Telmo Catarino

Telomerase promoter mutations in cancer: an emerging molecular biomarker?

Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the “alternative mechanism of telomere lengthening” (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target.

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.

The prognostic impact of TERT promoter mutations in glioblastomas is modified by the rs2853669 single nucleotide polymorphism

Human hotspot TERT promoter (TERTp) mutations have been reported in a wide range of tumours. Several studies have shown that TERTp mutations are associated with clinicopathological features; in some instances, TERTp mutations were considered as biomarkers of poor prognosis. The rs2853669 SNP, located in the TERT promoter region, was reported to modulate the increased TERT expression levels induced by the recurrent somatic mutations. In this study we aimed to determine the frequency and prognostic value of TERTp mutations and TERT rs2853669 SNP in 504 gliomas from Portuguese and Brazilian patients. TERTp mutations were detected in 47.8% of gliomas (216/452). Glioblastomas (GBM) exhibited the highest frequency of TERTp mutations (66.9%); in this glioma subtype, we found a significant association between TERTp mutations and poor prognosis, regardless of the population. Moreover, in a multivariate analysis, TERTp mutations were the only independent prognostic factor. Our data also showed that the poor prognosis conferred by TERTp mutations was restricted to GBM patients carrying the rs2853669 A allele and not in those carrying the G allele. In conclusion, the presence of TERTp mutations was associated with worse prognosis in GBM patients, although such association depended on the status of the rs2853669 SNP. The status of the rs2853669 SNP should be taken in consideration when assessing the prognostic value of TERTp mutations in GBM patients. TERTp mutations and the rs2853669 SNP can be used in the future as biomarkers of glioma prognosis.

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon14C‐BT uptake in tumoral (Caco‐2) and non‐tumoral (IEC‐6) intestinal epithelial cell lines. A 2‐day exposure to CDCA markedly and concentration‐dependently inhibited 14C‐BT uptake by IEC‐6 cells (IC50 = 120 µM), and, less potently, by Caco‐2 cells (IC50 = 402 µM). The inhibitory effect of CDCA upon 14C‐BT uptake did not result from a decrease in cell proliferation or viability. In IEC‐6 cells: (1) uptake of 14C‐BT involves both a high‐affinity and a low‐affinity transporter, and CDCA acted as a competitive inhibitor of the high‐affinity transporter; (2) CDCA inhibited both Na+‐coupled monocarboxylate cotransporter 1 (SMCT1)‐ and H+‐coupled monocarboxylate transporter 1 (MCT1)‐mediated uptake of 14C‐BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of 14C‐BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC‐6 cell viability and increased IEC‐6 cell differentiation, and CDCA (100 µM) reduced this effect. In conclusion, CDCA is an effective inhibitor of 14C‐BT uptake in tumoral and non‐tumoral intestinal epithelial cells, through inhibition of both H+‐coupled MCT1‐ and SMCT1‐mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level. J. Cell. Biochem. 113: 2937–2947, 2012.

The effect of oxidative stress upon the intestinal epithelial uptake of butyrate.

Our aim was to investigate the effect of oxidative stress upon butyrate uptake at the intestinal epithelial level. For this, IEC-6 cells were treated with tert-butylhydroperoxide 3000μM (tBOOH), which increased levels of oxidative stress biomarkers, while maintaining cellular viability. The effect of tBOOH upon uptake of [(14)C]butyrate ([(14)C]BT) (10μM) can be summarized as follows: (a) it caused a reduction in the intracellular accumulation of [(14)C]BT over time, (b) it strongly reduced total [(14)C]BT uptake but did not affect Na(+)-independent uptake of [(14)C]BT, and (c) it did not affect the kinetics of [(14)C]BT uptake at 37°C, but increased uptake at 4°C. Moreover, tBOOH increased the efflux of [(14)C]BT not mediated by breast cancer resistance protein. We thus conclude that tBOOH strongly inhibits Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)-mediated, but not H(+)-coupled monocarboxylate transporter (MCT1)-mediated butyrate uptake; moreover, it increases uptake and efflux of butyrate by passive diffusion. tBOOH did not affect the mRNA expression levels of MCT1 and SMCT1 nor their cell membrane insertion. Rather, its effect was dependent on extracellular signal regulated kinase 1/2 and protein tyrosine kinase activation and on the generation of reactive oxygen species by NADPH and xanthine oxidases and was partially prevented by the polyphenols quercetin and resveratrol. In conclusion, tBOOH is an effective inhibitor of SMCT1-mediated butyrate transport in non-tumoral intestinal epithelial cells. Given the important role played by butyrate in the intestine, this mechanism may contribute to the procarcinogenic and proinflammatory effect of oxidative stress at this level.

A founder SDHB mutation in Portuguese paraganglioma patients

This work was supported by Fundacao para a Ciencia e Tecnologia (Program Ciencia 2007 to V Maximo, Program Ciencia 2008 to J Lima, grant numbers SFRH/BPD/64233/2009 to Pedro Soares, PIC/IC/83037/2007 to V Maximo, and PTDC/CS-ANT/113832/2009 to T Rito). Further funding by Portuguese Foundation for Science and Technology by the project ‘Microenvironment, metabolism and cancer’ partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte) under the Quadro de Referencia Estrategico Nacional (QREN) and through the Fundo Europeu de Desenvolvimento Regional (FEDER).

Association between Polymorphisms in Antioxidant Genes and Inflammatory Bowel Disease

Inflammation is the driving force in inflammatory bowel disease (IBD) and its link to oxidative stress and carcinogenesis has long been accepted. The antioxidant system of the intestinal mucosa in IBD is compromised resulting in increased oxidative injury. This defective antioxidant system may be the result of genetic variants in antioxidant genes, which can represent susceptibility factors for IBD, namely Crohn’s disease (CD) and ulcerative colitis (UC). Single nucleotide polymorphisms (SNPs) in the antioxidant genes SOD2 (rs4880) and GPX1 (rs1050450) were genotyped in a Portuguese population comprising 436 Crohn’s disease and 367 ulcerative colitis patients, and 434 healthy controls. We found that the AA genotype in GPX1 is associated with ulcerative colitis (OR = 1.93, adjusted P-value = 0.037). Moreover, we found nominal significant associations between SOD2 and Crohn’s disease susceptibility and disease subphenotypes but these did not withstand the correction for multiple testing. These findings indicate a possible link between disease phenotypes and antioxidant genes. These results suggest a potential role for antioxidant genes in IBD pathogenesis and should be considered in future association studies.

Metabolic control of T cell immune response through glycans in inflammatory bowel disease

Significance Our findings demonstrate that metabolic supplementation of mucosal T cells, isolated from patients with active ulcerative colitis (UC), with N-acetylglucosamine (GlcNAc) leads to the enhancement of branched N-glycosylation on the T cell receptor, which was associated with the control of T cell activation and function. These results were validated in “glycoengineered” mouse models with severe colitis. Overall, our results open new avenues for a targeted-specific therapy in inflammatory bowel disease (IBD). The therapeutic use of GlcNAc (either alone or in combination with other antiinflammatory therapies) represents a simple immunomodulatory strategy in IBD, with absence of side effects, low costs, and the possibility of being used as a simple rescue therapy to avoid unnecessary toxic effects and step-up therapies in IBD. Mucosal T lymphocytes from patients with ulcerative colitis (UC) were previously shown to display a deficiency in branched N-glycosylation associated with disease severity. However, whether this glycosylation pathway shapes the course of the T cell response constituting a targeted-specific mechanism in UC remains largely unknown. In this study, we demonstrated that metabolic supplementation of ex vivo mucosal T cells from patients with active UC with N-acetylglucosamine (GlcNAc) resulted in enhancement of branched N-glycosylation in the T cell receptor (TCR), leading to suppression of T cell growth, inhibition of the T helper 1 (Th1)/Th17 immune response, and controlled T cell activity. We further demonstrated that mouse models displaying a deficiency in the branched N-glycosylation pathway (MGAT5−/−, MGAT5+/−) exhibited increased susceptibility to severe forms of colitis and early-onset disease. Importantly, the treatment of these mice with GlcNAc reduced disease severity and suppressed disease progression due to a controlled T cell-mediated immune response at the intestinal mucosa. In conclusion, our human ex vivo and preclinical results demonstrate the targeted-specific immunomodulatory properties of this simple glycan, proposing a therapeutic approach for patients with UC.

Abstract C118: TERT promoter mutations in human gliomas.

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Recently, two mutations (-124G>A and -146G>A) in the promoter region of the telomerase catalytic subunit (TERT) were found in a significant proportion of human tumors, including gliomas, thyroid carcinomas, bladder carcinomas and melanomas. These mutations resulted in increased expression of telomerase, both in vitro and in vivo. In order to further characterize the prevalence of TERT mutations in human tumors, we screened a cohort of 273 human gliomas and found an overall TERT mutation frequency of 50%. TERT mutations were most commonly found in high grade tumors such as glioblastoma multiforme (GBM) and gliosarcoma (65% and 89%, respectively), whereas low grade tumors such as pilocytic astrocytoma and diffuse astrocytoma showed a low frequency of TERT mutations (6% and 16%, respectively). Oligodendrogliomas (grade II) and anaplastic oligodendrogliomas (grade III) also harbored a high frequency of TERT mutations (44% and 52%, respectively). In GBM patients, the presence of TERT mutations was significantly associated with a higher mean age at diagnosis (p<0.0001) and worse survival (p=0.006). Our results show that TERT promoter mutations are frequent in human GBM and oligodendrogliomas and are associated with worse survival in GBM patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C118. Citation Format: Jorge Lima, Rui M. Reis, Telmo Catarino, Joana Peixoto, Joao Vinagre, Rui Batista, Ligia Castro, Fernando Pardal, Manuel Sobrinho-Simoes, Valdemar Maximo, Paula Soares. TERT promoter mutations in human gliomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C118.