Expression of ABCB1, ABCC1 and 3 and ABCG2 in glioblastoma and their relevance in relation to clinical survival surrogates.

PURPOSE: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumors in adults. Patients invariably relapse during or after first-line therapy and the median overall survival is 14.6 months. Such poor clinical response is partly ascribed to the activity of ATP-binding cassette (ABC) transporters. The activity of these proteins, severely reduces the amount of therapeutics that penetrates the tumor cells. We hypothesized that ABC transporter expression could correlate with survival surrogates. In this study, we assessed the expression of four commonly expressed ABC transporters in GBM samples and investigated if mRNA levels could serve as a prognostic biomarker. METHODS: Human specimens were analyzed by qPCR to assess ABCB1, ABCC1/3 and ABCG2 expression. Kaplan-Meier and multivariate analyses were then used to evaluate the correlation with overall survival (OS) and progression-free survival (PFS). RESULTS: Our cohort included 22 non-tumoral samples as well as 159 GBM tumor specimens. ABC transporters were significantly more expressed in GBM samples compared to non-tumoral tissue. Moreover ABCC1 and 3 mRNA expression were significantly increased at recurrence. Statistical analyses revealed that increased expression of either ABCC1 or ABCC3 did not confer a poorer prognosis. However, increased ABCC1 mRNA levels did correlate with a significantly shorter PFS. CONCLUSION: In this manuscript, the analyses we conducted suggest that the expression of the four ABC transporters evaluated would not be suitable prognostic biomarkers. We believe that, when estimating prognosis, the plethora of mechanisms implicated in chemoresistance should be analyzed as a multi-facetted entity rather than isolated units.

Differential Expression and Clinical Significance of Transforming Growth Factor-Beta Isoforms in GBM Tumors.

Glioblastoma (GBM) represents the most common and aggressive malignant primary brain tumors in adults. Response to standard treatment is transitory and the survival of clinical trial cohorts are little more than 14 months. GBM are characterized by excessive proliferation, invasiveness, and radio-/chemoresistance features; which are strongly upregulated by transforming growth factor-beta (TGF-ß). We hypothesized that TGF-ß gene expression could correlate with overall survival (OS) and serve as a prognostic biomarker. TGF-ß1 and -ß2 expression were analyzed by qPCR in 159 GBM tumor specimens. Kaplan-Meier and multivariate analyses were used to correlate expression with OS and progression-free survival (PFS). In GBM, TGF-ß1 and -ß2 levels were 33- and 11-fold higher respectively than in non-tumoral samples. Kaplan-Meier and multivariate analyses revealed that high to moderate expressions of TGF-ß1 significantly conferred a strikingly poorer OS and PFS in newly diagnosed patients. Interestingly, at relapse, neither isoforms had meaningful impact on clinical evolution. We demonstrate that TGF-ß1 is the dominant isoform in newly diagnosed GBM rather than the previously acknowledged TGF-ß2. We believe our study is the first to unveil a significant relationship between TGF-ß1 expression and OS or PFS in newly diagnosed GBM. TGF-ß1 could serve as a prognostic biomarker or target affecting treatment planning and patient follow-up.

Chloroquine inhibits the malignant phenotype of glioblastoma partially by suppressing TGF-beta.

BACKGROUND: Glioblastoma (GBM), the most common and aggressive primary brain tumor, is characterized by excessive brain infiltration which prevents the complete surgical resection. These tumors also display treatment non-compliance and responses to standard therapy are invariably transient; consequently, the prognosis barely exceeds 14 months and recurrence is inevitable. Accordingly, several new treatment strategies have been studied. One such option is the use of chloroquine (CQ), a lysosomotropic weak base and renowned antimalarial drug, that has shown promising results in several pre-clinical studies. In this paper, we investigate the efficiency of CQ to hinder the malignant phenotype of GBM, namely extensive proliferation, invasion and radio-resistance. RESULTS: In cell cycle analysis, proliferation assays and immunofluorescence, CQ treatments halved proliferation of primary cultures from GBM specimens and GBM cell lines (U-373 MG et U-87 MG). Gelatin zymography and Matrigel(TM)-coated transwell invasion assays also revealed a 50 % CQ induced inhibition of MMP-2 activity and GBM invasion. Concomitant treatment with CQ and radiation also radiosensitized GBM cells as shown by an accumulation in the G2/M phase, increased cell death and reduced clonogenic formation. Moreover, radiation-induced invasion was considerably restrained by CQ. We also observe that these effects are owed to CQ-induced inhibition of TGF-ß secretion and signaling pathway, a predominant growth factor in GBM progression. CONCLUSION: These results suggest that CQ, alone or as an adjuvant therapeutic, could be used to inhibit the GBM malignant phenotype and could benefit GBM afflicted patients.

Transforming growth factor-beta and its implication in the malignancy of gliomas.

Malignant gliomas are the most common type of primary malignant brain tumors. They are characterized by enhanced growing capabilities, neoangiogenic proliferation, and extensive infiltration of the brain parenchyma, which make their complete surgical resection impossible. Together with transient and refractory responses to standard therapy, these aggressive neoplasms are incurable and present a median survival of 12 to 14 months. Transforming growth factor-beta (TGF-ß) is a pleiotropic cytokine of which two of the three isoforms expressed in humans have been shown to be overexpressed proportionally to the histologic grade of glioma malignancy. The increase of chromosomal aberrations and genetic mutations observed in glioma cells turns TGF-ß into an oncogene. For that reason, it plays critical roles in glioma progression through induction of several genes implicated in many carcinogenic processes such as proliferation, angiogenesis, and invasion. Consequently, investigators have begun developing innovative therapeutics targeting this growth factor or its signaling pathway in an attempt to hinder TGF-ß's appalling effects in order to refine the treatment of malignant gliomas and improve their prognosis. In this paper, we extensively review the TGF-ß-induced oncogenic pathways and discuss the diverse new molecules targeting this growth factor.

Uptake and intracellular release kinetics of liposome formulations in glioma cells.

Glioblastoma (GBM) is the most malignant primary brain tumor in adults, and its prognosis remains very limited despite decades of research. Enhanced drug delivery to GBM using liposomes represents a promising therapeutic strategy. In this study, we describe a novel cationic and pH-sensitive liposome formulation composed of DPPC:DC-Chol:DOPE:DHPE Oregon Green producing efficient internalization and intracellular delivery to F98 and U-118 GBM cells. With a series of derived modifications of the lipid composition, we investigated the impact of membrane fluidity, steric stabilization and loss of both cationic and pH-sensitive components on cellular uptake and intracellular release kinetics by flow cytometry and confocal microscopy, respectively. DPPC:DC-Chol:DOPE:DHPE Oregon Green liposomes were strongly internalized in both cell lines within 6h. Following cellular uptake, liposomes traveled towards the nucleus (12h) and gradually released their cargo in the cytosol (over 24h). Modifications in liposomal composition of our original formulation had detrimental consequences on both the uptake and intracellular release kinetics in the two tested cell lines. Thus, we report a novel potent liposomal formulation for efficient cytosolic delivery of intracellular therapeutics such as chemotherapy agents and siRNAs to GBM cells.

The effect of RanBPM on the regulation of the hypothalamic-pituitary axis by thyroid hormone receptors is isoform-specific.

Although crucial for TH homeostasis, the molecular mechanisms responsible of thyroid hormone receptors (TRs)-mediated regulation of the hypothalamic-pituitary-thyroid axis (HPT) axis remain unclear. We examined the role played by TR-isoforms in combination with RanBPM, a novel coactivator of TRs. In transient transfections studies with the human TRH and TSH-alpha subunit promoters, we found that the overexpression of RanBPM increases the transcriptional activity of all TR-isoforms by a magnitude of 1.7- to 3-fold. The addition of RanBPM, in the absence of THs, increased the ligand-independent activation (LIA) of TRalpha1 and TRbeta1 on both promoters tested by 300% and 200%, respectively, whereas, the LIA of TRbeta2 was not significantly modified. This data reinforces the concept of isoform-specific regulation of genes of the HPT axis and demonstrates that RanBPM may be an important factor to achieve adequate regulation of nTREs in the presence of low TH levels.

Identification and characterization of RanBPM, a novel coactivator of thyroid hormone receptors.

Thyroid hormone receptors (TRs) are transcription factor members of the nuclear receptor superfamily. The transcriptional activity of TRs is controlled by thyroid hormones and cell-specific coregulators. Using the yeast two-hybrid system, we identified RanBPM as a new protein partner for TRs. RanBPM was initially discovered as an interacting partner for Ran, and was also shown to be a protein partner and coactivator of the androgen receptor. The novel interaction between RanBPM and TR isoforms was addressed by glutathione-S-transferase (GST) pull-down assays and co-immunoprecipitation in intact mammalian cells, where RanBPM was shown to bind TRs in a ligand-independent fashion. We also studied the regions implicated in the interaction with deletion mutants: the principal interacting region of RanBPM is comprised within its carboxyl-terminal end and the TR DNA-binding domain is sufficient to mediate the interaction. To investigate the potential role of RanBPM in thyroid hormone action, transient transfections with luciferase reporter genes were performed in CV-1 cells. We found that the over-expression of RanBPM increases the activation of TRETK- and DR+4-positive thyroid hormone response elements. Interestingly, over-expression of the truncated protein RanBPM55, which lacks the N-terminal polyglutaminated region but binds TRs, decreased the fold activation by almost 80%. Furthermore, we performed competition assays using transient transfection of RanBPM and increasing amounts of RanBPM55. This revealed that the stimulating effect on TR transactivation by the full-length protein is inhibited in a dose-dependent fashion by RanBPM55. This suggests that although the polyglutaminated region of RanBPM is not required for the binding to TRs, it is required for the stimulation of TR transactivation. Taken together, our results provide evidence that RanBPM is a potent novel coactivator for thyroid hormone receptors.

General receptor for phosphoinositides 1, a novel repressor of thyroid hormone receptor action that prevents deoxyribonucleic acid binding.

Thyroid hormone receptors (TRs) bind to response elements (TREs) located in the promoter region of target genes and modulate their transcription. The effects of TRs require the presence of coregulators that act as adaptor molecules between TRs and complexes that are involved in chromatin remodeling or that directly contact the basal transcription machinery. Using the yeast two-hybrid system, we identified a new interacting partner for TRs: GRP1 (general receptor for phosphoinositides-1), a nucleotide exchange factor, which had never been shown to interact with nuclear receptors. We reconfirmed the interaction between TRs and GRP1 in yeast and glutathione-S-transferase pull-down assays, and determined the areas of TRs and GRP1 involved in the interaction. Coimmunoprecipitation studies demonstrated that the interaction between GRP1 and TRs takes place in the cytoplasm and the nucleus of mammalian cells. To assess functional consequences of the interaction, we used transient transfection of CV-1 cells with TR and GRP1 expression vectors and luciferase reporter genes. On positive TREs, GRP1 decreased activation by 45-60%. On the negative TREs it increased repression by blunting the activation in the absence of T3, except for TRbeta2, which was not affected. Using EMSA, we have determined that addition of GRP1 diminishes the formation of TR/TR homodimers and TR/retinoid X receptor heterodimers on TREs, which could explain the effect of GRP1 on transcription. Furthermore, protein interaction assays using increasing concentrations of double-stranded TREs show a dose-dependent decrease of the interaction between GRP1 and TRs. The homo/heterodimers formed by TRs and retinoic X receptor-alpha were not influenced by the presence of GRP1, also suggesting that GRP1 interferes directly with DNA binding. Taken together, these data provide evidence that GRP1 is a new corepressor for TRs, which modulates both positive and negative regulation by T3 by decreasing TR-complex formation on TREs.