Marta Pojo

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

Background Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods Expression of hypoxia- and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (α-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.

Molecular Hallmarks of Gliomas

Tumors of the central nervous system (CNS) encompass a wide variety of entities, which span from benign to highly malignant. The classification of these tumors is typically based on their histopathological features or their location within the CNS. Despite these apparently simple criteria, there are a great number of independent CNS tumor types as defined by the most recent World Health Organization (WHO) classification of CNS tumors (Louis et al., 2007), which is the standard for the definition of CNS tumors worldwide. The WHO listing of CNS tumors is impressively vast and has, in fact, been surrounded by some controversy concerning the nosology of some tumor entities (e.g., the nosologic place of highly anaplastic oligoastrocytic tumors, glioblastoma with oligodendroglioma components). The agestandardized incidence rate of all primary non-malignant and malignant CNS tumors in the US is 16.5 per 100,000 person–years (9.2 per 100,000 person–years for non–malignant tumors and 7.3 per 100,000 person–years for malignant tumors) (CBTRUS, 2010). This rate is higher in females (17.2 per 100,000 person–years) than males (15.8 per 100,000 person–years). Worldwide data is available only for malignant primary CNS tumors; in this setting, the incidence rates are higher in males (approximately 3.7 per 100,000 person-years) than females (2.6 per 100,000 person–years) (Ferlay et al., 2008). In Western Europe, the male and female incidence rates of malignant CNS tumors are 6.7 per 100,000 person-years and 4.5 per 100,000 person-years, respectively. Very similar figures are observed in Northern America (6.0 and 4.5 per 100,000 person-years for males and females, respectively). Interestingly, the incidence rates are higher in more developed countries than in less developed ones, but these differences may be a consequence of differences in diagnostic practices, completeness of reporting and access to adequate health care, rather than attributable to geographic and genetic variation. CNS tumors are considered to be primary when the tumor originally initiates in the CNS, as opposed to the far more common brain metastases derived from malignant tumors located in other organs, which are considered secondary brain tumors. Primary brain and CNS tumors account for only approximately 2% of all primary tumors (Louis et al., 2007), but they rank first among tumor types for the average years of life lost (~20 years, compared, for example, with ~6 years for prostate cancer and ~12 years for lung cancer) (Burnet et al., 2005). These tumors are the most frequent solid malignancy in children, being the leading cause of cancer-related death in children under the age of 19 (Rickert & Paulus, 2001). The impact and nature of primary brain tumors in adults is somewhat different, but they still rank second as cause of cancer death in males aged 20 to 39 years, and fifth in females of

Effects of the functional HOTAIR rs920778 and rs12826786 genetic variants in glioma susceptibility and patient prognosis

Abnormal expression of the long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is oncogenic in several human cancers, including gliomas. The HOTAIR single nucleotide polymorphisms (SNPs) rs920778 (C > T) and rs12826786 (C > T) present in the intronic enhancer and promoter regions of HOTAIR, respectively, are associated with expression, cancer susceptibility, and patient prognosis in some tumor types. However, the relevance of these HOTAIR SNPs has not been studied in glioma. Here, we report a case-control study comprising 177 Portuguese glioma patients and 199 cancer-free controls. All subjects were genotyped by PCR and restriction fragment length polymorphism (RFLP). No statistically significant differences were found in the genotype or allele distributions of either rs920778 or rs12826786 between glioma patients and controls, suggesting these SNPs are not associated with glioma risk. No significant associations were found between rs920778 variants and HOTAIR expression levels, while rs12826786 CT genotype was associated with increased intratumoral HOTAIR RNA levels when compared to TT genotype (p-value = 0.04). Univariate (Log-rank) and multivariate (Cox proportional) analyses showed both rs920778 CT and rs12826786 CT genotypes were significantly associated with longer overall survival of WHO grade III anaplastic oligodendroglioma patients. Our results suggest that HOTAIR SNPs rs920778 and rs12826786 do not play a significant role in glioma susceptibility, but may be important prognostic factors in anaplastic oligodendroglioma patients. Future studies are warranted to validate and expand these findings, and to further dissect the importance of these SNPs in glioma.

Mechanisms of Aggressiveness in Glioblastoma: Prognostic and Potential Therapeutic Insights

Glioblastoma (GBM) is the most prevalent and most malignant (WHO grade IV) type of brain tumor in adults [1, 2]. In the United States, there are ~10,000 new cases diagnosed annually, and >50,000 patients living with the disease [2, 3]. The clinical responses of patients are particularly poor and vary greatly among individuals [4], and ~32% of all diagnosed cases survive less than a year [3]. This highly aggressive tumor develops either de novo (primary GBM), or as the result of the malignant progression from a lower-grade glioma (secondary GBM). In both cases, prognosis is very poor, and the median survival when radiotherapy and chemotherapy are combined is approximately 15 months [5]. Importantly, GBM is also characterized by extensive heterogeneity at the cellular and molecular levels. These tumors are highly diffuse, with extensive dissemination of tumor cells within the brain, which hinders complete surgical resection. These aggressive characteristics are associated with a remarkable resistance to therapies available today [6], which unfortunately are mostly palliative. In the context of their highest incidence of all malignant brain tumors in adults, their highly aggres‐ sive behavior and therapy-insensitive nature, which together account for a very poor prognosis of GBM patients, this chapter will focus specifically on GBM. In particular, it will review the different hypotheses of glioma/GBM-initiating cells, the major alterations at the levels of gene expression and signaling pathways found in GBM, as well as putative biomarkers of GBM prognosis, and current therapies currently available or under investigation for dealing with these tumors.

WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

Glioblastoma (GBM) is a universally fatal brain cancer, for which novel therapies targeting specific underlying oncogenic events are urgently needed. While the WNT pathway has been shown to be frequently activated in GBM, constituting a potential therapeutic target, the relevance of WNT6, an activator of this pathway, remains unknown. Methods: WNT6 protein and mRNA levels were evaluated in GBM. WNT6 levels were silenced or overexpressed in GBM cells to assess functional effects in vitro and in vivo. Phospho-kinase arrays and TCF/LEF reporter assays were used to identify WNT6-signaling pathways, and significant associations with stem cell features and cancer-related pathways were validated in patients. Survival analyses were performed with Cox regression and Log-rank tests. Meta-analyses were used to calculate the estimated pooled effect. Results: We show that WNT6 is significantly overexpressed in GBMs, as compared to lower-grade gliomas and normal brain, at mRNA and protein levels. Functionally, WNT6 increases typical oncogenic activities in GBM cells, including viability, proliferation, glioma stem cell capacity, invasion, migration, and resistance to temozolomide chemotherapy. Concordantly, in in vivo orthotopic GBM mice models, using both overexpressing and silencing models, WNT6 expression was associated with shorter overall survival, and increased features of tumor aggressiveness. Mechanistically, WNT6 contributes to activate typical oncogenic pathways, including Src and STAT, which intertwined with the WNT pathway may be critical effectors of WNT6-associated aggressiveness in GBM. Clinically, we establish WNT6 as an independent prognostic biomarker of shorter survival in GBM patients from several independent cohorts. Conclusion: Our findings establish WNT6 as a novel oncogene in GBM, opening opportunities to develop more rational therapies to treat this highly aggressive tumor.

PO-105 CDH3/P-cadherin as a novel biomarker in glioblastoma: functional and prognostic insights

Introduction Glioblastoma (GBM) is the most common and malignant primary brain tumour in adults. Patients diagnosed with GBM exhibit a poor prognosis with a median survival of approximately 15 months. P-cadherin (encoded by CDH3) is a classical cadherin that contributes for cell-cell adhesion by homophilic interactions, and is being studied and considered an attractive therapeutic target in several cancer types. Critically, its relevance in GBM is completely unknown. In this study, we investigate the functional roles and prognostic value of P-cadherin in this highly malignant form of gliomas. Material and methods CDH3 mRNA expression was evaluated in glioma patients available in TCGA database, and at the protein level by immunohistochemistry in our dataset of glioma patients from Hospital Braga. CDH3/P-cadherin functional roles were assessed in vitro using silencing (in two primary GBM cell lines) and overexpression (a commercially available GBM cell line) models. In vitro functional assays included evaluation of cell viability (trypan blue and MTS), migration (wound healing), invasion (Matrigel chamber), and stemness features (neurosphere formation). In vivo, subcutaneous and orthotopic intracranial GBM xenografts were established in Nude and NOD-scid gamma (NSG) mice, respectively. CDH3 expression in GBM samples was evaluated by RT-qPCR and its relevance in patients’ prognosis was evaluated using the Log-rank test. Results and discussions We found CDH3 expression is higher in high-grade gliomas. CDH3-silencing and overexpression GBM models show CDH3/P-Cadherin regulates GBM cell viability, invasion, migration and stemness capacity. In vivo CDH3-overexpressing cells generate larger subcutaneous tumours, and when orthotopically injected cause shorter overall survival of mice. Concordantly, higher CDH3 expression is predictive of shorter overall and recurrence-free survivals in various cohorts of GBM patients. Conclusion Together, our results show for the first time CDH3/P-cadherin is associated with GBM aggressiveness and patient prognosis, suggesting it may be an attractive therapeutic target in this deadly brain tumour.

The long non-coding RNA HOTAIR is transcriptionally activated by HOXA9 and is an independent prognostic marker in patients with malignant glioma

The lncRNA HOTAIR has been implicated in several human cancers. Here, we evaluated the molecular alterations and upstream regulatory mechanisms of HOTAIR in glioma, the most common primary brain tumors, and its clinical relevance. HOTAIR gene expression, methylation, copy-number and prognostic value were investigated in human gliomas integrating data from online datasets and our cohorts. High levels of HOTAIR were associated with higher grades of glioma, particularly IDH wild-type cases. Mechanistically, HOTAIR was overexpressed in a gene dosage-independent manner, while DNA methylation levels of particular CpGs in HOTAIR locus were associated with HOTAIR expression levels in GBM clinical specimens and cell lines. Concordantly, the demethylating agent 5-Aza-2′-deoxycytidine affected HOTAIR transcriptional levels in a cell line-dependent manner. Importantly, HOTAIR was frequently co-expressed with HOXA9 in high-grade gliomas from TCGA, Oncomine, and our Portuguese and French datasets. Integrated in silico analyses, chromatin immunoprecipitation, and qPCR data showed that HOXA9 binds directly to the promoter of HOTAIR. Clinically, GBM patients with high HOTAIR expression had a significantly reduced overall survival, independently of other prognostic variables. In summary, this work reveals HOXA9 as a novel direct regulator of HOTAIR, and establishes HOTAIR as an independent prognostic marker, providing new therapeutic opportunities to treat this highly aggressive cancer.