Nhan L. Tran

Molecular targets of glioma invasion

Abstract.Glioblastoma multiforme is the most common and lethal primary malignant brain tumor. Although considerable progress has been made in technical proficiencies of surgical and radiation treatment for brain tumor patients, the impact of these advances on clinical outcome has been disappointing, with median survival time not exceeding 15 months. Over the last 30 years, no significant increase in survival of patients suffering from this disease has been achieved. A fundamental source of the management challenge presented in glioma patients is the insidious propensity of tumor invasion into distant brain tissue. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure and chemotherapy. Recent improved understanding of biochemical and molecular determinants of glioma cell invasion provide valuable insight into the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. These findings are moving forward to translational research and clinical trials as novel antiglioma therapies.

Migrating glioma cells activate the PI3-K pathway and display decreased susceptibility to apoptosis

Glioma cells that migrate out of the main tumor mass into normal brain tissue contribute to the failure of most gliomas to respond to treatment. Treatments that target migratory glioma cells may enhance the therapeutic response. Multiple lines of evidence suggest that suppression of apoptosis accompanies activation of the migratory phenotype. Here, we determine whether migration and apoptosis are consistently linked in glioma cells and whether manipulation of migration influences cytotoxic therapy-induced apoptosis. Camptothecin and Trail-induced apoptosis were decreased 2-5-fold in actively migrating glioma cells relative to migration-restricted cells. Consistent with a mechanistic link between migration and apoptosis, the dose-response for stimulation of migration on laminin was inversely proportional to apoptosis induction. Treatment of glioma cells with migration inhibitors alone had little effect on basal rates of apoptosis and had little effect on Trail-induced or camptothecin-induced apoptosis in migration-restricted cells. By contrast, migration inhibitors increased camptothecin and Trail-induced apoptosis in actively migrating glioma cells. Migrating glioma cells have increased amounts of phosphorylated Akt and its downstream substrate glycogen synthase kinase-3 relative to migration restricted cells. Treatment of migrating cells with a specific inhibitor of phosphoinositide 3-kinase (PI3-K), LY294002, blocked the phosphorylation of Akt and increased the sensitivity to apoptosis. LY294002 had no effect on the migration of restricted cells. This suggests that migrating glioma cells activate the PI3-K survival pathway, protecting migrating cells from apoptosis. Taken together, these data provide support for a link between migration and apoptosis in glioma cells. In addition, evidence indicates that treatment with migration inhibitors, while not affecting apoptosis-induction in migration-restricted cells, can sensitize migrating glioma cells to cytotoxic agents.

Increased Fibroblast Growth Factor-Inducible 14 Expression Levels Promote Glioma Cell Invasion via Rac1 and Nuclear Factor-κB and Correlate with Poor Patient Outcome

Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-kappaB (NF-kappaB) activity. The Fn14 promoter region contains NF-kappaB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.

Role of Synaptojanin 2 in Glioma Cell Migration and Invasion

The small GTPase Rac1 is thought to play an important role in cell migration and invasion. We have previously identified synaptojanin 2, a phosphoinositide phosphatase, as an effector of Rac1. Here, we show that small interfering RNA-mediated depletion of either Rac1 or synaptojanin 2 inhibits invasion of SNB19 and U87MG glioblastoma cells through Matrigel and rat brain slices. Depletion of Rac1 or synaptojanin 2 also inhibits migration of SNB19 and U87MG cells on glioma-derived extracellular matrix. In addition, we found that depletion of Rac1 or synaptojanin 2 inhibits the formation of lamellipodia and invadopodia, specialized membrane structures that are thought to be involved in extracellular matrix degradation. These results suggest that synaptojanin 2 contributes to the role of Rac1 in cell invasion and migration by regulating the formation of invadopodia and lamellipodia. This study also identifies synaptojanin 2 as a novel potential target for therapeutic intervention in malignant tumors.

The Guanine Nucleotide Exchange Factors Trio, Ect2, and Vav3 Mediate the Invasive Behavior of Glioblastoma

Malignant gliomas are characterized by their ability to invade normal brain tissue. We have previously shown that the small GTPase Rac1 plays a role in both migration and invasion in gliomas. Here, we aim to identify Rac-activating guanine nucleotide exchange factors (GEFs) that mediate glioblastoma invasiveness. Using a brain tumor expression database, we identified three GEFs, Trio, Ect2, and Vav3, that are expressed at higher levels in glioblastoma versus low-grade glioma. The expression of these GEFs is also associated with poor patient survival. Quantitative real-time polymerase chain reaction and immunohistochemical analyses on an independent set of tumors confirmed that these GEFs are overexpressed in glioblastoma as compared with either nonneoplastic brain or low-grade gliomas. In addition, depletion of Trio, Ect2, and Vav3 by siRNA oligonucleotides suppresses glioblastoma cell migration and invasion. Depletion of either Ect2 or Trio also reduces the rate of cell proliferation. These results suggest that targeting GEFs may present novel strategies for anti-invasive therapy for malignant gliomas.

EphB2/R-ras signaling regulates glioma cell adhesion, growth, and invasion

Eph receptor tyrosine kinases mediate neurodevelopmental processes such as boundary formation, vasculogenesis, and cell migration. Recently, we found that overexpression of EphB2 in glioma cells results in reduced cell adhesion and increased cell invasion. Since R-Ras has been shown to play a critical role in EphB2 regulation of integrin activity, we explored whether the biological role of EphB2 in glioma invasion is mediated by downstream R-Ras activation. On EphB2 activation, R-Ras associated with the receptor and became highly phosphorylated. Depletion of endogenous R-Ras expression by siRNA abrogated EphB2 effects on glioma cell adhesion, proliferation, and invasion in ex vivo rat brain slices. Anti-proliferative responses to EphB2 activation were consistent with suppressed mitogen-activated protein kinase activity. Moreover, R-Ras was highly phosphorylated in the invading glioma cells. In human brain tumor specimens, R-Ras expression and phosphorylation correlated with increasing grade of gliomas. Laser capture microdissection of invading glioblastoma cells revealed elevated R-Ras mRNA (1.5- to 26-fold) in 100% (eight of eight) of biopsy specimens, and immunohistochemistry revealed high R-Ras localization primarily in glioblastoma cells. The phosphorylation ratio of R-Ras positively correlated with the phosphorylation ratio of EphB2 in glioblastoma tissues. These results demonstrate that R-Ras plays an important role in glioma pathology, further suggesting the EphB2/R-Ras signaling pathway as a potential therapeutic target.

MicroRNA-328 is associated with （non-small） cell lung cancer （NSCLC） brain metastasis and mediates NSCLC migration

Brain metastasis (BM) can affect ∼ 25% of nonsmall cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been disappointing. microRNAs (miRNAs) regulate the expression of target mRNAs. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which make them a powerful tool for early detection of disease, risk assessment, and prognosis. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from seven patients with BM (BM+) and six without BM (BM−). Using t‐test and further qRT‐PCR validation, eight miRNAs were confirmed to be significantly differentially expressed. Of these, expression of miR‐328 and miR‐330‐3p were able to correctly classify BM+ vs. BM− patients. This classifier was used on a validation cohort (n = 15), and it correctly classified 12/15 patients. Gene expression analysis comparing A549 parental and A549 cells stably transfected to over‐express miR‐328 (A549‐328) identified several significantly differentially expressed genes. PRKCA was one of the genes over‐expressed in A549‐328 cells. Additionally, A549‐328 cells had significantly increased cell migration compared to A549 cells, which was significantly reduced upon PRKCA knockdown. In summary, miR‐328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA and with further corroboration in additional independent cohorts, these miRNAs may be incorporated into clinical treatment decision making to stratify NSCLC patients at higher risk for developing BM.

Current approaches to the treatment of metastatic brain tumours

Metastatic tumours involving the brain overshadow primary brain neoplasms in frequency and are an important complication in the overall management of many cancers. Importantly, advances are being made in understanding the molecular biology underlying the initial development and eventual proliferation of brain metastases. Surgery and radiation remain the cornerstones of the therapy for symptomatic lesions; however, image-based guidance is improving surgical technique to maximize the preservation of normal tissue, while more sophisticated approaches to radiation therapy are being used to minimize the long-standing concerns over the toxicity of whole-brain radiation protocols used in the past. Furthermore, the burgeoning knowledge of tumour biology has facilitated the entry of systemically administered therapies into the clinic. Responses to these targeted interventions have ranged from substantial toxicity with no control of disease to periods of useful tumour control with no decrement in performance status of the treated individual. This experience enables recognition of the limits of targeted therapy, but has also informed methods to optimize this approach. This Review focuses on the clinically relevant molecular biology of brain metastases, and summarizes the current applications of these data to imaging, surgery, radiation therapy, cytotoxic chemotherapy and targeted therapy.

Toward precision medicine in glioblastoma: the promise and the challenges

Integrated sequencing strategies have provided a broader understanding of the genomic landscape and molecular classifications of multiple cancer types and have identified various therapeutic opportunities across cancer subsets. Despite pivotal advances in the characterization of genomic alterations in glioblastoma, targeted agents have shown minimal efficacy in clinical trials to date, and patient survival remains poor. In this review, we highlight potential reasons why targeting single alterations has yielded limited clinical efficacy in glioblastoma, focusing on issues of tumor heterogeneity and pharmacokinetic failure. We outline strategies to address these challenges in applying precision medicine to glioblastoma and the rationale for applying targeted combination therapy approaches that match genomic alterations with compounds accessible to the central nervous system.

The Fibroblast Growth Factor–Inducible 14 Receptor Is Highly Expressed in HER2-Positive Breast Tumors and Regulates Breast Cancer Cell Invasive Capacity

Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor–inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor–like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor–negative (HER2+/ER−) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER− breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-κB (NF-κB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-κB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference–mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-κB activity. Our findings support a role for Fn14-mediated NF-κB pathway activation in breast tumor invasion and metastasis. (Mol Cancer Res 2008;6(5):725–34)