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Objectives The relative contributions of inflammatory signalling and sequential oncogenic dysregulation driving liver cancer pathogenesis remain incompletely understood. Lymphotoxin-β receptor (LTβR) signalling is critically involved in hepatitis and liver tumorigenesis. Therefore, we explored the interdependence of inflammatory lymphotoxin signalling and specific oncogenic pathways in the progression of hepatic cancer. Design Pathologically distinct liver tumours were initiated by hydrodynamic transfection of oncogenic V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT)/β-catenin or AKT/Notch expressing plasmids. To investigate the relationship of LTβR signalling and specific oncogenic pathways, LTβR antagonist (LTβR-Fc) or agonist (anti-LTβR) were administered post oncogene transfection. Initiated livers/tumours were investigated for changes in oncogene expression, tumour proliferation, progression, latency and pathology. Moreover, specific LTβR-mediated molecular events were investigated in human liver cancer cell lines and through transcriptional analyses of samples from patients with intrahepatic cholangiocarcinoma (ICC). Results AKT/β-catenin-transfected livers displayed increased expression of LTβ and LTβR, with antagonism of LTβR signalling reducing tumour progression and enhancing survival. Conversely, enforced LTβR-activation of AKT/β-catenin-initiated tumours induced robust increases in proliferation and progression of hepatic tumour phenotypes in an AKT-dependent manner. LTβR-activation also rapidly accelerated ICC progression initiated by AKT/Notch, but not Notch alone. Moreover, LTβR-accelerated development coincides with increases of Notch, Hes1, c-MYC, pAKT and β-catenin. We further demonstrate LTβR signalling in human liver cancer cell lines to be a regulator of Notch, pAKTser473 and β-catenin. Transcriptome analysis of samples from patients with ICC links increased LTβR network expression with poor patient survival, increased Notch1 expression and Notch and AKT/PI3K signalling. Conclusions Our findings link LTβR and oncogenic AKT signalling in the development of ICC.

Using two MYCN transgenic mouse strains, we established 10 transplantable neuroblastoma cell lines via serial orthotopic passage in the adrenal gland. Tissue arrays demonstrate that by histochemistry, vascularity, immunohistochemical staining for neuroblastoma markers, catecholamine analysis, and concurrent cDNA microarray analysis, there is a close correspondence between the transplantable lines and the spontaneous tumors. Several genes closely associated with the pathobiology and immune evasion of neuroblastoma, novel targets that warrant evaluation, and decreased expression of tumor suppressor genes are demonstrated. These studies describe a unique and generalizable approach to expand the utility of transgenic models of spontaneous tumor, providing new tools for preclinical investigation.

IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8+ ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4+CD25+Foxp3+ regulatory and IL-17-expressing CD4+ cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

Obesity is a risk factor for development of certain cancers but the basis for this risk is unclear. In this study, we developed a novel mouse model that demonstrates directly how lipogenic phenotypes commonly associated with diet-induced metabolic syndromes can influence hepatic cancer development. Activated AKT and β-catenin (AKT/CAT) genes were hydrodynamically codelivered using the Sleeping Beauty transposon to initiate liver tumorigenesis. AKT/CAT and MET/CAT combination induced microscopic tumor foci by 4 weeks, whereas no tumorigenesis resulted from delivery of AKT, MET, or CAT alone. Primary AKT/CAT tumor cells were steatotic (fatty) hepatocellular adenomas which progressed to hepatocellular carcinomas (HCC) upon in vivo passage, whereas primary MET/CAT tumors emerged directly as frank HCC. Conversion of AKT/CAT tumor cells to frank HCC during passage was associated with induction of the human HCC marker α-fetoprotein and the stem cell marker CD133. Using hierarchical clustering and gene set enrichment analysis, we compared the primary murine AKT/CAT and MET/CAT tumors to a panel of 53 human HCCs and determined that these two mouse models could be stratified as distinct subtypes associated in humans with poor clinical prognosis. The chief molecular networks identified in primary and passaged AKT/CAT tumors were steatosis and lipid metabolic pathways, respectively. Our findings show how coactivation of the AKT and CAT pathways in hepatocytes can efficiently model development of a lipogenic tumor phenotype. Furthermore, we believe that our approach could speed the dissection of microenvironmental factors responsible for driving steatotic-neoplastic transformation to frank carcinoma, through genetic modification of existing immunodefined transgenic models. Cancer Res; 71(7); 2718–27. ©2011 AACR.

Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8 + T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2 −/− mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2 −/− mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-γ-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-γ-dependent reduction in CD4 + /FoxP3 + Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.

Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-γ plus TNF-α, we hypothesized that bortezomib also could sensitize these cells to IFN-γ plus TNF-α. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-γ and TNF-α on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN-γ plus TNF-α in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-γ/TNF-α-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma.

Neuroblastoma is one of the most common solid tumors in children. The prognosis of patients with advanced neuroblastoma is poor overall despite standard therapeutic modalities and has stimulated substantial interest in the potential role for biologics such as immunotherapeutic and/or antiangiogenic agents for the treatment of neuroblastoma. To facilitate preclinical investigation of the efficacy and mechanisms of action of new biologic agents for the treatment of neuroblastoma, a comprehensive panel of disease-specific fluorescence-based model systems has been developed by our group to image the growth, neovascularization, metastasis, and apoptosis of neuroblastoma tumors. These model systems use fluorescent proteins to monitor cytokine-induced alterations in the growth and metastasis of neuroblastoma and allow for monitoring and/or quantitation of even minimal residual disease that is localized to visceral organ sites such as the liver, lung, and/or bone marrow. Further, based on the differential spectra of red fluorescent protein, green fluorescent protein (GFP), and agents such as 4'-6-diamidino-2-phenylindole (DAPI) (blue) and fluorescein isothiocyanate-dextran (green), multicolor systems have now been established by our group that allow for combined assessment of parameters, including the macroscopic relation of tumors to their associated vasculature and, within tissue sections, simultaneous quantitation of tumor neovascularization and evaluation of therapy-induced apoptosis within the tumor and vascular endothelial compartments. Further, by engineering cells to express specific mediators of apoptosis that have been linked to GFP (ie, BID-EGFP), these systems can also be used to dissect mechanisms by which neuroblastoma cells are induced to undergo apoptosis in vitro as well as in vivo. Collectively, these model systems provide important tools for investigation of the biology of neuroblastoma tumors and evaluation of mechanisms that mediate the regression of these tumors in response to novel therapeutic agents, including cytokines such as interleukin-12.

Background: Patients with advanced neuroblastoma have a poor prognosis. The antiapoptotic protein Akt has been implicated as a possible mediator of the resistance of human neuroblastoma cells to apoptosis; the proapoptotic protein Bid, is inhibited by activated Akt. Neuroblastoma has demonstrated responsiveness to immunotherapeutic approaches in preclinical studies, prompting investigation of new therapeutic strategies based on potentiation of the host immune response, including the use of systemic cytokines. Methods: We examined the antitumor effi cacy and mechanisms of action of the central immunoregulatory cytokine interleukin12 (IL-12) in mice bearing established orthotopic neuroblastoma tumors derived from murine TBJ and Neuro-2a cells. Cohorts of mice (10 mice/group) bearing established orthotopic neuroblastoma tumors were injected intraperitoneally with IL-12 or vehicle and monitored for survival. IL-12 – induced apoptosis within the tumor microenvironment was investigated using ribonuclease protection assays, nuclear staining, and electron microscopy. Protein expression was de termined via Western blot analysis and enzyme-linked immunosorbent assays. Confocal microscopy was used to examine the distribution of overexpressed Bid – enhanced green fl uorescent protein fusion protein (Bid-EGFP) in TBJ cells. All statistical tests were two-sided. Results : IL-12 induced complete tumor regression and long-term survival of 8 (80%) of 10 mice bearing established neuroblastoma tumors compared with 1 (10%) of 10 control mice ( P = .0055) and profound tumor cell apoptosis in vivo despite the fact that TBJ and Neuro-2a cells were resistant to receptor- mediated apoptosis in vitro . These cells expressed high levels of phosphorylated Akt, a key prosurvival molecule, and Akt inhibitors sensitized neuroblastoma cells to apoptosis mediated by IL-12 – inducible cytokines including tumor necrosis factor- α and interferon- γ in vitro. IL-12 increased the expression of proapoptotic genes and decreased Akt phosphorylation within established TBJ tumors in conjunction with activation and subcellular translocation of Bid. Conclusions: Our results suggest that IL-12 overcomes a potentially critical mechanism of tumor self-defense in vivo by inhibiting Akt activity and imply that IL-12 may possess unique therapeutic activity against tumors that express high levels of activated Akt. [J Natl Cancer Inst 2006;98:190 – 202]

We have shown previously that IFN-γ-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8+ T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-γ. In addition, marked enhancement of local IFN-γ gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8+, but not CD4+ T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third most common cause of cancer related deaths worldwide. We set out to perform immune studies in HCC. In a recent published mouse model of HCC, the hydrodynamic injection of two plasmids, pT3-EF5-hMET (pMET) and pT3CAT led to prompt tumor growth. We modified the pT3CAT plasmid with conventional PCR cloning methods to create pCAT_LucOS, in which luciferase including T cell epitopes is expressed in addition to the onogene. Whereas co-delivery of pMET and pT3CAT in C57BL/6 mice led to rapid tumor development, that required euthanasia within 9 weeks, co-delivery of pMET and pCAT_LucOS did not lead to any signs of tumor burden over a course of 7 months. In contrast, survival studies with immunocompromised Rag1KO mice showed similar survival between mice that were injected with the same plasmid combinations. CAT gene expression was elevated over normal liver tissue level in pMET and pT3CAT injected C57BL/6 mice over the course of the 9 week experiment. In pMET and pCAT_LucOS injected mice the CAT expression level was elevated only in the first 2 weeks after the injection. The tumor bearing pMET and pT3CAT injected mice showed increased frequencies of CD11b+Gr-1+ MDSC and CD11b+F4/80+ Macrophages and decreased frequencies of CD4+ and CD8+ T cells in the liver. In pMET and pCAT_LucOS injected mice an increase in CD8+ T cells in the first two weeks was seen together with strong CD8 response against one of the tumor specific T cell epitopes.

Meeting abstracts To investigate the relationship between tumor-associated CD40 expression, RCC patient survival and tumor stage. The expression of CD40, TUNEL and CD8 in human renal cell carcinomas was analyzed by immunohistochemistry. Computer-assisted quantitation of protein expression was used

Many tumors have been demonstrated to contain a subpopulation of cancer stem cells (CSC) whose epigenetic modifications convey resistance to conventional therapies. Furthermore, tumor expression of genes associated with pluripotent embryonic cells, such as Oct-4, Sox-2 and Nanog, often correlates with poor prognosis. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) would be a possible alternative to cell surface phenotype for identification of CSC populations. Therefore we evaluated Nanog expression in 4T1.2 murine breast carcinoma cells, as a biomarker for CSC sub-populations. Introduction of a Nanog promoter reporter that drives destabilized GFP into 4T1.2 cells resulted in a stable expression of GFP in about 1% of the cells. These GFP+ cell could be easily isolated by FACS cell sorting. On subsequent in vitro cell culture of GFP+ populations, up to 80% of the cells became GFP- over a period of 4 weeks. This would be consistent with a subsequent differentiation of these cells. By contrast GFP- populations remained 100% GFP-. In vitro CSC surrogate assays show that Nanog-GFP+ cells produced more spheroids in soft agar and under non-adherent growth conditions. More importantly, in vivo cell transfer studies demonstrated that Nanog-GFP+ cells were more efficient at generating experimental lung metastases when compared to Nanog-GFP-cells. Interestingly Nanog-GFP+ cells were more resistant to the chemotherapeutic drugs paclitaxel and bortezomib. However targeting the extrinsic apoptosis pathway with a combination of bortezomib and agonist antibodies to the TRAIL death receptor DR5 was equally effective against both Nanog-GFP+ and Nanog-GFP- cells. These findings suggest that Nanog promoter activity is a robust marker of highly metastatic 4T1.2 CSC subpopulations, and suggests that eradication of such cells may be required for improved anti-cancer therapies. Funded by NCI Contract HHSN261200800001E Citation Format: Alan D. Brooks, Rachel L. de Kluyver, Jimmy K. Stauffer, Marcella Kaddoura, Thomas J. Sayers. Using Nanog expression to identify breast cancer stem cell populations. [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 2656. doi:10.1158/1538-7445.AM2013-2656

The development of accurate disease models is critically important in the ongoing search for better therapies and ultimately cures. Neoplasia manifests through the aberrant development of altered cellular phenotypes. Such phenotypes are characterized by the particular subset of genes expressed. The pathological regulation of gene expression is a function of both cell-innate and tissue-homeostatic responses including inflammation and regeneration. However, little is understood about the actual cellular and biochemical interrelationship between these mechanisms in guiding initiation, promotion, and progression of cancer. The experimental rationale we use in addressing this problem facilitates the interrogation and manipulation of microenvironmental signals (inflammation) and collaborating neoplastic-precursor cell oncogenic lesions resulting in hepatic tumorigenesis. By technical necessity, this approach employs the delivery of disease-specific oncogenes into relevant somatic tissues (hepatocytes). The gene delivery technique used in theses studies, Sleeping Beauty transposition, is highly versatile and efficient, shortening the “gene cocktail to tumor” testing cycle to levels not possible through germ-line transgenesis or any other current in vivo modality. We use this system to first identify collaborating oncogenic- genes/signaling pathways able to initiate hepatic tumorigenesis in vivo. We have found activated AKT, MET, and beta-catenin (CAT) are initiating oncogenes that efficiently induce hepatic tumorigenesis only when co-delivered but not as single agents. Additionally we have been able to establish transplantable hepatocellular carcinoma lines derived by serial transplantation from the AKT/CAT model in vivo. We are currently characterizing and comparing these models to Human hepatocellular carcinoma using QPCR, RNA microarray and immunohistochemical analysis. We will test these individual initiating oncogenes for collaboration with inflammatory microenvironments (hepatotoxins) in the induction of tumorigenesis in order to recapitulate inflammation induced cancer. We are testing these defined tumor initiating models in immunocompromised and transgenic mice to identify the role of host-derived microenvironmental interactions. We are also testing a highly sensitive reporter, Gaussia luciferase, along with the oncoproteins to prospectively monitor tumor growth that otherwise would be undetectable. We are also optimizing highly sensitive PCR based assays to define the kinetics of the molecular events necessary for tumor initiation and early establishment. By using this molecularly defined approach to hepatocellular oncogenesis we will be able to rapidly reproduce oncogene signaling pathways leading to tumorigenesis thereby providing an ideal test bed for therapeutic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4375.