Oxidative stress and the subcellular localization of the telomerase reverse transcriptase (TERT) in papillary thyroid cancer

Highlights

• The H₂O₂ generation activity is significantly higher in tumors than in normal tissues.

• In thyroid tumors TERT shuttles from the nucleus to the mitochondria.

• Mitochondrial H₂O₂ activity is not significantly different in tumors and normal tissues.

Abstract

During hormonogenesis, thyrocytes are physiologically exposed to high levels of oxidative stress (OS) which could either be involved in the pathogenesis of thyroid cancer or exert a cytotoxic effect. We analyzed the oxidative status of papillary thyroid cancer (PTC) both directly, by measuring H₂O₂ generation by NADPH oxidases (NOXs), and indirectly, by evaluating the antioxidant activity of glutathione peroxidase (GPX), which neutralizes H₂O₂ excess, and the lipid peroxidation (LP). Moreover, we investigated the subcellular localization of telomerase reverse transcriptase (TERT), and the H₂O₂ levels in the mitochondria of tumor and normal tissues.

The calcium-dependent and independent H₂O₂ generation activity was significantly higher in tumors than in normal tissues. The GPX activity was higher in PTCs than in normal tissues, and, consistently, no differences were found in LP levels. Moreover, while TERT nuclear expression was similar in tumor and normal tissues, the mitochondrial localization was significantly higher in tumors. At the mitochondrial level, no differences were found in H₂O₂ generation between tumor and normal tissues.

In conclusion, present data demonstrate that the intracellular H₂O₂ generation by NOXs is significantly higher in PTCs than in normal thyroid tissues. The increased GPX activity found in tumors counteracts the potential cytotoxic effects of high OS exposure. The significantly higher mitochondrial localization of TERT in tumors is consistent with its shuttling from the nucleus upon exposure to high OS. Finally, mitochondrial OS was not significantly different in tumors and normal tissues, supporting the postulated role of mitochondrial TERT in the control of local H₂O₂ production.

Introduction

Thyroid cancer (TC) is the most frequent endocrine tumor, and its incidence and prevalence are increasing worldwide (Pellegriti et al., 2013). Among environmental factors causing or predisposing to TC, oxidative stress (OS) can theoretically have a major impact on thyroid cells, since, due to the process of hormonogenesis, they are exposed to higher OS levels compared to other organs (Maier et al., 2006). OS is a biochemical state in which cells are exposed to high levels of reactive oxygen species (ROS), such as superoxide anion radical (O₂⁻), hydroxyl radical (OH) and hydrogen peroxide (H₂O₂), due to an imbalance between pro-oxidant compounds and antioxidant defenses. Beyond electron transport chain of the mitochondria, which generates ROS as a by-product, the main source of cellular ROS is represented by NADPH oxidases (NOXs). NOXs are transmembrane electron carriers that use NADPH as an electron source and molecular oxygen as an acceptor to generate O₂⁻ and H₂O₂. The NOXs family embraces 7 members (NOX1-5, DUOX1 and 2), involved in different biological functions (Bedard and Krause, 2007). In the thyroid gland, three different NOXs have been identified: DUOX1, DUOX2 and, more recently, NOX4. DUOX1 and 2, located at the apical membrane of the thyrocyte, together with the maturation factors DUOXA1 and 2, are responsible for the calcium-dependent H₂O₂ generation, which is used by thyroid peroxidase for iodide organification (Dupuy et al., 1999, Grasberger and Refetoff, 2006). Differently from DUOXs, NOX4 produces H₂O₂ and/or O₂⁻ in intracellular compartments, such as mitochondria, nucleus and endoplasmic reticulum, and is constitutively active (Weyemi et al., 2010). An increased expression of both DUOX and NOX4 has been documented in papillary thyroid cancer (PTC) (Gérard et al., 2003, Lacroix et al., 2001, Weyemi et al., 2010). Moreover, previous studies associated OS to TC, by showing abnormally regulated oxidative and antioxidant molecules (Akinci et al., 2008, Lassoued et al., 2010, Senthil and Manoharan, 2004) and an increased total oxidant status (Wang et al., 2011) in the serum of patients. This evidence indicated OS as a risk factor in TC though their precise relationship has not been yet elucidated. In particular, excessive ROS may result in genetic alterations, that could lead to the constitutive activation of the major signaling pathways involved in the pathogenesis of TC (such as MAP kinase pathway) (Aikawa et al., 1997, Guyton et al., 1996, Rao and Berk, 1992). Moreover, DUOX2 and NOX4 were shown to be involved in cell cycle entry through inactivation of p53, probably because the generated H₂O₂ oxidizes the cysteine residues of p53 preventing its DNA binding activity (Salmeen et al., 2010). Since all the available evidence, in the thyroid and in other organs, suggest that a moderate increase in ROS may play an important part in the initiation and progression of cancer, the OS is often viewed as an adverse event. However, excessive levels of ROS, not compensated by the antioxidant defenses, can also be toxic to the cells, causing oxidative damage to lipids (lipid peroxidation), proteins and DNA, leading to the death of malignant cells and thus limiting cancer progression. Moreover, cancer cells with increased oxidative stress are likely to be more vulnerable to damage by further ROS insults induced by exogenous agents. Consistently, most of the chemotherapeutic and radiotherapeutic agents kill cancer cells by increasing ROS stress (Trachootham et al., 2009).

Recent evidences demonstrated an important role of telomerase reverse transcriptase (TERT) in human cancer. TERT is the catalytic subunit of telomerase that maintains telomeres at the ends of chromosomes and is constitutively expressed in most cancer cells, allowing unlimited proliferation. Two mutations in the TERT promoter, C228T and C250T, corresponding to −124 C > T and −146 C > T from translational start site, have been implicated in TERT over-expression in tumors and were recently reported in TC, associated with a worst tumor outcome (Vinagre et al., 2013, Melo et al., 2014, Muzza et al., 2015, Xing et al., 2014). Moreover, TERT has been found to be involved in the protection towards OS in cancer cells upon its translocation in the mitochondria (Saretzki, 2009, Singhapol et al., 2013). Indeed, TERT is known to shuttle dynamically from nucleus to mitochondria, where it decreases mitochondrial ROS generation, mitochondrial and nuclear DNA damage and stress-induced apoptosis (Ahmed et al., 2008, Haendeler et al., 2009, Saretzki, 2009, Singhapol et al., 2013). TERT mitochondrial localization has been observed in either TERT over-expressing cancer cells, or in normal cell lines after induction of exogenous stress, such as hydrogen peroxide exposition or irradiation or anticancer treatments (Ahmed et al., 2008, Haendeler et al., 2003, Santos et al., 2004, Singhapol et al., 2013), or in endothelial cells approaching senescence due to increased oxidative stress (Haendeler et al., 2004). Moreover, a mitochondrial localization of TERT was detected in cord blood mononuclear cells under elevated OS, and was demonstrated to protect neonatal mtDNA from oxidative damage in gestational diabetes mellitus pregnancies (Li et al., 2014). Furthermore, a defensive role of mitochondrial TERT from the oxidative damage of mtDNA was reported in hepatocellular carcinoma tissues (Piciocchi et al., 2015). Finally, TERT mitochondrial translocation was shown to contribute to the drug resistance of tumor cells by reducing ROS production (Yan et al., 2015), suggesting that TERT mitochondrial localization could contribute to the progression of TC. In this context, at variance with the previously reported almost exclusive expression of TERT in the nuclei of TC cells (Ito et al., 2005, Wang et al., 2005), we recently showed also a cytoplasmic TERT localization. Interestingly, in thyroid tumors we found a dot pattern of distribution in the cytoplasm, consistent with the localization of TERT in subcellular structures, such as mitochondria (Muzza et al., 2015).

Aims of the present study were thus to investigate the whole intracellular and mitochondrial ROS generation activity and the anti-oxidant defenses in papillary thyroid cancer (PTC) compared to normal thyroid tissues. Moreover, we studied the subcellular TERT expression, in order to eventually confirm in TC its shuttling from the nucleus to the mitochondria upon the increase of intracellular OS.