EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition.
ABSTRACT: Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII+) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIII+ cells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIII+ tumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIII+ cells. As hypoxic tumor cells greatly contribute to therapy resistance, autophagy inhibition revokes the radioresistant phenotype of EGFRvIII+ tumors in (patient derived) xenograft tumors. In line with these findings, retrospective analysis of glioblastoma patients indicated that chloroquine treatment improves survival of all glioblastoma patients, but patients with EGFRvIII+ glioblastoma benefited most. Our findings disclose the unique autophagy dependency of EGFRvIII+ glioblastoma as a therapeutic opportunity. Chloroquine treatment may therefore be considered as an additional treatment strategy for glioblastoma patients and can reverse the worse prognosis of patients with EGFRvIII+ glioblastoma.
Project description:Hypoxia is negatively associated with glioblastoma patient survival and contributes strongly to tumor resistance. Unfortunately novel anti-angiogenic therapy increases hypoxia and activates survival pathways in tumor cells leading to inevitable tumor relapse. Here we demonstrate that primary glioma cultures and cell lines depend on autophagy to survive severe hypoxia but also at normal oxygen levels. Positive regulators of autophagy are expressed at higher levels in tumor cells and induction in severe hypoxia is more prominent compared to normal brain cells. We demonstrate that autophagy is an essential/critical target for novel treatment in glioblastoma. We show ATG9A is induced by severe hypoxia and could be a novel player of the autophagic response in glioma cells. ATG9A targeting inhibited tumor growth, suggesting an essential role in glioma cell survival in vivo. While autophagy induction was also observed in normal astrocytes, glioma cells displayed a higher sensitivity towards autophagy inhibitors. Importantly, patient-derived cultures exhibited varying sensitivity towards anti-autophagy treatment, where certain cultures were dependent on autophagy already in normoxic conditions. The treatment of tumor bearing mice with the autophagy inhibitor chloroquine significantly increased mice survival, but combination treatment of the agent with bevacizumab did not reveal additive or synergistic effect. The present study demonstrates that inhibition of autophagy using chloroquine as a single agent provides a novel treatment strategy against glioblastoma. It remains to be seen whether autophagy inhibition will improve current standard of care treatment of newly diagnosed glioblastoma patients and whether more specific inhibitors will lead to stronger therapeutic outcome. (Provisional)
Project description:Glioblastoma (GBM) is one of the most lethal brain tumors with a short survival time. EGFR amplification and mutation is the most significant genetic signature in GBM. About half of the GBMs with EGFR amplification express a constitutively autophosphorylated variant of EGFR, known as EGFRvIII. Our in vitro data demonstrated further enhanced EGFRvIII activity and tumor cell invasion in the tumor microenvironment of hypoxia plus extracellular matrix (ECM) vitronectin, in which EGFRvIII and integrin ?3 tended to form complexes. The treatment with ITGB3 siRNA or the integrin antagonist cilengetide preferentially interrupted the EGFRvIII/integrin ?3 complex, effectively reduced tumor cell invasion and activation of downstream signaling effectors. Cilengitide is recently failed in Phase III CENTRIC trial in unselected patients with GBM. However, we found that cilengitide demonstrated efficacious tumor regression via inhibition of tumor growth and angiogenesis in EGFRvIII orthotopic xenografts. Bioinformatics analysis emphasized key roles of integrin ?3, hypoxia and vitronectin and their strong correlations with EGFRvIII expression in malignant glioma patient samples in vivo. In conclusion, we demonstrate that EGFRvIII/integrin ?3 complexes promote GBM progression and metastasis in the environment of hypoxia and vitronectin-enrichment, and cilengitide may serve as a promising therapeutics for EGFRvIII-positive GBMs.
Project description:Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1? (HIF-1?)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.
Project description:Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ? 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ?EGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.
Project description:Tumor hypoxia, acidosis, and excessive reactive oxygen species (ROS) were the main characteristics of the bladder tumor microenvironment (TME), and abnormal TME led to autophagy activation, which facilitated cancer cell proliferation. The therapeutic efficacy of autophagy inhibitors might also be impeded by abnormal TME. To address these issues, we proposed a new strategy that utilized manganese dioxide (MnO2) nanoparticles to optimize the abnormal TME and revitalize autophagy inhibitors, and both oxygenation and autophagy inhibition may sensitize the tumor cells to radiation therapy. Methods: By taking advantage of the strong affinity between negatively charged MnO2 and positively charged chloroquine (CQ), the nanoparticles were fabricated by integrating MnO2 and CQ in human serum albumin (HSA)-based nanoplatform (HSA-MnO2-CQ NPs). Results: HSA-MnO2-CQ NPs NPs efficiently generated O2 and increased pH in vitro after reaction with H+/H2O2 and then released the encapsulated CQ in a H+/H2O2 concentration-dependent manner. The NPs restored the autophagy-inhibiting activity of chloroquine in acidic conditions by increasing its intracellular uptake, and markedly blocked hypoxia-induced autophagic flux. In vivo studies showed the NPs improved pharmacokinetic behavior of chloroquine and effectively accumulated in tumor tissues. The NPs exhibited significantly decreased tumor hypoxia areas and increased tumor pH, and had remarkable autophagy inhibition efficacy on bladder tumors. Finally, a significant anti-tumor effect achieved by the enhanced autophagy inhibition and radiation sensitization. Conclusions: HSA-MnO2-CQ NPs synergistically regulated the abnormal TME and inhibited autophagic flux, and effectively sensitized radiation therapy to treat bladder cancers.
Project description:Malignant brain tumor-glioblastoma is not only difficult to treat but also hard to study and model. One of the reasons for these is their heterogeneity, i.e., individual tumors consisting of cancer cells that are unlike each other. Such diverse cells can thrive due to the simultaneous co-evolution of anatomic niches and adaption into zones with distorted homeostasis of oxygen. It dampens cytotoxic and immune therapies as the response depends on the cellular composition and its adaptation to hypoxia. We explored what transcriptome reposition strategies are used by cells in the different areas of the tumor. We created the hypoxic map by differential expression analysis between hypoxic and cellular features using RNA sequencing data cross-referenced with the tumor's anatomic features (Ivy Glioblastoma Atlas Project). The molecular functions of genes differentially expressed in the hypoxic regions were analyzed by a systematic review of the gene ontology analysis. To put a hypoxic niche signature into a clinical context, we associated the model with patients' survival datasets (The Cancer Genome Atlas). The most unique class of genes in the hypoxic area of the tumor was associated with the process of autophagy. Both hypoxic and cellular anatomic features were enriched in immune response genes whose, along with autophagy cluster genes, had the power to predict glioblastoma patient survival. Our analysis revealed that transcriptome responsive to hypoxia predicted worse patients' outcomes by driving tumor cell adaptation to metabolic stress and immune escape.
Project description:: Amplification of the EGFR gene and its truncation mutant EGFRvIII are hallmarks of glioblastoma. Although coexpression of EGFR and EGFRvIII confers a growth advantage, how EGFR and EGFRvIII influence the tumor microenvironment remains incompletely understood. Here, we show that EGFR and EGFRvIII cooperate to induce macrophage infiltration via upregulation of the chemokine CCL2. EGFRvIII was significantly enriched in glioblastoma patient samples with high CCL2, and knockout of CCL2 in tumors coexpressing EGFR and EGFRvIII led to decreased infiltration of macrophages. KRAS was a critical signaling intermediate for EGFR- and EGFRvIII-induced expression of CCL2. Our results illustrate how EGFR and EGFRvIII direct the microenvironment in glioblastoma. SIGNIFICANCE: Full-length EGFR and truncated EGFRvIII work through KRAS to upregulate the chemokine CCL2 and drive macrophage infiltration in glioblastoma.
Project description:Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM.Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells.We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion.This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.
Project description:No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.
Project description:<h4>Background</h4>Epidermal growth factor receptor variant III (EGFRvIII) is a driver mutation and potential therapeutic target in glioblastoma. Non-invasive in vivo EGFRvIII determination, using clinically acquired multiparametric MRI sequences, could assist in assessing spatial heterogeneity related to EGFRvIII, currently not captured via single-specimen analyses. We hypothesize that integration of subtle, yet distinctive, quantitative imaging/radiomic patterns using machine learning may lead to non-invasively determining molecular characteristics, and particularly the EGFRvIII mutation.<h4>Methods</h4>We integrated diverse imaging features, including the tumor's spatial distribution pattern, via support vector machines, to construct an imaging signature of EGFRvIII. This signature was evaluated in independent discovery (n = 75) and replication (n = 54) cohorts of de novo glioblastoma, and compared with the EGFRvIII status obtained through an assay based on next-generation sequencing.<h4>Results</h4>The cross-validated accuracy of the EGFRvIII signature in classifying the mutation status in individual patients of the independent discovery and replication cohorts was 85.3% (specificity = 86.3%, sensitivity = 83.3%, area under the curve [AUC] = 0.85) and 87% (specificity = 90%, sensitivity = 78.6%, AUC = 0.86), respectively. The signature was consistent with EGFRvIII+ tumors having increased neovascularization and cell density, as well as a distinctive spatial pattern involving relatively more frontal and parietal regions compared with EGFRvIII- tumors.<h4>Conclusions</h4>An imaging signature of EGFRvIII was found, revealing a complex, yet distinct macroscopic glioblastoma phenotype. By non-invasively capturing the tumor in its entirety, the proposed methodology can assist in evaluating the tumor's spatial heterogeneity, hence overcoming common spatial sampling limitations of tissue-based analyses. This signature can preoperatively stratify patients for EGFRvIII-targeted therapies, and potentially monitor dynamic mutational changes during treatment.