Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells.
ABSTRACT: Glioblastoma (GBM) is the most malignant and lethal brain tumor harboring glioma stem cells (GSCs) that promote tumor propagation and therapeutic resistance. GSCs preferentially express several critical cell surface molecules that regulate the pro-survival signaling for maintaining the stem cell-like phenotype. Tetraspanin CD9 has recently been reported as a GSC biomarker that is relevant to the GSC maintenance. However, the underlying molecular mechanisms of CD9 in maintaining GSC property remain elusive. Herein, we report that CD9 stabilizes the IL-6 receptor glycoprotein 130 (gp130) by preventing its ubiquitin-dependent lysosomal degradation to facilitate the STAT3 activation in GSCs. CD9 is preferentially expressed in GSCs of human GBM tumors. Mass spectrometry analysis identified gp130 as an interacting protein of CD9 in GSCs, which was confirmed by immunoprecipitation and immunofluorescent analyses. Disrupting CD9 or gp130 by shRNA significantly inhibited the self-renewal and promoted the differentiation of GSCs. Moreover, CD9 disruption markedly reduced gp130 protein levels and STAT3 activating phosphorylation in GSCs. CD9 stabilized gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote the BMX-STAT3 signaling in GSCs. Importantly, targeting CD9 potently inhibited GSC tumor growth in vivo, while ectopic expression of the constitutively activated STAT3 (STAT3-C) restored the tumor growth impaired by CD9 disruption. Collectively, we uncovered a critical regulatory mechanism mediated by tetraspanin CD9 to maintain the stem cell-like property and tumorigenic potential of GSCs.
Project description:An important factor correlated with poor survival in glioblastoma (GBM) is the aberrant and persistent activation of STAT3, a critical transcription factor that regulates multiple genes with key roles in cell survival, proliferation, resistance to chemotherapy, and stem cell maintenance. The Interleukin-6 (IL6)-STAT3 signaling axis has been studied extensively in inflammation and cancer. However, it is not completely understood how high levels of activated STAT3 are sustained in tumors. Previously, we identified a novel mechanism of biphasic activation of STAT3 in response to gp130-linked cytokines, including IL6, in which activation of STAT3 is prolonged by circumventing the negative regulatory mechanisms induced by its initial activationTo target prolonged STAT3 activation, we used the small molecule inhibitor bazedoxifene (BZA), which blocks formation of the IL6 receptor-gp130 complex. Glioma stem-like cells (GSCs) are more tumorigenic and more resistant to therapy. STAT3 is a key driver of the expression of stem cell transcription factors, making it a therapeutically important target in GBM. We show that treating GSCs with BZA decreases their self-renewal capacity and the expression of GSC markers in vitro. Additionally, BZA crosses the blood-brain barrier and confers a survival advantage in an orthotopic syngeneic mouse model of GBM. Although IL6-STAT3 signaling is important for GSC survival, a therapeutic agent that inhibits this pathway without toxicity has yet to be identified. Our findings reveal a mechanism of sustained STAT3 signaling in GBM and reveal its role in GSC maintenance, and we identify BZA as a novel candidate for treating GBM.
Project description:Glioblastoma (GBM) stem cells (GSCs) represent tumor-propagating cells with stem-like characteristics (stemness) that contribute disproportionately to GBM drug resistance and tumor recurrence. Understanding the mechanisms supporting GSC stemness is important for developing therapeutic strategies for targeting GSC-dependent oncogenic mechanisms. Using GBM-derived neurospheres, we identified the cell surface tetraspanin family member CD151 as a novel regulator of glioma cell stemness, GSC self-renewal capacity, migration, and tumor growth. CD151 was found to be overexpressed in GBM tumors and GBM neurospheres enriched in GSCs. Silencing CD151 inhibited neurosphere forming capacity, neurosphere cell proliferation, and migration and attenuated the expression of markers and transcriptional drivers of the GSC phenotype. Conversely, forced CD151 expression promoted neurosphere self-renewal, cell migration, and expression of stemness-associated transcription factors. CD151 was found to complex with integrins ?3, ?6, and ?1 in neurosphere cells, and blocking CD151 interactions with integrins ?3 and ?6 inhibited AKT phosphorylation, a downstream effector of integrin signaling, and impaired sphere formation and neurosphere cell migration. Additionally, targeting CD151 in vivo inhibited the growth of GBM neurosphere-derived xenografts. These findings identify CD151 and its interactions with integrins ?3 and ?6 as potential therapeutic targets for inhibiting stemness-driving mechanisms and stem cell populations in GBM.
Project description:Malignant glioblastoma (GBM) is a highly aggressive brain tumor with a dismal prognosis and limited therapeutic options. Genomic profiling of GBM samples has identified four molecular subtypes (Proneural, Neural, Classical and Mesenchymal), which may arise from different glioblastoma stem-like cell (GSC) populations. We previously showed that adherent cultures of GSCs grown on laminin-coated plates (Ad-GSCs) and spheroid cultures of GSCs (Sp-GSCs) had high expression of stem cell markers (CD133, Sox2 and Nestin), but low expression of differentiation markers (?III-tubulin and glial fibrillary acid protein). In the present study, we characterized GBM tumors produced by subcutaneous and intracranial injection of Ad-GSCs and Sp-GSCs isolated from a patient-derived xenoline. Although they formed tumors with identical histological features, gene expression analysis revealed that xenografts of Sp-GSCs had a Classical molecular subtype similar to that of bulk tumor cells. In contrast xenografts of Ad-GSCs expressed a Mesenchymal gene signature. Adherent GSC-derived xenografts had high STAT3 and ANGPTL4 expression, and enrichment for stem cell markers, transcriptional networks and pro-angiogenic markers characteristic of the Mesenchymal subtype. Examination of clinical samples from GBM patients showed that STAT3 expression was directly correlated with ANGPTL4 expression, and that increased expression of these genes correlated with poor patient survival and performance. A pharmacological STAT3 inhibitor abrogated STAT3 binding to the ANGPTL4 promoter and exhibited anticancer activity in vivo. Therefore, Ad-GSCs and Sp-GSCs produced histologically identical tumors with different gene expression patterns, and a STAT3/ANGPTL4 pathway is identified in glioblastoma that may serve as a target for therapeutic intervention.
Project description:Malignant glioblastoma (GBM) is a highly aggressive brain tumor with a dismal prognosis and limited therapeutic options. Genomic profiling of GBM samples in the TCGA database has identified four molecular subtypes (Proneural, Neural, Classical and Mesenchymal), which may arise from different glioblastoma stem-like cell (GSC) populations. In the present study, we identify two GSC populations that produce GBM tumors by subcutaneous and intracranial injection with identical histological features. Gene expression analysis revealed that xenografts of GSCs grown as spheroid cultures had a Classical molecular subtype similar to that of bulk tumor cells. In contrast xenografts of GSCs grown as adherent cultures on laminin-coated plates expressed a Mesenchymal gene signature. Adherent GSC-derived xenografts had high STAT3 and ANGPTL4 expression as well as enrichment for stem cell markers, transcriptional networks and pro-angiogenic markers characteristic of the Mesenchymal subtype. Examination of clinical samples from GBM patients showed that STAT3 expression was directly correlated with ANGPTL4 expression, and that increased expression of these genes correlated with poor patient survival and performance. A pharmacological STAT3 inhibitor abrogated STAT3 binding to the ANGPTL4 promoter and exhibited anticancer activity in vivo. Taken together, we identified two distinct GSC populations that produce histologically identical tumors but with very different gene expression patterns, and a STAT3/ ANGPTL4 pathway in glioblastoma that may serve as a target for therapeutic intervention. 2 samples of each variable were analyzed. Cells were cultured under normal adherent conditon (Bulk tumor cells), non-adherent plates with stem cell medium (Sp-GSC) or laminin-coated plates with stem cell medium (Ad-GSC). Xenografts were generated in NSG mice by subcutaneous inoculation.
Project description:Glioblastoma (GBM) is the most malignant form of primary brain tumor, and GBM stem-like cells (GSCs) contribute to the rapid growth, therapeutic resistance, and clinical recurrence of these fatal tumors. STAT3 signaling supports the maintenance and proliferation of GSCs, yet regulatory mechanisms are not completely understood. Here, we report that tri-partite motif-containing protein 8 (TRIM8) activates STAT3 signaling to maintain stemness and self-renewing capabilities of GSCs. TRIM8 (also known as 'glioblastoma-expressed ring finger protein') is expressed equally in GBM and normal brain tissues, despite its hemizygous deletion in the large majority of GBMs, and its expression is highly correlated with stem cell markers. Experimental knockdown of TRIM8 reduced GSC self-renewal and expression of SOX2, NESTIN, and p-STAT3, and promoted glial differentiation. Overexpression of TRIM8 led to higher expression of p-STAT3, c-MYC, SOX2, NESTIN, and CD133, and enhanced GSC self-renewal. We found that TRIM8 activates STAT3 by suppressing the expression of PIAS3, an inhibitor of STAT3, most likely through E3-mediated ubiquitination and proteasomal degradation. Interestingly, we also found that STAT3 activation upregulates TRIM8, providing a mechanism for normalized TRIM8 expression in the setting of hemizygous gene deletion. These data demonstrate that bidirectional TRIM8-STAT3 signaling regulates stemness in GSC.
Project description:Glioblastoma multiforme (GBM), a lethal brain tumor developing in the white matter of the adult brain, contains a small population of GBM stem cells (GSCs), which potentially cause chemotherapeutic resistance and tumor recurrence. However, the mechanisms underlying the pathogenesis and maintenance of GSCs remain largely unknown. A recent study reported that incorporation of ribosomes and ribosomal proteins into somatic cells promoted lineage trans-differentiation toward multipotency. This study aimed to investigate the mechanism underlying stemness acquisition in GBM cells by focusing on 40S ribosomal protein S6 (RPS6). RPS6 was significantly upregulated in high-grade glioma and localized at perivascular, perinecrotic, and border niches in GBM tissues. siRNA-mediated RPS6 knock-down significantly suppressed the characteristics of GSCs, including their tumorsphere potential and GSC marker expression; STAT3 was downregulated in GBM cells. RPS6 overexpression enhanced the tumorsphere potential of GSCs and these effects were attenuated by STAT3 inhibitor (AG490). Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data herein indicated that RPS6 was predominant in GSC niches, concurrent with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were upregulated in GSC-predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemoradioresistance and GBM recurrence.
Project description:Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6R alpha) and glycoprotein 130 (gp130). Targeting IL6R alpha or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6R alpha or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.
Project description:Glioblastoma (GBM) is the most lethal primary brain tumor and is highly resistant to current treatments. GBM harbors glioma stem cells (GSCs) that not only initiate and maintain malignant growth but also promote therapeutic resistance including radioresistance. Thus, targeting GSCs is critical for overcoming the resistance to improve GBM treatment. Because the bone marrow and X-linked (BMX) nonreceptor tyrosine kinase is preferentially up-regulated in GSCs relative to nonstem tumor cells and the BMX-mediated activation of the signal transducer and activator of transcription 3 (STAT3) is required for maintaining GSC self-renewal and tumorigenic potential, pharmacological inhibition of BMX may suppress GBM growth and reduce therapeutic resistance. We demonstrate that BMX inhibition by ibrutinib potently disrupts GSCs, suppresses GBM malignant growth, and effectively combines with radiotherapy. Ibrutinib markedly disrupts the BMX-mediated STAT3 activation in GSCs but shows minimal effect on neural progenitor cells (NPCs) lacking BMX expression. Mechanistically, BMX bypasses the suppressor of cytokine signaling 3 (SOCS3)-mediated inhibition of Janus kinase 2 (JAK2), whereas NPCs dampen the JAK2-mediated STAT3 activation via the negative regulation by SOCS3, providing a molecular basis for targeting BMX by ibrutinib to specifically eliminate GSCs while preserving NPCs. Our preclinical data suggest that repurposing ibrutinib for targeting GSCs could effectively control GBM tumor growth both as monotherapy and as adjuvant with conventional therapies.
Project description:Glioblastoma (GBM) stem cells (GSC) are a subpopulation of tumor cells that display stem-like characteristics (stemness) and play unique roles in tumor propagation, therapeutic resistance, and tumor recurrence. Therapeutic targets in GSCs are a focus of increasing interest to improve GBM therapy. Here we report that the hyaluronan-mediated motility receptor (HMMR) is highly expressed in GBM tumors, where it supports the self-renewal and tumorigenic potential of GSCs. HMMR silencing impairs GSC self-renewal and inhibits the expression of GSC markers and regulators. Furthermore, HMMR silencing suppresses GSC-derived tumor growth and extends the survival of mice bearing GSC xenografts. Conversely, HMMR overexpression promotes GSC self-renewal and intracranial tumor propagation. In human GBM tumor specimens, HMMR expression is correlated positively with the expression of stemness-associated markers and regulators. Our findings identify HMMR as a candidate therapeutic target to GSCs as a GBM treatment strategy.
Project description:Glioblastoma (GBM) represents the most common and lethal primary malignant brain tumor. The standard treatment for glioblastoma patients involves surgical resection with concomitant radio and chemotherapy. Despite today's clinical protocol, the prognosis for patients remains very poor with a median survival of 15 months. Tumor resistance and recurrence is strongly correlated with a subpopulation of highly radioresistant and invasive cells termed Glioblastoma Stem Cells (GSCs). The transcription factor STAT3 has been found to be constitutively activated in different tumors including GBM and enhanced tumor radioresistance. In this study, we assessed radiosensitization of GSC lines isolated from patients by inhibition of STAT3 activation using Stattic or WP1066. We showed that inhibitor treatment before cell irradiation decreased the surviving fraction of GSCs suggesting that STAT3 inhibition could potentiate radiation effects. Finally, we investigated STAT3 activation status on 61 GBM clinical samples and found a preferential phosphorylation of STAT3 on Serine727 (pS727). Moreover, we found that pS727 was associated with a significant lower overall patient survival and progression-free survival but not pY705. Taken together, our results suggest that pS727-STAT3 could be a potential prognostic marker and could constitute a therapeutic target to sensitize highly radioresistant GSCs.