Khatri Latha

Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ΔeGFR

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal, we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.

Nuclear EGFRvIII-STAT5b complex contributes to glioblastoma cell survival by direct activation of the Bcl-XL promoter

Aberrant EGFR signaling strongly promotes glioma malignancy and treatment resistance. The most prevalent mutation, ΔEGFR/EGFRvIII, is an in‐frame deletion of the extracellular domain, which occurs in more than 25% of glioblastomas and enhances growth and survival of tumor cells. Paradoxically, the signaling of the potent oncogene ΔEGFR is of low intensity, raising the question of whether it exhibits preferential signaling to key downstream targets. We have observed levels of phosphorylation of STAT5 at position Y699 in cells expressing ΔEGFR that are similar or higher than in cells that overexpress EGFR and are acutely stimulated with EGF, prompting us to investigate the role of STAT5 activation in glioblastoma. Here, we show that in human glioblastoma samples, pSTAT5 levels correlated positively with EGFR expression and were associated with reduced survival. Interestingly, the activation of STAT5b downstream of ΔEGFR was dependent on SFKs, while the signal from acutely EGF‐stimulated EGFR to STAT5b involved other kinases. Phosphorylated STAT5b and ΔEGFR associated in the nucleus, bound DNA and were found on promoters known to be regulated by STAT5 including that of the Aurora A gene. ΔEGFR cooperated with STAT5b to regulate the Bcl‐XL promoter and knockdown of STAT5b suppressed anchorage independent growth, reduced the levels of Bcl‐XL and sensitized glioblastoma cells to cisplatin. Together these results delineate a novel association of nuclear ΔEGFR with STAT5b, which promotes oncogenesis and treatment resistance in glioblastoma by direct regulation of anti‐apoptotic gene, Bcl‐XL.

Combined action of the dinuclear platinum compound BBR3610 with the PI3-K inhibitor PX-866 in glioblastoma†

Polynuclear platinum compounds are more effective at killing glioblastoma cells than cisplatin, work by a different mechanism, and typically do not induce high levels of apoptosis at early time points after exposure. Here, we tested the hypothesis that combining BBR3610, the most potent polynuclear platinum, with a phosphoinositide‐3‐kinase (PI3K) inhibitor would promote apoptosis and enhance the impact on glioblastoma cells. The PI3K pathway is commonly activated in glioblastoma and promotes tumor cell survival, suggesting that its inhibition would make cells more sensitive to cytotoxic agents. We chose PX‐866 as a PI3K inhibitor as it is a clinically promising agent being evaluated for brain tumor therapy. Combining BBR3610 and PX‐866 resulted in synergistic killing of cultured glioma cells and an extension of survival in an orthotopic xenograft animal model. Both agents alone induced autophagy, and this appeared to be saturated, because when they were combined no additional autophagy was observed. However, the combination of PX‐866 and BBR3610 did induce statistically significant increases in the level of apoptosis, associated with a reduction in pAkt and pBad, as well as inhibition of transwell migration. We conclude that combining polynuclear platinums with PI3K inhibitors has translational potential and alters the cellular response to include early apoptosis.

The Duality of Fgl2 - Secreted Immune Checkpoint Regulator Versus Membrane-Associated Procoagulant: Therapeutic Potential and Implications

Fibrinogen-like protein 2 (Fgl2), a member of the fibrinogen family, can be expressed as a membrane-associated protein with coagulation activity or in a secreted form possessing unique immune suppressive functions. The biological importance of Fgl2 is evident within viral-induced fibrin depositing inflammatory diseases and malignancies and provides a compelling rationale for Fgl2 expression to not only be considered as a disease biomarker but also as a therapeutic target. This article will provide a comprehensive review of the currently known biological properties of Fgl2 and clarifies future scientific directives.

Analysis of the inhibitors of apoptosis identifies BIRC3 as a facilitator of malignant progression in glioma

Gliomas, the most common primary brain tumor in humans, include a spectrum of disease. High-grade gliomas (HGG), such as glioblastoma, may arise from low-grade gliomas (LGG) that have a more indolent course. The process of malignant transformation (MT) of LGG to HGG is poorly understood but likely involves the activation of signaling programs that suppress apoptosis. We previously showed that Survivin (BIRC5) plays a role in malignant progression of glioma. Here, we investigated the role of the remaining members of the Inhibitors of Apoptosis (IAP) family on promoting MT in glioma. Utilizing expression data from the cancer genome atlas (TCGA), we identified BIRC3 as a key facilitator of MT from LGG to HGG. TCGA HGGs with high expression of BIRC 3 demonstrated a survival disadvantage and expression levels of BIRC3 were also significantly higher in TCGA HGG compared to TCGA LGG cases. We validated our findings from TCGA by using matched human specimens to show that BIRC expression is increased in HGG compared to their precursor LGG lesions. Using a unique murine model of glioma, we show that overexpression of BIRC3 promotes higher grade glioma and significantly reduces tumor-free survival in mice.

Suppression of RAF/MEK or PI3K synergizes cytotoxicity of receptor tyrosine kinase inhibitors in glioma tumor-initiating cells

BackgroundThe majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these signaling pathways for their survival and proliferation. However, recent studies suggest that monotherapies or inappropriate combination therapies using the molecular targeted drugs have limited efficacy possibly because of tumor heterogeneities, signaling redundancy and crosstalk in intracellular signaling network, indicating necessity of rationale and methods for efficient personalized combination treatments. Here, we evaluated the growth of colonies obtained from glioma tumor-initiating cells (GICs) derived from glioma sphere culture (GSC) in agarose and examined the effects of combination treatments on GICs using targeted drugs that affect the signaling pathways to which most glioma cells are addicted.MethodsHuman GICs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinases or transcription factors. The colony number and volume were analyzed using a colony counter, and Chou-Talalay combination indices were evaluated. Autophagy and apoptosis were also analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting.ResultsIncreases of colony number and volume in agarose correlated with the Gompertz function. GICs showed diverse drug sensitivity, but inhibitions of RTK and RAF/MEK or PI3K by combinations such as EGFR inhibitor and MEK inhibitor, sorafenib and U0126, erlotinib and BKM120, and EGFR inhibitor and sorafenib showed synergy in different subtypes of GICs. Combination of erlotinib and sorafenib, synergistic in GSC11, induced apoptosis and autophagic cell death associated with suppressed Akt and ERK signaling pathways and decreased nuclear PKM2 and β-catenin in vitro, and tended to improve survival of nude mice bearing GSC11 brain tumor. Reverse phase protein array analysis of the synergistic treatment indicated involvement of not only MEK and PI3K signaling pathways but also others associated with glucose metabolism, fatty acid metabolism, gene transcription, histone methylation, iron transport, stress response, cell cycle, and apoptosis.ConclusionInhibiting RTK and RAF/MEK or PI3K could induce synergistic cytotoxicity but personalization is necessary. Examining colonies in agarose initiated by GICs from each patient may be useful for drug sensitivity testing in personalized cancer therapy.

Survivin transcript variant 2 drives angiogenesis and malignant progression in proneural gliomas

BACKGROUND The influence of survivin isoforms on outcome in glioblastoma is poorly understood. We analyzed the dominant anti-apoptotic transcript variants of survivin using expression data and modeled them in vivo to determine their impact on glioma formation and progression. METHODS Using data from low- and high-grade glioma knowledge bases, we expressed the anti-apoptotic isoforms of survivin (transcript variants 1 and 2) in vivo using the RCAS/Ntv-a model of murine glioma. RESULTS In low-grade gliomas, survivin RNA expression was increased in 22 of 167 (13.2%) of cases and was associated with shortened survival (P = .005). Survivin RNA was preferentially expressed in proneural (PN) relative to mesenchymal high-grade gliomas (P < .0001). In proneural gliomas, survivin was expressed in 94 of 141 (67%) of cases and was associated with shorter disease-free survival (P = .04). In a platelet-derived growth factor subunit B-dependent murine model of PN glioma, ectopic expression of variant 1 yielded tumors in 28 of 30 (93%) of mice, of which 25% were high-grade tumors, whereas ectopic expression of variant 2 yielded tumors in 27 of 28 (96%), of which 81% were high-grade tumors (P < .0001). Microvascular proliferation was significantly more prominent (P < .0001), and tumor-free survival was shorter in mice with variant 2 than variant 1-derived tumors (P = .01). CONCLUSIONS Survivin expression in low-grade gliomas is associated with poor survival and is preferentially expressed in PN gliomas. Compared with variant 1, variant 2 was associated with poorer survival and promoted malignant progression, angiogenesis, and shorter tumor-free survival in the PN murine model. Inhibiting survivin transcript variant 2, rather than variant 1 (the common isoform), may be an effective treatment strategy for glioma.

Access to the Nucleus and Functional Association with c-Myc Is Required for the Full Oncogenic Potential of ΔEGFR/EGFRvIII

Background: Expression of ΔEGFR, a mutant of EGFR in gliomas, correlates with poor prognosis. Results: Access to the nucleus is required for full oncogenicity of ΔEGFR, and nuclear ΔEGFR regulates transcription of target genes via c-Myc. Conclusion: Functional association of nuclear ΔEGFR with c-Myc is necessary for ΔEGFR-induced oncogenicity. Significance: These data show a novel activity of ΔEGFR and offer new opportunities for therapeutic intervention. ΔEGFR is a potent glioblastoma oncogene which has been studied primarily as a plasma membrane kinase. Using intracranial xenograft studies in mice, we show that blocking ΔEGFR access to the nucleus attenuates its tumorigenicity and, conversely, that promoting nuclear accumulation enhances this, providing the first in vivo evidence that the nuclear actions of ΔEGFR contribute strongly to its oncogenic function. Nuclear actions of ΔEGFR include regulation of gene expression by participation in chromatin-bound complexes, and genome-wide mapping of these sequences by chromatin immunoprecipitation and massively parallel sequencing identified 2294 peaks. Bioinformatic analysis showed enrichment of the E-box motif in the dataset, and c-Myc and ΔEGFR were corecruited to the promoters of and transcriptionally activated a subset of nuclear ΔEGFR chromatin targets. Knockdown of c-Myc decreased the expression of these targets and diminished ΔEGFR-stimulated anchorage-independent colony formation. We conclude that transcriptional regulation of target genes by association with gene regulatory chromatin in cooperation with c-Myc by nuclear ΔEGFR makes a unique contribution to its oncogenicity and propose that this venue provides new targets for therapeutic intervention.

Signal transducer and activator of transcription 5b drives malignant progression in a PDGFB‐dependent proneural glioma model by suppressing apoptosis

Signal transducer and activator of transcription 5b (STAT5b) is likely the relevant STAT5 isoform with respect to the process of malignant progression in gliomas. STAT5b is a latent cytoplasmic protein involved in cell signaling through the modulation of growth factors, apoptosis, and angiogenesis. Previous in vitro studies have shown increased STAT5b expression in glioblastomas relative to low‐grade tumors and normal brain. We recently demonstrated that phosphorylated STAT5b associates with delta epidermal growth factor receptor in the nucleus and subsequently binds the promoters of downstream effector molecules, including aurora kinase A. Analysis of TCGA dataset reveals that STAT5b is predominantly expressed in proneural (PN) gliomas relative to mesenchymal and neural gliomas. Here, we modeled ectopic expression of STAT5b in vivo using a platelet‐derived growth factor subunit B (PDGFB)‐dependent mouse model of PN glioma to determine its effect on tumor formation and progression. We showed that coexpression of STAT5b and PDGFB in mice yielded a significantly higher rate of high‐grade gliomas than PDGFB expression alone. We also observed shorter survival in the combined expression set. High‐grade tumors from the STAT5b + PDGFB expression set were found to have a lower rate of apoptosis than those from PDGFB alone. Furthermore, we showed that increased expression of STAT5b + PDGFB led to increased expression of downstream STAT5b targets, including Bcl‐xL, cyclin D1 and aurora kinase A in high‐grade tumors when compared to tumors derived from PDGFB alone. Our findings show that STAT5b promotes the malignant transformation of gliomas, particularly the PN subtype, and is a potential therapeutic target.

Nuclear Signaling of EGFR and EGFRvIII in Glioblastoma

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as therapeutic targets across a broad spectrum of cancers. The epidermal growth factor receptor (EGFR) was the first receptor tyrosine kinase to be discovered and remains the most investigated. Most of the mechanistic principles of receptor tyrosine kinases were first established with the EGFR family as a model. EGFR is a single pass transmembrane receptor with two extracellular, cysteine-rich regions involved in ligand binding, and intervening region important for receptor dimerization, an intracellular tyrosine kinase domain, and a number of intracellular sites for autophosphorylation, phosphorylation by other kinases, and docking of signaling components. Three additional EGFR family members have been identified, human epidermal growth factor receptor (erbB or HER) 2, 3 and 4. In many cell types, including those of epithelial and mesenchymal lineages, receptors of the HER family transduce signals from the cell surface to the intracellular domain, regulating normal cell growth, lineage determination, repair and functional differentiation. A range of growth factors serves as ligands for these receptors, although none have been identified for the HER2 receptor. Ligands for HER1/EGFR include epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), and heregulins serve as ligands for both HER3 and HER4. Binding of a ligand to a HER family member leads to receptor homodimerization, or heterodimerization with another HER receptor, bringing about receptor phosphorylation. Ligand binding to HER1, HER3 or HER4 induces rapid receptor dimerization, with a marked preference for HER2 as a partner(Graus-Porta et al., 1997). Moreover, HER2-containing heterodimers generate intracellular signals that are significantly more potent than signals emanating from other HER combinations. Of the receptor dimers, HER3 homodimers cannot initiate signal transduction.The differing signaling characteristics of the HER family members are thought to be due to their different ligand-binding affinities and the type of phosphorylated homoor heterodimer formed, and the resulting intracellular signaling events. Two of the key pathways involved in this cascade are the ras-raf-mitogen-activated protein kinase (MAPK) pathway, which affects DNA synthesis and cell proliferation, and the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which plays a role in cell metabolism and survival (Yarden and Sliwkowski, 2001; Bange et al., 2001).