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Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)–dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1–dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)–mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1–dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.Significance: This study reveals that GBM cells have devised a mechanism to subvert the normal pathways for feedback inhibition of cholesterol homeostasis via EGFRvIII and PI3K-dependent activation of SREBP-1. We show that an LXR agonist causes IDOL-mediated LDLR degradation and increases expression of the ABCA1 cholesterol efflux transporter, potently promoting GBM cell death in vivo. These results suggest a role for LXR agonists in the treatment of GBM patients. Cancer Discovery; 1(5): 442–56. ©2011 AACR.Read the Commentary on this article by Moschetta, p. 381This article is highlighted in the In This Issue feature, p. 367

PDF file - 212K, Fig. S2. Ligand-dependent PDGFRβ stimulation promotes the proliferation of erlotinib-treated glioblastoma cells.

mp3 file (9.8 MB). In the November edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with Paul S. Mischel about his paper, which identifies mTORC2 as a novel mediator of drug resistance and regulator of NF-κB signaling in glioblastoma.

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PDF file - 199K, Fig. S1. EGFRvIII and wild type EGFR signaling regulate PDGFRβ in vitro.

PDF file - 2561K, Treatment with CC214-1, leads to induction of autophagy in GBM39 cells and genetic inhibition of autophagy sensitizes U87EGFRvIII cells to apoptosis.

PDF file - 5321K, In vitro and in vivo pharmacological inhibition of autophagy sensitizes U87EGFRvIII cells and orthotopic xenografts to CC214-1/CC214-2 mediated apoptosis.

PDF file - 2080K, Tunable expression of EGFRvIII in genetically modified models correlates with autophagy induction upon CC214-1 treatment.

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PDF file - 5300K, CC214-1 synergizes at low doses with rapamycin. Quantification of the blots shown in Fig. 2B. PTEN detection on DEAL captured GBM39 cells upon CC214-1 treatment.

PDF file - 3457K, Time course dependent CC214-1 treatment in a panel of isogenic GBM cell lines, U87EGFRvIII, U251 and LN229.

PDF file - 1554K, Cap-dependent translation is enhanced by EGFRvIII expression and CC214-1 strongly inhibits P-4E-BP1 phosphorylation.

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.

PDF file - 1694K, EGFRvIII expression sensitizes cells to CC214-1 mediated inhibition of proliferation and to autophagy.

PDF File - 155K, The lipogenic program and growth of glioma cells is SCAP sensitive.

PDF File - 90K, Loss of SREBP signaling results in the uncoupling of saturated fatty acid biosynthesis from desaturase activity.

PDF File - 90K, Graphical representation of method used to determine the relative contribution of de novo cholesterol and long chain fatty acids synthesis to the total cellular pool.

PDF File - 92K, Gene set enrichment analysis on the ten most statistically significant down-regulated metabolic pathways in SCAP loss-of-function cells.

PDF File - 158K, Inhibition of lipogenic transcriptional program in various cancer cells.

PDF File - 118K, Legends for Supplementary Figures 1-8.

PDF File - 92K, Cholesterol content is unchanged in SREBP loss-of-function cells.

PDF File - 52K, Cholesterol content is unchanged in SREBP loss-of-function cells.

PDF File - 112K, Expression of mature SREBP1 alters the ratio of saturated to monounsaturated fatty acids.

Purpose There is currently no curative treatment for patients diagnosed with triple-negative breast cancer brain metastases (TNBC-BM). CAR T cells hold potential for curative treatment given they retain the cytolytic activity of a T cell combined with the specificity of an antibody. In this proposal we evaluated the potential of EGFR re-directed CAR T cells as a therapeutic treatment against TNBC cells in vitro and in vivo . Methods We leveraged a TNBC-BM tissue microarray and a large panel of TNBC cell lines and identified elevated epidermal growth factor receptor (EGFR) expression. Next, we designed a second-generation antiEGFR CAR T construct incorporating a clinically relevant mAb806 tumor specific single-chain variable fragment (scFv) and intracellular 4-1BB costimulatory domain and CD3ζ using a lentivirus system and evaluated in vitro and in vivo anti-tumor activity. Results We demonstrate EGFR is enriched in TNBC-BM patient tissue after neurosurgical resection, with six of 13 brain metastases demonstrating both membranous and cytoplasmic EGFR. Eleven of 13 TNBC cell lines have EGFR surface expression ≥85% by flow cytometry. EGFR806 CAR T treated mice effectively eradicated TNBC-BM and enhanced mouse survival (log rank pConclusion Our results demonstrates anti-tumor activity of EGFR CAR T cells against TNBC cells in vitro and in vivo . Given EGFR CAR T cells are currently undergoing clinical trials in primary brain tumor patients without obvious toxicity, our results are immediately actionable against the TNBC-BM patient population.

There is currently no curative treatment for patients diagnosed with triple-negative breast cancer brain metastases (TNBC-BM). CAR T cells hold potential for curative treatment given they retain the cytolytic activity of a T cell combined with the specificity of an antibody. In this proposal we evaluated the potential of EGFR re-directed CAR T cells as a therapeutic treatment against TNBC cells in vitro and in vivo.We leveraged a TNBC-BM tissue microarray and a large panel of TNBC cell lines and identified elevated epidermal growth factor receptor (EGFR) expression. Next, we designed a second-generation anti-EGFR CAR T construct incorporating a clinically relevant mAb806 tumor specific single-chain variable fragment (scFv) and intracellular 4-1BB costimulatory domain and CD3ζ using a lentivirus system and evaluated in vitro and in vivo anti-tumor activity.We demonstrate EGFR is enriched in TNBC-BM patient tissue after neurosurgical resection, with six of 13 brain metastases demonstrating both membranous and cytoplasmic EGFR. Eleven of 13 TNBC cell lines have EGFR surface expression ≥ 85% by flow cytometry. EGFR806 CAR T treated mice effectively eradicated TNBC-BM and enhanced mouse survival (log rank p 0.004).Our results demonstrates anti-tumor activity of EGFR806 CAR T cells against TNBC cells in vitro and in vivo. Given EGFR806 CAR T cells are currently undergoing clinical trials in primary brain tumor patients without obvious toxicity, our results are immediately actionable against the TNBC-BM patient population.

Glioblastoma multiforme (GBM) is the most aggressive primary malignant brain cancer with a median survival of 16-20 months. Maximal safe resection and adjuvant chemotherapy improve survival but there is no cure and hence improved therapies are urgently needed. CAR T cell therapy has been FDA approved for hematologic malignancies but there are some barriers to their application for solid tumors in the clinic such as heterogenous tumor antigen expression. Chimeric Antigen Receptor (CAR) T Cells combine the cytolytic potency of T cells with the tumor specificity of an antibody. The interleukin 13 receptor alpha 2 (IL13Rα2) is an important target of CAR T cell therapy in ongoing GBM clinical trials. However, regulation of IL13Ra2 expression in GBM is unclear. Identifying upstream regulators of IL13Rα2 will not only help delineate IL13Rα2 carcinogenesis but also may help improve CAR T cell therapy. Utilizing knockout and overexpression constructs, we have identified protein kinase C family as an upstream regulator of IL13Rα2 transcription and translation in GBM cell lines. We have also identified a small molecule regulator of IL13Rα2, Ingenol-3-Angelate (I3A), that inhibits U87 tumor cell proliferation. Furthermore, I3A enhances IL13Rα2 re-directed CAR T cell mediated tumor cytolysis in vitro. Importantly, I3A treatment increases interferon gamma secretion by T cells. Taken together, our data implicates PKC isoforms as an upstream regulator of IL13Rα2. I3A treatment may serve as a therapeutic strategy to enhance CAR T mediated killing. Citation Format: Siddharth Subham, John D. Jeppson, Bryan Schatmeyer, Stephen J. Forman, Christine E. Brown, David Akhavan. Protein kinase C is an upstream regulator of IL13Ra2 and small molecular activator enhances CAR T mediated killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3235.

AIMS The basis of this research revolves around the issue of limited expression of targetable tumour-specific antigens, heterogeneity, and mutation, which impede the development and success of CAR T therapy for glioblastoma. The research will identify an off-tumour cell target involved in tumour angiogenesis. The principal aim is to generate an off-tumour targeted CAR T and test their efficacy and safety in cell culture and animal models. METHOD Analyse the prevalence and distribution of the target molecules via immunohistochemistry (IHC). Confirm IHC results via mRNA extraction. Analyse the target molecule’s serum concentrations via the Luminex instrument. Generate the off-tumour targeted CAR T using lentivirus. Assess tumour burden and progression via a bioluminescent imaging system. Assess toxicity in vitro against human pericytes and in vivo by monitoring mouse weight and submitting histologic tissue for sectioning. RESULTS Utilise a scoring system to analyse the staining and correspond this to the serum concentrations to identify a blood-borne treatment response biomarker. If murine toxicity is observed, the CAR T construct will be affinity matured to selectively target molecules expressed at a cancerous level. A tandem bi-specific CAR T cells may also be created to mitigate immune escape and enhance anti-tumour efficacy. CONCLUSION The search for an off-tumour cell target for CAR T therapy in glioblastoma has not yet been completed. The discovery and validation of the novel molecule as a biomarker of treatment response would be a unique finding.

Malignant brain tumors, including glioblastoma, represent some of the most difficult to treat of solid tumors. Nevertheless, recent progress in immunotherapy, across a broad range of tumor types, provides hope that immunological approaches will have the potential to improve outcomes for patients with brain tumors. Chimeric antigen receptors (CAR) T cells, a promising immunotherapeutic modality, utilizes the tumor targeting specificity of any antibody or receptor ligand to redirect the cytolytic potency of T cells. The remarkable clinical response rates of CD19‐targeted CAR T cells and early clinical experiences in glioblastoma demonstrating safety and evidence for disease modifying activity support the potential of further advancements ultimately providing clinical benefit for patients. The brain, however, is an immune specialized organ presenting unique and specific challenges to immune‐based therapies. Remaining barriers to be overcome for achieving effective CAR T cell therapy in the central nervous system (CNS) include tumor antigenic heterogeneity, an immune‐suppressive microenvironment, unique properties of the CNS that limit T cell entry, and risks of immune‐based toxicities in this highly sensitive organ. This review will summarize preclinical and clinical data for CAR T cell immunotherapy in glioblastoma and other malignant brain tumors, including present obstacles to advancement.

e16254 Background: Neo-adjuvant therapy (NAT) and associated pathologic complete response (pCR) rates have correlated with improved survival in resected pancreatic ductal adenocarcinoma (PDAC). In this study, we explored the relationship between pathologic response, peri-operative therapy, and survival, especially the impact of change in adjuvant therapy in patients with no/poor path response to NAT. Methods: Retrospectively reviewed 66 PDAC patients who received NAT ± radiation and underwent resection at KU Cancer Center between 2011-2022. We compared DFS and OS between Path Responders vs Non-Responders based on standard Tumor Regression Scores from pathology reports. A subanalysis was performed in path non-responders based on switch in adjuvant therapy (AT) versus not. Results: Patient characteristics are summarized in the table. Among 66 PDAC patients, 50 (75.8%) achieved a path response (G0-G2), 16 (24.2%) experienced no/poor path response (G3). Of the 50 pts who achieved a path response, 4 (8.0%) had a complete path response (pCR; G0), 5 (10%) marked response (G1), 41 (82%) moderate response (G2). Median DFS (mDFS) was 17.3 months (95% CI: 12.7-22.4) in Path Responders vs 15.9m (95% CI: 9.6-35.8) in Non-Responders [p=0.59]. Median OS (mOS) was 32.9m (95% CI: 23.4-41.5) vs 27.7m (95% CI: 15.2-38.2), respectively [p=0.39). A sub-analysis in the Non-Responders (n=16) based on switch in AT (n=8) vs not (n=3), revealed mDFS 16.4m (95% CI: 9.6-41.8) when AT was switched vs mDFS 11.3m (95% CI: 5.9-16.6) when AT was not switched [p=0.24]; and mOS 30.6m (95% CI: 15.7-60.3) vs 17.2 months (95% CI: 6.7-27.7), respectively [p=0.18]. Conclusions: Our study found no statistical difference in DFS and OS between Pathologic Responders and Non-Responders to neo-adjuvant therapy. However, a sub-analysis within Pathologic Non-Responders revealed a longer DFS and OS after switching adjuvant therapy without reaching statistical significance, likely due to small sample size. Our findings warrant validation in a larger cohort as switch in adjuvant therapy could potentially change the treatment landscape for Pathologic Non-Responders.[Table: see text]

Background: M5A is a humanized monoclonal antibody (mAb) directed against carcinoembryonic antigen (CEA) The purpose of this first in human phase I dose-escalation trial was to characterize the toxicities and determine the maximum tolerated dose (MTD) of yttrium-90 ((90)Y)-DOTA-M5A as a single agent and in combination with gemcitabine (gem). Methods: Patients with advanced metastatic CEA-producing malignancies who had progressed on standard therapies were first administered indium-111 ((111)In)-DOTA-M5A. If tumor targeting was observed, the patient then received the therapy dose of (90)Y-DOTA-M5A. Serial scans, blood sampling, and 24 h urine collections were then performed to estimate radiation doses to organs and total body. Assays for human antihuman antibody (HAHA) responses were performed out to 6 months. Results: Of the 18 patients who received (111)In-DOTA-M5A, 16 received (90)Y-DOTA-M5A therapy; 1 patient at 14 mCi/m(2) with gem (150 mg/m(2) days 1and 3), 3 patients at 12 mCi/m(2) with gem, 6 patients at 12 mCi/m(2) without gem, and 6 at 10 mCi/m(2) without gem. Prolonged cytopenias resulted in discontinuation of dose escalation with gemcitabine. A single agent MTD of 10 mCi/m(2) was established based on dose-limiting hematopoietic toxicities. HAHA immune response was identified in 2 of 16 patients (12.5%). Stable disease at 3 months was seen in 10 patients and 2 patients demonstrated an 88% and 64% decrease in CEA back to normal levels. In 2 patients (111)In-DOTA-M5A imaging revealed previously unknown brain metastases. Conclusion: This study demonstrates the potential utility of the (90)Y-DOTA-M5A anti-CEA mAb as a therapeutic antibody. There is decreased immunogenicity compared with murine and chimeric mAbs, allowing for the potential of multiple administrations. Combined modality therapy approaches incorporating this agent should continue to be evaluated.

Glioblastoma (GBM) is the most common malignant primary brain tumor and remains challenging to treat due to its aggressive nature with high rate of recurrence. Despite advances in therapy, prognosis is poor with median survival of 19 months with maximal therapy. The normal astrocytes within the GBM TME undergo a phenotypic shift, promoting alterations supportive of tumor growth. There is increasing attention on the role of tumor-associated astrocytes (TAA) in GBM progression and resistance to therapy. Previous studies assessing astrocyte support of GBM employ astrocytes derived from murine or fetal sources. We have isolated TAAs from patients with GBM as well as normal human astrocytes (NHA) from cancer-free patients undergoing surgical treatment of epilepsy. The cells are maintained at a low passage number ( Citation Format: Joseph S. Domino, Anthony Alvarado, Levi Arnold, Sumedha Gunewardena, David Akhavan, Sufi M. Thomas. Targeting tumor-associated astrocyte dependence in glioblastoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3170.

The purpose of this study was to determine the effects of time from diagnosis to treatment (TTI) on survival in patients with nonmetastatic non-small-cell lung cancer (NSCLC).The National Cancer Database was queried for patients with stages 1 to 3 NSCLC between 2004 and 2013. Patients with missing survival status/time, unknown TTI, or receipt of palliative therapy were excluded. Multivariable Cox proportional hazards modeling, logistic regression, and recursive partitioning analysis were performed to determine associated variables and survival outcomes.Altogether, 1,393,232 patients met inclusion criteria. The median follow-up was 36 months. The median TTI increased between 2004 and 2013 from 35 to 39 days (P .001). On multivariable Cox proportional hazards modeling, TTI groups 31 to 60 days, 61 to 90 days, and90 days were independently related to poorer overall survival (OS) compared with TTI 1 to 30 days (hazard ratio, 1.04, 1.10, and 1.14; 95% confidence interval [CI], 1.02-1.06, 1.07-1.12, and 1.11-1.17, respectively; P .001 for all). Recursive partitioning analysis revealed that TTI of ≤ 45 days was the most optimal threshold for survival (P .001); patients with TTI ≤ 45 days had a median OS of 70.2 months (95% CI, 69.3-71.1 months) versus 61.5 months (95% CI, 60.5-62.4) (P .001). There were significant disparities by age, race, ethnicity, and income for delayed (45 days) TTI (P .001 for all). Subgroup analysis revealed that stage 1 and 2 patients with TTI45 days had a higher risk of mortality compared with TTI ≤ 45 days (hazard ratio, 1.15 and 1.05; 95% CI, 1.12-1.17 and 1.01-1.09, respectively) (P .001).Increased TTI is independently associated with poorer survival in non-metastatic NSCLC. TTI ≤ 45 days is a clinically targetable time frame associated with improved outcomes and ought to be considered for patients with lung cancer undergoing definitive therapy.

OBJECTIVES The treatment of choice for locally advanced cervical cancer is definitive chemoradiation (CRT). Hysterectomy is not indicated due to higher-rates of cut-through resections leaving gross disease behind, requiring additional therapy with increasing morbidity and no benefit in overall survival (OS). The objectives of this study were to determine factors associated with cut-through hysterectomies and evaluate OS outcomes. MATERIALS AND METHODS The National Cancer Database (NCDB) was queried for patients 18 years and older with clinical Federation of Gynecology and Obstetrics stage IB2 to IVA. All patients underwent upfront hysterectomy and had known margin status. Cut-through hysterectomy was classified as presence of microscopic or macroscopic disease at the margin. RESULTS A total of 11,638 patients were included; 993 (8.5%) had positive margins. In patients with positive margins, 560 (56.4%) received postoperative CRT and 148 (14.9%) underwent postoperative radiation. Five-year OS was worse for those with cut-through resections when compared with those with negative margins, 66.0% versus 86.7%, respectively (hazard ratios, 3.08; P

Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the first example of this alternate mechanism in brain tumors by showing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor β (PDGFRβ). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFRβ transcription in an mTORC1- and extracellular signal–regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo. These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy. Significance: These results provide the first clinical and biologic evidence for receptor tyrosine kinase (RTK) “switching” as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecular explanation of how tumors can become “addicted” to a nonamplified, nonmutated, physiologically regulated RTK to evade targeted treatment. Cancer Discov; 3(5); 534–47. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 471

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. Cancer Discovery; 1(6); 524–38. ©2011 AACR. Read the Commentary on this article by Wick et al., p. 475 This article is highlighted in the In This Issue feature, p. 457

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.

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 mTOR's role in glioblastoma. Mechanisms of escape from mTOR inhibition are also discussed, as well as future clinical strategies of mTOR inhibition.

We determined the x-ray structure of bovine aquaporin 0 (AQP0) to a resolution of 2.2 Å. The structure of this eukaryotic, integral membrane protein suggests that the selectivity of AQP0 for water transport is based on the identity and location of signature amino acid residues that are hallmarks of the water-selective arm of the AQP family of proteins. Furthermore, the channel lumen is narrowed only by two, quasi-2-fold related tyrosine side chains that might account for reduced water conductance relative to other AQPs. The channel is functionally open to the passage of water because there are eight discreet water molecules within the channel. Comparison of this structure with the recent electron-diffraction structure of the junctional form of sheep AQP0 at pH 6.0 that was interpreted as closed shows no global change in the structure of AQP0 and only small changes in side-chain positions. We observed no structural change to the channel or the molecule as a whole at pH 10, which could be interpreted as the postulated pH-gating mechanism of AQP0-mediated water transport at pH >6.5. Contrary to the electron-diffraction structure, the comparison shows no evidence of channel gating induced by association of the extracellular domains of AQP0 at pH 6.0. Our structure aids the analysis of the interaction of the extracellular domains and the possibility of a cell–cell adhesion role for AQP0. In addition, our structure illustrates the basis for formation of certain types of cataracts that are the result of mutations.

The aqua (glycero) porins conduct water (and glycerol) across cell membranes. The structure of these channels reveals a tripathic channel that supports a hydrophobic surface and, opposite to this, a line of eight hydrogen-bond acceptors and four hydrogen-bond donors. The eight carbonyls act as acceptors for water (or glycerol OH) molecules. The central water molecule in the channel is oriented to polarize hydrogen atoms outward from the center. This arrangement suggests how the structure prevents the potentially lethal conduction of protons across the membrane. The structure also suggests the mechanism behind the selectivity of aquaglyceroporins for glycerol, the basis for enantioselectivity among alditols, and the basis for the prevention of any leakage of the electrochemical gradient.