Mike A. Leonis

Mammary-Specific Ron Receptor Overexpression Induces Highly Metastatic Mammary Tumors Associated with β-Catenin Activation

Activated growth factor receptor tyrosine kinases (RTK) play pivotal roles in a variety of human cancers, including breast cancer. Ron, a member of the Met RTK proto-oncogene family, is overexpressed or constitutively active in 50% of human breast cancers. To define the significance of Ron overexpression and activation in vivo, we generated transgenic mice that overexpress a wild-type or constitutively active Ron receptor in the mammary epithelium. In these animals, Ron expression is significantly elevated in mammary glands and leads to a hyperplastic phenotype by 12 weeks of age. Ron overexpression is sufficient to induce mammary transformation in all transgenic animals and is associated with a high degree of metastasis, with metastatic foci detected in liver and lungs of >86% of all transgenic animals. Furthermore, we show that Ron overexpression leads to receptor phosphorylation and is associated with elevated levels of tyrosine phosphorylated beta-catenin and the up-regulation of genes, including cyclin D1 and c-myc, which are associated with poor prognosis in patients with human breast cancers. These studies suggest that Ron overexpression may be a causative factor in breast tumorigenesis and provides a model to dissect the mechanism by which the Ron induces transformation and metastasis.

Evaluation and Management of End-Stage Liver Disease in Children

End-stage liver disease in children presents a challenging array of medical and psychosocial problems for the health care delivery team. Many of these problems are similar to those encountered by caregivers of adults with end-stage liver disease, such as the development of complications of cirrhosis, including ascites, spontaneous bacterial peritonitis, and esophageal variceal hemorrhage. However, the natural history of disease progression in children and their responses to medical therapy can differ significantly from that of their adult counterparts. Children with end-stage liver disease are especially vulnerable to nutritional compromise; if not effectively managed, this can seriously impact long-term outcomes and survival both before and after liver transplantation. Moreover, close attention must be given to vaccination status and the clinical setting at which health care is delivered to optimize outcomes and the delivery of high-quality pediatric health care. In this review, we address important components of the evaluation and management of children with chronic end-stage liver disease.

Ron-receptor tyrosine kinase in tumorigenesis and metastasis

The Ron-receptor tyrosine kinase has been increasingly recognized for its tumorigenic potential in the last decade. Ron-receptor activation leads to the activation of common receptor tyrosine kinase downstream-signaling pathways, and most prominently in tumor models, activation of MAPK, PI3K and beta-catenin. Numerous experimental models of mammalian tumorigenesis have demonstrated that increased Ron-receptor activity correlates with increased tumorigenesis in a variety of organs of epithelial origin. The evidence for Ron as an oncogene in human tumor biology is growing. The Ron receptor is overexpressed and over activated in a large number of human tumors, and overexpression of Ron correlates with a worse clinical outcome for patients in at least two human cancer states, namely breast and bladder cancer. Several experimental approaches have been demonstrated to successfully block Ron activity and function, and given these convincing data, approaches to block Ron-receptor activity in targeted human cancers should prove to be fruitful in the setting of future clinical research trials.

Ron receptor regulates Kupffer cell‐dependent cytokine production and hepatocyte survival following endotoxin exposure in mice

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well‐characterized model of lipopolysaccharide (LPS)‐induced acute liver failure in D‐galactosamine (GalN)‐sensitized mice. Hepatocyte protection in TK−/− mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNF‐α). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wildtype (TK+/+) and TK−/− mice were studied. Utilizing quantitative reverse‐transcription polymerase chain reaction (RT‐PCR), we demonstrated that Ron is expressed in these cell types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK−/− Kupffer cells produce increased levels of TNF‐α and select cytokines compared to TK+/+ cells following LPS stimulation. We also show that conditioned media from LPS‐treated TK−/− Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK−/− Kupffer cells are detrimental to wildtype hepatocytes. In addition, we observed that TK−/− hepatocytes were more resistant to cell death compared to TK+/+ hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell‐type‐specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. Conclusion: We dissected cell‐type‐specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. (HEPATOLOGY 2011;)

Ron receptor tyrosine kinase-dependent hepatic neutrophil recruitment and survival benefit in a murine model of bacterial peritonitis

Objective:To determine whether Ron receptor tyrosine kinase signaling affects the in vivo response to bacterial peritonitis. Design:Experimental study. Setting:University laboratory. Subjects:Male mice 8–11 wks of age (22–28 g). Interventions:A genetic approach comparing wild-type mice to mice with a targeted deletion of the Ron tyrosine kinase signaling domain (TK−/−) was undertaken to determine the influence of Ron receptor in the in vivo response to a well-characterized model of bacterial peritonitis and sepsis induced by cecal ligation and puncture. Measurements and Main Results:Several clinical (i.e., survival curves, blood and tissue bacterial burdens, and neutrophil oxidative burst), morphologic (i.e., liver histology and leukocyte trafficking), and biochemical variables (i.e., serum aminotransferases and select serum cytokine and chemokine levels) important for assessing inflammatory responses to bacterial infection were assessed in mice following cecal ligation and puncture. Ron TK−/− mice had a significant decrease in survival time compared with controls, and this was associated with a significant increase in bacterial colony-forming units found in the blood and several end-organs. Moreover, this increased bacterial load was associated with increased liver necrosis and serum alanine aminotransferase levels. Neutrophils isolated from TK−/− mice exhibited decreased spontaneous oxidative burst capacity ex vivo, and by intravital microscopy, a reduced level of neutrophil migration to and translocation within the liver was observed. Loss of Ron signaling resulted in significantly reduced production of serum monocyte chemoattractant protein-1 and interleukin-6 levels following cecal ligation and puncture, and peritoneal macrophage isolated from TK−/− mice exhibited blunted production of monocyte chemoattractant protein-1, interleukin-6, and macrophage inflammatory protein-2 following stimulation with endotoxin ex vivo. Conclusions:Ron signaling negatively regulates the response to polymicrobial infection by regulating the activation and recruitment of inflammatory cells necessary for clearing a systemic bacterial burden. This effect may be regulated in part through the Ron-dependent, macrophage-mediated production of cytokines and chemokines, namely monocyte chemoattractant protein-1, interleukin-6, and macrophage inflammatory protein-2, important for neutrophil mobilization.

Chronic Acetaminophen Exposure in Pediatric Acute Liver Failure

BACKGROUND: Acetaminophen (N-acetyl-p-aminophenol [APAP]) is a widely used medication that can cause hepatotoxicity. We examined characteristics and outcomes of children with chronic exposure (CE) to APAP in the multinational Pediatric Acute Liver Failure (PALF) Study. METHODS: A total of 895 children enrolled from 2002 to 2009 were grouped by APAP exposure history as: CE (received multiple doses \x{2265}2 days; n = 83), single dose exposure (SE; n = 85), and no exposure (NE; n = 498). CE was the reference group for pairwise comparisons. Median values are shown. RESULTS: Patients with CE compared with those with SE were younger (3.5 vs 15.2 years, P < .0001), less likely to be female (46% vs 82%, P < .0001), and more likely to be Hispanic (25% vs 7%, P = .001), but they did not differ significantly from the NE group. At enrollment, total bilirubin was lower with CE than with NE (3.2 vs 13.1 mg/dL, P < .001). Alanine aminotransferase levels were higher with CE than with NE (2384 vs 855 IU/L, P < .0001), but lower than with SE (5140 IU/L, P < .0001). Survival without liver transplantation at 21 days was worse for CE than for SE (68% vs 92%, P = .0004) but better than for NE (49%, P = .008). CONCLUSIONS: Children in the PALF study with CE had lower bilirubin and higher alanine aminotransferase than those with NE. Outcomes with CE were worse than with SE but better than with NE. Potential reasons for this outcomes advantage over non–APAP-exposed subjects should be explored.

Neonatal hemochromatosis: It's OK to say “NO” to antioxidant‐chelator therapy

Neonatal hemochromatosis (NH) is a rare medical condition presenting as severe liver disease in the newborn period. The natural history of patients with NH is grim; when not stillborn, most infants present in the first days or weeks of life with clinical features of liver failure. Within the first few weeks of life, most succumb to the complications of end-stage liver disease if not successfully rescued by liver transplantation. Given its rarity and severity, NH has been difficult to study and frustrating to treat. Rarely, spontaneous recovery without disease-specific medical intervention or liver transplantation is observed in patients with NH. In this issue of Liver Transplantation, Rodrigues and co-workers describe the presentation, management and outcomes of the largest series of patients with NH reported to date: 19 infants evaluated over a 12-year time span at the King’s College Hospital, London. Very few centers in the world have reported on more than a few patients with NH. 1-3

Novel missense MTTP gene mutations causing abetalipoproteinemia.

OBJECTIVE The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients. APPROACH AND RESULTS In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity. CONCLUSIONS These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity.

Outcomes of Children With and Without Hepatic Encephalopathy From the Pediatric Acute Liver Failure Study Group.

Objectives: Hepatic encephalopathy (HE) is challenging to identify in children with acute liver failure and was not a requirement for enrollment into the Pediatric Acute Liver Failure Study Group (PALFSG). The outcomes of PALFSG participants presenting with and without HE are presented. Methods: PALFSG participants were classified based on daily assessment of HE during the first 7 days following study enrollment: group 1—never developed HE; group 2—no HE at enrollment with subsequent HE development; and group 3—HE at study enrollment. Clinical and biochemical parameters and outcomes of death, spontaneous recovery, or liver transplantation were compared between groups. Results: Data from 769 PALFSG (54% boys; median age 4.2 years; range 0–17.9 years) participants were analyzed, with 277 in group 1 (36%), 83 in group 2 (11%), and 409 in group 3 (53%). Mortality occurred in 11% of all participants and was highest among group 3 participants who demonstrated persistent grade III–IV HE (55%) or showed progression of HE (26%). Eleven (4%) group 1 participants died within 21 days of enrollment. Spontaneous recovery was highest in group 1 (79%) and lowest in group 2 (25%; P < 0.001). Conclusions: Mortality 21 days after enrollment was highest in participants enrolled with severe HE (grades III or IV) or demonstrating HE progression. Four percent of participants without recorded clinical HE in the 7 days after enrollment, however, died within 21 days. Improved assessment of neurological injury and pediatric acute liver failure prognostication schema are needed.

Chemoresistant hepatoblastoma in a patient with mosaic trisomy 18 treated with orthotopic liver transplantation

We present a 9‐month‐old male with mosaic trisomy 18 with a right hepatic lobe mass. The tumor was completely resected and identified as pure fetal histology hepatoblastoma but contained increased mitotic activity. Adjuvant chemotherapy consisted of cisplatin, vincristine, and 5‐fluorouracil. After the first and fourth cycles of chemotherapy, recurrent tumor developed. The patient underwent rescue orthotopic liver transplantation, and is currently alive without evidence of hepatoblastoma 28 months after transplantation. This report demonstrates the use of orthotopic liver transplantation in a child with mosaic trisomy 18 and hepatoblastoma. Pediatr Blood Cancer 2011;56:498–500.