Effects of Lactate Transport Inhibition by AZD3965 in Muscle-Invasive Urothelial Bladder Cancer.

The Warburg Effect is characterized by high rates of glucose uptake and lactate production. Monocarboxylate transporters (MCTs) are crucial to avoid cellular acidosis by internal lactate accumulation, being largely overexpressed by cancer cells and associated with cancer aggressiveness. The MCT1-specific inhibitor AZD3965 has shown encouraging results in different cancer models. However, it has not been tested in urothelial bladder cancer (UBC), a neoplasm where rates of recurrence, progression and platinum-based resistance are generally elevated. We used two muscle-invasive UBC cell lines to study AZD3965 activity regarding lactate production, UBC cells' viability and proliferation, cell cycle profile, and migration and invasion properties. An "in vivo" assay with the chick chorioallantoic membrane model was additionally performed, as well as the combination of the compound with cisplatin. AZD3965 demonstrated anticancer activity upon low levels of MCT4, while a general lack of sensitivity was observed under MCT4 high expression. Cell viability, proliferation and migration were reduced, cell cycle was arrested, and tumor growth "in vivo" was inhibited. The compound sensitized these MCT4-low-expressing cells to cisplatin. Thus, AZD3965 seems to display anticancer properties in UBC under a low MCT4-expression setting, but additional studies are necessary to confirm AZD3965 activity in this cancer model.

Glucose Metabolism Reprogramming in Bladder Cancer: Hexokinase 2 (HK2) as Prognostic Biomarker and Target for Bladder Cancer Therapy.

Proliferating cancer cells are able to reprogram their energy metabolism, favouring glycolysis even in the presence of oxygen and fully functioning mitochondria. Research is needed to validate the glycolysis-related proteins as prognostic/predictive biomarkers in urothelial bladder carcinoma (UBC), a malignancy tagged by high recurrence rates and poor response to chemotherapy. Here, we assessed GLUT1, HK2, PFKL, PKM2, phospho-PDH, and LDHA immunoexpression in 76 UBC samples, differentiating among urothelial, fibroblast, and endothelial cells and among normoxic versus hypoxic areas. We additionally studied the functional effects of the HK2 inhibitor 2-deoxy-D-glucose (2DG) in "in vitro" and "in vivo" preclinical UBC models. We showed that the expression of the glycolysis-related proteins is associated with UBC aggressiveness and poor prognosis. HK2 remained as an independent prognostic factor for disease-free and overall survival. 2DG decreased the UBC cell's viability, proliferation, migration, and invasion; the inhibition of cell cycle progression and apoptosis occurrence was also verified. A significant reduction in tumour growth and blood vessel formation upon 2DG treatment was observed in the chick chorioallantoic membrane assay. 2DG potentiated the cisplatin-induced inhibition of cell viability in a cisplatin-resistant subline. This study highlights HK2 as a prognostic biomarker for UBC patients and demonstrates the potential benefits of using 2DG as a glycolysis inhibitor. Future studies should focus on integrating 2DG into chemotherapy design, as an attempt to overcome cisplatin resistance.

Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness.

Clear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT.

Chronic Stress Does Not Influence the Survival of Mouse Models of Glioblastoma.

The existence of a clear association between stress and cancer is still a matter of debate. Recent studies suggest that chronic stress is associated with some cancer types and may influence tumor initiation and patient prognosis, but its role in brain tumors is not known. Glioblastoma (GBM) is a highly malignant primary brain cancer, for which effective treatments do not exist. Understanding how chronic stress, or its effector hormones glucocorticoids (GCs), may modulate GBM aggressiveness is of great importance. To address this, we used both syngeneic and xenograft in vivo orthotopic mouse models of GBM, in immunocompetent C57BL/6J or immunodeficient NSG mice, respectively, to evaluate how different paradigms of stress exposure could influence GBM aggressiveness and animals' overall survival (OS). Our results demonstrated that a previous exposure to exogenous corticosterone administration, chronic restraint stress, or chronic unpredictable stress do not impact the OS of these mice models of GBM. Concordantly, ex vivo analyses of various GBM-relevant genes showed similar intra-tumor expression levels across all experimental groups. These findings suggest that corticosterone and chronic stress do not significantly affect GBM aggressiveness in murine models.

Cadherin switches during epithelial-mesenchymal transition: CDH4/RCAD downregulation reduces bladder cancer progression.

PURPOSE: Non-muscle invasive bladder cancer (NMIBC) is a highly recurrent disease that progresses to muscle-invasive bladder cancer (MIBC) in 5-25% of the cases. Epithelial-mesenchymal transition (EMT) has been associated with features of disease progression. Thus, we aimed to characterize the cadherin switch (CS), an EMT hallmark, and its regulatory mechanisms in bladder cancer (BlCa) progression, as well as the biological role of RCAD, a lesser-known cadherin, in bladder carcinogenesis. METHODS: Cadherin mRNA and promoter methylation levels were retrieved from The Cancer Genome Atlas (TCGA). Validation was performed in an independent set of 121 primary BlCa (NMIBC and MIBC) and 40 normal bladder samples from IPO Porto, using RT-qPCR and qMSP. Immunohistochemistry was performed in these samples and in 14 additional sarcomatoid BlCa. CRISPR-Cas9 was performed to explore the potential in vitro impact of RCAD on BlCa cell migration and invasion. RESULTS: In both the TCGA and IPO Porto BlCa cohorts, cadherin gene deregulation was observed compared to normal tissue samples, independent of promoter methylation. At the protein level, decreased E-cadherin and increased P- and R-cadherin expression was noted in BlCa tissues. In sarcomatoid BlCa the same trend was observed, with a more intense staining compared to that in conventional MIBCs. RCAD knockout considerably reduced the malignant properties of BlCa cells. CONCLUSIONS: Our data indicate that E-, P- and R-cadherin switches occur in BlCa, being associated with tumor progression. Promoter methylation is not the likely mechanism underlying cadherin expression deregulation. Our findings suggest an oncogenic role of RCAD in BlCa progression.

Cadherin-3 is a novel oncogenic biomarker with prognostic value in glioblastoma.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. The prognosis of patients is very poor, with a median overall survival of ~ 15 months after diagnosis. Cadherin-3 (also known as P-cadherin), a cell-cell adhesion molecule encoded by the CDH3 gene, is deregulated in several cancer types, but its relevance in GBM is unknown. In this study, we investigated the functional roles, the associated molecular signatures, and the prognostic value of CDH3/P-cadherin in this highly malignant brain tumor. CDH3/P-cadherin mRNA and protein levels were evaluated in human glioma samples. Knockdown and overexpression models of P-cadherin in GBM were used to evaluate its functional role in vitro and in vivo. CDH3-associated gene signatures were identified by enrichment analyses and correlations. The impact of CDH3 in the survival of GBM patients was assessed in independent cohorts using both univariable and multivariable models. We found that P-cadherin protein is expressed in a subset of gliomas, with an increased percentage of positive samples in grade IV tumors. Concordantly, CDH3 mRNA levels in glioma samples from The Cancer Genome Atlas (TCGA) database are increased in high-grade gliomas. P-cadherin displays oncogenic functions in multiple knockdown and overexpression GBM cell models by affecting cell viability, cell cycle, cell invasion, migration, and neurosphere formation capacity. Genes that were positively correlated with CDH3 are enriched for oncogenic pathways commonly activated in GBM. In vivo, GBM cells expressing high levels of P-cadherin generate larger subcutaneous tumors and cause shorter survival of mice in an orthotopic intracranial model. Concomitantly, high CDH3 expression is predictive of shorter overall survival of GBM patients in independent cohorts. Together, our results show that CDH3/P-cadherin expression is associated with aggressiveness features of GBM and poor patient prognosis, suggesting that it may be a novel therapeutic target for this deadly brain tumor.

MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma.

Background: Glioblastomas (GBMs) present remarkable metabolism reprograming, in which many cells display the "Warburg effect", with the production of high levels of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). We described previously that MCT1 is up-regulated in human GBM samples, and MCT1 inhibition decreases glioma cell viability and aggressiveness. In the present study, we aimed to unveil the role of MCT1 in GBM prognosis and to explore it as a target for GBM therapy in vivo. Methods: MCT1 activity and protein expression were inhibited by AR-C155858 and CHC compounds or stable knockdown with shRNA, respectively, to assess in vitro and in vivo the effects of MCT1 inhibition and on response of GBM to temozolomide. Survival analyses on GBM patient cohorts were performed using Cox regression and Log-rank tests. Results: High levels of MCT1 expression were revealed to be a predictor of poor prognosis in multiple cohorts of GBM patients. Functionally, in U251 GBM cells, MCT1 stable knockdown decreased glucose consumption and lactate efflux, compromising the response to the MCT1 inhibitors CHC and AR-C155858. MCT1 knockdown significantly increased the survival of orthotopic GBM intracranial mice models when compared to their control counterparts. Furthermore, MCT1 downregulation increased the sensitivity to temozolomide in vitro and in vivo, resulting in significantly longer mice survival. Conclusions: This work provides first evidence for MCT1 as a new prognostic biomarker of GBM survival and further supports MCT1 targeting, alone or in combination with classical chemotherapy, for the treatment of GBM.

Fucoidan/chitosan nanoparticles functionalized with anti-ErbB-2 target breast cancer cells and impair tumor growth in vivo.

The work herein presented reports the development of fucoidan/chitosan nanoparticles (NPs) loaded with gemcitabine and functionalized with ErbB-2 antibody at their surface (NPs + Gem + Ab). The maximum immobilization of ErbB-2 on NPs' surface was set at 10 µg mL-1 and resulted in NPs with a size around 160 nm, a polydispersity index of 0.18, and a zeta potential of 21 mV. ErbB-2 is overexpressed in some subtypes of breast cancers, and the targeting capability of the NPs + Gem + Ab system was confirmed by an increased cellular uptake of SKBR3 cells (ErbB-2 positive) when compared to MDA-MB-231 (ErbB-2 negative). To validate the targeting efficacy of NPs + Gem + Ab, a co-culture system with human endothelial and SKBR3 cells was established. Cytotoxic effects over endothelial cells were similar for all the tested conditions (between 25 and 30%). However, the NPs + Gem + Ab system presented increased toxicity over breast cancer cells, above 80% after 24 h, when compared to free Gem and NPs + Gem (around 15% and 20%, respectively). In vivo studies demonstrated that the developed targeting system significantly reduced tumor growth and the appearance of lung metastasis compared to untreated controls. In summary, the efficacy of the NPs + Gem + Ab system to target cancer cells was established and validated both in vitro and in vivo, being a compelling alternative strategy to current chemotherapeutic approaches.

Intracellular Autofluorescence as a New Biomarker for Cancer Stem Cells in Glioblastoma.

The identification of cancer stem cells (CSCs), which are implicated in tumor initiation, progression, therapy resistance, and relapse, is of great biological and clinical relevance. In glioblastoma (GBM), this is still a challenge, as no single marker is able to universally identify populations of GBM cancer stem cells (GSCs). Indeed, there is still controversy on whether biomarker-expressing cells fulfill the functional criteria of bona fide GSCs, despite being widely used. Here, we describe a novel subpopulation of autofluorescent (Fluo+) cells in GBM that bear all the functional characteristics of GSCs, including higher capacity to grow as neurospheres, long-term self-renewal ability, increased expression of stem cell markers, and enhanced in vivo tumorigenicity. Mechanistically, the autofluorescent phenotype is largely due to the intracellular accumulation of riboflavin, mediated by the ABC transporter ABCG2. In summary, our work identifies an intrinsic cellular autofluorescent phenotype enriched in GBM cells with functional stem cells features that can be used as a novel, simple and reliable biomarker to target these highly malignant tumors, with implications for GBM biological and clinical research.

Exploiting the Complexities of Glioblastoma Stem Cells: Insights for Cancer Initiation and Therapeutic Targeting.

The discovery of glioblastoma stem cells (GSCs) in the 2000s revolutionized the cancer research field, raising new questions regarding the putative cell(s) of origin of this tumor type, and partly explaining the highly heterogeneous nature of glioblastoma (GBM). Increasing evidence has suggested that GSCs play critical roles in tumor initiation, progression, and resistance to conventional therapies. The remarkable oncogenic features of GSCs have generated significant interest in better defining and characterizing these cells and determining novel pathways driving GBM that could constitute attractive key therapeutic targets. While exciting breakthroughs have been achieved in the field, the characterization of GSCs is a challenge and the cell of origin of GBM remains controversial. For example, the use of several cell-surface molecular markers to identify and isolate GSCs has been a challenge. It is now widely accepted that none of these markers is, per se, sufficiently robust to distinguish GSCs from normal stem cells. Finding new strategies that are able to more efficiently and specifically target these niches could also prove invaluable against this devastating and therapy-insensitive tumor. In this review paper, we summarize the most relevant findings and discuss emerging concepts and open questions in the field of GSCs, some of which are, to some extent, pertinent to other cancer stem cells.

MicroRNA-30a-5pme: a novel diagnostic and prognostic biomarker for clear cell renal cell carcinoma in tissue and urine samples.

BACKGROUND: The rising incidence of renal cell carcinomas (RCC) constitutes a significant challenge owing to risk of overtreatment. Because aberrant microRNA (miR) promoter methylation contributes to cancer development, we investigated whether altered miR-30a-5p expression associates with DNA promoter methylation and evaluated the usefulness as clear cell RCC (ccRCC) diagnostic and prognostic markers. METHODS: Genome-wide methylome and RNA sequencing data from a set of ccRCC and normal tissue samples from The Cancer Genome Atlas (TCGA) database were integrated to identify candidate CpG loci involved in cancer onset. MiR-30a-5p expression and promoter methylation were quantitatively assessed by PCR in a tissue set (Cohort #1) and urine sets (Cohorts #2 and 3) from IPOPorto and Homburg University Hospital. Non-parametric tests were used for comparing continuous variables. MiR-30a-5p promoter methylation (miR-30a-5pme) performance as diagnostic (receiver operator characteristics [ROC] - validity estimates) and prognostic [metastasis-free (MFS) and disease-specific survival (DSS)] biomarker was further validated in urine samples from ccRCC patients by Kaplan Meier curves (with log rank) and both univariable and multivariable analysis. RESULTS: Two significant hypermethylated CpG loci in TCGA ccRCC samples, correlating with miR-30a-5p transcriptional downregulation, were disclosed. MiR-30a-5pme in ccRCC tissues was confirmed in an independent patient's cohort of IPOPorto and associated with shorter time to relapse. In urine samples, miR-30a-5pme levels identified cancer both in testing and validation cohorts, with 83% sensitivity/53% specificity and 63% sensitivity/67% specificity, respectively. Moreover, higher miR-30a-5pme levels independently predicted metastatic dissemination and survival. CONCLUSION: To the best of our knowledge, this is the first study validating the diagnostic and prognostic potential of miR-30a-5pme for ccRCC in urine samples, providing new insights for its clinical usefulness as non-invasive cancer biomarker.

Unravelling the anticancer potential of functionalized chromeno[2,3-b]pyridines for breast cancer treatment.

A selection of new chromeno[2,3-b]pyridines was prepared from chromenylacrylonitriles and N-substituted piperazines, using a novel and efficient synthetic procedure. The compounds were tested for their anticancer activity using breast cancer cell lines MCF-7, Hs578t and MDA-MB-231 and the non-neoplastic cell line MCF-10A for toxicity evaluation. In general, compounds showed higher activity towards the luminal breast cancer subtype (MCF-7), competitive with the reference compound Doxorubicin. The in vivo toxicity assay using C. elegans demonstrated a safe profile for the most active compounds. Chromene 3f revealed a promising drug profile, inhibiting cell growth and proliferation, inducing cell cycle arrest in G2/M phase, apoptosis and microtubule destabilization. The new compounds presented exciting bioactive features and may be used as lead compounds in cancer related drug discovery.

Sirtuins' Deregulation in Bladder Cancer: SIRT7 Is Implicated in Tumor Progression through Epithelial to Mesenchymal Transition Promotion.

Sirtuins are emerging players in cancer biology and other age-related disorders, and their putative role in bladder cancer (BlCa) remains elusive. Further understanding of disease biology may allow for generation of more effective pathway-based biomarkers and targeted therapies. Herein, we aimed to illuminate the role of sirtuins' family in BlCa and evaluate their potential as disease biomarkers and therapeutic targets. SIRT1-7 transcripts and protein levels were evaluated in a series of primary BlCa and normal bladder mucosa tissues. SIRT7 knockdown was performed through lentiviral transduction in MGHU3, 5637 and J82 cells and its functional role was assessed. SIRT1, 2, 4 and 5 expression levels were significantly lower in BlCa, whereas SIRT6 and 7 were overexpressed, and these results were corroborated by TCGA cohort analysis. SIRT7 transcript levels were significantly decreased in muscle-invasive vs. papillary BlCa. In vitro studies showed that SIRT7 downregulation promoted cells migration and invasion. Accordingly, increased EMT markers expression and decreased E-Cadherin (CDH1) was observed in those BlCa cells. Moreover, increased EZH2 expression and H3K27me3 deposition in E-Cadherin promoter was found in sh-SIRT7 cells. We demonstrated that sirtuins are globally deregulated in BlCa, and specifically SIRT7 downregulation is implicated in EMT, fostering BlCa invasiveness through EZH2-CDH1 axis.

HOX gene cluster (de)regulation in brain: from neurodevelopment to malignant glial tumours.

HOX genes encode a family of evolutionarily conserved homeodomain transcription factors that are crucial both during development and adult life. In humans, 39 HOX genes are arranged in four clusters (HOXA, B, C, and D) in chromosomes 7, 17, 12, and 2, respectively. During embryonic development, particular epigenetic states accompany their expression along the anterior-posterior body axis. This tightly regulated temporal-spatial expression pattern reflects their relative chromosomal localization, and is critical for normal embryonic brain development when HOX genes are mainly expressed in the hindbrain and mostly absent in the forebrain region. Epigenetic marks, mostly polycomb-associated, are dynamically regulated at HOX loci and regulatory regions to ensure the finely tuned HOX activation and repression, highlighting a crucial epigenetic plasticity necessary for homeostatic development. HOX genes are essentially absent in healthy adult brain, whereas they are detected in malignant brain tumours, namely gliomas, where HOX genes display critical roles by regulating several hallmarks of cancer. Here, we review the major mechanisms involved in HOX genes (de)regulation in the brain, from embryonic to adult stages, in physiological and oncologic conditions. We focus particularly on the emerging causes of HOX gene deregulation in glioma, as well as on their functional and clinical implications.

A novel molecular link between HOXA9 and WNT6 in glioblastoma identifies a subgroup of patients with particular poor prognosis.

Despite much effort to improve treatments, patients with malignant glioma still present a very poor prognosis that has not changed significantly in the last decades. In this context, it is crucial to better understand glioma pathogenesis to identify new molecular prognostic subgroups and therapeutic targets. WNT6 was recently identified as a new oncogenic molecule in glioblastoma (GBM), with prognostic value in patients, but the mechanisms underlying WNT6 aberrant expression in glioma are still unknown. WNT6 was overexpressed in a subset of gliomas independently of IDH mutations, 1p/19q codeletion status, and WNT6 gene copy number. Interestingly, WNT6 expression is associated with the DNA methylation levels of particular CpG regions at both the WNT6 promoter and the gene body in glioma patient samples. HOXA9, a transcription factor previously associated with poorer clinical outcome in GBM, was identified as a novel transcriptional regulator of WNT6, activating the WNT/ß-catenin pathway in vitro and in vivo. In various cohorts of glioma patients, mRNA levels of WNT6 and HOXA9 were significantly correlated, extending our in vitro and in vivo findings into the clinical setting. Interestingly, this novel molecular link between WNT6 and HOXA9 was not limited to glioma, as they were co-expressed also in patients with other tumor types. Clinically, WNT6 was a prognostic biomarker of shorter survival in GBM, independently of HOXA9 expression. Concomitant high expression of both WNT6 and HOXA9 identified a subgroup of patients with particularly dismal survival. These findings describe novel WNT6 regulatory mechanisms in GBM, establishing particular DNA methylation patterns and HOXA9 as critical regulators of WNT6 expression in glioma. This HOXA9-WNT6 molecular link supports WNT signaling in GBM cells and is a powerful prognostic biomarker, highlighting the clinical relevance of this axis in patients. Novel therapies targeting WNT6-HOXA9 signaling may thus be useful for this deadly disease.

Overexpression of circulating MiR-30b-5p identifies advanced breast cancer.

BACKGROUND: Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. METHODS: Selected microRNAs were assessed using a quantitative reverse transcription-polymerase chain reaction in a testing cohort of formalin-fixed paraffin-embedded primary (n = 16) and metastatic BrC tissues (n = 22). Then, miR-30b-5p and miR-200b-3p were assessed in a validation cohort #1 of formalin-fixed paraffin-embedded primary (n = 82) and metastatic BrC tissues (n = 93), whereas only miR-30b-5p was validated on a validation cohort #2 of liquid biopsies from BrC patients with localized (n = 20) and advanced (n = 25) disease. ROC curve was constructed to evaluate prognostic performance. RESULTS: MiR-30b-5p was differentially expressed in primary tumors and paired metastatic lesions, with bone metastases displaying significantly higher miR-30b-5p expression levels, paralleling the corresponding primary tumors. Interestingly, patients with advanced disease disclosed increased circulating miR-30b-5p expression compared to patients with localized BrC. CONCLUSIONS: MiR-30b-5p might identify BrC patients at higher risk of disease progression, thus, providing a useful clinical tool for patients' monitoring, entailing earlier and more effective treatment. Nonetheless, validation in larger multicentric cohorts is mandatory to confirm these findings.

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.

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.

A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97.

Background: Prostate cancer (PCa) is a major cause of morbidity and mortality in men worldwide. MicroRNAs are globally downregulated in PCa, especially in poorly differentiated tumors. Nonetheless, the underlying mechanisms are still elusive. Herein, using combined analysis of microRNAs expression and genomewide DNA methylation, we aimed to identify epigenetically downregulated microRNAs in PCa. Results: We found that miR-152-3p was underexpressed in PCa and that lower expression levels were associated with promoter hypermethylation in accordance with TCGA dataset analysis. Functional in vitro assays suggest that miR-152-3p suppresses cell viability and invasion potential, whereas it promotes cell cycle arrest at S and G2/M phases. Additionally, miR-152-3p expression was associated with longer disease-free survival in PCa patients from TCGA. Finally, TMEM97, which is overexpressed in PCa, was identified as a novel miR-152-3p target gene. Conclusions: Our findings demonstrate the advantages of using a combinatory approach to identify microRNAs downregulated due to aberrant promoter methylation. MiR-152-3p downregulation and promoter methylation was found to be prevalent in primary PCa, which impairs its role in control of cell viability, cell cycle regulation and invasion.