Background: A major challenge in cancer chemotherapy has been developing safe and clinically efficacious chemotherapeutic agents. With its low toxicity profile, curcumin (diferuloylmethane), a naturally occurring flavinoid derived from the rhizome of Curcuma longa, has great promise. In vitro and in vivo preclinical studies have shown its inhibitory anticancer, antioxidant, antiinflammatory, antiproliferative, and proapoptotic activities. The multiple mechanisms of the antitumor effect of curcumin putatively include down-regulating the expression of gene products such as nuclear factor-KB, growth suppression, inducing apoptosis, and modulating various signal transduction pathways and the expression of many oncogenes. The mechanisms underlying the antitumor activity of curcumin have not, however, been completely delineated. Methods: An oligonucleotide microarray chip was developed and used to profile microRNA (miRNA) expressions in pancreatic cells treated with curcumin. Transcripts with regulated expression patterns on the arrays were validated by real-time PCRs. Additionally, potential mRNA targets were analyzed bioinformatically and confirmed with flow cytometry experiments. Results: Curcumin alters miRNA expression in human pancreatic cells, up-regulating miRNA-22 and down-regulating

• A novel germ-l i n e PHD1 mutation causing heochromocytoma/paraganglioma and polycythemia. • Increased EPOR activity and inappropriate hypersensitivity of erythroid progenitors to EPO.

• Newly identified mutations in HIF2A result in polycythemia and neuroendocrine tumors.• Disruption of the hydroxylation domain in HIF-2a results in protein stabilization, pseudohypoxia, and tumorigenesis.Hypoxia-inducible factors (HIFs) control the cellular response to hypoxia and, when dysregulated, contribute to tumorigenesis. Previously, we identified 2 gain-of-function somatic mutations in patients presenting with multiple paragangliomas or somatostatinomas, and polycythemia. Here, we report 2 additional unique HIF2A mutations, which disrupt the hydroxylation domain recognized by prolyl hydroxylase domain-2 containing protein, leading to stabilization of HIF-2a and increased expression of hypoxia-related genes. (Blood. 2013;121(13):2563-2566 Introduction Hypoxia-inducible factors (HIFs) are central to the cellular hypoxic response. 1 HIFs are ab-heterodimers composed of a constitutively expressed HIF-b subunit and an oxygen-dependent HIF-a subunit. HIF-a is inducible during hypoxic conditions via reduced oxygendependent prolyl hydroxylation, decreased binding to the von HippelLindau protein (VHL), and thus decreased proteasomal degradation. 2,3Stabilized HIF-a translocates into the nucleus and leads to an increase in the expression of pro-survival genes such as EPO, EDN1, GLUT1, and VEGFA.4 HIF-a has been found to be critical for vasculogenesis and hematopoiesis during development.5 Abnormal HIF-a function enhances cell proliferation and growth, which is a recognized mechanism in aggressive tumors. 6 Polycythemia is a hematologic disorder defined by an increase in red cell mass. Genetic changes in key elements of hypoxia-sensing pathways, such as germline mutations in VHL or EGLN1, are essential for the establishment of polycythemia. 7,8 Percy et al 9 reported novel HIF2A mutations (M535V, G537R) associated with polycythemia as a result of constitutively activated hypoxic cell signaling. Paragangliomas and somatostatinomas are catecholamine-and somatostatin-producing neuroendocrine tumors, respectively. Mutations in SDHx, SDHAF2, TMEM127, MAX, EGLN1, and VHL have been implicated in the pathogenesis of these neuroendocrine tumors.10 These mutations indirectly lead to HIF stabilization and aberrant expression of hypoxia-related genes. These diseases share a disruption in HIF-a regulation. Study designThis study was approved by the institutional review board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The patients provided written informed consent in accordance with the Declaration of Helsinki. Peptides, plasmids, and mutagenesisWild-type HIF-2a ELDLETLAPYIPMDGEDFQ and HIF-2a mutants (L529P) ELDLETPAPYIPMDGEDFQ and (Y532C) ELDLETLAPCIPMDGEDFQ were synthesized, and the N terminus was biotinylated (Peptide 2.0). HA (hemagglutinin)-HIF2a-pcDNA3 plasmid (Addgene plasmid 18950 11 ) was obtained for generating HIF2A mutants. Site-directed mutagenesis was accomplished using the Quikchange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Hydrox...