The Status of EGFR Modulates the Effect of miRNA-200c on ZEB1 Expression and Cell Migration in Glioblastoma Cells.

Migration of glioblastoma cells into surrounding tissue is one of the main features that makes this tumor incurable. We evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns in 30 cases of primary glioblastoma. From the 64 miRNAs that showed differential expression between tumors with a high level of EGFR amplification and tumors without EGFR amplification, 40% were related with cell migration, being miR-200c the most differentially expressed between these two groups. We investigated the effect of miR-200c on ZEB1 expression and cell migration in an in vitro transfection model with a miR-200c mimic, a miR-200c inhibitor and siRNA targeting EGFR in three short-term cultures with different levels of EGFR amplification obtained from resected glioblastomas. The cell culture with the highest EGFR amplification level presented the lowest miR-200c expression and the status of EGFR modulated the effect of miR-200c on ZEB1 expression. Silencing EGFR led to miR-200c upregulation and ZEB1 downregulation in transfected cultures, except in the presence of high levels of EGFR. Likewise, miR-200c upregulation decreased ZEB1 expression and inhibited cell migration, especially when EGFR was not amplified. Our results suggest that modulating miR-200c may serve as a novel therapeutic approach for glioblastoma depending on EGFR status.

Pam3CSK4, a TLR2 ligand, induces differentiation of glioblastoma stem cells and confers susceptibility to temozolomide.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and GBM stem cells (GSC) may be responsible for its recurrence and therapeutic resistance. Toll-like receptors (TLRs), which recognize multiple ligands (endogenous and pathogen-associated) and trigger the immune response of mature immune cells, are also expressed by hematopoietic stem and progenitor cells, where their activation results in the differentiation of these cells into myeloid cells. Since TLR expression has been recently described in neural cells, including neural stem cells, we studied TLR expression by GSCs and the effect of stimulation by TLR ligands on promoting GSC differentiation into mature GBM cells. First, our results showed heterogeneous TLR expression by GBM cells from human tumors and, for the first time, by human GSCs defined by their CD133+ and CD44+ phenotypes. Next, the effect of TLR ligands was studied in in vitro cell cultures of neurospheres and CD44+ cells obtained from two GBM cell lines (U-87 and U-118). The expression of GSC markers diminished in the presence of Pam3CSK4 or LPS (TLR2 and TLR4 ligands, respectively), thus indicating TLR-dependent differentiation. Interestingly, simultaneous treatment with Pam3CSK4 plus temozolomide (TMZ), the reference drug in GBM treatment, significantly increased cell death compared to the effect of the ligand alone, which showed no toxicity, or TMZ alone. These results suggest a synergistic effect between Pam3CSK4 and TMZ based on the induction of TLR-dependent GSC differentiation towards mature GBM cells, which exhibited increased sensitivity to chemotherapy, and provide new perspectives in GBM therapy.

Characterization of a new glioblastoma cell line, GB-val4, with unusual TP53 mutation.

A novel cell line derived from a human glioblastoma (GB), named GB-val4, has been established and characterized. GB-val4 cells were hyperdiploid, with many numerical and structural chromosomal rearrangements. The cell line did not show mutations in IDH1/IDH2 genes or EGFR amplification, but it presented two missense mutations in TP53, which imply a very low p53 protein activity within the cell line. Cells also had gain of TP73 copies, hypermethylation of APC, CASP8 and RASSF1, increased expression of ARF1, CDH1 and NF-κB and decreased expression of CDKN2A. Tumorigenity was demonstrated by transplant of GB-val4 cells into athymic nude mice, where solid tumors were grown. Interestingly, a high percentage of GB-val4 cells presented expression of GSC markers CD133 or CD44. These GSC markers were increased in neurosphere cultures, which better mimic solid tumor conditions and maintain the genetic features of the tumor cells. In this study, we aimed to define the characteristics of this novel cell line and its applications in human cancer research. With its genetic features and a poor p53 activity, GB-val4 cells resemble GB tumors. Moreover, the important presence of GSCs in adherent cultures and especially in neurosphere cultures makes GB-val4 an attractive tool to study cancer stem cells, deepen in the knowledge the molecular pathways of GB and develop new therapeutic strategies for patients with these tumors.

Epigenetic changes underlie the aggressiveness of histologically benign meningiomas that recur.

Meningiomas are the most frequent primary brain tumor. Usually, they are curable by surgery, but even after seemingly complete resection, some low-grade lesions recur. Despite recent improvements, signatures having prognostic value in grade I tumors remain poorly characterized. The frequency and delicate location of these tumors suggest that the risk of recurrence might be more accurately predicted. Herein, we show an easy way to evaluate the methylation status of meningiomas and its correlation with the prognosis of the disease. A series of 120 meningiomas, including primary tumors and recurrences, were analyzed histopathologically, and 24 tumor suppressor genes (TSGs) were studied by methylation-specific multiple ligation probe amplification. Long-term follow-up was conducted to classify patients with grade I primary tumors according to their outcomes. We found that hypermethylation in at least one TSG is frequent. The number of hypermethylated TSG per case was significantly higher in recurrences than in primary tumors and in primary benign meningiomas that recurred than in tumors from patients who showed no evidence of disease during follow-up. Finally, hypermethylation in RASSF1A, MLH1, and CDKN2B was an independent prognostic factor associated with the time to recurrence of these benign tumors that were biologically aggressive. To our knowledge, this is one of the widest studies of primary grade I tumors of patients who developed a tumor recurrence. The frequency of epigenetic changes suggests that hypermethylation is an early event in meningiomas, whereas the accumulation of epigenetic changes is related to greater biological aggressiveness and may be a signature of potential clinical relevance.

Characterization of the new human pleomorphic undifferentiated sarcoma TP53-null cell line mfh-val2.

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm2, a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.

Association between epidermal growth factor receptor amplification and ADP-ribosylation factor 1 methylation in human glioblastoma.

PURPOSE: Glioblastoma (GB) is the most frequent and most malignant primary brain tumor in adults. Previously, it has been found that both genetic and epigenetic factors may play critical roles in its etiology and prognosis. In addition, it has been found that the epidermal growth factor receptor gene (EGFR) is frequently over-expressed and amplified in primary GBs. Here, we assessed the promoter methylation status of 10 genes relevant to GB and explored associations between these findings and the EGFR gene amplification status. METHODS: Tumor samples were obtained from 36 patients with primary GBs. In addition, 6 control specimens were included from patients who were operated for diseases other than brain tumors. The amplification status of the EGFR gene, and its deletion mutant EGFRvIII, were evaluated using FISH and MLPA, respectively. The IDH1/2 gene mutation status was verified using Sanger sequencing. A commercial DNA methylation kit was used to assess the promoter methylation status of 10 pre-selected genes. Metabolic profiles were measured using HR-MAS NMR spectroscopy. The EGFR and ARF1 mRNA expression levels were quantified using qRT-PCR. RESULTS: Of the 10 genes analyzed, we found that only ARF1 promoter hypermethylation was significantly associated with EGFR gene amplification. ARF1 is a GTPase that is involved in vesicle trafficking and the Golgi apparatus. Subsequent tumor metabolism measurements revealed a positive association between EGFR amplification and different membrane precursors and methyl-donor metabolites. Finally, we found that EGFR gene amplifications were associated with distinct tumor infiltration patterns, thus representing a putative novel functional association between EGFR gene amplification and ARF1 gene promoter methylation in GB. CONCLUSIONS: The results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism(s) underlying these connections and their functional consequences remain to be established.

Expression of the Chemokine Receptors CXCR3, CXCR4, CXCR7 and Their Ligands in Rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are soft tissue malignant tumors of childhood and adolescents. The mechanisms underlying their aggressiveness are still poorly understood. Chemokines are chemotactic proteins involved in pathological processes that have been intensely studied in several types of cancers because of their influence in migration, angiogenesis, or metastases. We analyzed the expression of the chemokine receptors CXCR3, CXCR4 and CXCR7 and their ligands CXCL9, CXCL10, CXCL11 and CXCL12, in 15 RMS samples derived from nine patients. Expression was measured in tumors and primary cultures of RMS by Real-Time Polymerase Chain Reaction, immunostaining and flow cytometry. Our results show that these receptors are widely expressed in RMS. A significant difference between CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR7 expression ratios was found in alveolar versus embryonal RMS and similarly between CXCL12/CXCR4 and CXCL11/CXCR3 ratios in primary versus recurrent tumors. These findings suggest a possible association between the interrelation of chemokine/chemokine-receptor and an aggressive biological behavior in RMS.

Correlation between EGFR amplification and the expression of microRNA-200c in primary glioblastoma multiforme.

Extensive infiltration of the surrounding healthy brain tissue is a critical feature in glioblastoma. Several miRNAs have been related to gliomagenesis, some of them related with the EGFR pathway. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns, studied by fluorescence in situ hybridization in tissue microarrays, of 30 cases of primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have also been analyzed. MicroRNA-200c showed a very significant difference between tumors having or not EGFR amplification. This microRNA plays an important role in epithelial-mesenchymal transition, but its implication in the behavior of glioblastoma is largely unknown. With respect to EGFR status our cases were categorized into three groups: high level EGFR amplification, low level EGFR amplification, and no EGFR amplification. Our results showed that microRNA-200c and E-cadherin expression are down-regulated, while ZEB1 is up-regulated, when tumors showed a high level of EGFR amplification. Conversely, ZEB1 mRNA expression levels were significantly lower in the group of tumors without EGFR amplification. Tumors with a low level of EGFR amplification showed ZEB1 expression levels comparable to those detected in the group with a high level of amplification. In this study we provide what is to our knowledge the first report of association between microRNA-200c and EGFR amplification in glioblastomas.

Monitoring of the action of drugs in melanoma cells by dynamic laser speckle.

This work presents the development of a protocol based on the dynamic laser speckle designed to monitor the reaction of cancer cells of line MEL-RC08 to the application of the drug Colcemid in two different concentrations: 0.2 and 0.4 µg/mL. The protocol was designed using the forward scattering approach with an He-Ne laser of 632.8 nm illuminating the samples, a control, and two variations of Colcemid, being monitored along 8 h. The data were analyzed numerically in the time and in the frequency domain, and the results presented the ability of the technique to monitor the action of the drug, particularly Colcemid (0.4 µg/mL).

Intracellular coexpression of CXC- and CC- chemokine receptors and their ligands in human melanoma cell lines and dynamic variations after xenotransplantation.

BACKGROUND: Chemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival. METHODS: In this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed. RESULTS: Our results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface. CONCLUSIONS: Coexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

Gene expression profiles of metabolic aggressiveness and tumor recurrence in benign meningioma.

Around 20% of meningiomas histologically benign may be clinically aggressive and recur. This strongly affects management of meningioma patients. There is a need to evaluate the potential aggressiveness of an individual meningioma. Additional criteria for better classification of meningiomas will improve clinical decisions as well as patient follow up strategy after surgery. The aim of this study was to determine the relationship between gene expression profiles and new metabolic subgroups of benign meningioma with potential clinical relevance. Forty benign and fourteen atypical meningioma tissue samples were included in the study. We obtained metabolic profiles by NMR and recurrence after surgery information for all of them. We measured gene expression by oligonucleotide microarray measurements on 19 of them. To our knowledge, this is the first time that distinct gene expression profiles are reported for benign meningioma molecular subgroups with clinical correlation. Our results show that metabolic aggressiveness in otherwise histological benign meningioma proceeds mostly through alterations in the expression of genes involved in the regulation of transcription, mainly the LMO3 gene. Genes involved in tumor metabolism, like IGF1R, are also differentially expressed in those meningioma subgroups with higher rates of membrane turnover, higher energy demand and increased resistance to apoptosis. These new subgroups of benign meningiomas exhibit different rates of recurrence. This work shows that benign meningioma with metabolic aggressiveness constitute a subgroup of potentially recurrent tumors in which alterations in genes regulating critical features of aggressiveness, like increased angiogenesis or cell invasion, are still no predominant. The determination of these gene expression biosignatures may allow the early detection of clinically aggressive tumors.

Genetic changes with prognostic value in histologically benign meningiomas.

Meningiomas add up to 25% of intracranial tumors. Although the majority is considered histologically benign, the prediction of their potential aggressiveness is still unclear. We studied the histopathology and aberrations of chromosomes 1p, 14, and 22 by FISH (fluorescence in situ hybridization) in histologically benign meningiomas of 70 patients for the purpose of defining the prognostic value of these alterations in tumoral progression and the risk of recurrence. According to the WHO histopathological criteria, the study set comprised 53 benign, 11 atypical, and 6 anaplastic meningiomas. In benign meningiomas, 25% of the cases displayed a normal karyotype, isolated monosomy 22 (36%), monosomy 22 + 1p deletion (14%), 1p deletion (10%), monosomy 22 + 14q deletion (5%), monosomy 22 + 1p deletion + 14q deletion (5%), or other alterations (5%). Grade II meningiomas presented losses in chromosome 14 in most of the cases (67%), and Grade III meningiomas showed alterations in chromosome 14 in all patients. We observed an overall relapse rate of 31%: recurrence was observed in 19% of Grade I meningiomas, 64% of Grade II, and 83% of Grade III. 9 out of 10 recurrent cases revealed abnormalities in chromosomes 1 and 14, which was a notably higher incidence compared to the series of tumors without relapse. Thus, benign meningiomas with cytogenetic alterations in chromosomes 1p and 14 may be more closely related to atypical meningiomas than benign meningiomas without these alterations, especially in terms of recurrence risk.

Characterization of a new human melanoma cell line with CD133 expression.

A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line.

Metabolic aggressiveness in benign meningiomas with chromosomal instabilities.

Meningiomas are often considered benign tumors curable by surgery, but most recurrent meningiomas correspond to histologic benign tumors. Because alterations in chromosome 14 among others have suggested clinical aggressiveness and recurrence, determining both the molecular phenotype and the genetic profile may help distinguish tumors with aggressive metabolism. The aim of this study was to achieve higher specificity in the detection of meningioma subgroups by measuring chromosomal instabilities by fluorescence in situ hybridization and cytogenetics and metabolic phenotypes by high-resolution magic angle spinning spectroscopy. We studied 46 meningioma biopsies with these methodologies. Of these, 34 were of WHO grade 1 and 12 were of WHO grade 2. Genetic analysis showed a subgroup of histologic benign meningioma with chromosomal instabilities. The metabolic phenotype of this subgroup indicated an aggressive metabolism resembling that observed for atypical meningioma. According to the metabolic profiles, these tumors had increased energy demand, higher hypoxic conditions, increased membrane turnover and cell proliferation, and possibly increased resistance to apoptosis. Taken together, our results identify distinct metabolic phenotypes for otherwise benign meningiomas based on cytogenetic studies and global metabolic profiles of intact tumors. Measuring the metabolic phenotype of meningioma intact biopsies at the same time as histopathologic analysis may allow the early detection of clinically aggressive tumors.

New pattern of EGFR amplification in glioblastoma and the relationship of gene copy number with gene expression profile.

Gene amplification is a process that is characterized by an increase in the copy number of a restricted region in a chromosome arm, and is frequently associated with an overexpression of the corresponding amplified gene. Amplified DNA can be organized either as extrachromosomal elements, repeated units at a single locus or scattered throughout the genome. The amplification of the gene for epidermal growth factor receptor (EGFR) is a common finding in glioblastomas and the amplified gene copies appears as double minutes. The aim of this study was to investigate the different patterns of EGFR amplification in 40 cases of glioblastoma using FISH analysis in metaphases and paraffin sections, and to investigate the relationship of gene copy number with gene expression profile. The analysis of copy number alterations of EGFR was validated by quantitative PCR and SNP microarrays. We observed that in 42% of the cases, the type of amplification of EGFR was as double minute chromosomes. In addition, we detected another type of amplification, with extra copies of EGFR inserted in different loci of chromosome 7, present in 28% of cases. In this form of amplification, the number of copies is small, and the percentage of cells with EGFR amplification is rarely more than 15%. This model of amplification could correspond to a variant of the insertion mechanism, or a consequence of a process of duplication. Our results suggest that this mechanism could represent an early stage of amplification in glioblastomas. Overall, we found a close correlation between EGFR gene copy-number alterations and the level of EGFR protein expression. However, all cases with a high level of mRNA exhibited strong expression for the EGFR protein, and most cases with a low level of mRNA showed no overexpression of EGFR protein.

Primary glioblastomas with and without EGFR amplification: relationship to genetic alterations and clinicopathological features.

Glioblastomas express a notable heterogeneity in both the histological and cell patterns with glial astrocytic differentiation. Primary glioblastoma, which is the most frequent presentation (90-95%), occurs mainly in older patients and arises de novo, without any clinical or histological evidence of a less malignant precursor lesion. EGFR amplification has been identified as a genetic hallmark of primary glioblastomas and occurs in 40-60% of cases. However, there exist primary glioblastomas without EGFR amplification/overexpression. The purpose of this study was to stabilize the association between cases with and without EGFR gene amplification with clinical and genetic parameters in 45 cases of primary glioblastomas. EGFR amplification was observed in 24 cases (53%), while in the remaining 21 cases (47%) this alteration was not displayed. And whereas EGFR was overexpressed in 79% of cases with EGFR amplification, only 33% of the cases without EGFR amplification showed overexpression. The amplification of EGFR was associated with amplifications in MDM2 and CDK4 and a higher percentage of cases with promoter methylation of INK4a. Only one case of glioblastoma with EGFR amplification presented TP53 mutation simultaneously. Seven remaining cases with TP53 mutations were glioblastomas without EGFR amplification. The INK4a, INK4b and ARF deletions were similar in the two groups. Primary glioblastomas with and without EGFR amplification did not show any significant differences in average survival. The genetic studies suggest the existence of molecular subtypes within primary glioblastoma that may, when fully defined, contribute toward the development of drugs that specifically target tumors with divergent genetic profiles.

Interaction of dicaffeoylquinic derivatives with peroxynitrite and other reactive nitrogen species.

Plant phenolic antioxidants, among them catechins and hydroxycinnamoyl conjugates, constitute a well defined class of inhibitors of reactive nitrogen species (RNS). To gain deeper insight in this field, we examined the effects of 3,5-di-O-caffeoylquinic acid (DCA), its methyl ester (DCE) and epigallocatechin gallate (EGCG) in nitrative and oxidative processes. These compounds were found to be strong inhibitors of the nitration of tyrosine residues induced by ONOO- in bovine seroalbumin, with their IC50 values (10-40 microM) notably decreasing in the presence of bicarbonate. When studied on the intracellular protein tyrosine nitration induced by ONOO- in cultured murine fibroblasts as well as that induced by phorbol ester (PMA) in nitrite-supplemented human neutrophils, all three phenolics were also effective (100% and over 75% inhibition for fibroblasts and neutrophils, respectively, at 25 microM). This ability seems to be due to a direct interaction with ONOO- or with the species generated by leukocytes. The possible interference with the production of NO was also studied: both DCA and EGCG inhibited nitrite production in LPS-stimulated macrophages by 24% and 40%, respectively, and the expression of nitric oxide synthase-2 (NOS-2), as well. DCA and EGCG reduced by 52% and 59%, respectively, the NF-kappaB transcriptional activity. In contrast, DCE did not show any effect. The assayed phenolics exert varying degrees of protection against the chemical modifications induced by RNS depending not only on the hydroxyl pattern, but also on the presence of bicarbonate.