Sunmin Lee

IL-1β-mediated up-regulation of HIF-1α via an NFκB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis

Growing evidence indicates that inflammation is a contributing factor leading to cancer development. However, pathways involved in this progression are not well understood. To examine whether HIF‐1α is a factor linking inflammation and tumorigenesis, we investigated whether the HIF‐1 signaling pathway was stimulated by the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) in A549 cells. We find that IL‐1β up‐regulated HIF‐1α protein under normoxia and activated the HIF‐1‐responsive gene vascular endothelial growth factor (VEGF) via a pathway dependent on nuclear factor κB (NFkB). Interestingly, although this pathway is stimulated by upstream signaling via AKT and mTOR and requires new transcription, IL‐1 mediated HIF‐1α induction also utilizes a post‐transcriptional mechanism that involves antagonism of VHL‐dependent HIF‐1α degradation, which results in increased HIF‐1α protein stability. IL‐1 mediated NFkB‐dependent cyclooxygenases‐2 (COX‐2) expression served as a positive effector for HIF‐1α induction. Although COX‐2 inhibitors attenuated IL‐1 mediated HIF‐1α induction, prostaglandin E2 (PGE2), a physiological product of COX‐2, induced HIF‐1α protein in a dose‐dependent manner. Our data, therefore, demonstrate that IL‐1β up‐regulates functional HIF‐1α protein through a classical inflammatory signaling pathway involving NFkB and COX‐2, culminating in up‐regulation of VEGF, a potent angiogenic factor required for tumor growth and metastasis. Thus, HIF‐1 is identified as a pivotal transcription factor linking the inflammatory and oncogenic pathways.

Curcumin is an inhibitor of p300 histone acetylatransferase.

Histone acetyltransferases (HATs), and p300/CBP in particular, have been implicated in cancer cell growth and survival, and as such, HATs represent novel, therapeutically relevant molecular targets for drug development. In this study, we demonstrate that the small molecule natural product curcumin, whose medicinal properties have long been recognized in India and Southeast Asia, is a selective HAT inhibitor. Furthermore the data indicate that alpha, beta unsaturated carbonyl groups in the curcumin side chain function as Michael reaction sites and that the Michael reaction acceptor functionality of curcumin is required for its HAT-inhibitory activity. In cells, curcumin promoted proteasome-dependent degradation of p300 and the closely related CBP protein without affecting the HATs PCAF or GCN5. In addition to inducing p300 degradation curcumin inhibited the acetyltransferase activity of purified p300 as assessed using either histone H3 or p53 as substrate. Radiolabeled curcumin formed a covalent association with p300, and tetrahydrocurcumin displayed no p300 inhibitory activity, consistent with a Michael reaction-dependent mechanism. Finally, curcumin was able to effectively block histone hyperacetylation in both PC3-M prostate cancer cells and peripheral blood lymphocytes induced by the histone deacetylase inhibitor MS-275. These data thus identify the medicinal natural product curcumin as a novel lead compound for development of possibly therapeutic, p300/CBP-specific HAT inhibitors.

Hypoxia-inducible factor induction by tumour necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor kappa B activation.

Tumour necrosis factor alpha (TNF-alpha) binds to its receptor (TNFR1) and activates both death- and inflammation/survival-related signalling pathways. The inflammation and survival-related signalling cascade results in the activation of the transcription factor, nuclear factor kappa B (NF-kappa B) and requires recruitment of receptor-interacting protein (RIP) to TNFR1. The indispensable role of RIP in TNF-induced NF-kappa B activation has been demonstrated in RIP(-/-) mice and in cell lines derived from such mice. In the present study, we show that the TNF-alpha-induced accumulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein in normoxic cells is RIP-dependent. Exposing fibroblasts derived from RIP(-/-) mice to either cobalt or PMA resulted in an equivalent HIF-1 alpha induction to that seen in RIP(+/+) fibroblasts. In contrast, RIP(-/-) cells were unable to induce HIF-1 alpha in response to TNF-alpha. Further, transient transfection of NIH 3T3 cells with an NF-kappa B super-repressor plasmid (an inhibitor of NF-kappa B activation) also prevented HIF-1 alpha induction by TNF-alpha. Surprisingly, although HIF-1 alpha mRNA levels remained unchanged after induction by TNF, induction of HIF-1 alpha protein by the cytokine was completely blocked by pretreatment with the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside. Finally, TNF failed to induce both HIF-1 alpha, made resistant to von Hippel-Lindau (VHL), and wild-type HIF-1 alpha transfected into VHL(-/-) cells. These results indicate that HIF-1 alpha induction by TNF-alpha in normoxic cells is mediated by protein stabilization but is nonetheless uniquely dependent on NF-kappa B-driven transcription. Thus the results describe a novel mechanism of HIF-1 alpha up-regulation and they identify HIF-1 alpha as a unique component of the NF-kappa B-mediated inflammatory/survival response.

The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels

Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells.

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis.

Significance TNF receptor-associated protein (TRAP1) is found predominantly in mitochondria. A possible direct impact of TRAP1 on mitochondrial metabolism remains unexplored. We used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1 deficiency promotes increased mitochondrial respiration, fatty acid oxidation, tricarboxylic acid cycle intermediates, ATP and reactive oxygen species, while concomitantly suppressing glucose metabolism. TRAP1 deficiency also results in strikingly enhanced cell motility and invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

Microtubule Disruption Utilizes an NFκB-dependent Pathway to Stabilize HIF-1α Protein

Hypoxia-inducible factor (HIF)-1α levels are elevated in normoxic cells undergoing physiological processes involving large scale microtubule reorganization, such as embryonic development, wound healing, and tumor cell metastasis. Although alterations in microtubules affect numerous cellular responses, no data have yet implicated microtubule dynamics in HIF-1α regulation. To investigate the effect of microtubule change upon HIF-1α regulation, we treated cells with the microtubule-depolymerizing agents (MDAs) colchicine, vinblastine or nocodazole. We demonstrate that these agents are able to induce transcriptionally active HIF-1. MDA-mediated induction of HIF-1α required microtubule depolymerization, since HIF-1α levels were unchanged in cells treated with either the microtubule-stabilizing agent paclitaxel, or an inactive form of colchicine, or in colchicine-resistant cells. HIF-1 induction was dependent upon cellular transcription, as transcription inhibitors abrogated HIF-1α protein up-regulation. The ability of transcriptional inhibitors to interfere with HIF-1α accumulation was specific to the MDA-initiated pathway, as they were ineffective in preventing hypoxia-mediated HIF-1 induction, which occurs by a distinct post-translational pathway. Moreover, we provide evidence implicating a requirement for NFκB transcription in the HIF-1 induction mediated by MDAs. The ability of MDAs to induce HIF-1α is dependent upon activation of NFκB, since inhibition of NFκB either pharmacologically or by transfection of an NFκB super-repressor plasmid abrogated this induction. Collectively, these data support a model in which NFκB is a focal point for the convergence of MDA-mediated signaling events leading to HIF-1 induction, thus revealing a novel aspect of HIF-1α regulation and function.

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

Sunitinib in patients with chemotherapy-refractory thymoma and thymic carcinoma: an open-label phase 2 trial

BACKGROUND No standard treatments are available for advanced thymic epithelial tumours after failure of platinum-based chemotherapy. We investigated the activity of sunitinib, an orally administered tyrosine kinase inhibitor. METHODS Between May 15, 2012, and Oct 2, 2013, we did an open-label phase 2 trial in patients with histologically confirmed chemotherapy-refractory thymic epithelial tumours. Patients were eligible if they had disease progression after at least one previous regimen of platinum-containing chemotherapy, an Eastern Cooperative Oncology Group performance status of two or lower, measurable disease, and adequate organ function. Patients received 50 mg of sunitinib orally once a day, in 6-week cycles (ie, 4 weeks of treatment followed by 2 weeks without treatment), until tumour progression or unacceptable toxic effects arose. The primary endpoint was investigator-assessed best tumour response at any point, which we analysed separately in thymoma and thymic carcinoma cohorts. Patients who had received at least one cycle of treatment and had their disease reassessed were included in the analyses of response. The trial was registered with ClinicalTrials.gov, number NCT01621568. FINDINGS 41 patients were enrolled, 25 with thymic carcinoma and 16 with thymoma. One patient with thymic carcinoma was deemed ineligible after enrolment and did not receive protocol treatment. Of patients who received treatment, one individual with thymic carcinoma was not assessable because she died. Median follow-up on trial was 17 months (IQR 14.0-18.4). Of 23 assessable patients with thymic carcinoma, six (26%, 90% CI 12.1-45.3, 95% CI 10.2-48.4) had partial responses, 15 (65%, 95% CI 42.7-83.6) achieved stable disease, and two (9%, 1.1-28.0) had progressive disease. Of 16 patients with thymoma, one (6%, 95% CI 0.2-30.2) had a partial response, 12 (75%, 47.6-92.7) had stable disease, and three (19%, 4.1-45.7) had progressive disease. The most common grade 3 and 4 treatment-related adverse events were lymphocytopenia (eight [20%] of 40 patients), fatigue (eight [20%]), and oral mucositis (eight [20%]). Five (13%) patients had decreases in left-ventricular ejection fraction, of which three (8%) were grade 3 events. Three (8%) patients died during treatment, including one individual who died of cardiac arrest that was possibly treatment-related. INTERPRETATION Sunitinib is active in previously treated patients with thymic carcinoma. Further studies are needed to identify potential biomarkers of activity. FUNDING National Cancer Institute (Cancer Therapy Evaluation Program).

Sequential 5-Aza 2'-deoxycytidine/depsipeptide FK228 treatment induces tissue factor pathway inhibitor 2 (TFPI-2) expression in cancer cells.

cDNA arrays were used to examine gene induction in CALU-6 and H460 lung cancer cells mediated by sequential 5-aza 2′-deoxycytidine (DAC)/depsipeptide FK228 (DP) exposure in order to identify translational end points for clinical trials evaluating these agents. In both cell lines, sequential DAC/DP treatment induced expression of tissue factor pathway inhibitor-2 (TFPI-2), an inhibitor of Factor VII: tissue factor signal transduction known to diminish the malignant phenotype of cancer cells. TFPI-2 expression was diminished or absent in 16 of 32 cell lines established from thoracic malignancies. Sequential DAC/DP treatment induced TFPI-2 in cancer cells deficient for TFPI-2 expression in the basal state. Promoter methylation coincided with loss of TFPI-2 expression in a number of cancer lines. TFPI-2 promoter methylation was observed in one of five pulmonary adenocarcinomas, and seven of seven esophageal adenocarcinomas, but not corresponding normal tissues. DP enhanced acetylation of TFPI-2-associated histones in CALU-6 cells. DP or PDBU, alone, induced TFPI-2 expression in cancer cells deficient for TFPI-2 expression in the absence of promoter methylation. In these cells, DP-mediated TFPI-2 induction was abrogated by calphostin. Induction of TFPI-2 by distinct, yet cooperative mechanisms involving chromatin remodeling and PKC signaling strengthens the preclinical rationale for sequential administration of DNA demethylating agents and HDAC inhibitors in cancer patients. Furthermore, induction of TFPI-2 may be a useful surrogate marker of treatment response in individuals receiving sequential DAC/DP infusions.

Englerin A stimulates PKCθ to inhibit insulin signaling and to simultaneously activate HSF1: pharmacologically induced synthetic lethality.

The natural product englerin A (EA) binds to and activates protein kinase C-θ (PKCθ). EA-dependent activation of PKCθ induces an insulin-resistant phenotype, limiting the access of tumor cells to glucose. At the same time, EA causes PKCθ-mediated phosphorylation and activation of the transcription factor heat shock factor 1, an inducer of glucose dependence. By promoting glucose addiction, while simultaneously starving cells of glucose, EA proves to be synthetically lethal to highly glycolytic tumors.