Esophageal squamous cell carcinoma (ESCC) is the second common cancer in Henan province and is well-known for aggressiveness and dismal prognosis. Adjuvant therapies, chemotherapy, radiotherapy and endoscopic treatment have not improved survival rates in patients with late stage esophageal carcinoma. All-trans retinoic acid (ATRA) is the active ingredient of Vitamin A and affects a wide spectrum of biological processes including development, growth, neural function, immune function, reproduction, and vision. It is one of the most potent therapeutic agents used for treating cancers, especially lung adenocarcinomas. ATRA inhibits metastatic potential and angiogenesis in several tumor models. We investigated the effects of ATRA on the expression of angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and receptor Tie-2 in EC1 cells in vitro. We also assessed the growth and migration of EC1 cells in vitro. ATRA treatment caused 29.5% and 40.3% reduction of the growth of EC1 cells after 24 hours and 48 hours, relative to the control. ATRA plus fluorouracil treatment reduced the viability more strongly than either drug alone, indicating an additive effect. Moreover, ATRA decreased EC1 migration by 87%. Furthermore, ATRA treatment led to a marked decrease of the transcript levels of Ang-1, Ang-2, Tie-2, VEGF, and VEGF receptors, as assessed by real-time RT-PCR. Importantly, the protein levels of Ang-1, Ang-2 and Tie-2 were reduced by ATRA treatment. In vivo, we found ATRA treatment suppressed the tumor growth and improved the cachexia of mice. Importantly, ATRA treatment decreased the expression of CD31, Ang-1, Ang-2 and Tie-2 in subcutaneous tumors of EC1 cells. Collectively, our findings demonstrate that ATRA exhibits a dose- and temporal-dependent effect on the metastatic behavior, suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and the progression of xenograft tumors of EC1 cells.

Tuberous sclerosis complex (TSC) is a genetic disorder that cause tumors to form in many organs. These lesions may lead to epilepsy, autism, developmental delay, renal, and pulmonary failure. Loss of function mutations in TSC1 and TSC2 genes by aberrant activation of the mechanistic target of rapamycin (mTORC1) signaling pathway are the known causes of TSC. Therefore, targeting mTORC1 becomes a most available therapeutic strategy for TSC. Although mTORC1 inhibitor rapamycin and Rapalogs have demonstrated exciting results in the recent clinical trials, however, tumors rebound and upon the discontinuation of the mTORC1 inhibition. Thus, understanding the underlying molecular mechanisms responsible for rapamycin-induced cell survival becomes an urgent need. Identification of additional molecular targets and development more effective remission-inducing therapeutic strategies are necessary for TSC patients.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Cancer stem cells (CSCs) are defined as a rare subpopulation of undifferentiated cells with biological characteristics that include the capacity for self-renewal, differentiation into various lineages, and tumor initiation. To explore the mechanism of CSCs in esophageal squamous cell carcinoma (ESCC), we focused on Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), a target gene of the Wnt signaling pathway, which has been identified as a marker of intestinal stem cells and shown to be overexpressed in several human malignancies. Lgr5 expression was significantly correlated with lymph node metastasis, increased depth of invasion, increased tumor size, advanced differentiation, higher AJCC stage and poorer survival. Silencing of Lgr5 expression in the ESCC cell line KYSE450 by small interfering RNA (siRNA) strongly inhibited cell proliferation, migration and invasion ability, the expression of CSCs-related genes and Wnt/β-catenin signaling. In addition, Lgr5 was highly expressed in ESCC spheroid body cells, which were identified by high expression of CSCs-related genes, and high tumorigenicity in vivo. Taken together, these results demonstrate that Lgr5 activation of Wnt/β-catenin signaling is a potential mechanism to promote the progression of ESCC and ESCC stem cell renewal, and Lgr5 may be used as a molecular target for the development of treatments for ESCC.

Tuberous sclerosis complex (TSC) is characterized by multisystem, low-grade neoplasia involving the lung, kidneys, brain, and heart. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting almost exclusively women. TSC and LAM are both caused by mutations in TSC1 and TSC2 that result in mTORC1 hyperactivation. Here, we report that single-cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid biosynthesis pathway. Accordingly, the expression of acid ceramidase (ASAH1) and dihydroceramide desaturase (DEGS1), key enzymes controlling sphingolipid and ceramide metabolism, was significantly increased in TSC2-null cells. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids, and suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) resulted in markedly decreased TSC2-null cell viability. In vivo, 17a significantly decreased the growth of TSC2-null cell-derived mouse xenografts and short-term lung colonization by TSC2-null cells. Combined rapamycin and 17a treatment synergistically inhibited renal cystadenoma growth in Tsc2+/- mice, consistent with increased ASAH1 expression and activity being rapamycin insensitive. Collectively, the present study identifies rapamycin-insensitive ASAH1 upregulation in TSC2-null cells and tumors and provides evidence that targeting aberrant sphingolipid biosynthesis pathways has potential therapeutic value in mechanistic target of rapamycin complex 1-hyperactive neoplasms, including TSC and LAM.

Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle-like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC(-)) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient-derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.

Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms.

Germline loss-of-function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt-Hogg-Dube (BHD) gene, has been linked to altered cell-cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho-S6, a marker of mTORC1 activation, highlighting the cell type-dependent functions of FLCN. Cell-cell adhesion forces were significantly increased in FLCN-deficient HBE cells, consistent with prior findings in FLCN-deficient human kidney-derived cells. To determine how these altered cell-cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd(+/-) mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell-cell adhesion in a cell type-dependent manner. FLCN deficiency may impact the physiologic response to inflation-induced mechanical stress, but further investigation is required. We hypothesize that FLCN-dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients.

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/- mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2-/-MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived "mesenchymal" cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease that can arise sporadically or in adults suffering from the tumor syndrome tuberous sclerosis complex (TSC). Microscopic tumors ('LAM nodules') in the lung interstitium arise from lymphatic invasion and metastasis. These consist of smooth muscle-like cells (LAM cells) that exhibit markers of neural crest differentiation and loss of the tumor suppressor protein 'tuberous sclerosis complex-2' (TSC2). Consistent with a neural phenotype, expression of the neuropeptide urotensin-II and its receptor was detected in LAM nodules. We hypothesized that loss of TSC2 sensitizes cells to the oncogenic effects of urotensin-II. TSC2-deficient Eker rat uterine leiomyoma ELT3 cells were stably transfected with empty vector or plasmid for the expression of TSC2. Urotensin-II increased cell viability and proliferation in TSC2-deficient cells, but not in TSC2-reconstituted cells. When exposed to urotensin-II, TSC2-deficient cells exhibited greater migration, anchorage-independent cell growth, and matrix invasion. The effects of urotensin-II on TSC2-deficient cells were blocked by the urotensin receptor antagonist SB657510, and accompanied by activation of Erk mitogen-activated protein kinase and focal adhesion kinase. Urotensin-II-induced proliferation and migration were reproduced in TSC2-deficient human angiomyolipoma cells, but not in those stably expressing TSC2. In a mouse xenograft model, SB657510 blocked the growth of established ELT3 tumors, reduced the number of circulating tumor cells, and attenuated the production of VEGF-D, a clinical biomarker of LAM. Urotensin receptor antagonists may be selective therapeutic agents for the treatment of LAM or other neural crest-derived neoplasms featuring loss of TSC2 or increased expression of the urotensin receptor.

Estrogen-related receptor alpha (ERRα) is an orphan nuclear factor that is a master regulator of cellular energy metabolism. ERRα is overexpressed in a variety of tumors, including ovarian, prostate, colorectal, cervical and breast, and is associated with a more aggressive tumor and a worse outcome. In breast cancer, specifically, high ERRα expression is associated with an increased rate of recurrence and a poor prognosis. Because of the common functions of ERRα and the mTORC1/S6K1 signaling pathway in regulation of cellular metabolism and breast cancer pathogenesis, we focused on investigating the biochemical relationship between ERRα and S6K1. We found that ERRα negatively regulates S6K1 expression by directly binding to its promoter. Downregulation of ERRα expression sensitized ERα-negative breast cancer cells to mTORC1/S6K1 inhibitors. Therefore, our results show that combinatorial inhibition of ERRα and mTORC1/S6K1 may have clinical utility in treatment of triple-negative breast cancer, and warrants further investigation.

Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease that almost exclusively affects women. LAM cells migrate to the lungs, where they cause cystic destruction of lung parenchyma. Mutations in TSC1 or TSC2 lead to the activation of the mammalian target of rapamycin complex-1, a kinase that regulates growth factor-dependent protein translation, cell growth, and metabolism. Insulin-like growth factor binding protein 2 (IGFBP2) binds insulin, IGF1 and IGF2 in circulation, thereby modulating cell survival, migration, and invasion in neoplasms. In this study, we identified that IGFBP2 primarily localized in the nucleus of TSC2-null LAM patient-derived cells in vitro and in vivo. We also showed that nuclear accumulation of IGFBP2 is closely associated with estrogen receptor alpha (ERa) expression. Furthermore, estrogen treatment induced IGFBP2 nuclear translocation in TSC2-null LAM patient-derived cells. Importantly, depletion of IGFBP2 by siRNA reduced cell proliferation, enhanced apoptosis, and decreased migration and invasion of TSC2-null LAM patient-derived cells. More interestingly, depletion of IGFBP2 markedly decreased the phosphorylation of MAPK in LAM patient-derived TSC2-null cells. Collectively, these results suggest that IGFBP2 plays an important role in promoting tumorigenesis, through estrogen and ERalpha signaling pathway. Thus, targeting IGFBP2 may serve as a potential therapeutic strategy for women with LAM and other female gender specific neoplasms.

Schools are salient locations for children with disabilities to accrue physical activity (PA) and to diminish sedentary time (ST). We examined seasonal variation in accelerometer-assessed PA and ST among children with disabilities during the school day in three school settings (physical education (PE) lessons, recess and lunchtime). Children (n = 270) from 13 special schools for those with five disability types (visual impairments, hearing impairments, physical disabilities, intellectual disabilities (ID), and social development problems) participated. Their PA and ST were assessed during three winter and three summer school days using accelerometry. Linear mixed models were performed to determine seasonal variation in the proportion of time they spent in moderate-to-vigorous physical activity (MVPA) and ST in the three settings. On average, the children spent 4.5% (18.6 min) and 4.0% (15.6 min) in MVPA at school during winter and summer days, respectively. They were more physically active during winter (especially during recess and lunchtime), but there were no seasonal differences for ST. Thus, children's year-round engagement in PA needs to be promoted, especially during summer.

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease that predominantly affects women. LAM cells carry TSC1/TSC2 mutations, causing mTORC1 hyperactivation and uncontrolled cell growth. mTORC1 inhibitors stabilize lung function; however, sustained efficacy requires long-term administration, and some patients fail to tolerate or respond to therapy. Although the genetic basis of LAM is known, mechanisms underlying LAM pathogenesis remain elusive. We integrated single-cell RNA sequencing and single-nuclei ATAC-seq of LAM lungs to construct a gene regulatory network controlling the transcriptional program of LAM cells. We identified activation of uterine-specific HOX-PBX transcriptional programs in pulmonary LAMCORE cells as regulators of cell survival depending upon HOXD11-PBX1 dimerization. Accordingly, blockage of HOXD11-PBX1 dimerization by HXR9 suppressed LAM cell survival in vitro and in vivo. PBX1 regulated STAT1/3, increased the expression of antiapoptotic genes, and promoted LAM cell survival in vitro. The HOX-PBX gene network provides promising targets for treatment of LAM/TSC mTORC1-hyperactive cancers.

Our study aims to investigate the impact of miR-224 on cell migration and invasion in colorectal cancer (CRC) as well as its molecular mechanisms. The results showed that miR-224 was significantly upregulated in CRC compared to normal tissues via the TCGA database. Overexpression of miR-224 promoted CRC cell migration and invasion, while inhibition of miR-224 demonstrated the opposite result via transwell assays. In addition, we found that BTRC was a target gene of miR-224 through the miRecords database and dual-luciferase assay, while western blot together with RT-qPCR showed that inhibition of miR-224 led to elevated BTRC expression in protein level but not in mRNA level, and also decreased the expression of β-catenin. In reference to the Human Protein Atlas, BTRC protein expression was higher in normal tissues than in CRC tissues. In conclusion, miR-224 regulates its target BTRC protein expression and its related Wnt/β-catenin pathway. Its impact on cell migration and invasion in CRC cells suggested that miR-224 could be a prospective therapeutic target for early-stage non-metastatic CRC.

Autophagy is a "self-feeding" phenomenon of cells, which is crucial in mammalian development. Long non-coding RNA (lncRNA) is a new regulatory factor for cell autophagy, which can regulate the process of autophagy to affect tumor progression. However, poor attention has been paid to the roles of autophagy-related lncRNAs in breast cancer.

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

In the connectivity map (CMap) approach to drug repositioning and development, transcriptional signature of disease is constructed by differential gene expression analysis between the diseased tissue or cells and the control. The negative correlation between the transcriptional disease signature and the transcriptional signature of the drug, or a bioactive compound, is assumed to indicate its ability to "reverse" the disease process. A major limitation of traditional CMaP analysis is the use of signatures derived from bulk disease tissues. Since the key driver pathways are most likely dysregulated in only a subset of cells, the "averaged" transcriptional signatures resulting from bulk analysis lack the resolution to effectively identify effective therapeutic agents. The use of single-cell RNA-seq (scRNA-seq) transcriptomic assay facilitates construction of disease signatures that are specific to individual cell types, but methods for using scRNA-seq data in the context of CMaP analysis are lacking. Lymphangioleiomyomatosis (LAM) mutations in TSC1 or TSC2 genes result in the activation of the mTOR complex 1 (mTORC1). The mTORC1 inhibitor Sirolimus is the only FDA-approved drug to treat LAM. Novel therapies for LAM are urgently needed as the disease recurs with discontinuation of the treatment and some patients are insensitive to the drug. We developed methods for constructing disease transcriptional signatures and CMaP analysis using scRNA-seq profiling and applied them in the analysis of scRNA-seq data of lung tissue from naïve and sirolimus-treated LAM patients. New methods successfully implicated mTORC1 inhibitors, including Sirolimus, as capable of reverting the LAM transcriptional signatures. The CMaP analysis mimicking standard bulk-tissue approach failed to detect any connection between the LAM signature and mTORC1 signaling. This indicates that the precise signature derived from scRNA-seq data using our methods is the crucial difference between the success and the failure to identify effective therapeutic treatments in CMaP analysis.

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

An up-regulation of COX-2 in malignant gliomas causes excessive synthesis of PGE2 , which is thought to facilitate brain tumour growth and invasion. However, which downstream PGE2 receptor subtype (i.e., EP1 -EP4 ) directly contributes to COX activity-promoted glioma growth remains largely unknown.