Expression Profiling Of Nuclear Receptors In Human And Mouse Embryonic Stem Cells

Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression of NRs between the two human cell lines was highly concordant, whereas in contrast, expression of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that have been implicated previously as crucial regulators of mouse ESC biology, including ERRβ, DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct species-specific functions. Taken together, these results highlight the complexity of the transcrip- tional hierarchy that exists between species and governs early development. These data should provide a unique resource for further exploration of the species-specific roles of NRs in ESC self-renewal and differentiation.Copyright © 2009 by The Endocrine Society

Nuclear receptor expression and function in human lung cancer pathogenesis

Lung cancer is caused by combinations of diverse genetic mutations. Here, to understand the relevance of nuclear receptors (NRs) in the oncogene-associated lung cancer pathogenesis, we investigated the expression profile of the entire 48 NR members by using QPCR analysis in a panel of human bronchial epithelial cells (HBECs) that included precancerous and tumorigenic HBECs harboring oncogenic K-ras(V12) and/or p53 alterations. The analysis of the profile revealed that oncogenic alterations accompanied transcriptional changes in the expression of 19 NRs in precancerous HBECs and 15 NRs according to the malignant progression of HBECs. Amongst these, peroxisome proliferator-activated receptor gamma (PPAR gamma), a NR chosen as a proof-of-principle study, showed increased expression in precancerous HBECs, which was surprisingly reversed when these HBECs acquired full in vivo tumorigenicity. Notably, PPAR gamma activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs. In fully tumorigenic HBECs with inducible expression of PPAR gamma, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPAR gamma-sumoylation dependent manner. Mechanistically, the sumoylation of liganded-PPAR gamma decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression. This suggests that ligand-mediated sumoylation of PPAR gamma plays an important role in lung cancer pathogenesis by modulating prostaglandin metabolism

Nuclear receptor expression defines a set of prognostic biomarkers for lung cancer.

The identification of prognostic tumor biomarkers that also would have potential as therapeutic targets, particularly in patients with early stage disease, has been a long sought-after goal in the management and treatment of lung cancer. The nuclear receptor (NR) superfamily, which is composed of 48 transcription factors that govern complex physiologic and pathophysiologic processes, could represent a unique subset of these biomarkers. In fact, many members of this family are the targets of already identified selective receptor modulators, providing a direct link between individual tumor NR quantitation and selection of therapy. The goal of this study, which begins this overall strategy, was to investigate the association between mRNA expression of the NR superfamily and the clinical outcome for patients with lung cancer, and to test whether a tumor NR gene signature provided useful information (over available clinical data) for patients with lung cancer.Using quantitative real-time PCR to study NR expression in 30 microdissected non-small-cell lung cancers (NSCLCs) and their pair-matched normal lung epithelium, we found great variability in NR expression among patients' tumor and non-involved lung epithelium, found a strong association between NR expression and clinical outcome, and identified an NR gene signature from both normal and tumor tissues that predicted patient survival time and disease recurrence. The NR signature derived from the initial 30 NSCLC samples was validated in two independent microarray datasets derived from 442 and 117 resected lung adenocarcinomas. The NR gene signature was also validated in 130 squamous cell carcinomas. The prognostic signature in tumors could be distilled to expression of two NRs, short heterodimer partner and progesterone receptor, as single gene predictors of NSCLC patient survival time, including for patients with stage I disease. Of equal interest, the studies of microdissected histologically normal epithelium and matched tumors identified expression in normal (but not tumor) epithelium of NGFIB3 and mineralocorticoid receptor as single gene predictors of good prognosis.NR expression is strongly associated with clinical outcomes for patients with lung cancer, and this expression profile provides a unique prognostic signature for lung cancer patient survival time, particularly for those with early stage disease. This study highlights the potential use of NRs as a rational set of therapeutically tractable genes as theragnostic biomarkers, and specifically identifies short heterodimer partner and progesterone receptor in tumors, and NGFIB3 and MR in non-neoplastic lung epithelium, for future detailed translational study in lung cancer. Please see later in the article for the Editors' Summary

Peroxisome proliferator-activated receptor gamma as a theragnostic target for mesenchymal-type glioblastoma patients

Glioblastomas (GBMs) are characterized by four subtypes, proneural (PN), neural, classical, and mesenchymal (MES) GBMs, and they all have distinct activated signaling pathways. Among the subtypes, PN and MES GBMs show mutually exclusive genetic signatures, and the MES phenotype is, in general, believed to be associated with more aggressive features of GBM: tumor recurrence and drug resistance. Therefore, targeting MES GBMs would improve the overall prognosis of patients with fatal tumors. In this study, we propose peroxisome proliferator-activated receptor gamma (PPARγ) as a potential diagnostic and prognostic biomarker as well as therapeutic target for MES GBM; we used multiple approaches to assess PPARγ, including biostatistics analysis and assessment of preclinical studies. First, we found that PPARγ was exclusively expressed in MES glioblastoma stem cells (GSCs), and ligand activation of endogenous PPARγ suppressed cell growth and stemness in MES GSCs. Further in vivo studies involving orthotopic and heterotopic xenograft mouse models confirmed the therapeutic efficacy of targeting PPARγ; compared to control mice, those that received ligand treatment exhibited longer survival as well as decreased tumor burden. Mechanistically, PPARγ activation suppressed proneural–mesenchymal transition (PMT) by inhibiting the STAT3 signaling pathway. Biostatistical analysis using The Cancer Genomics Atlas (TCGA, n = 206) and REMBRANDT (n = 329) revealed that PPARγ upregulation is linked to poor overall survival and disease-free survival of GBM patients. Analysis was performed on prospective (n = 2) and retrospective (n = 6) GBM patient tissues, and we finally confirmed that PPARγ expression was distinctly upregulated in MES GBM. Collectively, this study provides insight into PPARγ as a potential therapeutic target for patients with MES GBM

TRAP1 Inhibition Increases Glutamine Synthetase Activity in Glutamine Auxotrophic Non-small Cell Lung Cancer Cells

Background/Aim: Cancer cells are distinct in terms of glutamine dependence. Here we investigated the different susceptibility of glutamine-independent and glutamine-dependent non-small cell lung cancer (NSCLC) to treatment with tumor necrosis factor receptor-associated protein 1 (TRAP1) inhibitor gamitrinib-triphenylphosphonium (G-TPP). Materials and Methods: Cell viability and proliferation under glutamine deprivation and G-TPP treatment were determined by the MTT and colony-formation assays. Protein and mRNA expression were determined by western blot and quantitative polymerase chain reaction. Colorimetric-based assay was performed to check for glutamine synthetase (GS) activity. Results: NSCLC cells showed diverse adaptation under glutamine-depleted condition and were categorized into glutamine-independent and glutamine-dependent cells. Treatment with G-TPP particularly increased GS activity and induced cell death due to energy shortage indicated by phosphorylated AMP-activated protein kinase (AMPK) in glutamine-dependent cells. Conclusion: This finding provides better understanding of TRAP1-mediated glutamine metabolism through GS activity, and evidence that TRAP1 could be a promising therapeutic target for glutamine-addicted cancer

Functional evaluation of PPARγ in tumorigenic lung cancer cells.

<p>Growth response and cell migration of lung cancer were analyzed to treatment with PPARγ ligands. (A) Cell growth response. HBEC-C1-wt-PPARγ and HBEC-C1-SUMO-PPARγ cell lines were treated with 3 μM of pioglitazone or troglitazone for 3 days with or without tetracycline induction and followed by cell counting assay. Note that the result represents three independent sets of experiments. Liquid colony formation (B,C) and cell migration (D) assays were performed as described in Materials and Methods. Cells were treated with 3 μM of TZDs, or 3 and 10 μM of WY-14643 under the condition of PPARγ-ON or -OFF. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by <i>ANOVA</i>. *<i>P</i> < 0.001 compared to HBEC-KT control.</p

Characterization of wildtype and sumoylation mutant PPARγ.

<p>(A) Luciferase reporter assay of wildtype and sumoylation mutant PPARγ plasmids. RLU, relative luciferase unit. (B) Tetracycline-induced expression of PPARγ and EGFP protein in stably transfected HBEC-C1-wt-PPARγ clone. A microscopic view of tetracycline-induced EFGP expression (top in B). Immunoblot assays for the expression of lamin A/C and tetracycline-induced PPARγ (bottom in B). A bicistronic construct of PPARγ and EGFP was stably introduced into a tumorigenic HBEC clone to generate HBEC-C1-PPARγ cell lines as described in Materials and Methods. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by <i>ANOVA</i>. *<i>P</i> < 0.001 compared to HBEC-KT control.</p

Expression profile of the NR superfamily in tumorigenic HBEC clones.

<p>The QPCR assay was performed for mRNA expression of the entire NR superfamily in non-tumorigenic HBEC-KTR_L53 cells with <i>p53</i> and <i>K-ras</i>^<i>V12</i> changes, two tumorigenic clones C1 and C5, and xenograft C5 tumor tissue. (A) Quantitative mRNA expression profiles of the NR subgroups with distinct expression pattern across the panel. Note that the rest of the NR profile was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134842#pone.0134842.s004" target="_blank">S4 Fig</a>. (B) Summary of NR expression from panel A and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134842#pone.0134842.g002" target="_blank">Fig 2</a> to show NR expression cascades correlated with tumorigenic progression. Data represent the mean ± SD (n = 3). Asterisks show statistically significant points as evaluated by <i>ANOVA</i>. *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 compared to HBEC-KTR_L53.</p