mTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals

Background: mTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling. Methodology/Principal Findings: Mice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1b, IFN-c and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways. Conclusions/Significance: We conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation an

Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

<p>Abstract</p> <p>Background</p> <p>The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach.</p> <p>Results</p> <p>We used the class comparison analysis and survival analysis algorithms to identify differentially expressed genes and their associated transcription factors, pathways and microRNAs among normal vs. tumor or good prognosis vs. poor prognosis phenotypes classes based on numerous human cancer gene expression data. We found that most of the human embryonic stem cell- associated signatures were frequently identified in the analysis, suggesting a strong linkage between human embryonic stem cells and cancer cells.</p> <p>Conclusions</p> <p>The present study revealed the close linkage between the human embryonic stem cell associated gene expression profiles and cancer-associated gene expression profiles, and therefore offered an indirect support for the cancer stem cell theory. However, many interest issues remain to be addressed further.</p

A Bioinformatics Filtering Strategy for Identifying Radiation Response Biomarker Candidates

The number of biomarker candidates is often much larger than the number of clinical patient data points available, which motivates the use of a rational candidate variable filtering methodology. The goal of this paper is to apply such a bioinformatics filtering process to isolate a modest number (<10) of key interacting genes and their associated single nucleotide polymorphisms involved in radiation response, and to ultimately serve as a basis for using clinical datasets to identify new biomarkers. In step 1, we surveyed the literature on genetic and protein correlates to radiation response, in vivo or in vitro, across cellular, animal, and human studies. In step 2, we analyzed two publicly available microarray datasets and identified genes in which mRNA expression changed in response to radiation. Combining results from Step 1 and Step 2, we identified 20 genes that were common to all three sources. As a final step, a curated database of protein interactions was used to generate the most statistically reliable protein interaction network among any subset of the 20 genes resulting from Steps 1 and 2, resulting in identification of a small, tightly interacting network with 7 out of 20 input genes. We further ranked the genes in terms of likely importance, based on their location within the network using a graph-based scoring function. The resulting core interacting network provides an attractive set of genes likely to be important to radiation response

IKKβ was not required for LPS-induced mTORC1-S6K activation.

<p>IKKβ was suppressed in RAW cells by siRNA (IK) 48 hr prior to LPS stimulation. Nonsense siRNA constructs (NS) served as controls. Representative immunoblots show that IKKβ suppression had no effect on mTORC1-S6K activation in LPS-treated RAW cells as reflected by pS6 levels.</p

Rapamycin treatment effectively suppressed mTORC1-S6K signaling.

<p>Following LPS challenge in vivo, mice were treated with vehicle or 6 mg/kg rapamycin 30 min prior to 25 mg/kg LPS challenge. Organs were harvested from mice at 2, 6, 24 hr post challenge. Immunoblots of organs extracted from 3 mice from each treatment group probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. Unchallenged mice with or without rapamycin served as controls (time 0 hr). Rapamycin treatment successfully suppressed mTORC1-S6K up-regulation in endotoxemic mice as reflected by the near-absent pS6 signals.</p

mTORC1-S6K up-regulation in the livers, lungs, and kidneys of mice treated with endotoxin.

<p>Representative immunoblots of normalized organ protein extracts probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. Mice were subjected to 25 mg/kg of LPS i.p. and sacrificed at 2,6, and 24 hr later. Untreated mice served as time 0 hr controls. mTORC1-S6K activation could be seen as early as 2 hr after LPS challenge in the livers and lungs of endotoxemic mice. mTORC1-S6K was also activated in the kidneys at 24 hr post challenge. Increased pAkt (S473) was seen only in the lung extracts post LPS challenged, occurring at 6 hr post LPS, after mTORC1-S6K activation, and was not seen in either liver or kidney.</p

Rapamycin suppression of mTORC1-S6K enhanced early survival of endotoxemic mice.

<p>Kaplan-Meier curves of LPS-challenged mice treated with vehicle control (N = 10) or rapamycin (N = 9) are shown here. Rapamycin-treated mice had significantly better survival compared to vehicle-treated (P = 0.033, logrank test). However, all mice had died by 72 hr after LPS challenge.</p

Rapamycin suppression of mTORC1-S6K was associated with lower cytokine levels.

<p>Following LPS treatment in vivo, circulating cytokine levels of LPS-challenged mice pre-treated with vehicle (solid bar) or rapamycin (open bar) at 0 (no LPS), 2, 6, and 24 hr post LPS are shown here. Data are expressed as mean of three mice in each group at each time point, with error bars representing standard deviations. Statistical analysis was performed using Repeated Measures ANOVA, and p values<0.05 are considered as significant (*). mTORC1-S6K suppression by rapamycin led to decreased circulation levels of IL1-β, VEGF, IFN-γ and IL-5. There was a trend toward lower TNF-α levels with a p value of 0.058.</p

mTORC1-S6K activation in endotoxemic TNFR-null mice.

<p>Transgenic mice deficient in TNF-α receptors (TNFR) were given LPS and sacrificed at 2, 6, and 24 hr later for organ extraction. Representative immunoblots probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. mTORC1-S6K activation could be observed in the livers, lungs and kidneys of these mice similar to wildtype mice.</p

TNF-α secretion of LPS-treated macrophages occurred after mTORC1-S6K activation.

<p>Conditioned media and cell lysates of RAW cells treated with 100 ng/ml of LPS were collected at various time points. The immunoblots probing for pS6(S240/244) (A.), and the averages of TNF-α levels in the conditioned media from triplicate experiments with error bars representing standard deviations (B.) are shown here. mTORC1-S6K activation occurred as soon as 30 min after LPS when TNF-α levels were not detectably increased at that time point.</p