Ben Z. Stanger

The Death Domain Kinase RIP Mediates the TNF-Induced NF-κB Signal

The death domain serine/threonine kinase RIP interacts with the death receptors Fas and tumor necrosis receptor 1 (TNFR1). In vitro, RIP stimulates apoptosis, SAPK/JNK, and NF-kappaB activation. To define the physiologic role(s) that RIP plays in regulating apoptosis in vivo, we introduced a rip null mutation in mice through homologous recombination. RIP-deficient mice appear normal at birth but fail to thrive, displaying extensive apoptosis in both the lymphoid and adipose tissue and dying at 1-3 days of age. In contrast to a normal thymic anti-Fas response, rip-/- cells are highly sensitive to TNFalpha-induced cell death. Sensitivity to TNFalpha-mediated cell death in rip-/- cells is accompanied by a failure to activate the transcription factor NF-kappaB.

RIP: A novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death

Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic programmed death response in susceptible cells. Using a genetic selection based on protein-protein interaction in yeast, we have identified two gene products that associate with the intracellular domain of Fas: Fas itself, and a novel 74 kDa protein we have named RIP, for receptor interacting protein. RIP also interacts weakly with the p55 tumor necrosis factor receptor (TNFR1) intracellular domain, but not with a mutant version of Fas corresponding to the murine lprcg mutation. RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Taken together, these properties indicate that RIP is a novel form of apoptosis-inducing protein.

Genetics and biology of pancreatic ductal adenocarcinoma

With 5-year survival rates remaining constant at 6% and rising incidences associated with an epidemic in obesity and metabolic syndrome, pancreatic ductal adenocarcinoma (PDAC) is on track to become the second most common cause of cancer-related deaths by 2030. The high mortality rate of PDAC stems primarily from the lack of early diagnosis and ineffective treatment for advanced tumors. During the past decade, the comprehensive atlas of genomic alterations, the prominence of specific pathways, the preclinical validation of such emerging targets, sophisticated preclinical model systems, and the molecular classification of PDAC into specific disease subtypes have all converged to illuminate drug discovery programs with clearer clinical path hypotheses. A deeper understanding of cancer cell biology, particularly altered cancer cell metabolism and impaired DNA repair processes, is providing novel therapeutic strategies that show strong preclinical activity. Elucidation of tumor biology principles, most notably a deeper understanding of the complexity of immune regulation in the tumor microenvironment, has provided an exciting framework to reawaken the immune system to attack PDAC cancer cells. While the long road of translation lies ahead, the path to meaningful clinical progress has never been clearer to improve PDAC patient survival.

Notch signaling controls multiple steps of pancreatic differentiation.

Multiple cell types of the pancreas appear asynchronously during embryogenesis, which requires that pancreatic progenitor cell potential changes over time. Loss-of-function studies have shown that Notch signaling modulates the differentiation of these progenitors, but it remains unclear how and when the Notch pathway acts. We established a modular transgenic system to heritably activate mouse Notch1 in multiple types of progenitors and differentiated cells. We find that misexpression of activated Notch in Pdx1-expressing progenitor cells prevents differentiation of both exocrine and endocrine lineages. Progenitors remain trapped in an undifferentiated state even if Notch activation occurs long after the pancreas has been specified. Furthermore, endocrine differentiation is associated with escape from this activity, because Ngn3-expressing endocrine precursors are susceptible to Notch inhibition, whereas fully differentiated endocrine cells are resistant.

Stromal Elements Act to Restrain, Rather Than Support, Pancreatic Ductal Adenocarcinoma

Sonic hedgehog (Shh), a soluble ligand overexpressed by neoplastic cells in pancreatic ductal adenocarcinoma (PDAC), drives formation of a fibroblast-rich desmoplastic stroma. To better understand its role in malignant progression, we deleted Shh in a well-defined mouse model of PDAC. As predicted, Shh-deficient tumors had reduced stromal content. Surprisingly, such tumors were more aggressive and exhibited undifferentiated histology, increased vascularity, and heightened proliferation--features that were fully recapitulated in control mice treated with a Smoothened inhibitor. Furthermore, administration of VEGFR blocking antibody selectively improved survival of Shh-deficient tumors, indicating that Hedgehog-driven stroma suppresses tumor growth in part by restraining tumor angiogenesis. Together, these data demonstrate that some components of the tumor stroma can act to restrain tumor growth.

Tumor-Derived Granulocyte-Macrophage Colony-Stimulating Factor Regulates Myeloid Inflammation and T Cell Immunity in Pancreatic Cancer

Cancer-associated inflammation is thought to be a barrier to immune surveillance, particularly in pancreatic ductal adenocarcinoma (PDA). Gr-1(+) CD11b(+) cells are a key feature of cancer inflammation in PDA, but remain poorly understood. Using a genetically engineered mouse model of PDA, we show that tumor-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) is necessary and sufficient to drive the development of Gr-1(+) CD11b(+) cells that suppressed antigen-specific T cells. In vivo, abrogation of tumor-derived GM-CSF inhibited the recruitment of Gr-1(+) CD11b(+) cells to the tumor microenvironment and blocked tumor development-a finding that was dependent on CD8(+) T cells. In humans, PDA tumor cells prominently expressed GM-CSF in vivo. Thus, tumor-derived GM-CSF is an important regulator of inflammation and immune suppression within the tumor microenvironment.

IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice

We have assessed the biologic role of IL-4 by fusing its gene to an immunoglobulin promoter/enhancer and introducing it into transgenic mice. By attenuating the transgene promoter through the insertion of E. coli lac operator sequences, we have created a series of animals that constitutively express varying amounts of IL-4. Overexpression of IL-4 results in a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion (blepharitis) with characteristic histopathologic features seen in allergic reactions. In addition, expression of the IL-4 transgene in the thymus perturbs T cell maturation, reducing the population of immature CD4+CD8+ thymocytes and peripheral T cells while increasing the population of mature CD8+ thymocytes. These results demonstrate that deregulation of a single cytokine gene in vivo can induce a complex inflammatory reaction resembling that observed in human allergic disease.

Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver

The determinants of vertebrate organ size are poorly understood, but the process is thought to depend heavily on growth factors and other environmental cues. In the blood and central nervous system, for example, organ mass is determined primarily by growth-factor-regulated cell proliferation and apoptosis to achieve a final target size. Here, we report that the size of the mouse pancreas is constrained by an intrinsic programme established early in development, one that is essentially not subject to growth compensation. Specifically, final pancreas size is limited by the size of the progenitor cell pool that is set aside in the developing pancreatic bud. By contrast, the size of the liver is not constrained by reductions in the progenitor cell pool. These findings show that progenitor cell number, independently of regulation by growth factors, can be a key determinant of organ size.

Mapping the gene for hereditary cutaneous malignant melanoma-dysplastic nevus to chromosome 1p

We used molecular genetic techniques and multipoint linkage analyses to locate the gene responsible for cutaneous malignant melanoma-dysplastic nevus. We evaluated 99 relatives and 26 spouses in six families with a predisposition to melanoma. Thirty-four family members had cutaneous malignant melanoma, and 31 of these 34 also had histologically confirmed dysplastic nevi. Twenty-four family members had dysplastic nevi alone. An analysis of the cosegregation of the cutaneous malignant melanoma-dysplastic nevus trait with 26 polymorphic DNA markers on the short arm of chromosome 1 demonstrated the presence of a gene for susceptibility to melanoma. The gene was located between an anonymous DNA marker (D1S47) and the gene locus for pronatrodilatin, a commonly used reference gene (PND), in chromosome band 1p36. The odds were greater than 260,000:1 in favor of linkage at this location.

Notch signaling controls liver development by regulating biliary differentiation

In the mammalian liver, bile is transported to the intestine through an intricate network of bile ducts. Notch signaling is required for normal duct formation, but its mode of action has been unclear. Here, we show in mice that bile ducts arise through a novel mechanism of tubulogenesis involving sequential radial differentiation. Notch signaling is activated in a subset of liver progenitor cells fated to become ductal cells, and pathway activation is necessary for biliary fate. Notch signals are also required for bile duct morphogenesis, and activation of Notch signaling in the hepatic lobule promotes ectopic biliary differentiation and tubule formation in a dose-dependent manner. Remarkably, activation of Notch signaling in postnatal hepatocytes causes them to adopt a biliary fate through a process of reprogramming that recapitulates normal bile duct development. These results reconcile previous conflicting reports about the role of Notch during liver development and suggest that Notch acts by coordinating biliary differentiation and morphogenesis.