Hypoxia, inflammation and necrosis as determinants of glioblastoma cancer stem cells progression

Tumor hypoxic microenvironment causes hypoxia inducible factor 1 alpha (HIF-1ff) activation and necrosis with alarmins release. Importantly, HIF-1ff also controls the expression of alarmin receptors in tumor cells that can bind to and be activated by alarmins. Human tumor tissues possess 1-2% of cancer stem cells (CSCs) residing in hypoxic niches and responsible for the metastatic potential of tumors. Our hypothesis is that hypoxic CSCs express alarmin receptors that can bind alarmins released during necrosis, an event favoring CSCs migration. To investigate this aspect, glioblastoma stem-like cell (GSC) lines were kept under hypoxia to determine the expression of hypoxic markers as well as receptor for advanced glycation end products (RAGE). The presence of necrotic extracts increased migration, invasion and cellular adhesion. Importantly, HIF-1ff inhibition by digoxin or acriflavine prevented the response of GSCs to hypoxia alone or plus necrotic extracts. In vivo, GSCs injected in one brain hemisphere of NOD/SCID mice were induced to migrate to the other one in which a necrotic extract was previously injected. In conclusion, our results show that hypoxia is important not only for GSCs maintenance but also for guiding their response to external necrosis. Inhibition of hypoxic pathway may therefore represent a target for preventing brain invasion by glioblastoma stem cells (GSCs)

Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: Tumor suppressor role of lncRNA MEG3

Background: Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis. Methods: Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3). Results: Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan-Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT). Conclusion: In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies

miR-135b suppresses tumorigenesis in glioblastoma stem-like cells impairing proliferation, migration and self-renewal

Glioblastoma multiforme (GBM) is the most common and fatal malignant adult primary brain tumor. Currently, the overall prognosis for GBM patients remains poor despite advances in neurosurgery and adjuvant treatments. MicroRNAs (miRNAs) contribute to the pathogenesis of various types of tumor, including GBM. In this study we analyzed the expression of a panel of miRNAs, which are known to be differentially expressed by the brain and GBM tumor, in a collection of patient-derived GBM stem- like cells (GSCs). Notably, the average expression level of miR-135b, was the most downregulated compared to its normal counterpart, suggesting a potential role as anti-oncogene. Restoration of miR-135b in GSCs signi cantly decreased proliferation, migration and clonogenic abilities. More importantly, miR-135b restoration was able to signi cantly reduce brain in ltration in mouse models of GBM obtained by intracerebral injection of GSC lines. We identi ed ADAM12 and con rmed SMAD5 and GSK3\u3b2 as miR-135b targets and potential mediators of its effects. The whole transcriptome analysis ascertained that the expression of miR-135b downmodulated additional genes driving key pathways in GBM survival and in ltration capabilities. Our results identify a critical role of miR-135b in the regulation of GBM development, suggesting that miR-135b might act as a tumor-suppressor factor and thus providing a potential candidate for the treatment of GBM patients

G-quadruplex ligand RHPS4 radiosensitizes glioblastoma xenograft in vivo through a differential targeting of bulky differentiated- and stem-cancer cells

Background: Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far.Methods: RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments.Results: RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair.Conclusions: We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults

Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423, and Hey2

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high-grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2-deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma-derived CSC-like lines. These experiments identify a network of critical tumor suppressive Sox2-targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets

Development and Validation of a HPLC–MS/MS Method to Measure Nifuroxazide and Its Application in Healthy and Glioblastoma-Bearing Mice

Nifuroxazide (NAZ), a nitrofuran derivative used to treat diarrhea, has been recently shown to possess anticancer activity. However, its pharmacokinetic profile is poorly known. The pharmacokinetic profile of NAZ was thus investigated in mice using a newly developed method based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). We determined the concentrations of NAZ in the plasma and brain tissue of mice treated with the drug. The method proved to be specific, reproducible, precise, and accurate. It also demonstrated high sensitivity, reaching an LOQ in the order of ppb for both matrices, using samples of 100 µL or 0.2 g. The new HPLC–MS/MS assay was successfully applied to study the pharmacokinetics of NAZ after chronic intraperitoneal administration in mice at a dose of 30 mg/kg. One hour after treatment, plasma concentrations of NAZ were in the range of 336–2640 ng/mL. Moreover, unlike the brains of healthy mice or those with healed mechanical injuries, we found that NAZ was able to cross the injured blood–brain barrier of tumor-infiltrated brains. Thus, following i.p. administration, NAZ reaches systemic levels suitable for testing its efficacy in preclinical models of glioblastoma. Overall, these pharmacokinetic data provide robust evidence supporting the repositioning of NAZ as an antitumor drug

Rapid and Efficient Invasion Assay of Glioblastoma in Human Brain Organoids

Glioblastoma (GBM) possesses glioma stem cells (GSCs) that exhibit aggressive invasion behavior in the brain. Current preclinical GBM invasion assays using mouse brain xenografts are time consuming and less efficient. Here, we demonstrate an array of methods that allow rapid and efficient assaying of GSCs invasion in human brain organoids. The assays are versatile to characterize various aspects of GSCs, such as invasion, integration, and interaction with mature neurons of brain organoids. Tissue clearing and quantitative 3D imaging of GSCs in host organoids reveal that invasiveness is inversely correlated with the organoids' age. Importantly, the described invasion assays can distinguish the invasive behaviors of primary and recurrent GSCs. The assays are also amenable to test pharmacological agents. As an example, we show that GI254023X, an inhibitor of ADAM10, could prevent the integration of GSCs into the organoids

Effects of the combined treatment with a g‐quadruplex‐stabilizing ligand and photon beams on glioblastoma stem‐like cells: A magnetic resonance study

Glioblastoma multiforme is a malignant primary brain tumor with a poor prognosis and high rates of chemo‐radiotherapy failure, mainly due to a small cell fraction with stem‐like properties (GSCs). The mechanisms underlying GSC response to radiation need to be elucidated to enhance sensitivity to treatments and to develop new therapeutic strategies. In a previous study, two GSC lines, named line #1 and line #83, responded differently to carbon ions and photon beams, with the differences likely attributable to their own different metabolic fingerprint rather than to radiation type. Data from the literature showed the capability of RHPS4, a G‐quadruplex stabilizing ligand, to sensitize the glioblastoma radioresistant U251MG cells to X‐rays. The combined metabolic effect of ligand #190, a new RHPS4‐derivative showing reduced cardiotoxicity, and a photon beam has been monitored by magnetic resonance (MR) spectroscopy for the two GSC lines, #1 and #83, to reveal whether a synergistic response occurs. MR spectra from both lines were affected by single and combined treatments, but the variations of the analysed metabolites were statistically significant mainly in line #1, without synergistic effects due to combination. The multivariate analysis of ten metabolites shows a separation between control and treated samples in line #1 regardless of treatment type, while separation was not detected in line #83

Glioblastoma Stem-Like Cells (GSCs) with Mesenchymal Signature: Lipid Profiles of Mobile Lipids Obtained with MRS before and after Radio/Chemical Treatments

Glioblastoma is the most common and lethal primary malignant brain tumor in adults. Glioblastoma stem cells (GSCs) promote and are responsible for glioblastoma intratumoral heterogeneity and therapy resistance, due to their two main features: self-renewal and differentiation. Lipids have important biological and physiological functions that are critical for understanding the regulation and control of stem cell fate; lipid metabolism and related unsaturation levels play a possible role as the target of therapeutics to overcome glioblastoma radioresistance. This paper aimed at an in-depth analysis of 13 GSC mesenchymal (MES) lines, two subclones, and a stabilized glioblastoma line (T98G) by magnetic resonance spectroscopy (MRS). Particularly, 2D MRS was used to investigate lipid unsaturation behavior during growth in culture and after treatment with etomoxir and photon beams. MES lines, although belonging to the same genetic and metabolic cluster, showed metabolic heterogeneity when observed by MRS, focusing on lipid signals. Nonetheless, the observed unsaturation level stability for two representative lines after stressful treatments suggests unusual robustness of the unsaturation levels for each line, as a peculiar and intrinsic characteristic of GSCs

FMRP modulates the Wnt signalling pathway in glioblastoma.

Converging evidence indicates that the Fragile X Messenger Ribonucleoprotein (FMRP), which absent or mutated in Fragile X Syndrome (FXS), plays a role in many types of cancers. However, while FMRP roles in brain development and function have been extensively studied, its involvement in the biology of brain tumors remains largely unexplored. Here we show, in human glioblastoma (GBM) biopsies, that increased expression of FMRP directly correlates with a worse patient outcome. In contrast, reductions in FMRP correlate with a diminished tumor growth and proliferation of human GBM stem-like cells (GSCs) in vitro in a cell culture model and in vivo in mouse brain GSC xenografts. Consistently, increased FMRP levels promote GSC proliferation. To characterize the mechanism(s) by which FMRP regulates GSC proliferation, we performed GSC transcriptome analyses in GSCs expressing high levels of FMRP, and in these GSCs after knockdown of FMRP. We show that the WNT signalling is the most significantly enriched among the published FMRP target genes and genes involved in ASD. Consistently, we find that reductions in FMRP downregulate both the canonical WNT/β-Catenin and the non-canonical WNT-ERK1/2 signalling pathways, reducing the stability of several key transcription factors (i.e. β-Catenin, CREB and ETS1) previously implicated in the modulation of malignant features of glioma cells. Our findings support a key role for FMRP in GBM cancer progression, acting via regulation of WNT signalling