Glioblastoma vulnerability to neddylation inhibition is dependent on PTEN status, and dysregulation of the cell cycle and DNA replication.

Background: Neddylation (NAE) inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report the cytotoxic vulnerability to NAE inhibitors in a subset of glioblastoma (GBM) preclinical models and identify genetic alterations and biological processes underlying differential response. Methods: GBM DNA sequencing and transcriptomic data were queried for genes associated with response to NAE inhibition; candidates were validated by molecular techniques. Multi-omics and functional assays revealed processes implicated in NAE inhibition response. Results: Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and DNA repair pathways as significant differentiators between sensitive and resistant models. Vulnerability to MLN4924, a NAE inhibitor, is associated with elevated S-phase populations, DNA re-replication, and DNA damage. In a panel of GBM models, loss of WT PTEN is associated with resistance to different NAE inhibitors. A NAE inhibition response gene set could segregate the GBM cell lines that are most resistant to MLN4924. Conclusions: Loss of WT PTEN is associated with non-sensitivity to 3 different compounds that inhibit NAE in GBM. A NAE inhibition response gene set largely consisting of DNA replication genes could segregate GBM cell lines most resistant to NAEi and may be the basis for future development of NAE inhibition signatures of vulnerability and clinical trial enrollment within a precision medicine paradigm.

PTEN loss drives resistance to the neddylation inhibitor MLN4924 in glioblastoma and can be overcome with TOP2A inhibitors.

BACKGROUND: Neddylation inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report vulnerability to MLN4924 or pevonedistat (a neddylation inhibitor) in a subset of glioblastoma (GBM) preclinical models and identify biomarkers, mechanisms, and signatures of differential response. METHODS: GBM sequencing data were queried for genes associated with MLN4924 response status; candidates were validated by molecular techniques. Time-course transcriptomics and proteomics revealed processes implicated in MLN4924 response. RESULTS: Vulnerability to MLN4924 is associated with elevated S-phase populations, re-replication, and DNA damage. Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and chromatin instability pathways as significant differentiators between sensitive and resistant models. Loss of PTEN and its nuclear functions is associated with resistance to MLN4924. Time-course proteomics identified elevated TOP2A in resistant models through treatment. TOP2A inhibitors combined with MLN4924 prove synergistic. CONCLUSIONS: We show that PTEN status serves as both a novel biomarker for MLN4924 response in GBM and reveals a vulnerability to TOP2A inhibitors in combination with MLN4924.

Genomic and Phenotypic Characterization of a Broad Panel of Patient-Derived Xenografts Reflects the Diversity of Glioblastoma.

PURPOSE: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. EXPERIMENTAL DESIGN: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. RESULTS: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient-PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. CONCLUSIONS: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

EGFRvIII-Stat5 Signaling Enhances Glioblastoma Cell Migration and Survival.

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.Implications: Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. Mol Cancer Res; 16(7); 1185-95. ©2018 AACR.

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.

Phase 0 Trial of AZD1775 in First-Recurrence Glioblastoma Patients.

Purpose: AZD1775 is a first-in-class Wee1 inhibitor with dual function as a DNA damage sensitizer and cytotoxic agent. A phase I study of AZD1775 for solid tumors suggested activity against brain tumors, but a preclinical study indicated minimal blood-brain barrier penetration in mice. To resolve this controversy, we examined the pharmacokinetics and pharmacodynamics of AZD1775 in patients with first-recurrence, glioblastoma.Patients and Methods: Twenty adult patients received a single dose of AZD1775 prior to tumor resection and enrolled in either a dose-escalation arm or a time-escalation arm. Sparse pharmacokinetic blood samples were collected, and contrast-enhancing tumor samples were collected intraoperatively. AZD1775 total and unbound concentrations were determined by a validated LC/MS-MS method. Population pharmacokinetic analysis was performed to characterize AZD1775 plasma pharmacokinetic profiles. Pharmacodynamic endpoints were compared to matched archival tissue.Results: The AZD1775 plasma concentration-time profile following a single oral dose in patients with glioblastoma was well-described by a one-compartment model. Glomerular filtration rate was identified as a significant covariate on AZD1775 apparent clearance. AZD1775 showed good brain tumor penetration, with a median unbound tumor-to-plasma concentration ratio of 3.2, and achieved potential pharmacologically active tumor concentrations. Wee1 pathway suppression was inferred by abrogation of G2 arrest, intensified double-strand DNA breakage, and programmed cell death. No drug-related adverse events were associated with this study.Conclusions: In contrast to recent preclinical data, our phase 0 study of AZD 1775 in recurrent glioblastoma indicates good human brain tumor penetration, provides the first evidence of clinical biological activity in human glioblastoma, and confirms the utility of phase 0 trials as part of an accelerated paradigm for drug development in patients with glioma. Clin Cancer Res; 24(16); 3820-8. ©2018 AACRSee related commentary by Vogelbaum, p. 3790.

When the Ends Are Really the Beginnings: Targeting Telomerase for Treatment of GBM.

PURPOSE OF REVIEW: High-throughput genomic sequencing has identified alterations in the gene encoding human telomerase reverse transcriptase (TERT) as points of interest for elucidating the oncogenic mechanism of multiple different cancer types, including gliomas. In gliomas, the TERT promoter mutation (TPM) and resultant overexpression of TERT are observed mainly in the most aggressive (primary glioblastoma/grade IV astrocytoma) and the least aggressive (grade II oligodendroglioma) cases. This article reviews recent research on (1) the mechanism of TERT activation in glioma, (2) downstream consequences of TERT overexpression on glioma pathogenesis, and (3) targeting TPMs as a therapeutic strategy. RECENT FINDINGS: New molecular classifications for gliomas include using TPMs, where the mutant group demonstrates the worst prognosis. Though a canonical function of TERT is established in regard to telomere maintenance, recent studies on non-canonical functions of TERT explore varied roles of telomerase in tumor progression and maintenance. Somatic alterations of the TERT promoter present a promising target for novel therapeutics development in primary glioma treatment.

Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation.

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood-brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1-eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATO's effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival.Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32-46. ©2017 AACR.

Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Combination therapy with potent PI3K and MAPK inhibitors overcomes adaptive kinome resistance to single agents in preclinical models of glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. METHODS: We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. RESULTS: PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. CONCLUSION: Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

Genomic profiles of low-grade murine gliomas evolve during progression to glioblastoma.

BACKGROUND: Gliomas are diverse neoplasms with multiple molecular subtypes. How tumor-initiating mutations relate to molecular subtypes as these tumors evolve during malignant progression remains unclear. METHODS: We used genetically engineered mouse models, histopathology, genetic lineage tracing, expression profiling, and copy number analyses to examine how genomic tumor diversity evolves during the course of malignant progression from low- to high-grade disease. RESULTS: Knockout of all 3 retinoblastoma (Rb) family proteins was required to initiate low-grade tumors in adult mouse astrocytes. Mutations activating mitogen-activated protein kinase signaling, specifically KrasG12D, potentiated Rb-mediated tumorigenesis. Low-grade tumors showed mutant Kras-specific transcriptome profiles but lacked copy number mutations. These tumors stochastically progressed to high-grade, in part through acquisition of copy number mutations. High-grade tumor transcriptomes were heterogeneous and consisted of 3 subtypes that mimicked human mesenchymal, proneural, and neural glioblastomas. Subtypes were confirmed in validation sets of high-grade mouse tumors initiated by different driver mutations as well as human patient-derived xenograft models and glioblastoma tumors. CONCLUSION: These results suggest that oncogenic driver mutations influence the genomic profiles of low-grade tumors and that these, as well as progression-acquired mutations, contribute strongly to the genomic heterogeneity across high-grade tumors.

Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells.

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

SGEF Is Regulated via TWEAK/Fn14/NF-κB Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide.

UNLABELLED: Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide, GB tumors develop treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly, conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance to develop targeted therapies to combat this refractory disease. Previous observations indicated that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and plays a role in promoting TWEAK-Fn14-mediated glioma invasion. Here, further investigation revealed an important role for SGEF in glioma cell survival. SGEF expression is upregulated by TWEAK-Fn14 signaling via NF-κB activity while shRNA-mediated reduction of SGEF expression sensitizes glioma cells to temozolomide-induced apoptosis and suppresses colony formation following temozolomide treatment. Nuclear SGEF is activated following temozolomide exposure and complexes with the DNA damage repair (DDR) protein BRCA1. Moreover, BRCA1 phosphorylation in response to temozolomide treatment is hindered by SGEF knockdown. The role of SGEF in promoting chemotherapeutic resistance highlights a heretofore unappreciated driver, and suggests its candidacy for development of novel targeted therapeutics for temozolomide-refractory, invasive GB cells. IMPLICATION: SGEF, as a dual process modulator of cell survival and invasion, represents a novel target for treatment refractory glioblastoma.

Propentofylline inhibits glioblastoma cell invasion and survival by targeting the TROY signaling pathway.

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.

Intravenous delivery of camptothecin-loaded PLGA nanoparticles for the treatment of intracranial glioma.

Effective treatment of glioblastoma multiforme remains a major clinical challenge, due in part to the difficulty of delivering chemotherapeutics across the blood-brain barrier. Systemically administered drugs are often poorly bioavailable in the brain, and drug efficacy within the central nervous system can be limited by peripheral toxicity. Here, we investigate the ability of systemically administered poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) to deliver hydrophobic payloads to intracranial glioma. Hydrophobic payload encapsulated within PLGA NPs accumulated at ∼10× higher levels in tumor compared to healthy brain. Tolerability of the chemotherapeutic camptothecin (CPT) was improved by encapsulation, enabling safe administration of up to 20mg/kg drug when encapsulated within NPs. Immunohistochemistry staining for γ-H2AFX, a marker for double-strand breaks, demonstrated higher levels of drug activity in tumors treated with CPT-loaded NPs compared to free drug. CPT-loaded NPs were effective in slowing the growth of intracranial GL261 tumors in immune competent C57 albino mice, providing a significant survival benefit compared to mice receiving saline, free CPT or low dose CPT NPs (median survival of 36.5 days compared to 28, 32, 33.5 days respectively). In sum, these data demonstrate the feasibility of treating intracranial glioma with systemically administered nanoparticles loaded with the otherwise ineffective chemotherapeutic CPT.

FN14 expression correlates with MET in NSCLC and promotes MET-driven cell invasion.

The five-year survival rate in advanced non-small cell lung cancer (NSCLC) remains below ten percent. The invasive and metastatic nature of NSCLC tumor cells contributes to the high mortality rate, and as such the mechanisms that govern NSCLC metastasis is an active area of investigation. Two surface receptors that influence NSCLC invasion and metastasis are the hepatocyte growth factor receptor (HGFR/MET) and fibroblast growth factor-inducible 14 (FN14). MET protein is over-expressed in NSCLC tumors and associated with poor clinical outcome and metastasis. FN14 protein is also elevated in NSCLC tumors and positively correlates with tumor cell migration and invasion. In this report, we show that MET and FN14 protein expressions are significantly correlated in human primary NSCLC tumors, and the protein levels of MET and FN14 are elevated in metastatic lesions relative to patient-matched primary tumors. In vitro, HGF/MET activation significantly enhances FN14 mRNA and protein expression. Importantly, depletion of FN14 is sufficient to inhibit MET-driven NSCLC tumor cell migration and invasion in vitro. This work suggests that MET and FN14 protein expressions are associated with the invasive and metastatic potential of NSCLC. Receptor-targeted therapeutics for both MET and FN14 are in clinical development, the use of which may mitigate the metastatic potential of NSCLC.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioblastoma cell chemotaxis via Lyn activation.

The long-term survival of patients with glioblastoma is compromised by the proclivity for local invasion into the surrounding normal brain, escaping surgical resection and contributing to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the Rho guanosine triphosphatase family member Rac1. Here, we demonstrate that TWEAK acts as a chemotactic factor for glioma cells, a potential process for driving cell invasion into the surrounding brain tissue. TWEAK exposure induced the activation of Src family kinases (SFKs), and pharmacologic suppression of SFK activity inhibited TWEAK-induced chemotactic migration. We employed a multiplexed Luminex assay and identified Lyn as a candidate SFK activated by TWEAK. Depletion of Lyn suppressed TWEAK-induced chemotaxis and Rac1 activity. Furthermore, Lyn gene expression levels increase with primary glioma tumor grade and inversely correlate with patient survival. These results show that TWEAK-induced glioma cell chemotaxis is dependent upon Lyn kinase function and, thus, provides opportunities for therapeutic targeting of this deadly disease.

Reciprocal activation of transcription factors underlies the dichotomy between proliferation and invasion of glioma cells.

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.

Molecular determinants of lung cancer metastasis to the central nervous system.

Lung cancer remains the leading cause of cancer-related mortality worldwide. The propensity for metastasis to the central nervous system (CNS) is a major clinical hurdle contributing to the low five-year survival rate of advanced disease. CNS metastases significantly outnumber primary brain tumors and carry a dismal prognosis in part due to the inability of therapeutic agents to cross the blood brain barrier. Standard treatment using radiation has been largely ineffective in improving mortality, suggesting the need for new agents targeting the critical metastatic drivers. The genetic and molecular events governing CNS metastasis from the lung are poorly understood at this time. This review highlights genetic events associated with CNS dissemination from the lung and molecular mechanisms associated with CNS metastasis. In vivo model systems that faithfully recapitulate escape from the lung and colonization of the CNS are described as tools for understanding the metastatic phenotype and for testing new therapeutic agents. A deeper understanding of the mechanisms of lung cancer metastasis to the CNS is needed to elucidate novel therapeutic avenues towards the improvement of the mortality associated with advanced stage lung cancer.