Karthigayan Shanmugasundaram

Nox4 Mediates Renal Cell Carcinoma Cell Invasion through Hypoxia-Induced Interleukin 6- and 8- Production

Background Inflammatory cytokines are detected in the plasma of patients with renal cell carcinoma (RCC) and are associated with poor prognosis. However, the primary cell type involved in producing inflammatory cytokines and the biological significance in RCC remain unknown. Inflammation is associated with oxidative stress, upregulation of hypoxia inducible factor 1-alpha, and production of pro-inflammatory gene products. Solid tumors are often heterogeneous in oxygen tension together suggesting that hypoxia may play a role in inflammatory processes in RCC. Epithelial cells have been implicated in cytokine release, although the stimuli to release and molecular mechanisms by which they are released remain unclear. AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status and a role for AMPK in the regulation of cell inflammatory processes has recently been demonstrated. Methods and Principal Findings We have identified for the first time that interleukin-6 and interleukin-8 (IL-6 and IL-8) are secreted solely from RCC cells exposed to hypoxia. Furthermore, we demonstrate that the NADPH oxidase isoform, Nox4, play a key role in hypoxia-induced IL-6 and IL-8 production in RCC. Finally, we have characterized that enhanced levels of IL-6 and IL-8 result in RCC cell invasion and that activation of AMPK reduces Nox4 expression, IL-6 and IL-8 production, and RCC cell invasion. Conclusions/Significance Together, our data identify novel mechanisms by which AMPK and Nox4 may be linked to inflammation-induced RCC metastasis and that pharmacological activation of AMPK and/or antioxidants targeting Nox4 may represent a relevant therapeutic intervention to reduce IL-6- and IL-8-induced inflammation and cell invasion in RCC.

NF-κB activity in muscle from obese and type 2 diabetic subjects under basal and exercise-stimulated conditions

NF-κB is a transcription factor that controls the gene expression of several proinflammatory proteins. Cell culture and animal studies have implicated increased NF-κB activity in the pathogenesis of insulin resistance and muscle atrophy. However, it is unclear whether insulin-resistant human subjects have abnormal NF-κB activity in muscle. The effect that exercise has on NF-κB activity/signaling also is not clear. We measured NF-κB DNA-binding activity and the mRNA level of putative NF-κB-regulated myokines interleukin (IL)-6 and monocyte chemotactic protein-1 (MCP-1) in muscle samples from T2DM, obese, and lean subjects immediately before, during (40 min), and after (210 min) a bout of moderate-intensity cycle exercise. At baseline, NF-κB activity was elevated 2.1- and 2.7-fold in obese nondiabetic and T2DM subjects, respectively. NF-κB activity was increased significantly at 210 min following exercise in lean (1.9-fold) and obese (2.6-fold) subjects, but NF-κB activity did not change in T2DM. Exercise increased MCP-1 mRNA levels significantly in the three groups, whereas IL-6 gene expression increased significantly only in lean and obese subjects. MCP-1 and IL-6 gene expression peaked at the 40-min exercise time point. We conclude that insulin-resistant subjects have increased basal NF-κB activity in muscle. Acute exercise stimulates NF-κB in muscle from nondiabetic subjects. In T2DM subjects, exercise had no effect on NF-κB activity, which could be explained by the already elevated NF-κB activity at baseline. Exercise-induced MCP-1 and IL-6 gene expression precedes increases in NF-κB activity, suggesting that other factors promote gene expression of these cytokines during exercise.

Reduced expression of fumarate hydratase in clear cell renal cancer mediates HIF-2α accumulation and promotes migration and invasion.

Germline mutations of FH, the gene that encodes for the tricarboxylic acid TCA (TCA) cycle enzyme fumarate hydratase, are associated with an inherited form of cancer referred to as Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). Individuals with HLRCC are predisposed to the development of highly malignant and lethal renal cell carcinoma (RCC). The mechanisms of tumorigenesis proposed have largely focused on the biochemical consequences of loss of FH enzymatic activity. While loss of the tumor suppressor gene von Hippel Lindau (VHL) is thought to be an initiating event for the majority of RCCs, a role for FH in sporadic renal cancer has not been explored. Here we report that FH mRNA and protein expression are reduced in clear cell renal cancer, the most common histologic variant of kidney cancer. Moreover, we demonstrate that reduced FH leads to the accumulation of hypoxia inducible factor- 2α (HIF-2α), a transcription factor known to promote renal carcinogenesis. Finally, we demonstrate that overexpression of FH in renal cancer cells inhibits cellular migration and invasion. These data provide novel insights into the tumor suppressor functions of FH in sporadic kidney cancer.

Effect of a sustained reduction in plasma free fatty acid concentration on insulin signalling and inflammation in skeletal muscle from human subjects

•  Reducing free fatty acids in the circulation gives protection against muscle insulin resistance. •  In the present study, we investigated the mechanism by which free fatty acid reduction improves muscle insulin sensitivity. •  The antilipolytic drug acipimox reduced the plasma concentration of unsaturated and saturated fatty acids in insulin‐resistant (obese and type 2 diabetic) subjects. •  The reduction in plasma free fatty acid concentration caused by acipimox led to an improvement in local inflammation and insulin signalling in skeletal muscle. •  The improvements in local inflammation and insulin signalling were more pronounced in obese type 2 diabetic subjects than obese non‐diabetic individuals, suggesting that diabetic subjects are more susceptible to the toxic effect of circulating free fatty acids.

PI3K regulation of the SKP-2/p27 axis through mTORC2

The cyclin-dependent kinase inhibitor p27 is a key regulator of cell-cycle progression. Its expression and localization are altered in several types of malignancies, which has prognostic significance in cancers such as renal cell carcinoma (RCC). S-phase kinase-associated protein 2 (SKP-2) is an F-box protein that is part of the SKP-1/Cul1/F-box ubiquitin ligase complex that targets nuclear p27 among many other cell-cycle proteins for proteosomal degradation. Its overexpression has been observed in several tumor types. Signaling by phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) has previously been shown to regulate the SKP-2/p27 axis. Recent evidence suggests that PI3K signaling may activate mammalian target of rapamycin complex 2 (mTORC2) activity. As PI3K signaling is known to regulate SKP-2 and p27, we sought to determine whether these effects were mediated by mTORC2. Here we provide additional genetic evidence that PI3K signaling activates mTORC2 kinase activity. We also demonstrate a novel role for mTORC2 in the modulation of nuclear p27 levels. In particular, mTORC2 signaling promotes the reduction of nuclear p27 protein levels through the increased protein expression of SKP-2. These are the first data to demonstrate a role for mTOR in the regulation of SKP-2. In concordance with these findings, mTORC2 activity promotes cell proliferation of RCC cells at the G1–S interphase of the cell cycle. Collectively, these data implicate mTORC2 signaling in the regulation of the SKP-2/p27 axis, a signaling node commonly altered in cancer.

HIF-1 mediates renal fibrosis in OVE26 type 1 diabetic mice

Hypoxia-inducible factor (HIF)-1 mediates hypoxia- and chronic kidney disease–induced fibrotic events. Here, we assessed whether HIF-1 blockade attenuates the manifestations of diabetic nephropathy in a type 1 diabetic animal model, OVE26. YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole], an HIF-1 inhibitor, reduced whole kidney glomerular hypertrophy, mesangial matrix expansion, extracellular matrix accumulation, and urinary albumin excretion as well as NOX4 protein expression and NADPH-dependent reactive oxygen species production, while blood glucose levels remained unchanged. The role of NOX oxidases in HIF-1–mediated extracellular matrix accumulation was explored in vitro using glomerular mesangial cells. Through a series of genetic silencing and adenoviral overexpression studies, we have defined GLUT1 as a critical downstream target of HIF-1α mediating high glucose–induced matrix expression through the NADPH oxidase isoform, NOX4. Together, our data suggest that pharmacological inhibition of HIF-1 may improve clinical manifestations of diabetic nephropathy.

The Oncometabolite Fumarate Promotes Pseudohypoxia Through Noncanonical Activation of NF-κB Signaling

Background: We examined alternative mechanisms by which fumarate levels contribute to hypoxia inducible factor (HIF)-1α accumulation and fumarate hydratase (FH)-deficient renal carcinogenesis. Results: Fumarate promotes HIF-1α transcription through Tank binding kinase 1 (TBK1)-dependent noncannonical activation of NF-κB signaling. Conclusion: Fumarate-mediated, TBK-dependent accumulation of HIF-1α mediates cell invasion in FH-deficient RCC. Significance: TBK is a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors. Inactivating mutations of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH) have been linked to an aggressive variant of hereditary kidney cancer (hereditary leiomyomatosis and renal cell cancer). These tumors accumulate markedly elevated levels of fumarate. Fumarate is among a growing list of oncometabolites identified in cancers with mutations of genes involved in intermediary metabolism. FH-deficient tumors are notable for their pronounced accumulation of the transcription factor hypoxia inducible factor-1α (HIF-1α) and aggressive behavior. To date, HIF-1α accumulation in hereditary leiomyomatosis and renal cell cancer tumors is thought to result from fumarate-dependent inhibition of prolyl hydroxylases and subsequent evasion from von Hippel-Lindau-dependent degradation. Here, we demonstrate a novel mechanism by which fumarate promotes HIF-1α mRNA and protein accumulation independent of the von Hippel-Lindau pathway. Here we demonstrate that fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank binding kinase 1 (TBK1). Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1α accumulation in cellular models of FH loss and markedly reduces cell invasion of FH-deficient RCC cancer cells. Collectively, our data demonstrate a novel mechanism by which pseudohypoxia is promoted in FH-deficient tumors and identifies TBK1 as a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors.

NOX4 functions as a mitochondrial energetic sensor coupling cancer metabolic reprogramming to drug resistance

The molecular mechanisms that couple glycolysis to cancer drug resistance remain unclear. Here we identify an ATP-binding motif within the NADPH oxidase isoform, NOX4, and show that ATP directly binds and negatively regulates NOX4 activity. We find that NOX4 localizes to the inner mitochondria membrane and that subcellular redistribution of ATP levels from the mitochondria act as an allosteric switch to activate NOX4. We provide evidence that NOX4-derived reactive oxygen species (ROS) inhibits P300/CBP-associated factor (PCAF)-dependent acetylation and lysosomal degradation of the pyruvate kinase-M2 isoform (PKM2). Finally, we show that NOX4 silencing, through PKM2, sensitizes cultured and ex vivo freshly isolated human-renal carcinoma cells to drug-induced cell death in xenograft models and ex vivo cultures. These findings highlight yet unidentified insights into the molecular events driving cancer evasive resistance and suggest modulation of ATP levels together with cytotoxic drugs could overcome drug-resistance in glycolytic cancers.NADPH oxidase NOX4 has been linked to poor cancer survival. Here the authors show that NOX4 regulates drug resistance in renal cancer carcinoma by regulating PKM2 and that NOX4 activity is allosterically activated by reduced mitochondrial ATP levels thus coupling energy metabolism to drug resistance.

141 PI3K REGULATES P27 THROUGH MTORC2 IN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most lethal of all urologic malignancies. Moreover, its incidence is rising and it is among the ten most common malignancies in both men and women. Dysregulation of the cell cycle regulator p27, particularly reduced nuclear expression has been shown to inversely correlate with RCC tumor stage and grade. The PI3K/AKT signaling axis has been implicated in the molecular pathogenesis of RCC and has been previously shown to regulate p27 through a variety of mechanisms. For example, PI3K signaling may regulate AKT-mediated phosphorylation of p27 which can impact p27 localization and steadystate levels. In addition PI3K/ AKT signaling may regulate levels of SKP2, a component of the SCFSKP2 ubiquitin ligase complex that mediates p27 proteolysis. Recent evidence suggests that the mTOR signaling complex 2 (mTORC2), in conjunction with PI3K signaling, regulates AKT activation. Based on these data, we investigated the role of mTORC2 in p27 regulation in the context of RCC. METHODS: Both pharmacologic and genetic techniques were utilized to determine the effects of mTORC2 signaling on p27. Effects on p27 steady state levels were analyzed through the combined use of real time RT-PCR as well as immunoblotting. Effects on localization were determined through cell fractionation studies in addition to immunofluorescence. The impact of mTORC2 on cell proliferation was determined through cell cycle analysis in addition to thymidine incorporation assays. RESULTS: Similar to inhibition of PI3K signaling, inhibition of mTORC2, either via genetic or pharmacologic means, led to an increase in both total as well as nuclear p27 protein levels. mTORC2 signaling, through AKT activation, mediates p27 levels in part through the regulation of SKP2. Finally, mTORC2 blockade led to inhibition of cell cycle progression with concomitant reduced cellular proliferation in RCC cells. CONCLUSIONS: Our data demonstrate a role for mTORC2 in the regulation of p27 levels as well as localization in RCC cells. Furthermore, our data demonstrate that these effects mediate the proliferation of RCC cells. Given the established role of p27 as a prognostic biomarker for RCC, our data suggest a role for targeting mTORC2 in the treatment of kidney cancer.