Estrogen-related receptor ? activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.
ABSTRACT: Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor ? (ERR-?) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3' end of the pre-mRNA leads to the production of 3 validated ERR-? protein products: ERR-? short form (ERR-?sf), ERR-?2, and ERR-? exon 10 deleted. Our prior studies have shown the ERR-?2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR-?sf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the proapoptotic ERR-?2 isoform in GBM. We show that the ERR-?2 isoform is located not only in the nucleus but also in the cytoplasm. ERR-?2 suppresses GBM cell migration and interacts with the actin nucleation-promoting factor cortactin, and an ERR-? agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases in combination with an ERR-? agonist shifts isoform expression in favor of ERR-?2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors.-Tiek, D. M., Khatib, S. A., Trepicchio, C. J., Heckler, M. M., Divekar, S. D., Sarkaria, J. N., Glasgow, E., Riggins, R. B. Estrogen-related receptor ? activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.
Project description:Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line data sets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined polypyrimidine tract-binding protein 1 (PTBP1) levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each data set. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5' splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.
Project description:Orphan receptors comprise nearly half of all members of the nuclear receptor superfamily. Despite having broad structural similarities to the classical estrogen receptors, estrogen-related receptors (ERRs) have their own unique DNA response elements and functions. In this study, we focus on 2 ERR? splice variants, short form ERR? (ERR?sf) and ERR?2, and identify their differing roles in cell cycle regulation. Using DY131 (a synthetic agonist of ERR?), splice-variant selective shRNA, and exogenous ERR?sf and ERR?2 cDNAs, we demonstrate the role of ERR?sf in mediating the G1 checkpoint through p21. We also show ERR?sf is required for DY131-induced cellular senescence. A key novel finding of this study is that ERR?2 can mediate a G2/M arrest in response to DY131. In the absence of ERR?2, the DY131-induced G2/M arrest is reversed, and this is accompanied by p21 induction and a G1 arrest. This study illustrates novel functions for ERR? splice variants and provides evidence for splice variant interaction.
Project description:Circular RNAs (circRNAs) have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM) pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5): it was significantly downregulated in GBM biopsies as compared to normal brain tissues (p-value < 0.00001, student's t-test), contrary to its linear isoform counterpart that did not show any differential expression (p-value = 0.694, student's t-test). Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma's histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs), specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1), a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE). Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3) RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD) substrate. Interestingly, SRSF3 is known to interplay with two other splicing factors, polypyrimidine tract binding protein 1 (PTBP1) and polypyrimidine tract binding protein 2 (PTBP2), that positively regulate glioma cells migration. Collectively, our data show circSMARCA5 as a promising druggable tumor suppressor in GBM and suggest that it may exert its function by tethering the RBP SRSF1.
Project description:Breast cancer remains a leading cause of cancer-related death in women, and triple negative breast cancer (TNBC) lacks clinically actionable therapeutic targets. Death in mitosis is a tumor suppressive mechanism that occurs in cancer cells experiencing a defective M phase. The orphan estrogen-related receptor beta (ERR?) is a key reprogramming factor in murine embryonic and induced pluripotent stem cells. In primates, ERR? is alternatively spliced to produce several receptor isoforms. In cellular models of glioblastoma, short form (ERR?sf) and beta2 (ERR?2) splice variants differentially regulate cell cycle progression in response to the synthetic agonist DY131, with ERR?2 driving arrest in G2/M.The goals of the present study are to determine the cellular function(s) of ligand-activated ERR? splice variants in breast cancer and evaluate the potential of DY131 to serve as an antimitotic agent, particularly in TNBC. DY131 inhibits growth in a diverse panel of breast cancer cell lines, causing cell death that involves the p38 stress kinase pathway and a bimodal cell cycle arrest. ERR?2 facilitates the block in G2/M, and DY131 delays progression from prophase to anaphase. Finally, ERR?2 localizes to centrosomes and DY131 causes mitotic spindle defects. Targeting ERR?2 may therefore be a promising therapeutic strategy in breast cancer.
Project description:In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-?/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d'Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness.
Project description:Sphingosine-1-phosphate is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce sphingosine-1-phosphate, is up-regulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and nonestablished human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of extracellular signal-regulated kinase 1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the c-Jun-NH(2)-kinase pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced the tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization, and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease.
Project description:The N6-methyladenosine (m6A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m6A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m6A modification peaks were enriched at metabolic pathway-related transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m6A modification levels of serine- and arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m6A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression. SIGNIFICANCE: These findings establish the oncogenic role of m6A writer METTL3 in glioblastoma stem cells.
Project description:Background:Oncogenic activation of phosphatidylinositol-3 kinase (PI3K) signaling plays a pivotal role in the development of glioblastoma (GBM). However, pharmacological inhibition of PI3K has so far not been therapeutically successful due to adaptive resistance through a rapid rewiring of cancer cell signaling. Here we identified that WEE1 is activated after transient exposure to PI3K inhibition and confers resistance to PI3K inhibition in GBM. Methods:Patient-derived glioma-initiating cells and established GBM cells were treated with PI3K inhibitor or WEE1 inhibitor alone or in combination, and cell proliferation was evaluated by CellTiter-Blue assay. Cell apoptosis was analyzed by TUNEL, annexin V staining, and blotting of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Both subcutaneous xenograft and orthotropic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis; the tumor growth was monitored by bioluminescence imaging, and tumor tissue was analyzed by immunohistochemistry to validate signaling changes. Results:PI3K inhibition activates WEE1 kinase, which in turn phosphorylates cell division control protein 2 homolog (Cdc2) at Tyr15 and inhibits Cdc2 activity, leading to G2/M arrest in a p53-independent manner. WEE1 inhibition abrogated the G2/M arrest and propelled cells to prematurely enter into mitosis and consequent cell death through mitotic catastrophe and apoptosis. Additionally, combination treatment significantly suppressed tumor growth in a subcutaneous model but not in an intracranial model due to limited blood-brain barrier penetration. Conclusions:Our findings highlight WEE1 as an adaptive resistant gene activated after PI3K inhibition, and inhibition of WEE1 potentiated the effectiveness of PI3K targeted inhibition, suggesting that a combinational inhibition of WEE1 and PI3K might allow successful targeted therapy in GBM.
Project description:Grade IV astrocytoma, also known as glioblastoma multiforme (GBM), is the most common and aggressive intracranial glial tumor. GBM is associated with very poor survival and effective treatments have remained elusive so far. Mounting evidence indicates that CD164 contributes to stemness and tumorigenesis in normal cells and is overexpressed in various tumor types, including glioblastoma. Using tissue microarray immunohistochemistry, we show that there is a significant correlation between CD164 expression and glioma type and grade. Depletion of CD164 expression in human glioblastoma cells with siRNA reduced proliferation, migration, and invasiveness. In parallel, immunoblotting showed that downregulation of CD164 expression decreased Akt activation and modified the expression of autophagy markers by upregulating Beclin-1 and LC3B and downregulating p62. These effects were mimicked by inhibition of Akt with MK2206, which suggests that CD164 induces autophagy via Akt/Beclin-1 signaling. We propose that CD164 may serve as a GBM molecular marker and a potential target in therapeutic strategies aimed to improve outcomes for this devastating brain tumor.
Project description:HOTAIR is a negative prognostic factor and is overexpressed in multiple human cancers including glioblastoma multiform (GBM). Survival analysis of Chinese Glioma Genome Atlas (CGGA) patient data indicated that high HOTAIR expression was associated with poor outcome in GBM patients. NLK (Nemo-like kinase), a negative regulator of the ?-catenin pathway, was negatively correlated with HOTAIR expression. When the ?-catenin pathway was inhibited, GBM cells became susceptible to cell cycle arrest and inhibition of invasion. Introduction of the HOTAIR 5' domain in human glioma-derived astrocytoma induced ?-catenin. An intracranial animal model was used to confirm that HOTAIR depletion inhibited GBM cell migration/invasion. In the orthotopic model, HOTAIR was required for GBM formation in vivo. In summary, HOTAIR is a potential therapeutic target in GBM.