Clear cell renal cell carcinoma (ccRCC), the most frequent form of kidney cancer1, is characterized by elevated glycogen and fat deposition2. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia inducible factors (HIFs)3, secondary to von hippel-lindau (VHL) mutations that occur in over 90% of ccRCC tumours4. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation5, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed loss of several chromatin remodelling enzymes in a subset of ccRCC (polybromo 1 [PBRM1] ~40%, SET domain containing 2 [SETD2] ~15%, BRCA1 associated protein-1 [BAP1] ~15%, etc.)6–9, indicating that epigenetic perturbations are likely important contributors to the natural history of this disease. Here we utilized an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis, and determined that the gluconeogenic enzyme fructose-1, 6-bisphosphatase 1 (FBP1)10 is uniformly depleted in over six hundred ccRCC tumours examined. Importantly, the human FBP1 locus resides on chromosome 9q22, whose loss is associated with poor prognosis for ccRCC patients11. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms: 1) FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin12, thereby inhibiting a potential “Warburg effect”13,14, and 2) in pVHL-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis, and the pentose phosphate pathway in a catalytic activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF “inhibitory domain”. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously-identified tumour suppressors (PBRM1, SETD2, BAP1, etc.) which are not consistently mutated in all tumours6,7,15.

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.

Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxiainducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α-deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α-dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α-dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.

BackgroundThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases.Methodology/Principal FindingsDuring a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR.ConclusionsThese data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders.

Glioblastoma multiforme (GBM) and the mesenchymal GBM subtype in particular are highly malignant tumors that frequently exhibit regions of severe hypoxia and necrosis. Because these features correlate with poor prognosis, we investigated microRNAs whose expression might regulate hypoxic GBM cell survival and growth. We determined that the expression of microRNA-218 (miR-218) is decreased significantly in highly necrotic mesenchymal GBM, and orthotopic tumor studies revealed that reduced miR-218 levels confer GBM resistance to chemotherapy. Importantly, miR-218 targets multiple components of receptor tyrosine kinase (RTK) signaling pathways, and miR-218 repression increases the abundance and activity of multiple RTK effectors. This elevated RTK signaling also promotes the activation of hypoxia-inducible factor (HIF), most notably HIF2α. We further show that RTK-mediated HIF2α regulation is JNK dependent, via jun proto-oncogene. Collectively, our results identify an miR-218-RTK-HIF2α signaling axis that promotes GBM cell survival and tumor angiogenesis, particularly in necrotic mesenchymal tumors.

Exposure to dioxins, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wide array of toxicities in vertebrates, which are mostly considered to be mediated through the inappropriate activation of the aryl hydrocarbon receptor (AHR) signaling pathway. Although transcriptional regulation by AHR is widely studied, the molecular mechanisms responsible for the adverse outcomes after AHR activation are largely unknown. To identify the important downstream events of AHR activation, we employed the zebrafish caudal fin regeneration model, where AHR activation blocks the regenerative process. Comparative toxicogenomic analysis revealed that both adult and larval fins respond to TCDD during regeneration with misexpression of Wnt signaling pathway members and Wnt target genes. R-Spondin1, a novel ligand for the Wnt coreceptor, was highly induced, and we hypothesized that misexpression of R-Spondin1 is necessary for AHR activation to block regeneration. Partial antisense repression of R-Spondin1 reversed the inhibitory effect of TCDD, and tissue regeneration was restored. This finding demonstrates that inhibition of regeneration by TCDD is mediated by misinduction of R-Spondin1. Because R-Spondin1 signals through the Wnt coreceptor LRP6, we further demonstrated that the TCDD-mediated block in regeneration is also LRP6 dependent. Collectively, these results indicate that inappropriate regulation of R-Spondin/LRP6 is absolutely required for TCDD to inhibit fin regeneration.

Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.

Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial wound cap over the amputation site by 12 h post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were ip injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3, and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two-fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two-fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b.