Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the ...tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src-MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations.
Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival.
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Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a discouraging prognosis. Its aggressive and highly infiltrative nature renders the current standard ...treatment of maximal surgical resection, radiation, and chemotherapy relatively ineffective. Identifying the signaling pathways that regulate GBM migration/invasion and resistance is required to develop more effective therapeutic regimens to treat GBM. Expression of TROY, an orphan receptor of the TNF receptor superfamily, increases with glial tumor grade, inversely correlates with patient overall survival, stimulates GBM cell invasion in vitro and in vivo, and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in a preclinical intracranial xenograft model. Here, we have identified for the first time that TROY interacts with JAK1. Increased TROY expression increases JAK1 phosphorylation. In addition, increased TROY expression promotes STAT3 phosphorylation and STAT3 transcriptional activity that is dependent upon JAK1. TROY-mediated activation of STAT3 is independent of its ability to stimulate activity of NF-κB. Inhibition of JAK1 activity by ruxolitinib or knockdown of JAK1 expression by siRNA significantly inhibits TROY-induced STAT3 activation, GBM cell migration, and decreases resistance to temozolomide. Taken together, our data indicate that the TROY signaling complex may represent a potential therapeutic target with the distinctive capacity to exert effects on multiple pathways mediating GBM cell invasion and resistance.
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with few effective treatments. EGFR alterations, including expression of the truncated variant EGFRvIII, are among the ...most frequent genomic changes in these tumors. EGFRvIII is known to preferentially signal through STAT5 for oncogenic activation in GBM, yet targeting EGFRvIII has yielded limited clinical success to date. In this study, we employed patient-derived xenograft (PDX) models expressing EGFRvIII to determine the key points of therapeutic vulnerability within the EGFRvIII-STAT5 signaling axis in GBM. Our findings reveal that exogenous expression of paralogs STAT5A and STAT5B augments cell proliferation and that inhibition of STAT5 phosphorylation in vivo improves overall survival in combination with temozolomide (TMZ). STAT5 phosphorylation is independent of JAK1 and JAK2 signaling, instead requiring Src family kinase (SFK) activity. Saracatinib, an SFK inhibitor, attenuates phosphorylation of STAT5 and preferentially sensitizes EGFRvIII+ GBM cells to undergo apoptotic cell death relative to wild-type EGFR. Constitutively active STAT5A or STAT5B mitigates saracatinib sensitivity in EGFRvIII+ cells. In vivo, saracatinib treatment decreased survival in mice bearing EGFR WT tumors compared to the control, yet in EGFRvIII+ tumors, treatment with saracatinib in combination with TMZ preferentially improves survival.
Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger ...transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the "Go or Grow" hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the "Go or Grow" hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-κB may underlie the shift of glioma cells from a "growing-to-going" phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection ...impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6 months and declines to 8 months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. Knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer’s disease and vascular dementia where it has demonstrated blood–brain permeability and minimal adverse side effects. Here we showed that PPF decreased GBM cell expression of TROY, inhibited glioma cell invasion, and sensitized GBM cells to TMZ. Mechanistically, PPF decreased glioma cell invasion by modulating TROY expression and downstream signaling, including AKT, NF-κB, and Rac1 activation. Thus, PPF may provide a pharmacologic approach to target TROY, inhibit cell invasion, and reduce therapeutic resistance in GBM.
Abstract
Glioblastoma (GBM) is the most common and deadly malignancy of the central nervous system in adults with a median survival of about 15 months after diagnosis. Its aggressive and highly ...infiltrative nature renders the current standard treatment of maximal surgical resection, radiation, and chemotherapy relatively ineffective. Identifying the molecular events that regulate GBM migration/invasion is required to develop more effective therapeutic regimens to treat GBM. Expression of TROY, an orphan receptor of the TNFR superfamily, increases with increasing glial tumor grade, inversely correlates with patient survival, stimulates GBM cell invasion in vitro and in vivo, and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in a preclinical intracranial xenograft model. Thus, the TROY signaling pathway represents an attractive therapeutic target. We previously reported that increased TROY expression strongly activates NF-kB. We have identified for the first time that TROY forms a unique signaling complex with JAK1. Interaction with TROY stimulates phosphorylation of JAK1 and significantly increases the activation of STAT3. TROY specifically associates with JAK1 as it does not associate with other JAK family members. Co-immunoprecipitation analysis demonstrated that TROY interacts with JAK1 through its cytoplasmic domain as a TROY variant without its extracellular domain associates with JAK1 while a TROY variant without a cytoplasmic domain fails to bind JAK1. Luciferase reporter assay confirmed that full length TROY or a variant lacking the extracellular domain are able to induce STAT3 activation, but not a TROY variant lacking the cytoplasmic domain. Pharmacological inhibition of JAK1 by the FDA approved inhibitor ruxolitinib or knockdown of JAK1 by siRNAs significantly inhibited TROY-induced STAT3 activation and GBM cell migration. Together, our data indicate that the TROY-JAK1 complex represents an unappreciated therapeutic target to inhibit glioma invasion and decrease therapeutic resistance.
Abstract
Glial tumors progress to malignant grades by heightened proliferation and aggressive invasion of the surrounding normal brain. Understanding the genetic and biochemical processes that foster ...these behaviors is likely to reveal specific and effective targets for therapeutic intervention. We have identified TROY / TNFRSF19 as a novel transmembrane receptor that is overexpressed in glioblastoma multiforme. TROY is an orphan member of the tumor necrosis factor receptor (TNFR) superfamily that lacks a cytoplasmic death domain. Our previous data demonstrates that the level of TROY mRNA expression directly correlates with increasing glial tumor grade and inversely with overall patient survival. In addition, TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive invasion and migration. Here, we show that TROY over-expression enhances glioma cell survival against chemotherapy-induced cell death. TROY expressing glioma cells also displayed an increased resistance to temozolomide (TMZ) treatment. Conversely, siRNA mediated-depletion of TROY expression enhanced TMZ-induced cell death. To investigate the mechanism of TROY-induced cell survival, we surveyed a panel of known cancer signaling pathways by immunoblot analysis of cell lysates from untransfected glioma cells and lysates from glioma cells overexpressing TROY. Of the signaling pathways surveyed, we observed an increased activation of the Akt and NFκB pathways in TROY overexpressing cells relative to the untransfected cells. Inhibition of the NFκB pathway by expression of the super-repressor IκBα mutant abrogated TROY-induced cell survival to TMZ treatment. Similarly, inhibition of the Akt pathway by treatment of cells with the pharmacological inhibitor, LY294002, increased TMZ-induced cell death in TROY expressing cells. Together, these data indicate that TROY-stimulated glioma cell migration and invasion increases resistance to chemotherapy-induced cell death in glioma. Understanding the function of TROY in glioma biology may lead to the development of effective therapies against invasive gliomas.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2357. doi:10.1158/1538-7445.AM2011-2357
Abstract
Glioblastoma (GBM) is among the most genetically heterogeneous, treatment resistant, and lethal of all human cancers. A significant hurdle to effective treatment of GBM is the aggressive ...invasion of tumor cells into surrounding healthy brain tissue that invariably leads to tumor recurrence, brain injury, and patient death. These invasive cells preclude complete surgical resection and exhibit marked resistance to chemotherapeutics. As invasion is a universal property of GBM, studies focused on the invasive cell population and on the development of therapies that target them are greatly needed in order to significantly improve the survival of GBM patients. We have previously showed that SGEF (ARHGEF26), a RhoG-specific guanine exchange factor is overexpressed in high-grade brain tumors and correlates with poor patient survival. Here we report that SGEF is critical for promoting GBM invasion and survival by modulation of the DNA repair mechanism. Upon TMZ treatment, SGEF accumulated in the nucleus and mediated BRCA1 binding to γH2AX, and knockdown of SGEF expression in GBM cells impaired the phosphorylation of BRCA1 and CHK1. In addition, GBM cells with stable knockdown of SGEF expression showed enhanced susceptibility to TMZ induced cell death. Re-expression of SGEF in these cells rescued BRCA1 phosphorylation and glioma cell resistance to TMZ. Thus, our data showed an important role for SGEF in mediating GBM cell survival.
Abstract
Glioblastoma (GBM), the most common primary brain tumor in adults, comprises all WHO Grade IV gliomas of astrocytic origin. Despite extensive characterization of the molecular and genetic ...features of GBM, there remains a paucity of viable treatment options, and this disease confers a dismal 5% 5-year survival rate. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. EGFR and EGFRvIII transduce oncogenic signals via cytoplasmic mediators, including the JAK/STAT signal transduction pathway. The oncogenic role of STAT3 has been well-studied in CNS malignancies; STAT3 has been found to induce the expression of target genes involved in the proliferative and invasive phenotypes of GBM. However, little is known about the mechanism and role of STAT5 activation in GBM progression. Microenvironmental cues and EGFRvIII signaling, lost in adherent cell culture, are required for STAT5 activation, making STAT5 difficult to study by conventional cell culture methods. Our recent data from patient-derived xenograft (PDX) cultures demonstrates that activation of STAT5 is important in GBM cell invasion and survival. Further, STAT5 activation is present predominantly at the invasive periphery of the tumor. In this study, we seek to characterize the mechanism and role of STAT5 activation downstream of EGFRvIII in GBM. RNA sequencing of PDX tumors expressing stable shRNA knockdown of STAT3 or STAT5 indicate both redundant and distinct transcriptional changes induced by STAT3 and STAT5 in GBM. We also find that while STAT3 activation is dependent upon JAK1/2, STAT5 activation is JAK-independent downstream of EGFRvIII in GBM. Depletion of JAK1/2 expression by siRNA oligonucleotides and pharmacologic inhibition of JAK1/2 suppressed STAT3 activation but not STAT5. In fact, targeting JAK2 with kinase inhibitor WP1066 suppressed STAT3 activation, but increased activation of STAT5 in GBM cells expressing EGFR/EGFRvIII. Inhibition of Src family kinases (SFK), a family of upstream signaling effectors of the JAK/STAT pathway, attenuates both STAT3 and STAT5 activation. These results suggest that activation of STAT5 may provide a therapeutic escape mechanism to JAK-STAT3 inhibition in GBM, necessitating dual STAT3 and STAT5 inhibition to successfully inhibit the distinct oncogenic signals transmitted by STAT3 and STAT5 to promote GBM cell survival and resistance.
Citation Format: Mylan Blomquist, Serdar Tuncali, Christopher Sereduk, Jean Kloss, Joseph Loftus, Nhan Tran. JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 101.
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