Implementing a Digital Deteriorating Patient Pathway to improve the safety and effectiveness of care of the adult deteriorating patient.

Identification, escalation and clinical review of the deteriorating patient is essential for a safe and effective hospital. We present a deteriorating patient pathway developed within our electronic patient record, including implementation of a digital escalation and senior review process, triggered from a logic algorithm and vital signs. The pathway is activated by an average 43 patients per day with median mortality of 13.3%. Our Trust has seen a significant improvement in escalation and senior review and increased use of treatment escalation plans. The pathway has facilitated a cultural shift in the Trust towards the deteriorating patient. The new pathway is transferrable to both other digital Trusts as well as maternity and paediatric practice.

PDEF promotes luminal differentiation and acts as a survival factor for ER-positive breast cancer cells.

Breast cancer is a heterogeneous disease and can be classified based on gene expression profiles that reflect distinct epithelial subtypes. We identify prostate-derived ETS factor (PDEF) as a mediator of mammary luminal epithelial lineage-specific gene expression and as a factor required for tumorigenesis in a subset of breast cancers. PDEF levels strongly correlate with estrogen receptor (ER)-positive luminal breast cancer, and PDEF transcription is inversely regulated by ER and GATA3. Furthermore, PDEF is essential for luminal breast cancer cell survival and is required in models of endocrine resistance. These results offer insights into the function of this ETS factor that are clinically relevant and may be of therapeutic value for patients with breast cancer treated with endocrine therapy.

Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia.

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.

HIF-2alpha deletion promotes Kras-driven lung tumor development.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. The oxygen-sensitive hypoxia inducible factor (HIF) transcriptional regulators HIF-1alpha and HIF-2alpha are overexpressed in many human NSCLCs, and constitutive HIF-2alpha activity can promote murine lung tumor progression, suggesting that HIF proteins may be effective NSCLC therapeutic targets. To investigate the consequences of inhibiting HIF activity in lung cancers, we deleted Hif-1alpha or Hif-2alpha in an established Kras(G12D)-driven murine NSCLC model. Deletion of Hif-1alpha had no obvious effect on tumor growth, whereas Hif-2alpha deletion resulted in an unexpected increase in tumor burden that correlated with reduced expression of the candidate tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1 as a direct HIF-2alpha target gene and demonstrate that HIF-2alpha regulates Scgb3a1 expression and tumor formation in human Kras(G12D)-driven NSCLC cells. AKT pathway activity, reported to be repressed by Scgb3a1, was enhanced in HIF-2alpha-deficient human NSCLC cells and xenografts. Finally, a direct correlation between HIF-2alpha and SCGB3a1 expression was observed in approximately 70% of human NSCLC samples analyzed. These data suggest that, whereas HIF-2alpha overexpression can contribute to NSCLC progression, therapeutic inhibition of HIF-2alpha below a critical threshold may paradoxically promote tumor growth by reducing expression of tumor suppressor genes, including Scgb3a1.

Hypoxia-inducible factor 2alpha regulates macrophage function in mouse models of acute and tumor inflammation.

Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1alpha and HIF-2alpha, and overexpression of HIF-2alpha in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2alpha during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1alpha versus HIF-2alpha must be elucidated. We demonstrate here that mice lacking HIF-2alpha in myeloid cells (Hif2aDelta/Delta mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2alpha directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aDelta/Delta mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2alpha modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2alpha as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.

The von Hippel-Lindau Chuvash mutation promotes pulmonary hypertension and fibrosis in mice.

Mutation of the von Hippel-Lindau (VHL) tumor suppressor protein at codon 200 (R200W) is associated with a disease known as Chuvash polycythemia. In addition to polycythemia, Chuvash patients have pulmonary hypertension and increased respiratory rates, although the pathophysiological basis of these symptoms is unclear. Here we sought to address this issue by studying mice homozygous for the R200W Vhl mutation (VhlR/R mice) as a model for Chuvash disease. These mice developed pulmonary hypertension independently of polycythemia and enhanced normoxic respiration similar to Chuvash patients, further validating VhlR/R mice as a model for Chuvash disease. Lungs from VhlR/R mice exhibited pulmonary vascular remodeling, hemorrhage, edema, and macrophage infiltration, and lungs from older mice also exhibited fibrosis. HIF-2alpha activity was increased in lungs from VhlR/R mice, and heterozygosity for Hif2a, but not Hif1a, genetically suppressed both the polycythemia and pulmonary hypertension in the VhlR/R mice. Furthermore, Hif2a heterozygosity resulted in partial protection against vascular remodeling, hemorrhage, and edema, but not inflammation, in VhlR/R lungs, suggesting a selective role for HIF-2alpha in the pulmonary pathology and thereby providing insight into the mechanisms underlying pulmonary hypertension. These findings strongly support a dependency of the Chuvash phenotype on HIF-2alpha and suggest potential treatments for Chuvash patients.

von Hippel-Lindau mutation in mice recapitulates Chuvash polycythemia via hypoxia-inducible factor-2alpha signaling and splenic erythropoiesis.

The R200W mutation in the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) is unique in that it is not associated with tumor development, but rather with Chuvash polycythemia, a heritable disease characterized by elevated hematocrit and increased serum levels of erythropoietin and VEGF. Previous studies have implicated hypoxia-inducible factor-1alpha (HIF-1alpha) signaling in this disorder, although the effects of this mutation on pVHL function are not fully understood. In order to explore the mechanisms underlying the development of this polycythemia, we generated mice homozygous for the R200W mutation (Vhl(R/R)). Vhl(R/R) mice developed polycythemia highly similar to the human disease. The activity of HIF proteins, specifically the HIF-2alpha isoform, was upregulated in ES cells and tissues from Vhl(R/R) mice. Furthermore, we observed a striking phenotype in Vhl(R/R) spleens, with greater numbers of erythroid progenitors and megakaryocytes and increased erythroid differentiation of Vhl(R/R) splenic cells in vitro. These findings suggest that enhanced expression of key HIF-2alpha genes promotes splenic erythropoiesis, resulting in the development of polycythemia in Vhl(R/R) mice. This mouse model is a faithful recapitulation of this VHL-associated syndrome and represents a useful tool for studying polycythemias and investigating potential therapeutics.

Regulation of angiogenesis by hypoxia and hypoxia-inducible factors.

Maintenance of oxygen homeostasis is critical for the survival of multicellular organs. As a result, both invertebrates and vertebrates have developed highly specialized mechanisms to sense changes in oxygen levels and to mount adequate cellular and systemic responses to these changes. Hypoxia, or low oxygen tension, occurs in physiological situations such as during embryonic development, as well as in pathological conditions such as ischemia, wound healing, and cancer. A primary effector of the adaptive response to hypoxia in mammals is the hypoxia-inducible factor (HIF) family of transcription regulators. These proteins activate the expression of a broad range of genes that mediate many of the responses to decreased oxygen concentration, including enhanced glucose uptake, increased red blood cell production, and the formation of new blood vessels via angiogenesis. This latter process is dynamic and results in the establishment of a mature vascular system that is indispensable for proper delivery of oxygen and nutrients to all cells in both normal tissue and hypoxic regions. Angiogenesis is essential for normal development and neoplastic disease as tumors must develop mechanisms to stimulate vascularization to meet increasing metabolic demands. The link between hypoxia and the regulation of angiogenesis is an area of intense research and the molecular details of this connection are still being elaborated. This chapter will provide an overview of current knowledge and highlight new insights into the importance of HIF and hypoxia in angiogenesis in both physiological and pathophysiological conditions.

Activation of human T cell leukemia virus type 1 LTR promoter and cellular promoter elements by T cell receptor signaling and HTLV-1 Tax expression.

Human T cell leukemia virus 1 (HTLV-1) gene expression is regulated by both the viral Tax protein and by cellular transcriptional factors. We have previously shown that immune activation stimuli such as phorbol esters (PMA) and phytohemagglutinin (PHA) cooperate with HTLV-1 Tax expression to enhance HTLV-1 gene expression in infected T cells through increased activity of the HTLV-1 LTR. We now extend these studies to demonstrate roles for the T cell receptor complex, Lck, and Ras molecules in the coactivation of the HTLV-1 LTR by Tax and T cell activation stimuli. We also observe coactivation of Tax-responsive cellular promoter elements containing NF-kappaB and serum response factor (SRF) binding sites by Tax and T cell activation stimuli. These results suggest a model whereby T cell receptor stimulation and Tax expression coactivate HTLV-1 gene expression and cellular gene expression, enhancing activation of latent HTLV-1 and expression of cellular genes involved in disease pathogenesis.

In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause tissue-specific tumors, with a striking genotype-phenotype correlation. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to pheochromocytoma, polycythemia, or combinations of hemangioblastoma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein (pVHL) has been implicated in many cellular activities including the hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix remodeling. We have expressed missense pVHL mutations in Vhl(-/-) murine embryonic stem cells to test genotype-phenotype correlations in euploid cells. We first examined the ability of mutant pVHL to direct degradation of the hypoxia inducible factor (HIF) subunits HIF1alpha and HIF2alpha. All mutant pVHL proteins restored proper hypoxic regulation of HIF1alpha, although one VHL mutation (VHL(R167Q)) displayed impaired binding to Elongin C. This mutation also failed to restore HIF2alpha regulation. In separate assays, these embryonic stem cells were used to generate teratomas in immunocompromised mice, allowing independent assessment of the effects of specific VHL mutations on tumor growth. Surprisingly, teratomas expressing the VHL(Y112H) mutant protein displayed a growth disadvantage, despite restoring HIFalpha regulation. Finally, we observed increased microvessel density in teratomas derived from Vhl(-/-) as well as VHL(Y112H), VHL(R167Q), and VHL(R200W) embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the cell, and that these activities can be separated via discrete VHL point mutations. The ability to dissect specific VHL functions with missense mutations in a euploid model offers a novel opportunity to elucidate the activities of VHL as a tumor suppressor.