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

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.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.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

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

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

Effects of IL-6 and IL-8 on RCC cell invasion.

<p>A) RCC 786-O cells were treated with recombinant IL-6 and/or IL-8 for 18 hours and cell invasion was analyzed by Boyden chamber as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#s2" target="_blank">materials and methods</a>. <i>Left panel,</i> the invaded cells were photographed and <i>right panel,</i> invaded cells were counted and quantified. The graph is representative of ten independent experiments and the results are expressed as the means <u>+</u> S.E. ***, p<0.001 versus buffer control (-). B) RCC 786-O cells were incubated with condition media from RCC 786-O cells subjected to 48hr norm or hypoxic conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#pone-0030712-g004" target="_blank">Fig. 1C,E</a>) for 18 hours and cell invasion was analyzed by Boyden chamber. C) RCC 786-O cells were incubated with condition media from RCC 786-O cells in normoxic media (norm) or hypoxic media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#pone-0030712-g004" target="_blank">Fig. 2 D</a>, E transfected with scrambled control (scr) or siRNA for Nox4 (siNox4). D) RCC 786-O cells were incubated with condition media from RCC 786-O cells in normoxic media (norm) or hypoxic media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#pone-0030712-g004" target="_blank">Fig. 3A</a>,B incubated with buffer control (-) or AICAR. B, C, D, Invaded cells were quantified from three independent experiments and expressed as the means <u>+</u> S.E. **, p<0.01 versus norm, <i>##</i>, p<0.01 versus hypoxic control (-).</p