David Akhavan

EGFR Signaling Through an Akt-SREBP-1–Dependent, Rapamycin-Resistant Pathway Sensitizes Glioblastomas to Antilipogenic Therapy

Inhibitors of fatty acid signaling promote apoptosis in glioblastoma cells with highly active EGFR signaling. Inhibiting Lipid Metabolism to Combat Glioblastoma Glioblastoma, the most common form of brain cancer, is frequently lethal. Glioblastoma is often associated with increased signaling through the epidermal growth factor receptor (EGFR); however, therapeutic efforts focused on inhibiting EGFR signaling have been disappointing. Guo et al. analyzed tumor tissue removed from glioblastoma patients before and during treatment with the EGFR inhibitor lapatinib and found that EGFR signaling activated sterol regulatory element–binding protein 1 (SREBP-1), a key regulator of lipid metabolism, and increased the cellular concentrations of fatty acids. Intriguingly, inhibiting fatty acid synthesis promoted apoptosis in glioblastoma cells with substantial EGFR signaling both in vitro and when transplanted into immunodeficient mice, but not in glioblastoma cells with little EGFR signaling. Thus, inhibition of fatty acid synthesis may represent a new avenue toward treating glioblastomas driven by EGFR signaling. Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element–binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin’s poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.

Oncogenic EGFR signaling activates an mTORC2-NF-κB pathway that promotes chemotherapy resistance

UNLABELLED Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-κB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-κB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.

mTOR Complex 2 Controls Glycolytic Metabolism in Glioblastoma through FoxO Acetylation and Upregulation of c-Myc

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

mTOR Signaling in Glioblastoma: Lessons Learned from Bench to Bedside

Phosphatidyl-inositol-3 kinases (PI3Ks) constitute a family of intracellular lipid kinases that are frequently hyperactivated in glioblastoma. The PI3K complex links growth factor signaling with cellular proliferation, differentiation, metabolism, and survival. Mammalian target of rapamycin (mTOR) acts both as a downstream effector and upstream regulator of PI3K, thus highlighting its importance in glioblastoma. This review highlights laboratory and clinical evidence of mTORs role in glioblastoma. Mechanisms of escape from mTOR inhibition are also discussed, as well as future clinical strategies of mTOR inhibition.

An Essential Requirement for the SCAP/SREBP Signaling Axis to Protect Cancer Cells from Lipotoxicity

The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.

EGFR Mutation-Induced Alternative Splicing of Max Contributes to Growth of Glycolytic Tumors in Brain Cancer

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

The mTOR Kinase Inhibitors, CC214-1 and CC214-2, Preferentially Block the Growth of EGFRvIII-Activated Glioblastomas

Purpose: mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it. Experimental Design: We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology—DNA Encoded Antibody Libraries—was used to identify mechanisms of resistance. Results: Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2–induced cell death. Conclusions: These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it. Clin Cancer Res; 19(20); 5722–32. ©2013 AACR.

Activation of Src induces mitochondrial localisation of de2-7EGFR (EGFRvIII) in glioma cells: implications for glucose metabolism

A common mutation of the epidermal growth factor receptor in glioma is the de2-7EGFR (or EGFRvIII). Glioma cells expressing de2-7EGFR contain an intracellular pool of receptor with high levels of mannose glycosylation, which is consistent with delayed processing. We now show that this delay occurs in the Golgi complex. Low levels of de2-7EGFR were also seen within the mitochondria. Src activation dramatically increased the amount of mitochondrial de2-7EGFR, whereas its pharmacological inhibition caused a significant reduction. Because de2-7EGFR is phosphorylated by Src at Y845, we generated glioma cells expressing a Y845F-modified de2-7EGFR. The de2-7EGFR(845F) mutant failed to show mitochondrial localisation, even when co-expressed with constitutive active Src. Low levels of glucose enhanced mitochondrial localisation of de2-7EGFR, and glioma cells expressing the receptor showed increased survival and proliferation under these conditions. Consistent with this, de2-7EGFR reduced glucose dependency by stimulating mitochondrial oxidative metabolism. Thus, the mitochondrial localisation of de2-7EGFR contributes to its tumorigenicity and might help to explain its resistance to some EGFR-targeted therapeutics.

Pilot Study on “Pericytic Mimicry” and Potential Embryonic/Stem Cell Properties of Angiotropic Melanoma Cells Interacting with the Abluminal Vascular Surface

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the “pericytic mimicry” by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

Abstract 1923: The mTOR kinase inhibitor, CC214, preferentially blocks the growth of EGFRvIII-activated glioblastomas

mTOR pathway hyper activation is a common feature in many cancers, including in nearly 90% of Glioblastomas. mTOR exists in two multiprotein complexes, which differ in regulation, function, and response to the allosteric mTOR inhibitor rapamycin. The failure of glioblastomas to clinically respond to rapamycin appears to be mediated both by inability of rapamycin to inhibit mTORC2 signaling, and by failure to fully suppress p4E-BP1, a critical effector of mTORC1. The new generation of mTOR kinase inhibitors (mTORki) has emerged as a new class of targeted cancer therapies capable of blocking mTOR more potently than rapalogs (such as Rapamycin). Here we demonstrate that CC214-1 and CC214-2, mTOR kinase inhibitors, have potent effects on the growth of glioblastoma in vitro and in vivo. Across a panel of GBM cell lines, CC214-1 was significantly more effective at suppressing 4E-BP1 phosphorylation and protein translation, and mTORC2 signaling than rapamycin, resulting in significantly more growth inhibition at equal levels of suppression of S6 phosphorylation. EGFRvIII, the constitutively active EGFR mutant frequently detected in GBMs strongly activates mTORC1 and mTORC2 signaling, potently sensitizing tumors to CC214-1. In contrast, PTEN expression suppressed mTOR signaling and reduced the response to CC214-1. In vivo CC214-2 significantly inhibited mTORC1 and mTORC2 signaling, significantly suppressing tumor growth by 50%. CC214-1 also potently activated autophagy. Addition of chloroquine suppressed autophagy resulting in significant CC214-1-dependent apoptotic cell death. These results demonstrate the potential activity of mTOR kinase inhibitors in GBM, and identify EGFRvIII expression and PTEN loss as molecular determinants of response. These data also suggest that mTOR kinase inhibitors, alone or in combination with autophagy inhibiting agents, are likely to have significant anti-tumor efficacy in GBM patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1923. doi:1538-7445.AM2012-1923