Ronald J. Marler

Reprogrammed FoxP3+ T Regulatory Cells Become IL-17+ Antigen-Specific Autoimmune Effectors In Vitro and In Vivo

In the course of investigating suspicious patterns of experimental results in the laboratory, a systematic and in-depth study of key findings in this article was carried out using blinded protocols. In these repeat studies, no evidence was found to support our original conclusions that B7-DC XAb modulates dendritic cell functions. We do not believe our failure to reproduce our earlier findings is the result of a technical problem. A member of the B7-DC XAb investigative team, Dr. Suresh Radhakrishnan, who was involved in or had access to all the work on this subject, was found in a formal investigation to have engaged in scientific misconduct in unpublished experiments involving the B7-DC XAb reagent. This finding of misconduct and our inability to reproduce key findings using blinded protocols has undermined our confidence in our published reports. We seek, therefore, to retract this body of work.

Loss of the Transcription Factor GLI1 Identifies a Signaling Network in the Tumor Microenvironment Mediating KRAS Oncogene-induced Transformation

Background: KRAS is a known oncogene driving transformation in multiple tissues. Results: We demonstrate a role for the transcription factor GLI1 in KRAS-induced transformation through regulation of the IL-6/STAT3 axis in the tumor microenvironment. Conclusion: This study defines a novel oncogenic network downstream of KRAS modulating transformation. Significance: This knowledge will contribute to the understanding of the pathogenesis of tumors driven by KRAS. Although the biological role of KRAS is clearly established in carcinogenesis, the molecular mechanisms underlying this phenomenon are not completely understood. In this study, we provide evidence of a novel signaling network regulated by the transcription factor GLI1 mediating KRAS-induced carcinogenesis. Using pancreatic cancer (a disease with high prevalence of KRAS mutations) as a model, we show that loss of GLI1 blocks the progression of KRAS-induced pancreatic preneoplastic lesions in mice with pancreas-specific Cre-activated oncogenic mutant kras. Mice lacking GLI1 develop only low-grade lesions at low frequency, and in most cases, the pancreata are histologically normal. Further characterization of the phenotype showed a decrease in the activation of STAT3 in pancreatic preneoplastic lesions; STAT3 is a transcription factor required for the development of premalignant lesions and their progression into pancreatic cancer. Analysis of the mechanisms revealed a key role for GLI1 in maintaining the levels of activated STAT3 through the modulation of IL-6 signaling. GLI1 binds to the IL-6 mouse promoter and regulates the activity and expression of this cytokine. This newly identified GLI1/IL-6 axis is active in fibroblasts, a known source of IL-6 in the tumor microenvironment. Sonic hedgehog induces GLI1 binding to the IL-6 promoter and increases IL-6 expression in fibroblasts in a paracrine manner. Finally, we demonstrate that mutant KRAS initiates this cascade by inducing the expression of Sonic hedgehog in cancer cells. Collectively, these results define a novel role for GLI1 in carcinogenesis acting as a downstream effector of oncogenic KRAS in the tumor microenvironment.

Longevity and Age-Related Pathology of Mice Deficient in Pregnancy-Associated Plasma Protein-A

The pregnancy-associated plasma protein-A knockout (PAPP-A KO) mouse is a model of reduced local insulin-like growth factor (IGF)-I activity with normal circulating IGF-I levels. In this study, PAPP-A KO mice had significantly increased mean (27%), median (27%), and maximum (35%) life span compared with wild-type (WT) littermates. End-of-life pathology indicated that the incidence of neoplastic disease was not significantly different in the two groups of mice; however, it occurred in older aged PAPP-A KO compared with WT mice. Furthermore, PAPP-A KO mice were less likely to show degenerative changes of age. Scheduled pathologies at 78, 104, and 130 weeks of age indicated that WT mice, in general, had more degenerative changes and tumors earlier than PAPP-A KO mice. This was particularly true for abnormalities in heart, testes, brain, kidney, spleen, and thymus. In summary, the major contributors to the extended life span of PAPP-A KO mice are delayed occurrence of fatal neoplasias and decreased incidence of age-related degenerative changes.

Absence of histologic retinal toxicity of intravitreal nanogold in a rabbit model

Purpose: To evaluate the retinal toxicity of intravitreal nanogold, a novel antiangiogenic and long-term delivery agent. Methods: One eye of each of 16 Dutch-belted rabbits was injected with intravitreal nanogold; the other eye served as a control. Eight rabbits received a dose of 67 &mgr;mol/0.1 mL of nanogold intravitreally into 1 eye; the other 8 rabbits received a dose of 670 &mgr;mol/0.1 mL of nanogold intravitreally into 1 eye. Eight rabbits were killed at 1 week, and eight were killed at 1 month; both eyes of each rabbit were enucleated. The eyes were fixed with 2% paraformaldehyde and sectioned for histologic examination. Results: In all injected and control eyes, there was mild vacuolization in the inner plexiform and ganglion cell layers. The retina and retinal pigment epithelium were otherwise histologically normal. Conclusion: Intravitreal nanogold at concentrations of 67 &mgr;mol/0.1 mL and 670 &mgr;mol/0.1 mL showed no signs of retinal or optic nerve toxicity by light microscopy during histologic examination at 1 month.

Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease

Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH(+/-) pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH(-/-) pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, the animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopathy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH(-/-) pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH(-/-) pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of the efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes.

Mice deficient in PAPP - a show resistance to the development of diabetic nephropathy

We investigated pregnancy-associated plasma protein-A (PAPP-A) in diabetic nephropathy. Normal human kidney showed specific staining for PAPP-A in glomeruli, and this staining was markedly increased in diabetic kidney. To assess the possible contribution of PAPP-A in the development of diabetic nephropathy, we induced diabetes with streptozotocin in 14-month-old WT and Papp-A knockout (KO) mice. Renal histopathology was evaluated after 4 months of stable hyperglycemia. Kidneys from diabetic WT mice showed multiple abnormalities including thickening of Bowmans capsule (100% of mice), increased glomerular size (80% of mice), tubule dilation (80% of mice), and mononuclear cell infiltration (90% of mice). Kidneys of age-matched non-diabetic WT mice had similar evidence of tubule dilation and mononuclear cell infiltration to those of diabetic WT mice, indicating that these changes were predominantly age-related. However, thickened Bowmans capsule and increased glomerular size appeared specific for the experimental diabetes. Kidneys from diabetic Papp-A KO mice had significantly reduced or no evidence of changes in Bowmans capsule thickening and glomerular size. There was also a shift to larger mesangial area and increased macrophage staining in diabetic WT mice compared with Papp-A KO mice. In summary, elevated PAPP-A expression in glomeruli is associated with diabetic nephropathy in humans and absence of PAPP-A is associated with resistance to the development of indicators of diabetic nephropathy in mice. These data suggest PAPP-A as a potential therapeutic target for diabetic nephropathy.

Curative ex vivo liver-directed gene therapy in a pig model of hereditary tyrosinemia type 1.

Transplantation of gene-corrected autologous hepatocytes can cure metabolic disease in a preclinical pig model of hereditary tyrosinemia type 1. Skipping the waiting list The only cure for hereditary tyrosinemia type 1 (HT1)—an inherited metabolic disease—is a liver transplant. However, owing to the shortage of liver donors, Hickey et al. turned to gene therapy as a way to cure HT1. The authors took liver cells from pigs that have HT (through a defect in the gene Fah), transduced them with the correct Fah, and then put the cells back into the same animals. The ex vivo gene therapy approach prevented liver failure and fibrosis and also restored metabolic function, which is deteriorated in HT1 disease. Having demonstrated in large animals the use of materials that are safe for use in people, the technology is now poised to move into patients, to regenerate their own livers and spare them the long wait times on the liver transplant list. We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase–deficient (Fah−/−) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah−/− pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide symporter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH+ cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah−/− liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah−/− pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use.

Inactivation of the transcription factor GLI1 accelerates pancreatic cancer progression

Background: GLI1 is required for pancreatic tumor initiation; however, its role at later stages of carcinogenesis remains elusive. Results: Genetic inactivation of GLI1 accelerates pancreatic cancer progression. Conclusion: GLI1 can act as both a promoter and suppressor of pancreatic carcinogenesis depending on the tumor stage. Significance: This knowledge increases our understanding of pancreatic carcinogenesis and may help the design of therapies targeting GLI1-related pathways. The role of GLI1 in pancreatic tumor initiation promoting the progression of preneoplastic lesions into tumors is well established. However, its function at later stages of pancreatic carcinogenesis remains poorly understood. To address this issue, we crossed the gli1 knock-out (GKO) animal with cre-dependent pancreatic activation of oncogenic kras concomitant with loss of the tumor suppressor tp53 (KPC). Interestingly, in this model, GLI1 played a tumor-protective function, where survival of GKO/KPC mice was reduced compared with KPC littermates. Both cohorts developed pancreatic cancer without significant histopathological differences in survival studies. However, analysis of mice using ultrasound-based imaging at earlier time points showed increased tumor burden in GKO/KPC mice. These animals have larger tumors, decreased body weight, increased lactate dehydrogenase production, and severe leukopenia. In vivo and in vitro expression studies identified FAS and FAS ligand (FASL) as potential mediators of this phenomenon. The FAS/FASL axis, an apoptotic inducer, plays a role in the progression of pancreatic cancer, where its expression is usually lost or significantly reduced in advanced stages of the disease. Chromatin immunoprecipitation and reporter assays identified FAS and FASL as direct targets of GLI1, whereas GKO/KPC mice showed lower levels of this ligand compared with KPC animals. Finally, decreased levels of apoptosis were detected in tumor tissue in the absence of GLI1 by TUNEL staining. Together, these findings define a novel pathway regulated by GLI1 controlling pancreatic tumor progression and provide a new theoretical framework to help with the design and analysis of trials targeting GLI1-related pathways.

Downregulation of Hematopoietic MUC1 during Experimental Colitis Increases Tumor-Promoting Myeloid-Derived Suppressor Cells

Purpose: MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that downregulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. Experimental Design: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT). Results: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. Conclusion: Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. In addition, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers. Clin Cancer Res; 19(18); 5039–52. ©2013 AACR.

The MUC1 Cytoplasmic Tail and Tandem Repeat Domains Contribute to Mammary Oncogenesis in FVB Mice.

Background Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood. Methods To address this, mouse models of mammary carcinogenesis were created expressing full-length cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs. Results Overexpression of full-length MUC1 resulted in tumor formation in young mice (≤ 12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background. Conclusions This study is the first spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation.