Sarangarajan Ranganathan

Activation of β-Catenin and Yap1 in Human Hepatoblastoma and Induction of Hepatocarcinogenesis in Mice

BACKGROUND & AIMS Aberrant activation of β-catenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with the development of multiple tumor types. Yap functions as a transcriptional coactivator by interacting with TEA domain DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. METHODS We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the United States, consisting of 104 hepatocellular carcinoma, 62 intrahepatic cholangiocarcinoma, and 94 hepatoblastoma samples. We assessed β-catenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering RNAs, and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (ΔN90/β-catenin) and Yap1 (YapS127A) in livers of mice; tissues were collected, and histological and immunohistochemical analyses were performed. RESULTS We observed nuclear localization of β-catenin and Yap1 in 79% of hepatoblastoma samples but not in most hepatocellular carcinoma or intrahepatic cholangiocarcinoma samples. Yap1 and β-catenin coprecipitated in hepatoblastoma but not hepatocellular carcinoma cells. Small interfering RNA-mediated knockdown of Yap1 or β-catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to coactivate transcription with β-catenin; β-catenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and β-catenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumor cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEA domain factors was required for tumorigenesis in mice. CONCLUSIONS β-catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets.

Deregulation of Hippo kinase signalling in human hepatic malignancies.

Hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and hepatoblastoma (HB) are the main hepatic malignancies with limited treatment options and high mortality. Recent studies have implicated Hippo kinase pathway in cancer development, but detailed analysis of Hippo kinase signalling in human hepatic malignancies, especially CC and HB, is lacking.

Pediatric small bowel transplantation

The multivisceral liver-intestine-pancreas-stomach allograft was first described by Starzl nearly 50 years ago. Since then, over 1000 children have received small bowel transplantation (SBTx), alone or with the liver and other organs, for refractory short gut syndrome (SGS) because of a variety of congenital conditions. In 2001, SBTx was approved as definitive therapy for SGS by Medicare. Currently, 1- and 5-year graft survival routinely exceeds 90% and 80%, respectively. The expected outcomes also include freedom from parenteral nutrition, normalization of growth parameters, and quality of life. However, recurrent rejection, complications of high-dose immunosuppression, or chronic rejection, which is more likely to occur after SBTx without a liver graft, account for differences between early and late survival. Future efforts aimed at overcoming such challenges include preventing SBTx through early referral to comprehensive SGS management programs and understanding why the liver protects the small bowel allograft from rejection. Finally, inflammatory mechanisms, which predispose the highly immunogenic small bowel allograft to a protracted risk of resistant rejection must be elucidated, in order to ensure durable success.

Towards an international pediatric liver tumor consensus classification: proceedings of the Los Angeles COG liver tumors symposium

Liver tumors are rare in children, and their diagnoses may be challenging particularly because of the lack of a current consensus classification system. Systematic central histopathological review of these tumors performed as part of the pediatric collaborative therapeutic protocols has allowed the identification of histologic subtypes with distinct clinical associations. As a result, histopathology has been incorporated within the Children’s Oncology Group (COG) protocols, and only in the United States, as a risk-stratification parameter and for patient management. Therefore, the COG Liver Tumor Committee sponsored an International Pathology Symposium in March 2011 to discuss the histopathology and classification of pediatric liver tumors, and hepatoblastoma in particular, and work towards an International Pediatric Liver Tumors Consensus Classification that would be required for international collaborative projects. Twenty-two pathologists and experts in pediatric liver tumors, including those serving as central reviewers for the COG, European Société Internationale d’Oncologie Pédiatrique, Gesellschaft für Pädiatrische Onkologie und Hämatologie, and Japanese Study Group for Pediatric Liver Tumors protocols, as well as pediatric oncologists and surgeons specialized in this field, reviewed more than 50 pediatric liver tumor cases and discussed classic and newly reported entities, as well as criteria for their classification. This symposium represented the first collaborative step to develop a classification that may lead to a common treatment-stratification system incorporating tumor histopathology. A standardized, clinically meaningful classification will also be necessary to allow the integration of new biological parameters and to move towards clinical algorithms based on patient characteristics and tumor genetics, which should improve future patient management and outcome.

Genetic polymorphisms impact the risk of acute rejection in pediatric heart transplantation: a multi-institutional study.

Objective. The objective of this study was to determine the association between the genetic polymorphisms of proinflammatory and regulatory cytokines and long-term rates of repeat and late acute rejection episodes in pediatric heart transplant (PHTx) recipients. Methods. Three hundred twenty-three PHTx recipients: 205 White non-Hispanic, 43 Black non-Hispanic, and 75 Hispanic were analyzed for time to first repeat and late acute rejection episodes by race, age at transplantation, and gene polymorphism (interleukin [IL]-6, −174 G/C, IL-10, −1082 G/A, −819 C/T, 592 C/A; vascular endothelial growth factor (VEGF) −2578 C/A, −460 C/T, +405 C/G; tumor necrosis factor alpha (TNF-&agr;)−308 G/A). Results. Recipient black race and older age at transplant were risk factors for both repeat and late rejections, though black race was more significantly related to late rejection (P=0.006). Individually, TNF-&agr; high, IL-6 high, VEGF high, and IL-10 low phenotypes did not impact the risk of repeat or late rejection. However, the combination VEGF high/IL-6 high and IL-10 low was associated with increased estimated risk of late rejection (P=0.0004) and only marginally with repeat rejection (P=0.051). In a multivariate analysis, adjusting for age and race, VEGF high/IL-6 high and IL-10 low still remained an independent risk factor for late acute rejection (RR=1.91, P<0.001). Conclusion. This is the largest multicenter study to document the impact of genetic polymorphism combinations on PHTx recipients’ outcome. The high proinflammatory (VEGF high/IL-6 high) and lower regulatory (IL-10 low) cytokine gene polymorphism profile exhibited increased risk for late rejection, irrespective of age and race/ethnicity.

Vaccine against MUC1 Antigen Expressed in Inflammatory Bowel Disease and Cancer Lessens Colonic Inflammation and Prevents Progression to Colitis-Associated Colon Cancer

Association of chronic inflammation with an increased risk of cancer is well established, but the contributions of innate versus adaptive immunity are not fully delineated. There has furthermore been little consideration of the role played by chronic inflammation–associated antigens, including cancer antigens, and the possibility of using them as vaccines to lower the cancer risk. We studied the human tumor antigen MUC1 which is abnormally expressed in colon cancers and also in inflammatory bowel disease (IBD) that gives rise to colitis-associated colon cancer (CACC). Using our new mouse model of MUC1+ IBD that progresses to CACC, interleukin-10 knockout mice crossed with MUC1 transgenic mice, we show that vaccination against MUC1 delays IBD and prevents progression to CACC. One mechanism is the induction of MUC1-specific adaptive immunity (anti-MUC1 IgG and anti-MUC1 CTL), which seems to eliminate abnormal MUC1+ cells in IBD colons. The other mechanism is the change in the local and the systemic microenvironments. Compared with IBD in vaccinated mice, IBD in control mice is dominated by larger numbers of neutrophils in the colon and myeloid-derived suppressor cells in the spleen, which can compromise adaptive immunity and facilitate tumor growth. This suggests that the tumor-promoting microenvironment of chronic inflammation can be converted to a tumor-inhibiting environment by increasing adaptive immunity against a disease-associated antigen. Cancer Prev Res; 3(4); 438–46. ©2010 AACR.

β-Catenin and Met Deregulation in Childhood Hepatoblastomas

ABSTRACTActivation of the Wnt/β-catenin and hepatocyte growth factor/Met signaling has been implicated in various tumors. Owing to the cross-talk between these pathways and aberrant redistribution of β-catenin in hepatoblastomas, we examined their status in this tumor. This study examined changes in β-catenin and Met in paired pretreatment and post-treatment hepatoblastoma tissues in relation to their effects on proliferation and target genes such as c-myc and cyclin-D1. In this study we compared proliferation indices, β-catenin staining and its known molecular targets, c-myc and cyclin-D1, and Met, a tyrosine kinase receptor for hepatocyte growth factor in pretreatment and post-treatment specimens. Pretreatment and post-treatment sections from 13 children, ages 11 weeks to 9 years, were analyzed for these markers by immunohistochemistry. All tumors (13 of 13) displayed increased proliferation and β-catenin (cytoplasmic and nuclear) staining in pretreatment biopsies that remained relatively unaffected after treatment. Aberrant Met staining (cytoplasmic) was observed in all pretreatment samples that decreased considerably after treatment in 11 of 13 patients. A significant subset of these tumors showed increased c-myc and cyclin-D1 staining in pretreatment biopsies that decreased after chemotherapy in most cases. β-Catenin redistribution in tumor cells corresponds to proliferation in hepatoblastomas. However, β-catenin nuclear localization remains unaffected in viable hepatoblastoma tissue after chemotherapy. In contrast, Met undergoes a prominent decrease after treatment and thus might be important in pathogenesis of hepatoblastoma.

Assaying macrophage activity in a murine model of inflammatory bowel disease using fluorine-19 MRI.

Macrophages have an important role in the pathogenesis of most chronic inflammatory diseases. A means of non-invasively quantifying macrophage migration would contribute significantly towards our understanding of chronic inflammatory processes and aid the evaluation of novel therapeutic strategies. We describe the use of a perfluorocarbon tracer reagent and in vivo 19F magnetic resonance imaging (MRI) to quantify macrophage burden longitudinally. We apply these methods to evaluate the severity and three-dimensional distribution of macrophages in a murine model of inflammatory bowel disease (IBD). MRI results were validated by histological analysis, immunofluorescence and quantitative real-time polymerase chain reaction. Selective depletion of macrophages in vivo was also performed, further validating that macrophage accumulation of perfluorocarbon tracers was the basis of 19F MRI signals observed in the bowel. We tested the effects of two common clinical drugs, dexamethasone and cyclosporine A, on IBD progression. Whereas cyclosporine A provided mild therapeutic effect, unexpectedly dexamethasone enhanced colon inflammation, especially in the descending colon. Overall, 19F MRI can be used to evaluate early-stage inflammation in IBD and is suitable for evaluating putative therapeutics. Due to its high macrophage specificity and quantitative ability, we envisage 19F MRI having an important role in evaluating a wide range of chronic inflammatory conditions mediated by macrophages.

A Pilot Study of Low-Dose Anti-Angiogenic Chemotherapy in Combination With Standard Multiagent Chemotherapy for Patients With Newly Diagnosed Metastatic Ewing Sarcoma Family of Tumors: A Children's Oncology Group (COG) Phase II Study NCT00061893

The aims of this study were to determine the feasibility of the combination of low dose, anti‐angiogenic chemotherapy with standard therapy for patients with metastatic Ewing sarcoma (ES), and to obtain preliminary outcome data.

Hodgkin-like Posttransplant Lymphoproliferative Disorder in Children: Does It Differ from Posttransplant Hodgkin Lymphoma?

Epstein-Barr virus (EBV)-mediated lymphoid proliferations occur in patients who are immunocompromised and are reported following bone marrow or solid organ transplantation. Most post-transplant lymphoproliferative disorders (PTLD) are polymorphic in appearance; some are monomorphic and can resemble conventional malignant lymphomas. PTLD that resembles Hodgkin lymphoma has been reported infrequently. We herein report seven cases of PTLD that have large numbers of Reed-Sternberg-like (RS-like) cells and highlight differences in the phenotype of these cases that may distinguish Hodgkin-like PTLD (HL-PTLD) from true Hodgkin lymphoma/disease (HD). All patients were in the second decade of life and were 8 months to 13 years following transplant. HL-PTLD involves lymph nodes that contain a mixed population of small to intermediate-sized lymphocytes with large mononuclear and occasionally binucleate RS-like cells. The large cells of HL-PTLD are pleomorphic B cells that react strongly for CD20 and/or CD79a, express CD30, but are usually negative for CD15 and have few mitoses. They are positive for EBV early RNA (EBER) using an EBER-1 probe, as are some of the background small lymphocytes. A single case of true Hodgkin lymphoma has highly atypical RS-like cells that contain numerous mitoses, does not have CD20 or CD79a reactivity, has CD15 and CD30 staining, and the EBER-1 probe is confined to the large cells only. All patients were managed by withdrawal of immunosuppression and variably treated with either antiviral or anti-CD20 monoclonal antibody, or with chemotherapy. A unique instance of evolution from a HL-PTLD to true HD is also illustrated. In conclusion, HL-PTLD and HD appear to be two related but immunophenotypically and biologically distinct forms of lymphoproliferation in post-transplant patients and may require different protocols for their management.