Iron commensalism of mesenchymal glioblastoma promotes ferroptosis susceptibility upon dopamine treatment.

The heterogeneity of glioblastoma multiforme (GBM) leads to poor patient prognosis. Here, we aim to investigate the mechanism through which GBM heterogeneity is coordinated to promote tumor progression. We find that proneural (PN)-GBM stem cells (GSCs) secreted dopamine (DA) and transferrin (TF), inducing the proliferation of mesenchymal (MES)-GSCs and enhancing their susceptibility toward ferroptosis. PN-GSC-derived TF stimulates MES-GSC proliferation in an iron-dependent manner. DA acts in an autocrine on PN-GSC growth in a DA receptor D1-dependent manner, while in a paracrine it induces TF receptor 1 expression in MES-GSCs to assist iron uptake and thus enhance ferroptotic vulnerability. Analysis of public datasets reveals worse prognosis of patients with heterogeneous GBM with high iron uptake than those with other GBM subtypes. Collectively, the findings here provide evidence of commensalism symbiosis that causes MES-GSCs to become iron-addicted, which in turn provides a rationale for targeting ferroptosis to treat resistant MES GBM.

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.

LOX family and ZFPM2 as novel diagnostic biomarkers for malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer that develops in the pleural and outer layer of tissues surrounding the lungs. MPM is primarily caused by occupational exposure to asbestos and results in a poor prognosis. Effective therapeutics as well as early diagnostics for the MPM are still lacking. To identify potential diagnostic biomarkers for MPM, we performed bioinformatics analysis of public database. METHODS: Utilizing databases from Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO), we identified several potential candidates that could act as MPM biomarkers. We carried out additional molecular analyses of these potential markers using MPM patient tissue samples via quantitative polymerase chain reaction. RESULTS: We identified Lysyl oxidase (LOX), Lysyl oxidase homologs 1&2 (LOXL1& LOXL2) Zinc Finger Protein, FOG Family Member 2 (ZFPM2) as potential diagnostic biomarkers for MPM. In this study, we found that the LOX family and ZFPM2 showed comparable diagnostic ability to Fibulin-3 or mesothelin (MSLN) and would be better potential biomarkers than Sulfatase 1 (SULF1), Thrombospondin 2 (THBS2) and Cadherin 11 (CDH11). CONCLUSIONS: LOX family and ZPFM2 were identified as novel MPM diagnostic biomarkers which could strengthen MPM clinical diagnostic capabilities.

WNK1 promotes renal tumor progression by activating TRPC6-NFAT pathway.

Deregulation of Ca2+ signaling has been regarded as one of the key features of cancer progression. Lysine-deficient protein kinase 1 (WNK1), a major regulator of renal ion transport, regulates Ca2+ signaling through stimulating the phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to activate Gαq-coupled receptor/PLC-ß signaling. However, the contribution of WNK1-mediated Ca2+ signaling in the development of clear-cell renal-cell carcinoma (ccRCC) is yet unknown. We found that the canonical transient receptor potential channel (TRPC)6 was widely expressed in ccRCC tissues and functioned as a primary Ca2+ influx mechanism. We further identified that the expressions of WNK1, PI4KIIIα, TRPC6, and the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were elevated in the tumor tissues compared with the adjacent normal tissues. WNK1 expression was directly associated with the nuclear grade of ccRCC tissues. Functional experiments showed that WNK1 activated TRPC6-mediated Ca2+ influx and current by stimulating PI4KIIIα. Notably, the inhibition of WNK1-mediated TRPC6 activation and its downstream substrate calcineurin attenuated NFATc1 activation and the subsequent migration and proliferation of ccRCC. These findings revealed a novel perspective of WNK1 signaling in targeting the TRPC6-NFATc1 pathway as a therapeutic potential for renal-cell carcinoma.-Kim, J.-H., Hwang, K.-H., Eom, M., Kim, M., Park, E. Y., Jeong, Y., Park, K.-S., Cha, S.-K. WNK1 promotes renal tumor progression by activating TRPC6-NFAT pathway.

Correction: PPARγ sumoylation-mediated lipid accumulation in lung cancer.

[This corrects the article DOI: 10.18632/oncotarget.19700.].

Inhibition of oncogenic Src induces FABP4-mediated lipolysis via PPARγ activation exerting cancer growth suppression.

BACKGROUND: c-Src is a driver oncogene well-known for tumorigenic signaling, but little for metabolic function. Previous reports about c-Src regulation of glucose metabolism prompted us to investigate its function in other nutrient modulation, particularly in lipid metabolism. METHODS: Oil-red O staining, cell growth assay, and tumor volume measurement were performed to determine lipid amount and growth inhibitory effect of treatments in lung cancer cells and xenograft model. Gene expression was evaluated by immunoblotting and relative RT-PCR. Transcriptional activity of peroxisome proliferator-activated receptor gamma (PPARγ) was assessed by luciferase assay. Reactive oxygen species (ROS) was measured using ROS sensing dye. Oxygen consumption rate was evaluated by Seahorse XF Mito Stress Test. Clinical relevance of candidate proteins was examined using patient samples and public database analysis. FINDINGS: Inhibition of Src induced lipolysis and increased intracellular ROS. Src inhibition derepressed PPARγ transcriptional activity leading to induced expression of lipolytic gene fatty acid binding protein (FABP) 4 which accompanies reduced lipid droplets and decreased tumor growth. The reverse correlation of Src and FABP4 was confirmed in pair-matched lung cancer patient samples, and further analysis using public datasets revealed upregulation of lipolytic genes is associated with better prognosis of cancer patients. INTERPRETATION: This study provides an insight of how oncogenic factor Src concurrently regulates both cellular signaling pathways and metabolic plasticity to drive cancer progression. FUND: National Research Foundation of Korea and Korea Health Industry Development Institute.

PPARgamma-mediated ALDH1A3 suppression exerts anti-proliferative effects in lung cancer by inducing lipid peroxidation.

CONTEXT: The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear. OBJECTIVES: To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth. MATERIALS AND METHODS: In silico analysis using microarray dataset was performed to screen the positive correlation between PPARγ and all ALDH isoforms. NUBIscan software and ChIP assay were used to identify the binding sites (BSs) of PPARγ on ALDH1A3 promoter. The expression of ALDH1A3 under thiazolidinedione (TZD) treatment was evaluated by QPCR and Western Blot in HBEC and H1993 cell lines. Upon treatment of TZD, colony formation assay was used to check cell growth inhibition and 4-hydroxy-2-nonenal (4HNE) production as lipid peroxidation marker was determined by Western Blot in PPARγ positive cell H1993 and PPARγ negative cell H1299. RESULTS: Compared to other ALDH isoforms, ALDH1A3 showed the highest positive correlation to PPARγ expression. ALDH1A3 upregulated PPARγ expression while PPARγ activation suppressed ALDH1A3. Among 2 potential screened PPARγ response elements, BS 1 and 2 in the promoter of ALDH1A3 gene, PPARγ bound directly to BS2. Ligand activation of PPARγ suppressed mRNA and protein expression of ALDH1A3. Growth inhibition was observed in H1993 (PPARγ positive cell) treated with PPARγ activator and ALDH inhibitor compared to H1299 (PPARγ negative cell). PPARγ activation increased 4HNE which is known to be suppressed by ALDH1A3. CONCLUSIONS: ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.

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.

PPARγ sumoylation-mediated lipid accumulation in lung cancer.

Metabolic reprogramming as a crucial emerging hallmark of cancer is critical for tumor cells to maintain cellular bioenergetics, biosynthesis and reduction/oxidation (REDOX) balance. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor regulating transcription of diverse gene sets involved in inflammation, metabolism, and suppressing tumor growth. Thiazolidinediones (TZDs), as selective PPARγ ligands, are insulin-sensitizing drugs widely prescribed for type 2 diabetic patients in the clinic. Here, we report that sumoylation of PPARγ couples lipid metabolism to tumor suppressive function of the receptor in lung cancer. We found that ligand activation of PPARγ dramatically induced de novo lipid synthesis as well as fatty acid beta (ß)-oxidation in lung cancer both in vitro and in vivo. More importantly, it turns out that PPARγ regulation of lipid metabolism was dependent on sumoylation of PPARγ. Further biochemical analysis revealed that PPARγ-mediated lipid synthesis depletes nicotinamide adenine dinucleotide phosphate (NADPH), consequently resulting in increased mitochondrial reactive oxygen species (ROS) level that subsequently disrupted REDOX balance in lung cancer. Therefore, liganded PPARγ sumoylation is not only critical for cellular lipid metabolism but also induces oxidative stress that contributes to tumor suppressive function of PPARγ. This study provides an important insight of future translational and clinical research into targeting PPARγ regulation of lipid metabolism in lung cancer patients accompanying type 2 diabetes.

Gallic acid inhibition of Src-Stat3 signaling overcomes acquired resistance to EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer.

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) have clinically benefited to lung cancer patients harboring a subset of activating EGFR mutations. However, even with the remarkable therapeutic response at the initial TKI treatment, most lung cancer patients eventually have relapsed aggressive tumors due to acquired resistance to the TKIs. Here, we report that 3, 4, 5-trihydroxybenzoic acid or gallic acid (GA), a natural polyphenolic compound, shows anti-tumorigenic effects in TKI-resistant non-small cell lung cancer (NSCLC). Using both in vitro growth assay and in vivo xenograft animal model, we demonstrated tumor suppressive effect of GA was more selective for the TKI-resistant cancer compared to the TKI-sensitive one. Mechanistically, GA treatment inhibited Src-Stat3-mediated signaling and decreased the expression of Stat3-regulated tumor promoting genes, subsequently inducing apoptosis and cell cycle arrest in the TKI-resistant lung cancer but not in the TKI-sensitive one. Consistent with the in vitro results, in vivo xenograft experiments showed the TKI-resistant tumor-selective growth inhibition and suppression of Src-Stat3-dependent signaling in the GA-treated tumors isolated from the xenograft model. This finding identified an importance of Src-Stat3 signaling cascade in GA-mediated tumor-suppression activity and, more importantly, provides a novel therapeutic insight of GA for advanced TKI-resistant lung cancer.

Licochalcone F alleviates glucose tolerance and chronic inflammation in diet-induced obese mice through Akt and p38 MAPK.

BACKGROUND & AIMS: Licochalcone (lico) F is a novel synthetic retrochalcone. In this study, we investigated the anti-inflammatory effects of lico F in vitro, and its effects on obesity-induced chronic inflammation, glucose intolerance, and fatty liver in vivo. METHODS: The inhibitory effects of lico F on TNFα-induced inflammation were investigated using NF-κB luciferase reporter assay and RT-PCR. Diet-induced obese mice were treated orally, once per day, with vehicle or lico F (10 mg/kg/day), for 3 weeks, and blood, liver, and adipose tissues were analyzed. RESULTS: Lico F inhibited TNFα-induced NF-κB activation and mRNA expression of TNFα, COX-2, IL-6, IL-1ß, and NOS2. In obese mice, lico F administration significantly alleviated glucose tolerance without changes in body weight gain and food intake. Lico F reduced adipocyte size and macrophage infiltration into white adipose tissue and improved hepatic lesions, by decreasing fat droplets and glycogen deposition. The mRNA expression levels of TNFα, MCP-1, and CD68 in white adipose tissue also decreased markedly. Moreover, lico F enhanced Akt signaling, but reduced p38 MAPK signaling in white adipose tissue. CONCLUSIONS: Lico F had anti-inflammatory effects and showed beneficial effects on glucose metabolism, which could be partially caused by activation of the Akt signal pathway and obesity-induced chronic inflammation, probably by downregulating p38 signal pathway. Moreover, lico F could be used as a potential novel therapeutic compound against type 2 diabetes and obesity-induced chronic inflammation without the deleterious effects of body weight gain and fatty liver.

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-rasV12 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 (PPARγ), 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, PPARγ 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 PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner. Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression. This suggests that ligand-mediated sumoylation of PPARγ plays an important role in lung cancer pathogenesis by modulating prostaglandin metabolism.

Insulin priming effect on estradiol-induced breast cancer metabolism and growth.

Diabetes is a risk factor for breast cancer development and is associated with poor prognosis for breast cancer patients. However, the molecular and biochemical mechanisms underlying the association between diabetes and breast cancer have not been fully elucidated. Here, we investigated estradiol response in MCF-7 breast cancer cells with or without chronic exposure to insulin. We found that insulin priming is necessary and specific for estradiol-induced cancer cell growth, and induces anaplerotic shunting of glucose into macromolecule biosynthesis in the estradiol treated cells. Treatment with ERK or Akt specific inhibitors, U0126 or LY294002, respectively, suppressed estradiol-induced growth. Interestingly, molecular analysis revealed that estradiol treatment markedly increases expression of cyclin A and B, and decreases p21 and p27 in the insulin-primed cells. In addition, estradiol treatment activated metabolic genes in pentose phosphate (PPP) and serine biosynthesis pathways in the insulin-primed cells while insulin priming decreased metabolic gene expression associated with glucose catabolism in the breast cancer cells. Finally, we found that anti-diabetic drug metformin and AMPK ligand AICAR, but not thiazolidinediones (TZDs), specifically suppress the estradiol-induced cellular growth in the insulin-primed cells. These findings suggest that estrogen receptor (ER) activation under chronic hyperinsulinemic condition increases breast cancer growth through the modulation of cell cycle and apoptotic factors and nutrient metabolism, and further provide a mechanistic evidence for the clinical benefit of metformin use for ER-positive breast cancer patients with diabetes.

Gallic acid regulates body weight and glucose homeostasis through AMPK activation.

Gallic acid [3,4,5-trihydroxybenzoic acid (GA)], a natural phytochemical, is known to have a variety of cellular functions including beneficial effects on metabolic syndromes. However, the molecular mechanism by which GA exerts its beneficial effects is not known. Here we report that GA plays its role through the activation of AMP-activated protein kinase (AMPK) and by regulating mitochondrial function via the activation of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Sirtuin 1 (Sirt1) knockdown significantly blunted GA's effect on PGC1α activation and downstream genes, suggesting a critical role of the AMPK/Sirt1/PGC1α pathway in GA's action. Moreover, diet-induced obese mice treated with GA showed significantly improved glucose and insulin homeostasis. In addition, the administration of GA protected diet-induced body weight gain without a change in food intake. Biochemical analyses revealed a marked activation of AMPK in the liver, muscle, and interscapular brown adipose tissue of the GA-treated mice. Moreover, uncoupling protein 1 together with other genes related to energy expenditure was significantly elevated in the interscapular brown adipose tissue. Taken together, these results indicate that GA plays its beneficial metabolic roles by activating the AMPK/Sirt1/PGC1α pathway and by changing the interscapular brown adipose tissue genes related to thermogenesis. Our study points out that targeting the activation of the AMPK/Sirt1/PGC1α pathway by GA or its derivatives might be a potential therapeutic intervention for insulin resistance in metabolic diseases.

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer.

Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.

Expression of adiponectin receptor 1 is indicative of favorable prognosis in non-small cell lung carcinoma.

Lung cancer is a major cause of cancer-related death worldwide. It is believed that obesity-related malignancies such as breast, endometrial, colorectal, and kidney carcinomas have lower plasma level and/or tissue expression of adiponectin receptors. However, the association between adiponectin receptors and lung cancer, a non obesity-related malignancy, is still unknown. We evaluated the tissue expression of adiponectin receptor (AdipoR) 1 and AdipoR2 in 83 cases of non-small cell lung carcinoma (NSCLC) and matched non-neoplastic lung tissues by immunohistochemistry and real-time polymerase chain reaction (PCR). Clinicopathological data, including smoking history, smoker's bronchiolitis, emphysema, lymph node metastasis, and T-stage were collected and evaluated. Expression of immunohistochemically stained AdipoR1 and AdipoR2 was observed in all samples of non-neoplastic lung tissues. Both receptors showed higher mRNA expression in non-neoplastic than neoplastic tissues (p < 0.05). In NSCLC tissues, AdipoR1 immunohistochemical expression was not observed in most of patients with squamous cell carcinoma and current smoking history (31/42, p = 0.04 and 25/29, p = 0.003, respectively). Additionally, AdipoR1 mRNA expression was significantly lower in patients with lymph node metastasis (p = 0.05). Meanwhile, AdipoR2 immunohistochemical stain expression was inversely correlated with T-stage (p = 0.05) and AdipoR2 mRNA expression was significantly lower in patients with smoker's bronchiolitis (p = 0.01) and emphysema (p = 0.03). Patients with expression of AdipoR1 had longer overall survival. AdipoR2 expression was not correlated with patients' survival. In conclusion, we suggest that expression of AdipoR1 is indicative of favorable prognosis and may be used as prognostic marker in NSCLC.

Research resource: Diagnostic and therapeutic potential of nuclear receptor expression in lung cancer.

Lung cancer is the leading cause of cancer-related death. Despite a number of studies that have provided prognostic biomarkers for lung cancer, a paucity of reliable markers and therapeutic targets exist to diagnose and treat this aggressive disease. In this study we investigated the potential of nuclear receptors (NRs), many of which are well-established drug targets, as therapeutic markers in lung cancer. Using quantitative real-time PCR, we analyzed the expression of the 48 members of the NR superfamily in a human panel of 55 normal and lung cancer cell lines. Unsupervised cluster analysis of the NR expression profile segregated normal from tumor cell lines and grouped lung cancers according to type (i.e. small vs. non-small cell lung cancers). Moreover, we found that the NR signature was 79% accurate in diagnosing lung cancer incidence in smokers (n = 129). Finally, the evaluation of a subset of NRs (androgen receptor, estrogen receptor, vitamin D receptor, and peroxisome proliferator-activated receptor-γ) demonstrated the therapeutic potential of using NR expression to predict ligand-dependent growth responses in individual lung cancer cells. Preclinical evaluation of one of these receptors (peroxisome proliferator activated receptor-γ) in mouse xenografts confirmed that ligand-dependent inhibitory growth responses in lung cancer can be predicted based on a tumor's receptor expression status. Taken together, this study establishes NRs as theragnostic markers for predicting lung cancer incidence and further strengthens their potential as therapeutic targets for individualized treatment.

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

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

Nuclear receptor regulation of stemness and stem cell differentiation.

Stem cells include a diverse number of toti-, pluri-, and multi-potent cells that play important roles in cellular genesis and differentiation, tissue development, and organogenesis. Genetic regulation involving various transcription factors results in the self-renewal and differentiation properties of stem cells. The nuclear receptor (NR) superfamily is composed of 48 ligand-activated transcription factors involved in diverse physiological functions such as metabolism, development, and reproduction. Increasing evidence shows that certain NRs function in regulating stemness or differentiation of embryonic stem (ES) cells and tissue-specific adult stem cells. Here, we review the role of the NR superfamily in various aspects of stem cell biology, including their regulation of stemness, forward- and trans-differentiation events; reprogramming of terminally differentiated cells; and interspecies differences. These studies provide insights into the therapeutic potential of the NR superfamily in stem cell therapy and in treating stem cell-associated diseases (e.g., cancer stem cell).

Suppression of beta-catenin signaling by liver X receptor ligands.

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and play an important role in the regulation of lipid metabolism. We investigated the potential effects of LXRs on pathways of colon carcinogenesis and found that LXR activation suppresses the transactivation activity of beta-catenin, a key molecule in Wnt signaling. LXRalpha and LXRbeta inhibited beta-catenin transactivation of T cell factor-mediated transcription in a ligand-dependent manner. LXR activation suppressed an oncogenic beta-catenin, which has phosphorylation site mutations, and did not change beta-catenin protein expression in cells. In contrast, beta-catenin enhanced LXR transactivation activity. Nuclear LXRs and beta-catenin were coimmunoprecipitated in colon cancer HCT116 cells, and in vitro experiments showed that LXRs bind directly to the Armadillo repeat region of beta-catenin in a ligand-independent manner. LXR ligand decreased mRNA expression of beta-catenin targets, MYC, MMP7 and BMP4, and recruited LXRs to MYC and MMP7 promoters. Transfection of a dominant negative LXR to HCT116 cells and experiments using LXR-null cells showed the involvement of cellular LXRs in beta-catenin suppression and proliferation inhibition. The results show lipid-sensing receptor LXRs regulate the beta-catenin activity and cellular proliferation.