Interleukin-13 receptor alpha 2, EphA2, and Fos-related antigen 1 as molecular denominators of high-grade astrocytomas and specific targets for combinatorial therapy.

PURPOSE: We investigated the expression of interleukin-13 receptor alpha2 (IL-13R alpha 2), EphA2, and Fos-related antigen 1 (Fra-1) in astrocytomas and normal brain. We sought to document whether the expression of the three factors changed with progression to higher grade malignancy and whether two or three targets in combination might be sufficient to target all patients with high-grade astrocytomas. EXPERIMENTAL DESIGN: Immunohistochemistry was done for IL-13R alpha 2, EphA2, and Fra-1 using human brain tumor tissue microarrays containing 30 specimens of WHO grades II and III astrocytomas, 46 glioblastoma multiformes (GBM), and 9 normal brain samples. Sections were scored based on frequency and intensity of expression. Western blotting was done for all three markers using GBM tumor specimens and xenograft cell lines. Two cytotoxins, IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, which target IL-13R alpha 2 or EphA2, respectively, were tested for cytotoxicity against human GBM primary explant cells and established cells. RESULTS: Expression of all three proteins was significantly higher in GBM compared with normal brain, low-grade, and anaplastic astrocytomas. Greater than 95% of GBM overexpressed at least two of the three markers. Importantly, every GBM overexpressed at least one marker. Human GBM primary explant cells and cell lines were potently killed by IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, in accordance with their level of expression of IL-13R alpha 2 and EphA2, respectively. CONCLUSIONS: IL-13R alpha 2, EphA2, and Fra-1 are attractive therapeutic targets representing molecular denominators of high-grade astrocytomas. One hundred percent of GBM tumors overexpress at least one of these proteins, providing the basis for rational combinatorial targeted therapies/diagnostics suitable for all patients with this disease.     

EphA2及其配体EphrinA1在恶性脑胶质瘤中的表达及与血管生成的关系

  目的  检测酪氨酸激酶受体EphA2及其配体EphrinA1在恶性脑胶质瘤中的表达,并探讨其与胶质瘤血管生成的关系。  方法  采用免疫组织化学S-P法检测62例胶质瘤组织及8例正常脑组织中EphA2、EphrinA1的表达情况,并采用CD105抗体标记微血管内皮细胞,计算微血管密度（microvesseldensity,MVD）。探讨EphA2、EphrinA1的表达水平与胶质瘤的微血管生成之间可能存在的关系。  结果  EphA2、MVD在胶质瘤中阳性表达明显高于正常脑组织,二者差异显著（P<0.01）。而且随着肿瘤恶性程度增加,EphA2及MVD蛋白染色强度和阳性细胞数均明显升高,高级别脑胶质瘤（Ⅲ~Ⅳ）中EphA2及MVD强阳性表达明显高于低级别胶质瘤组,差异有显著统计学意义（P<0.01）。EphrinA1则具有相反的趋势,在大多数恶性脑胶质瘤中呈低水平表达,在低级别胶质瘤和正常组织中呈较高水平表达,且胶质瘤级别越低EphrinA1表达越高。EphA2表达与MVD呈显著正相关（r=0.713,P<0.01）,EphrinA1表达与MVD呈显著负相关（r=-0.772,P<0.01）,EphA2的表达与EphrinA1呈显著负相关（r=-0.912,P<0.01）。  结论  EphA2在恶性脑胶质瘤中特异性高表达、EphrinA1特异性低表达与胶质瘤的侵袭性和恶性程度密切相关,且EphA2过表达和EphrinA1的缺陷表达可能协同促进胶质瘤组织新生血管的生成,在胶质瘤的发病及恶性进展中发挥重要作用。      

A kinase-dependent role for EphA2 receptor in promoting tumor growth and metastasis

Receptor tyrosine kinases of the Eph family are upregulated in several different types of cancer. One family member in particular, the EphA2 receptor, has been linked to breast, prostate, lung and colon cancer, as well as melanoma. However, mechanisms by which EphA2 contributes to tumor progression are far from clear. In certain tumor cell lines, EphA2 receptor is underphosphorylated, raising the question of whether ligand-induced receptor phosphorylation and its kinase activity play a role in oncogenesis. To test directly the role of EphA2 receptor phosphorylation/kinase activity in tumor progression, we generated EphA2 receptor variants that were either lacking the cytoplasmic domain or carrying a point mutation that inhibits its kinase activity. Expression of these EphA2 mutants in breast cancer cells resulted in decreased tumor volume and increased tumor apoptosis in primary tumors. In addition, the numbers of lung metastases were significantly reduced in both experimental and spontaneous metastasis models. Reduced tumor volume and metastasis are not due to defects in tumor angiogenesis, as there is no significant difference in tumor vessel density between wild-type tumors and tumors expressing EphA2-signaling-defective mutants. In contrast, tumor cells expressing the EphA2 mutants are defective in RhoA GTPase activation and cell migration. Taken together, these results suggest that receptor phosphorylation and kinase activity of the EphA2 receptor, at least in part, contribute to tumor malignancy.     

Gene expression microarray analysis reveals YKL-40 to be a potential serum marker for malignant character in human glioma

We have identified a potential serum marker for glioblastoma multiforme (GBM) using microarray analysis from samples of GBM. We compared the gene expression profile of 19 gliomas to pooled normal brain by competitive hybridization of RNA from each tumor sample to a pooled sample of RNA isolated from normal brain tissue using the Incyte 10,000 gene expression array. The most differentially expressed gene in this analysis encodes a secreted glycoprotein and is referred to as YKL-40. YKL-40 mRNA was detected in the GBM samples with a range of 3- to 62-fold elevation over normal brain. It has been reported previously that this protein is expressed in pathologic conditions of extracellular matrix degradation and angiogenesis, such as rheumatoid arthritis, severe osteoarthritis, hepatic fibrosis, primary colorectal cancer, and metastatic breast cancer. These data suggest YKL-40 may be involved in extracellular matrix degradation and/or angiogenesis. Western blot analysis of glioma samples for YKL-40 protein levels revealed substantial elevation in approximately 65% of GBMs and undetectable levels in lower-grade gliomas (grade II and III) or normal brain tissue. We performed ELISA analysis on serum samples of glioma patients to determine whether this protein would correlate with the presence of tumor and tumor grade or burden. YKL-40 serum levels were substantially elevated in many of the GBM patients. Statistical analysis of these data indicates that in patients with glioma, serum YKL-40 levels correlate with tumor grade and potentially tumor burden in GBM.     

Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin

EphA2 receptor tyrosine kinase is frequently overexpressed in different human cancers, suggesting that it may promote tumor development and progression. However, evidence also exists that EphA2 may possess antitumorigenic properties, raising a critical question on the role of EphA2 kinase in tumorigenesis in vivo. We report here that deletion of EphA2 in mouse led to markedly enhanced susceptibility to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. EphA2-null mice developed skin tumors with an increased frequency and shortened latency. Moreover, tumors in homozygous knockout mice grew faster and were twice as likely to show invasive malignant progression. Haploinsufficiency of EphA2 caused an intermediate phenotype in tumor development but had little effects on invasive progression. EphA2 and ephrin-A1 exhibited compartmentalized expression pattern in mouse skin that localized EphA2/ephrin-A1 interactions to the basal layer of epidermis, which was disrupted in tumors. Loss of EphA2 increased tumor cell proliferation, whereas apoptosis was not affected. In vitro, treatment of primary keratinocytes from wild-type mice with ephrin-A1 suppressed cell proliferation and inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) activities. Both effects were abolished in EphA2-null keratinocytes, suggesting that loss of ERK inhibition by EphA2 may be one of the contributing mechanisms for increased tumor susceptibility. Interestingly, despite its tumor suppressive function, EphA2 was overexpressed in skin tumors compared with surrounding normal skin in wild-type mice, similar to the observations in human cancers. EphA2 overexpression may represent a compensatory feedback mechanism during tumorigenesis. Together, these results show that EphA2 is a novel tumor suppressor gene in mammalian skin.     

Up-regulation of EphA2 and down-regulation of EphrinA1 are associated with the aggressive phenotype and poor prognosis of malignant glioma

Malignant gliomas display over-expression of the receptor tyrosine kinase EphA2. However, expression levels of the EphA2 ligand, EphrinA1, have not been fully elucidated. Seventy-eight patients with primary gliomas were included in this study who underwent surgical resection, radiation, and chemotherapy. The expression of EphA2 and EphrinA1 in tumors was assessed by immunohistochemistry and was statistically analyzed in combination with the follow-up data of patients. EphA2 was highly expressed in most malignant gliomas, but EphrinA1 was expressed at low levels in these tumors. The increased EphA2 expression is associated with higher-grade histology and poor patient prognosis. Contrary to this, the increased EphrinA1 expression is associated with lower-grade histology, but not associated with poor patient prognosis. Moreover, patients with tumors positive for EphA2 and negative for EphrinA1 had significantly shorter overall and progression-free survival than patients with tumors positive for both EphA2 and EphrinA1, negative for both EphA2 and EphrinA1, or negative for EphA2 and positive for EphrinA1. RNAi-mediated suppression of endogenous EphA2 in human glioblastoma multiforme cells resulted in increased EphrinA1 levels, as well as decreased cell viability, anchorage independence and in vitro invasion, and increased apoptosis. Furthermore, suppression of EphA2 resulted in delayed tumor growth in mice xenografts. Together, these data indicate that up-regulation of EphA2 and down-regulation of Ephrina1 may correlate with poor prognosis for patients with high-grade glioma. EphA2 suppression partially reversed the aggressive phenotypes of malignant gliomas, possibly through up-regulating EphrinA1 expression, which may help explain how EphA2 modulates the malignant progression of gliomas.     

Involvement of EphA2 in head and neck squamous cell carcinoma: mRNA expression, loss of heterozygosity and immunohistochemical studies

EphA2 is a 130-kDa transmembrane protein primarily found in adult human epithelial cells and is a member of one of the largest receptor tyrosine kinases. It is located on 1p36.1, a genetic hot spot in cancer. EphA2 overexpression has been observed in aggressive solid tumors and its potential role in tumorigenesis, which includes cell growth, survival, migration and angiogenesis have been reported. However, the role of EphA2 remains unknown in head and neck cancer. In this study, we investigated the genetic profile of EphA2 in primary head and neck squamous cell carcinoma (HNSCC) by determining mRNA level, status of loss of heterozygosity and protein expression. mRNA expression was also correlated with clinicopathological data. Infrequent loss of heterozygosity (20%) was observed, though a 10-fold increase of mRNA expression in tumors compared to normal tissues was noted. A significant number of samples with normal to high mRNA expression was observed among patients with regional metastasis, with T3-T4 tumor size and with moderate to poor differentiation. However, statistical studies did not show any correlation between mRNA expression and any of the clinicopathological parameters. Tumor cells expressed EphA2 protein, but only weakly. These results suggest that EphA2 might be involved in the early development of HNSCC although not directly responsible for its progression.     

EphA2在人膀胱移行细胞癌中的表达及其临床意义

目的研究EphA2在在人膀胱移行细胞癌中的表达及临床意义。方法选取全膀胱切除后癌旁2.5 cm以上镜下证实为对照组的膀胱黏膜11例,移行细胞癌标本48例,采用免疫组化1二步法,每个石蜡块标本切取5张切片。其中的4片分别用于EphA2、VE-cadherin、K i67作为一抗的研究,另外1片用于HE染色以确定该病例的WHO分级及分期。另外对患者的预后进行随访,随访6~50个月,对复发者与未复发者的EphA2的阳性表达情况进行统计,并对患者术后生存时间进行分析。结果EphA2在膀胱TCC病理G1中的阳性表达率较低,只有14.3%;在G2级中阳性染色达到36.8%;在G3中阳性染色达到66.7%。通过两两比较,差异有显著性（P〈0.01）,而在正常膀胱黏膜组织中都是阴性。结论EphA2过度表达可以促进细胞的增殖,血管形成,可能是通过VE-cadherin而起作用。复发患者的EphA2的阳性表达率明显高于未复发者,可以将其作为评估患者术后是否容易复发的一个指标,EphA2还可能是评估患者术后生存时间的一个指标,并且可能成为肿瘤靶向治疗的新靶点。     

Inhibition of VEGF-dependent multistage carcinogenesis by soluble EphA receptors

Elevated expression of Eph receptors has long been correlated with the growth of solid tumors. However, the functional role of this family of receptor tyrosine kinases in carcinogenesis and tumor angiogenesis has not been well characterized. Here we report that soluble EphA receptors inhibit tumor angiogenesis and tumor progression in vivo in the RIP-Tag transgenic model of vascular endothelial growth factor (VEGF)-dependent multistage pancreatic islet cell carcinoma. Soluble EphA receptors delivered either by a transgene or an osmotic minipump inhibited the formation of angiogenic islet, a premalignant lesion, and reduced tumor volume of solid islet cell carcinoma. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor volume but increased tumor and endothelial cell apoptosis in vivo. In addition, soluble EphA receptors inhibited VEGF and betaTC tumor cell-conditioned medium-induced endothelial cell migration in vitro and VEGF-induced cornea angiogenesis in vivo. A dominant negative EphA2 mutant inhibited--whereas a gain-of-function EphA2 mutant enhanced--tumor cell-induced endothelial cell migration, suggesting that EphA2 receptor activation is required for tumor cell-endothelial cell interaction. These data provide functional evidence for EphA class receptor regulation of VEGF-dependent tumor angiogenesis, suggesting that the EphA signaling pathway may represent an attractive novel target for antiangiogenic therapy in cancer.     

Increased expression of EphA7 correlates with adverse outcome in primary and recurrent glioblastoma multiforme patients

Background  

Malignant gliomas are lethal cancers, highly dependent on angiogenesis and treatment options and prognosis still remain poor for patients with recurrent glioblastoma multiforme (GBM). Ephs and ephrins have many well-defined functions during embryonic development of central nervous system such as axon mapping, neural crest cell migration, hindbrain segmentation and synapse formation as well as physiological and abnormal angiogenesis. Accumulating evidence indicates that Eph and ephrins are frequently overexpressed in different tumor types including GBM. However, their role in tumorigenesis remains controversial, as both tumor growth promoter and suppressor potential have been ascribed to Eph and ephrins while the function of EphA7 in GBM pathogenesis remains largely unknown.     

Tyrosine kinase Eph receptor A6 sensitizes glioma‐initiating cells towards bone morphogenetic protein‐induced apoptosis

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation‐ and apoptosis‐inducing effects on glioma‐initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin‐producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP‐2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP‐2‐induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK‐2, which was associated with BMP‐induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain‐of‐function of EPHA6 together with BMP‐2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP‐2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.     

An LXR agonist promotes glioblastoma cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway

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.     

Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo

The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.     

EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human cancers. We sought to characterize the role of EphA2 in pancreatic adenocarcinoma and, using RNA interference (RNAi) mediated by small interfering RNA (siRNA), we determined the effects of suppressing EphA2 expression in vitro and in vivo. EphA2 expression in PANC1, MIAPaCa2, BxPC3 and Capan2 cells was assessed by Northern and Western blot. We artificially overexpressed EphA2 by transient transfection and suppressed EphA2 expression using RNAi. Cellular invasiveness was quantified by modified Boyden chamber assay. Anoikis was induced by anchorage-independent polyHEMA culture and caspase 3 activity was quantified fluorometrically. Focal adhesion kinase (FAK) phosphorylation was assessed by immunoprecipitation. EphA2 siRNA treatment was assessed in a nude mouse xenograft model. Pancreatic adenocarcinoma cells differentially express EphA2. Inherent and induced EphA2 overexpression is associated with increased cellular invasiveness and anoikis resistance. EphA2 siRNA suppresses EphA2 expression, cellular invasiveness, anoikis resistance and FAK phosphorylation in vitro and retards tumor growth and inhibits metastasis in vivo. EphA2 is both a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma.     

EphA2在乳腺癌治疗耐药中作用的研究进展

促红细胞生成素诱导肝细胞受体（erythropoietin-producing hepatocyte receptor,Eph）是目前已知的受体型酪氨酸激酶（receptor tyrosine kinase,RTK）中最庞大的家族。该家族与经典RTK的不同之处在于其配体Ephrin也是一种膜定位分子。因此,Ephrin-Eph介导的信号传导依赖细胞与细胞间的直接接触。生理情况下,该信号系统在调控胚胎发育和维持内环境稳态中发挥重要作用。Ephrin-Eph信号系统失调与肿瘤发生和发展密切相关。研究提示,在所有的Eph家族成员中,EphA2与肿瘤的关系最为密切。EphA2表达上调能够增强乳腺癌细胞的增殖、存活、侵袭、耐药和血管生成等多种恶性生物学行为。EphA2在乳腺癌组织中表达增高与患者预后不良密切相关。近年研究发现,EphA2有望成为一个预测乳腺癌治疗效果的分子标志物,而且很有可能成为一个治疗乳腺癌的分子靶标。本文主要针对EphA2在乳腺癌治疗耐药中的作用进行综述。      

A combination of tyrosine kinase inhibitors,crizotinib and dasatinib for the treatment of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM.     

EphA2 overexpression is associated with lack of hormone receptor expression and poor outcome in endometrial cancer

BACKGROUND:

EphA2 is a tyrosine kinase receptor in the ephrin family that is implicated in oncogenesis and angiogenesis. The objective of the current investigation was to study the role of EphA2 in endometrial cancer and its relation to steroid hormone receptor expression.

METHODS:

EphA2, estrogen receptor (ER), progesterone receptor (PR), and Ki‐67 expression levels were evaluated using immunohistochemistry in 139 endometrioid endometrial carcinoma (EEC) samples and in 10 benign endometrial samples. Samples were scored by 2 investigators who were blinded to clinical outcome. The results were correlated with clinicopathologic characteristics using univariate and multivariate analysis. A P value <.05 was considered statistically significant.

RESULTS:

High expression of EphA2 was detected in 48% of EEC samples versus 10% of benign samples. EphA2 overexpression was associated significantly with high disease stage (P = .04), high tumor grade (P = .003), increased depth of myometrial invasion (P = .05), low ER expression (P = .01), low PR expression (P = .006), and high Ki‐67 expression (P = .04). Low ER and PR expression levels were associated with high tumor grade, positive lymph nodes, high Ki‐67 expression, and high EphA2 expression. On univariate analysis of all patients, high EphA2 expression was associated significantly with shorter disease‐specific survival (DSS) (P < .001). On multivariate analysis, age (P < .001), high disease stage (P = .002), and high EphA2 expression (P = .04) were independent predictors of poor DSS.

CONCLUSIONS:

EphA2 overexpression was associated with aggressive phenotypic features in EEC and was associated inversely with ER and PR expression. Thus, EphA2 may be an important therapeutic target, especially in patients with hormone receptor‐negative endometrial carcinoma. Cancer 2009. © 2009 American Cancer Society.     

Matrix metalloproteinase-2 regulates vascular patterning and growth affecting tumor cell survival and invasion in GBM

Glioblastoma multiforme (GBM) is one the most aggressive brain tumors due to the fast and invasive growth that is partly supported by the presence of extensive neovascularization. The matrix metalloproteinase MMP-2 has been associated with invasive and angiogenic properties in gliomas and is a marker of poor prognosis. Since MMP-2 is expressed in both tumor cells and endothelial cells in GBM, we generated genetically engineered MMP-2 knockout (MMP-2ko) GBM to examine the importance of the spatial expression of MMP-2 in tumor and/or normal host-derived cells. MMP-2-dependent effects appeared to be dose-dependent irrespective of its expression pattern. GBM completely devoid of MMP-2 exhibited markedly increased vascular density associated with vascular endothelial growth factor receptor 2 (VEGFR2) activation and enhanced vascular branching and sprouting. Surprisingly, despite the high vascular density, tumor cells were more prone to apoptosis, which led to prolonged survival of tumor-bearing mice, suggesting that the increased vascularity is not functional. Congruently, tumor vessels were poorly perfused, exhibited lower levels of VEGFR2, and did not undergo proper maturation because pericytes of MMP-2ko tumors were not activated and were less abundant. As a result of impaired and dysfunctional angiogenesis, MMP-2ko GBM became more invasive, predominantly by migrating along blood vessels into the brain parenchyma.     

Bevacizumab impairs oxidative energy metabolism and shows antitumoral effects in recurrent glioblastomas: a 31P/1H MRSI and quantitative magnetic resonance imaging study

Bevacizumab shows unprecedented rates of response in recurrent glioblastomas (GBM), but the detailed mechanisms are still unclear. We employed in vivo magnetic resonance spectroscopic imaging (MRSI) and quantitative magnetic resonance imaging to investigate whether bevacizumab alters oxygen and energy metabolism and whether this effect has antitumoral activity in recurrent GBM. (31)P and (1)H MRSI, apparent diffusion coefficient (ADC), and high-resolution T2 and T2' mapping (indirect marker of oxygen extraction) were investigated in 16 patients with recurrent GBM at 3 Tesla before and 1.5-2 months after initiation of therapy with bevacizumab. Changes of metabolite concentrations and of the quantitative values in the tumor and normal appearing brain tissue were calculated. The Wilcoxon signed-ranks test was used to evaluate differences for tumor/edema versus control as well as changes before versus after commencement of therapy. Survival analyses were performed for significant parameters. Tumor T2', pH, ADC, and T2 decreased significantly in patients responding to bevacizumab therapy (n = 10). Patients with at least 25% T2' decrease during treatment showed longer progression-free and overall survival durations. Levels of high-energy metabolites were lower at baseline; these persisted under therapy. Glycerophosphoethanolamine as catabolic phospholipid metabolite increased in responders. The MRSI data support the hypothesis that bevacizumab induces relative tumor hypoxia (T2' decrease) and affects energy homeostasis in recurrent GBM, suggesting that bevacizumab impairs vascular function. The antiangiogenic effect of bevacizumab is predictive of better outcome and seems to induce antitumoral activity in the responding GBMs.