The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is not a single disease, but several histologically defined cancers with different genetic drivers, clinical courses, and therapeutic responses. The current study evaluated 843 RCC from the three major histologic subtypes, including 488 clear cell RCC, 274 papillary RCC, and 81 chromophobe RCC. Comprehensive genomic and phenotypic analysis of the RCC subtypes reveals distinctive features of each subtype that provide the foundation for the development of subtype-specific therapeutic and management strategies for patients affected with these cancers. Somatic alteration of BAP1, PBRM1, and PTEN and altered metabolic pathways correlated with subtype-specific decreased survival, while CDKN2A alteration, increased DNA hypermethylation, and increases in the immune-related Th2 gene expression signature correlated with decreased survival within all major histologic subtypes. CIMP-RCC demonstrated an increased immune signature, and a uniform and distinct metabolic expression pattern identified a subset of metabolically divergent (MD) ChRCC that associated with extremely poor survival.

Identification of Pur alpha as a new hypoxia response factor responsible for coordinated induction of the beta 2 integrin family.

Central to the process of inflammation are hypoxic conditions that lead to the binding of circulating leukocytes to the endothelium. We have previously shown that such binding is mediated by monocytes being able to directly sense hypoxic conditions and respond by inducing their surface expression of the beta(2) integrin family of adhesion molecules. In this study, we show that coordinated induction of the beta(2) integrins during direct hypoxia-sensing occurs through transcriptional activation of each of the genes by which they are encoded. Certain of the molecular mechanisms that mediate this activation in transcription are dependent upon hypoxia-inducible factor-1 (HIF-1), whereas others are HIF-1 independent. In search of these HIF-1-independent mechanisms, we identified Pur alpha as a new hypoxia-response factor. Binding of Pur alpha to the HIF-1-independent beta(2) integrin promoters is induced by hypoxia and mutagenesis of these Pur alpha-binding sites almost completely abolishes the ability of the promoters to respond to hypoxic conditions. Additional studies using siRNA directed against Pur alpha also revealed a loss in the hypoxic response of the beta(2) integrin promoters. Taken together, our findings demonstrate that hypoxia induces a coordinated up-regulation in beta(2) integrin expression that is dependent upon transcriptional mechanisms mediated by HIF-1 and Pur alpha.

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

Leukocyte adhesion during hypoxia is mediated by HIF-1-dependent induction of beta2 integrin gene expression.

Inflammatory responses are associated with significant changes in tissue metabolism. In particular, metabolic shifts during inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia-responsive genes. Given this association, we hypothesized that leukocyte functional responses are influenced by hypoxia. Initial experiments revealed that exposure of the promonocytic cell line U937 to hypoxia resulted in increased adhesion to activated endothelia. Such increases were transcription-dependent and were blocked by antibodies directed against beta2, but not beta1, integrins. Analysis of beta2 integrin mRNA and protein in U937 cells revealed a 5- to 6-fold increase with hypoxia. Extension of this analysis to hypoxic human whole blood revealed prominent induction of beta2 integrin mRNA and protein ex vivo. Furthermore, murine beta2 integrin mRNA was found to be significantly induced during hypoxia in vivo. Subsequent studies identified a binding site for hypoxia-inducible factor 1 (HIF-1) in the CD18 gene. This gene encodes the subunit common to all four known types of beta2 integrin heterodimer. HIF-1 binding was demonstrated in vivo, and mutational analysis of the HIF-1 site within the CD18 promoter resulted in a loss of hypoxia inducibility. Taken together, these results demonstrate that hypoxia induces leukocyte beta2 integrin expression and function by transcriptional mechanisms dependent upon HIF-1.

CD11c gene expression in hairy cell leukemia is dependent upon activation of the proto-oncogenes ras and junD.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the beta2 integrin CD11c. In order to determine the cause of CD11c gene expression in HCL the CD11c gene promoter was characterized. Transfection of the CD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reduced CD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11c promoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11c gene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.

The zinc finger transcription factor ZBP-89 is a repressor of the human beta 2-integrin CD11b gene.

Integrin CD11b is a differentiation marker of the myelomonocytic lineage and an important mediator of inflammation. Expression of the CD11b gene is transcriptionally induced as myeloid precursors differentiate into mature cells, then drops as monocytes further differentiate into macrophages. Previous studies have identified elements and factors involved in the transcriptional activation of the CD11b gene during myeloid differentiation, but no data exist regarding potential down-regulatory factors, especially in the later stages of differentiation. Using 2 copies of a GC-rich element (-141 to -110) in the CD11b promoter, we probed a cDNA expression library for interacting proteins. Three clones were identified among 9.1 million screened, all encoding the DNA-binding domain of the zinc finger factor ZBP-89. Overexpression of ZBP-89 in the monocyte precursor cell line U937 reduced CD11b promoter-driven luciferase activity when U937 cells were induced to differentiate into monocytelike cells using phorbol esters. To identify the differentiation stage at which ZBP-89 repression of the CD11b gene is exerted, the protein level of ZBP-89 was correlated with that of CD11b mRNA in differentiating U937 as well as in normal human monocytes undergoing in vitro differentiation into macrophages. A clear inverse relationship was observed in the latter but not the former state, suggesting that ZBP-89 represses CD11b gene expression during the further differentiation of monocytes into macrophages.

During differentiation of the monocytic cell line U937, Pur alpha mediates induction of the CD11c beta 2 integrin gene promoter.

CD11c is a member of the beta(2) integrin family of adhesion molecules that, together with CD18, forms a heterodimeric receptor on the surface of myeloid, NK, dendritic, and certain leukemic, lymphoma, and activated lymphoid cells. Monocytic differentiation is associated with an induction of both CD11c and CD18 gene expression. The resulting CD11c/CD18 receptor mediates firm adhesion to the vascular endothelium, transendothelial migration, chemotaxis, and phagocytosis. Monocytic differentiation can be mimicked in vitro by treatment of the promonocytic cell line U937 with PMA. Recently, we reported that in U937 cells, expression of the CD11c gene is controlled by an unidentified transcription factor that binds ssDNA. This finding suggested that DNA secondary structure plays an important role in controlling the CD11c gene and prompted us to search for additional ssDNA-binding activities with which this gene interacts. In this study, we report that in U937 cells, expression of the CD11c gene is mediated by the ssDNA-binding protein Puralpha. During PMA-induced differentiation, the ability of Puralpha to activate the CD11c promoter in U937 cells increases, as does that of Sp1. Together, these increases in the functional activity of both Puralpha and Sp1 combine to induce CD11c expression.