SUMMARY T cell differentiation into distinct functional effector and inhibitory subsets is regulated in part by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between T regulatory (Treg) and TH17 differentiation. HIF-1α enhances TH17 development through direct transcriptional activation of RORvt, and via tertiary complex formation with RORvt and p300 recruitment to the IL17 promoter, thereby regulating TH17 signature genes. Concurrently, HIF-1α attenuates Treg development by binding Foxp3 and targeting it for proteasomal degradation. Importantly this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α deficient T cells are resistant to induction of TH17-dependent experimental autoimmune encephalitis associated with diminished TH17 and increased Treg cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

SUMMARY The pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by hypoxia-inducible factor 1 (HIF-1). PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells.

Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxiainducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.orthotopic transplantation | triple negative breast cancer | oxygen | tumor microenvironment | mammary fat pad implantation

Increased conversion of glucose to lactate is a key feature of many cancer cells that promotes rapid growth. Pyruvate kinase M2 (PKM2) expression is increased and facilitates lactate production in cancer cells. Modulation of PKM2 catalytic activity also regulates synthesis of DNA and lipids required for cell proliferation and NADPH required for redox homeostasis. In addition to its role as a pyruvate kinase, PKM2 also functions as a protein kinase and as a transcriptional coactivator. These biochemical activities are controlled by allosteric regulators and post-translational modifications of PKM2 that include acetylation, oxidation, phosphorylation, prolyl hydroxylation, and sumoylation. Given its pleiotropic effects on cancer biology, PKM2 represents an attractive target for cancer therapy.

SUMMARY Regulatory T (Treg) cells suppress inflammatory immune responses and autoimmunity caused by self-reactive T cells. The key Treg cell transcription factor Foxp3 is downregulated during inflammation to allow for the acquisition of effector T cell-like functions. Here, we demonstrate that stress signals elicited by proinflammatory cytokines and lipopolysaccharide lead to the degradation of Foxp3 through the action of the E3 ubiquitin ligase Stub1. Stub1 interacted with Foxp3 to promote its K48-linked polyubiquitination in an Hsp70-dependent manner. Knockdown of endogenous Stub1 or Hsp70 prevented Foxp3 degradation. Furthermore, the overexpression of Stub1 in Treg cells abrogated their ability to suppress inflammatory immune responses in vitro and in vivo, and conferred a T helper 1 (Th1) cell-like phenotype. Our results demonstrate the critical role of the stress-activated Stub1-Hsp70 complex in promoting Treg cell inactivation, thus providing a potential therapeutic target for the intervention against autoimmune disease, infection and cancer.

Sleep-disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro-oxidant enzymes, especially NADPH oxidase-2 (Nox2). Previous studies showed that (i) IH activates hypoxia-inducible factor 1 (HIF-1) in a ROS-dependent manner and (ii) HIF-1 is required for IH-induced ROS generation, indicating the existence of a feed-forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH-induced expression of Nox2 is mediated by HIF-1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild-type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF-1 activity through RNA interference or pharmacologic inhibition (digoxin or YC-1) or by genetic knockout of HIF-1α in MEFs. Increasing HIF-1α expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF-1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild-type mice to IH (8 h/day for 10 days) up-regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a+/− mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF-1, ROS generation, and cardiovascular pathology in response to IH.

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Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that play key roles in breast cancer biology. We hypothesized that interaction of HIF-1 with epigenetic regulators may increase HIF-1 transcriptional activity, and thereby promote breast cancer progression. We report that the histone demethylase jumonji domain containing protein 2C (JMJD2C) selectively interacts with HIF-1α, but not HIF-2α, and that HIF-1α mediates recruitment of JMJD2C to the hypoxia response elements of HIF-1 target genes. JMJD2C decreases trimethylation of histone H3 at lysine 9, and enhances HIF-1 binding to hypoxia response elements, thereby activating transcription of BNIP3, LDHA, PDK1, and SLC2A1, which encode proteins that are required for metabolic reprogramming, as well as LOXL2 and L1CAM, which encode proteins that are required for lung metastasis. JMJD2C expression is significantly associated with expression of GLUT1, LDHA, PDK1, LOX, LOXL2, and L1CAM mRNA in human breast cancer biopsies. JMJD2C knockdown inhibits breast tumor growth and spontaneous metastasis to the lungs of mice following mammary fat pad injection. Taken together, these findings establish an important epigenetic mechanism that stimulates HIF-1-mediated transactivation of genes encoding proteins involved in metabolic reprogramming and lung metastasis in breast cancer.H3K9me3 | SILAC | chromatin immunoprecipitation | gene expression