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/β-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.info:eu-repo/semantics/publishedVersio

Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication

Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of three genes-SMYD2, SETD3, and NO66-was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity (AUC=0.959), and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC: 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.This study was funded by research grants from Research Center of Portuguese Oncology Institute – Porto (CI-IPOP 4-2012) and European Community’s Seventh Framework Program – Grant number FP7-HEALTH-F5-2009-241783. ASP-L and FQV are and were supported by FCT-Fundação para a Ciência e a Tecnologia grants (SFRH/SINTD/94217/2013 and SFRH/ BD/70564/2010, respectively)

SETDB2 and RIOX2 are differentially expressed among renal cell tumor subtypes, associating with prognosis and metastization

Increasing detection of small renal masses by imaging techniques entails the need for accurate discrimination between benign and malignant renal cell tumors (RCTs) as well as among malignant RCTs, owing to differential risk of progression through metastization. Although histone methylation has been implicated in renal tumorigenesis, its potential as biomarker for renal cell carcinoma (RCC) progression remains largely unexplored. Thus, we aimed to characterize the differential expression of histone methyltransferases (HMTs) and histone demethylases (HDMs) in RCTs to assess their potential as metastasis biomarkers. We found that SETDB2 and RIOX2 (encoding for an HMT and an HDM, respectively) expression levels was significantly altered in RCTs; these genes were further selected for validation by quantitative RT-PCR in 160 RCTs. Moreover, SETDB2, RIOX2, and three genes encoding for enzymes involved in histone methylation (NO66, SETD3, and SMYD2), previously reported by our group, were quantified (RT-PCR) in an independent series of 62 clear cell renal cell carcinoma (ccRCC) to assess its potential role in ccRCC metastasis development. Additional validation was performed using TCGA dataset. SETDB2 and RIOX2 transcripts were overexpressed in RCTs compared to renal normal tissues (RNTs) and in oncocytomas vs. RCCs, with ccRCC and papillary renal cell carcinoma (pRCC) displaying the lowest levels. Low SETDB2 expression levels and higher stage independently predicted shorter disease-free survival. In our 62 ccRCC cohort, significantly higher RIOX2, but not SETDB2, expression levels were depicted in cases that developed metastasis during follow-up. These findings were not apparent in TCGA dataset. We concluded that SETDB2 and RIOX2 might be involved in renal tumorigenesis and RCC progression, especially in metastatic spread. Moreover, SETDB2 expression levels might independently discriminate among RCC subgroups with distinct outcome, whereas higher RIOX2 transcript levels might identify ccRCC cases with more propensity to endure metastatic dissemination.This study was funded by research grants from Research Center of Portuguese Oncology Institute - Porto (CI-IPOP 4-2012 and CI-IPOP 27) and from Associacao Portuguesa de Urologia (APU-2010). ASP-L was supported by FCT-Fundacao para a Ciencia e a Tecnologia fellowship (SFRH/SINTD/94217/2013). CSG is supported by FCT- Fundacao para a Ciencia e Tecnologia PhD fellowships (SFRH/BD/92786/2013) and BMC is funded by FCT-Fundacao para a Ciencia e a Tecnologia (IF/00601/2012).info:eu-repo/semantics/publishedVersio

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.FCT - Foundation for Science and Technology (PTDC/SAU-GMG/113795/2009 and IF/00601/2012 to B.M.C.; SFRH/BD/92786/2013 to C.S.G.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/81042/2011 to M.P.; SFRH/BD/93443/2013 to S.Q.) and Fundação Calouste Gulbenkian (B.M.C.), by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and National Funds through FCT under the project POCI-01-0145-FEDER-007038; by the project NORTE-01-0145-FEDER-000013 and NORTE-01-0246-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); and by the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)info:eu-repo/semantics/publishedVersio

The metabolic microenvironment of melanomas: prognostic value of MCT1 and MCT4

BRAF mutations are known drivers of melanoma development and, recently, were also described as players in the Warburg effect, while this reprogramming of energy metabolism has been identified as a possible strategy for treating melanoma patients. Therefore, the aim of this work was to evaluate the expression and prognostic value of a panel of glycolytic metabolism-related proteins in a series of melanomas. The immunohistochemical expression of MCT1, MCT4, GLUT1, and CAIX was evaluated in 356 patients presenting melanoma and 20 patients presenting benign nevi. Samples included 20 benign nevi, 282 primary melanomas, 117 lymph node and 54 distant metastases samples. BRAF mutation was observed in 29/92 (31.5%) melanoma patients and 17/20 (85%) benign nevi samples. NRAS mutation was observed in 4/36 (11.1%) melanoma patients and 1/19 (5.3%) benign nevi samples. MCT4 and GLUT1 expression was significantly increased in metastatic samples, and MCT1, MCT4 and GLUT1 were significantly associated with poor prognostic variables. Importantly, MCT1 and MCT4 were associated with shorter overall survival. In conclusion, the present study brings new insights on metabolic aspects of melanoma, paving the way for the development of new-targeted therapies.This work was supported by FAPESP grant to VLV (2012/04194-1) and CP (2015/25351-6). VMG received a doctoral fellowship (SFRH/BD/51997/2012) from Fundacao para a Ciencia e a Tecnologia (FCT) and ON. 2 SR&TD Integrated Program (NORTE-07-0124-FEDER-000017) co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER).info:eu-repo/semantics/publishedVersio

HOXA9 promotes glioblastoma initiation, aggressiveness and resistance to therapy

Glioblastoma is the most common and malignant subtype of glioma, exhibiting remarkable resistance to treatment. Here we investigated the oncogenic potential of HOXA9 in gliomagenesis, the molecular and cellular mechanisms by which HOXA9 may render glioblastoma more aggressive, and how HOXA9 affects response to chemotherapy and prognosis. Expression microarrays were used to identify HOXA9 target genes. Stable glioblastoma cell lines with ectopic HOXA9 overexpression or shRNA-­mediated knockdown of HOXA9 were established to evaluate the roles of HOXA9 in cell viability, death, invasion, and response to temozolomide. Subcutaneous and orthotopic intracranial xenograft models of glioblastoma were established to evaluate the oncogenic potential of HOXA9 in vivo, and its role in response to temozolomide and overall survival. Transcriptomic analyses identified novel HOXA9-­target genes that have key roles in critical cancer processes, including cell proliferation, adhesion, DNA metabolism and repair, and stem cell maintenance. Functional assays with a variety of glioblastoma cells revealed that HOXA9 promotes cell viability, stemness, and invasion; conversely, HOXA9 displayed anti-­apoptotic functions. Additionally, ectopic expression of HOXA9 promoted the malignant transformation of human immortalized astrocytes in an intracranial orthotopic mouse model of glioblastoma, and caused tumor-­associated death. HOXA9 also mediated resistance to temozolomide treatment both in vitro and in vivo. Mechanistically, BCL2 was identified as a novel HOXA9 target that may be therapeutically targeted. Indeed, the pharmacological inhibition of BCL2 with ABT-­737 specifically reverted temozolomide resistance in HOXA9-­positive cells. These data establish HOXA9 as a critical driver of glioma initiation, aggressiveness and resistance to therapy

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

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. 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.Research Center of Portuguese Oncology Institute of Porto (PI 74-CI-IPOP-19-2016). JL and CSG are supported by a PhD fellowship from FCT - Fundação para a Ciência e Tecnologia (SFRH/ BD/132751/2017 and SFRH/BD/92786/2013, respectively). SS is supported by a PhD fellowship IPO/ESTIMA-1 NORTE-01-0145-FEDER-000027. BMC is funded by FCT-Fundação para a Ciência e a Tecnologia (IF/00601/2012

Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer

BACKGROUND. Monocarboxylate transporters (MCTs) are transmembrane proteins involved in the transport of monocarboxylates across the plasma membrane, which appear to play an important role in solid tumours, however the role of MCTs in prostate cancer is largely unknown.The aim of the present work was to evaluate the clinico-pathological value of monocarboxylate transporters (MCTs) expression, namely MCT1, MCT2 and MCT4, together with CD147 and gp70 as MCT1/4 and MCT2 chaperones, respectively, in prostate carcinoma. METHODS. Prostate tissues were obtained from 171 patients, who performed radical prostatectomy and 14 patients who performed cystoprostatectomy. Samples and clinico-pathological data were retrieved and organized into tissue microarray (TMAs) blocks. Protein expression was evaluated by immunohistochemistry in neoplastic (n= 171), adjacent non-neoplastic tissues (n= 135), PIN lesions (n=40) and normal prostatic tissue (n=14). Protein expression was correlated with patients' clinicopathologic characteristics. RESULTS. In the present study, a significant increase of MCT2 and MCT4 expression in the cytoplasm of tumour cells and a significant decrease in both MCT1 and CD147 expression in prostate tumour cells was observed when compared to normal tissue. All MCT isoforms and CD147 were expressed in PIN lesions. Importantly, for MCT2 and MCT4 the expression levels in PIN lesions were between normal and tumour tissue, which might indicate a role for these MCTs in the malignant transformation. Associations were found between MCT1, MCT4 and CD147 expressions and poor prognosis markers; importantly MCT4 and CD147 overexpression correlated with higher PSA levels, Gleason score and pT stage, as well as with perineural invasion and biochemical recurrence. CONCLUSIONS. Our data provides novel evidence for the involvement of MCTs in prostate cancer. According to our results, we consider that MCT2 should be further explored as tumour marker and both MCT4 and CD147 as markers of poor prognosis in prostate cancer.NPG, CP and VMG received fellowships from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009, SFRH/BPD/69479/ 2010 and SFRH/BI/33503/2008, respectively. This work was supported by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of Programa Operacional Temático Factores de Competitividade” (COMPETE) of Quadro Comunitário de Apoio III and co-financed by Fundo Comunitário Europeu FEDER

Impact of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma risk and patient prognosis

Transforming growth factor beta (TGF-ß) plays an important role in carcinogenesis. Two polymorphisms in the TGF-ß1 gene (-509C/T and 869T/C) were described to influence susceptibility to gastric and breast cancers. The 869T/C polymorphism was also associated with overall survival in breast cancer patients. In the present study, we investigated the relevance of these TGF-ß1 polymorphism in glioma risk and prognosis. A case-control study that included 114 glioma patients and 138 cancer-free controls was performed. Single nucleotide polymorphisms (SNPs) were evaluated by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95 % confidence intervals (95 % CI). The influence of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma patient survival was evaluated by a Cox regression model adjusted for patients' age and sex and represented in Kaplan-Meier curves. Our results demonstrated that TGF-ß1 gene polymorphisms -509C/T and 869T/C are not significantly associated with glioma risk. Survival analyses showed that the homozygous -509TT genotype associates with longer overall survival of glioblastoma (GBM) patients when compared with patients carrying CC + CT genotypes (OR, 2.41; 95 % CI, 1.06-5.50; p = 0.036). In addition, the homozygous 869CC genotype is associated with increased overall survival of GBM patients when compared with 869TT + TC genotypes (OR, 2.62; 95 % CI, 1.11-6.17; p = 0.027). In conclusion, this study suggests that TGF-ß1 -509C/T and 869T/C polymorphisms are not significantly associated with risk for developing gliomas but may be relevant prognostic biomarkers in GBM patients.This work was supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/113795/2009 and SFRH/BPD/33612/2009 to B.M.C.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/92786/2013 to C.S.G.; PTDC/SAU-ONC/115513/2009 to R.R.)