Involvement of estrogen-related receptors in transcriptional response to hypoxia and growth of solid tumors.

The development of intratumoral hypoxia is a universal hallmark of rapidly growing solid tumors. Adaptation to the hypoxic environment, which is critical for tumor cell survival and growth, is mediated primarily through a hypoxia-inducible factor (HIF)-dependent transcriptional program. HIF activates genes that facilitate crucial adaptive mechanisms including increased glucose uptake and glycolysis and tumor angiogenesis, making it an important therapeutic target. However, the HIF-dependent transcriptional mechanism remains incompletely understood, and targeting HIF is a difficult endeavor. Here, we show that the orphan nuclear receptor estrogen-related receptors (ERRs) physically interact with HIF and stimulate HIF-induced transcription. Importantly, ERRs appear to be essential for HIF's function. Transcriptional activation of hypoxic genes in cells cultured under hypoxia is largely blocked by suppression of ERRs through expression of a dominant negative form of ERR or treatment with a pharmacological ERR inhibitor, diethylstilbestrol. Systematic administration of diethylstilbestrol severely diminished growth and angiogenesis of tumor xenografts in vivo. Because nuclear receptors are outstanding targets for drug discovery, the findings not only may offer mechanistic insights into HIF-mediated transcription but also may open new avenues for targeting the HIF pathway for cancer therapy.

Warburg effect in chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-resistant cancer cells to taxol.

BACKGROUND: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. RESULTS: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. CONCLUSION: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

Evidence for a Novel Regulatory Interaction Involving Cyclin D1, Lipid Droplets, Lipolysis, and Cell Cycle Progression in Hepatocytes.

During normal proliferation, hepatocytes accumulate triglycerides (TGs) in lipid droplets (LDs), but the underlying mechanisms and functional significance of this steatosis are unknown. In the current study, we examined the coordinated regulation of cell cycle progression and LD accumulation. As previously shown, hepatocytes develop increased LD content after mitogen stimulation. Cyclin D1, in addition to regulating proliferation, was both necessary and sufficient to promote LD accumulation in response to mitogens. Interestingly, cyclin D1 promotes LD accumulation by inhibiting the breakdown of TGs by lipolysis through a mechanism involving decreased lipophagy, the autophagic degradation of LDs. To examine whether inhibition of lipolysis is important for cell cycle progression, we overexpressed adipose TG lipase (ATGL), a key enzyme involved in TG breakdown. As expected, ATGL reduced LD content but also markedly inhibited hepatocyte proliferation, suggesting that lipolysis regulates a previously uncharacterized cell cycle checkpoint. Consistent with this, in mitogen-stimulated cells with small interfering RNA-mediated depletion of cyclin D1 (which inhibits proliferation and stimulates lipolysis), concurrent ATGL knockdown restored progression into S phase. Following partial hepatectomy, a model of robust hepatocyte proliferation in vivo, ATGL overexpression led to decreased LD content, cell cycle inhibition, and marked liver injury, further indicating that down-regulation of lipolysis is important for normal hepatocyte proliferation. Conclusion: We suggest a new relationship between steatosis and proliferation in hepatocytes: cyclin D1 inhibits lipolysis, resulting in LD accumulation, and suppression of lipolysis is necessary for cell cycle progression.

Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation.

Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation.

Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine-289 phosphorylation.

The Warburg effect, which reflects cancer cells' preference for aerobic glycolysis over glucose oxidation, contributes to tumor growth, progression and therapy resistance. The restraint on pyruvate flux into mitochondrial oxidative metabolism in cancer cells is in part attributed to the inhibition of pyruvate dehydrogenase (PDH) complex. Src is a prominent oncogenic non-receptor tyrosine kinase that promotes cancer cell proliferation, invasion, metastasis and resistance to conventional and targeted therapies. However, the potential role of Src in tumor metabolism remained unclear. Here we report that activation of Src attenuated PDH activity and generation of reactive oxygen species (ROS). Conversely, Src inhibitors activated PDH and increased cellular ROS levels. Src inactivated PDH through direct phosphorylation of tyrosine-289 of PDH E1α subunit (PDHA1). Indeed, Src was the main kinase responsible for PDHA1 tyrosine phosphorylation in cancer cells. Expression of a tyrosine-289 non-phosphorable PDHA1 mutant in Src-hyperactivated cancer cells restored PDH activity, increased mitochondrial respiration and oxidative stress, decreased experimental metastasis, and sensitized cancer cells to pro-oxidant treatment. The results suggest that Src contributes to the Warburg phenotype by inactivating PDH through tyrosine phosphorylation, and the metabolic effect of Src is essential for Src-driven malignancy and therapy resistance. Combination therapies consisting of both Src inhibitors and pro-oxidants may improve anticancer efficacy.

Glucose oxidation modulates anoikis and tumor metastasis.

Cancer cells exhibit altered glucose metabolism characterized by a preference for aerobic glycolysis or the Warburg effect, and the cells resist matrix detachment-induced apoptosis, which is called anoikis, a barrier to metastasis. It remains largely unclear whether tumor metabolism influences anoikis and metastasis. Here we show that when detached from the matrix, untransformed mammary epithelial cells undergo metabolic reprogramming by markedly upregulating pyruvate dehydrogenase (PDH) kinase 4 (PDK4) through estrogen-related receptor gamma (ERRγ), thereby inhibiting PDH and attenuating the flux of glycolytic carbon into mitochondrial oxidation. To decipher the significance of this metabolic response, we found that depletion of PDK4 or activation of PDH increased mitochondrial respiration and oxidative stress in suspended cells, resulting in heightened anoikis. Conversely, overexpression of PDKs prolonged survival of cells in suspension. Therefore, decreased glucose oxidation following cell detachment confers anoikis resistance. Unlike untransformed cells, most cancer cells demonstrate reduced glucose oxidation even under attached conditions, and thus they inherently possess a survival advantage when suspended. Normalization of glucose metabolism by stimulating PDH in cancer cells restores their susceptibility to anoikis and impairs their metastatic potential. These results suggest that the Warburg effect, more specifically, diminished glucose oxidation, promotes anoikis resistance and metastasis and that PDKs are potential targets for antimetastasis therapy.

Phosphorylation of Y372 is critical for Jak2 tyrosine kinase activation.

Jak2 tyrosine kinase plays an important role in cytokine mediated signal transduction. There are 49 tyrosine residues in Jak2 and phosphorylation of some of these are known to play important roles in the regulation of Jak2 kinase activity. Here, using mass spectrometry, we identified tyrosine residues Y372 and Y373 as novel sites of Jak2 phosphorylation. Mutation of Y372 to F (Y372F) significantly inhibited Jak2 phosphorylation, including that of Y1007, whereas the Jak2-Y373F mutant displayed only modest reduction in phosphorylation. Relative to Jak2-WT, the ability of Jak2-Y372F to bind to and phosphorylate STAT1 was decreased, resulting in reduced Jak2-mediated downstream gene transcription. While the Y372F mutation had no effect on receptor-independent, hydrogen peroxide-mediated Jak2 activation, it impaired interferon-gamma (IFNγ) and epidermal growth factor (EGF)-dependent Jak2 activation. Interestingly however, the Y372F mutant exhibited normal receptor binding properties. Finally, co-expression of SH2-Bß only partially restored the activation of the Jak2-Y372F mutant suggesting that the mechanism whereby phosphorylation of Y372 is important for Jak2 activation is via dimerization. As such, our results indicate that Y372 plays a critical yet differential role in Jak2 activation and function via a mechanism involving Jak2 dimerization and stabilization of the active conformation.

Chloroplast-derived anthrax and other vaccine antigens: their immunogenic and immunoprotective properties.

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation, heat stability, absence of human pathogens, protection of antigens in the stomach through bioencapsulation (when delivered orally), elimination of the need for expensive purification and sterile injections and generation of both systemic and mucosal immunity. Recent studies have demonstrated that chloroplast-derived anthrax-protective antigen elicits effective immune responses, develops neutralizing antibodies, confers complete protection against anthrax lethal toxin challenge and produces 360 million doses of vaccine in one acre of transgenic plants. Chloroplast-derived vaccine antigens are efficacious against bacterial, fungal, viral and protozoan pathogens.