment is considerably more effective than either single agents in suppressing tumor growth, angiogenesis, cell proliferation and cell survival. Our findings underscore the potential of sorafenib/BAY 86-9766 combination in the treatment of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B3.

Dual targeting of mTORC1/C2 complexes enhances histone deacetylase inhibitor-mediated anti-tumor efficacy in primary HCC cancer in vitro and in vivo

The mammalian target of rapamycin (mTOR) plays a pivotal role in hepatocellular carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity, accompanied with feedback activation of AKT. Therefore, dual targeting of mTORC1/C2 should be more efficient in suppressing AKT activity and in enhancing the anti-tumor activity of HDACi in HCC. The interactions between mTOR kinase inhibitors (mTORKis) (i.e., Pp242, AZD8055, OSI027) and HDACis (i.e., SAHA, LBH589) were examined in vitro using HCC cell lines and in vivo using patient-derived primary HCC xenografts on SCID mice. mTORKis significantly enhanced HDACi-induced apoptosis in HCC cells. The inhibition of both mTORC1/2 not only efficiently blocked mTORC1 signaling, but also abrogated AKT-feedback activation caused by selective mTORC1 inhibition. The co-treatment of mTORKi and HDACi further inhibited AKT signaling and upregulated Bim. Dysfunction of mTORC2 by shRNA significantly lowered the threshold of HDACi-induced cytotoxicity by abrogating AKT activation. Knockdown of AKT1 sensitized Pp242/HDACi-induced apoptosis and ectopic expression of constitutively active AKT1 abrogated the combination-induced cytotoxicity, indicating AKT plays a vital role in the combination-induced effects. Knockdown of Bim prevented Pp242/HDACis-induced cytotoxicity in HCC. Lastly, in vivo studies indicated that the combination of AZD8055 and SAHA almost completely inhibited tumor-growth, without obvious adverse effects, by abrogating AKT and upregulating Bim; while either agent alone shows only 30% inhibition in primary HCC xenografts. Our findings suggest that a combining-regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC.

Abstract LB-396: FGFR4-antagonistic CovX-Body™ inhibits tumor growth

Abstract FGFR4 is the receptor for FGF19. Published data has shown FGF19 transgenic animals develop hepatocellular carcinoma (HCC), suggesting that the FGFR4:FGF19 axis may play a role in HCC. FGFR4 upregulation has been shown to be a negative prognostic indicator in multiple tumor types. CVX-63, an FGFR4-specific CovX-Body, was produced by fusing a chemically modified peptide to the specific Fab binding site of a specially designed antibody. CVX-63 was shown to inhibit FGF19 binding to FGFR4 in a competition ELISA with an IC50 of 10 nM, and had no cross reactivity with FGFR1–3. CVX-63 has a beta half life of 55 hrs in the mouse. The anti-tumor activity of CVX-63 was demonstrated in a staged Colo-205 xenograft model, with CVX-63 administered i.v. weekly. Maximal activity of 30% tumor growth inhibition was seen at dosing 10 mg/kg. Immunohistochemistry analysis of treated tumors shows a >90% reduction in phospho-ERK and 50% downregulation of FGFR4 on tumor cells. Ki67, a marker of proliferation was reduced, but active caspase 3 was not found in treated tumors. A patient-derived HCC xenograft was identified which expresses both FGFR4 and FGF19. When tested in this HCC model, CVX-63 did not show any effect inhibiting tumor growth. These data demonstrate that CVX-63 effectively inhibits FGFR4 activity in xenografts. FGFR4 inhibition has a modest effect on human tumor progression and FGFR4 does not seem to be a critical driver of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-396. doi:10.1158/1538-7445.AM2011-LB-396

155 EML4-ALK signaling is required for the maintenance of neoplastic phenotype of non-small cell lung cancer cells: novel strategy for lung cancer tailored therapies

Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic potential.We treated patient-derived HCC xenografts with (i) sorafenib, (ii) AZD6244 (ARRY-142886), and (iii) sorafenib plus AZD6244. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry.We report here that sorafenib treatment resulted in suppression of tumor growth, reduction in cell proliferation, induction of apoptosis and inhibition of mTOR targets. Sorafenib-induced elevation of the insulin-like growth factor receptor 1 (IGF-1R), phospho-c-Raf Ser338, phospho-MEK Ser217/221 and phospho-ERK Thr202/Tyr204 was attenuated by co-treating cells with anti-human IGF-1R antibody or over-expression of activated mutant p70S6K. Pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the anti-tumor effect of sorafenib in both orthotopic and ectopic models of HCC. Such inhibition led to a further increase in pro-apoptotic Bim, apoptosis and a profound inhibition of cell proliferation.Our findings underscore the potential of a combined therapeutic approach with sorafenib and MEK inhibitors in the treatment of HCC.