Abstract
Breast cancer and thyroid dysfunctions have been associated for decades. Although many studies suggest a biological correlation, the mechanisms linking these two pathologies have not been elucidated. Reactive oxygen species (ROS) can oxidize lipids, proteins, and DNA molecules and may promote tumor initiation. Hence, we aimed at evaluating the mammary redox balance and genomic instability in a model of experimental hypothyroidism. Female Wistar rats were treated with 0.03% methimazole for 7 or 21 days to evaluate ROS generation, antioxidant enzyme activities, and oxidative stress biomarkers, as well as genomic instability. After 7 days, lower catalase, GPX, and DUOX activities were detected in the breast of hypothyroid group compared to the control while the levels of 4-hydroxynonenal (HNE) were higher. In addition, hypothyroid group showed an increase in γH2Ax/H2Ax ratio. Twenty-one days hypothyroid group had increased catalase and SOD activities, without significant differences between groups in the levels of oxidative stress biomarkers and DNA damage. TSH-treated MCF10A cells showed a higher extracellular, intracellular, and mitochondrial ROS production. Additionally, greater DNA damage was observed in these cells, demonstrated by a higher comet tail DNA percentage and increased 53BP1 foci. Finally, we found that TSH treatment was not able to alter cell viability. The Genome Cancer Atlas (TGCA) data showed that high TSHR expression is associated with more invasive breast cancer types. In conclusion, we demonstrate that oxidative stress and DNA damage in breast are early events of experimental hypothyroidism. Moreover, high TSH levels induce oxidative stress and genomic instability in mammary cells.
Introduction
Breast cancer is the most common malignancy in women worldwide (Jemal et al. 2011, Siegel et al. 2016), representing 23% of the total cancer diagnoses in women and 14% of cancer deaths in women in the USA (Jemal et al. 2011). It represents the main cause of death from cancer in women in developing countries and the second main cause in developed regions (WHO 2017). In Brazil, 16,724 deaths from female breast cancer occurred in 2017, the equivalent to a risk of 16.16 per 100 thousand (INCA 2019). Breast cancer is a highly heterogeneous disease and has an extensive list of risk factors associated with its development, such as age, sex, genetic predisposition, breast density, personal and family history of breast cancer, obesity, early menarche, among others (Devita et al. 2015). Prolonged exposure to estrogens, due to early menarche and/or late menopause, and late age at first delivery, is among the most important risk factors for breast cancer, but other hormonal factors also seem to be associated with this disease, including thyroid dysfunction (Bernstein & Ross 1993, ESHRE Capri Workshop Group et al. 2004).
Epidemiological studies are conflicting, with both hypo and hyperthyroidism being associated with breast cancer, while other works do not show an association between breast cancer and thyroid dysfunction (Itoh & Maruchi 1975, Maruchi et al. 1976, Perry et al. 1978, Hedley et al. 1981, Sarlis et al. 2002, Simon et al. 2002, Turken et al. 2003, Hardefeldt et al. 2012, Prinzi et al. 2014, Fang et al. 2017). Recently, a retrospective cohort study analyzed 33,325 women with or without hypothyroidism and found that hypothyroid women were at a higher risk of breast cancer (Huang et al. 2021). This discrepancy can be attributed to the use of different diagnostic criteria for existing thyroid disease, and the heterogeneity of breast carcinoma and its subtypes.
The role of thyroid hormones on breast carcinogenesis has been suggested by studies that demonstrated that they increase cancer cell proliferation through several mechanisms, including angiogenesis, immunoreactivity, and estrogen-like pathways (Dinda et al. 2002, Pinto et al. 2011, Moeller & Führer 2013). On the other hand, experimental hypothyroidism increased the latency of tumor appearance, reduced tumor incidence and retarded tumor growth in rats with dimethylbenzanthracene-induced mammary tumors (López-Fontana et al. 2013). Sterle et al. (2021) recently showed a higher tumor growth rate and an immunosuppressive microenvironment in hyperthyroid mice inoculated with metastatic breast cancer cells, while the hypothyroid animals had lower tumor growth but a higher number of lung metastasis. It is important to note that serum thyroid-stimulating hormone (TSH) is generally high in the hypothyroid state, but its specific role in breast was not elucidated. Although the influence of thyroid dysfunctions on breast cancer promotion and progression is covered by the literature, to the best of our knowledge, little is known regarding breast cancer initiation. The acquisition of irreversible changes in DNA, such as point mutations or chromosomal aberrations, is related to tumor initiation, and higher reactive oxygen species (ROS) availability is strongly associated with this phenomenon (Hecht et al. 2016). Thus, our aim in the present study was to evaluate in the mammary tissue of rats submitted to experimental hypothyroidism the activity and expression of enzymes involved in the production and degradation of ROS, as well as DNA damage biomarker. Moreover, the role of TSH in ROS production and DNA damage was also evaluated through in vitro experiments.
Materials and methods
Animal and treatments
Three-month-old female adult Wistar rats were housed under controlled conditions of temperature (24 ± 1°C) and light (12 h light starting at 07:00 h). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication number 85-23, revised 1996) and was approved by the Institutional Committee for Evaluation of Animal Use in Research (Comissão de Ética com o Uso de Animais (CEUA) em Experimentação Científica da Universidade Federal do Rio de Janeiro, number: 032/18).
All female rats showed a regular 4–5 days estrous cycle, monitored by vaginal smears collected each morning for at least 2 consecutive weeks before starting the experiments. Rats were divided into the following groups (n = 10 for each group): control and hypothyroid (0.03% 2-mercapto-1-methylimidazole, MMI, Sigma) in drinking water for 7 or 21 days (Ferreira et al. 2005). For all groups, water and standard rat chow were available ad libitum. All animals were weighed prior to and following the treatment. After that, the rats were euthanized by decapitation, and the breast tissue and thyroid were excised. Serum was obtained after centrifugation of the blood at 1500 g for 15 min and stored at −20°C.
Hormonal measurements
Serum TSH was assayed using commercial kits (MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel, RTHYMAG-30K, Merck), following manufacture’s protocol. Serum total T4 was performed with a specific RIA using primary antibodies for T4 (Total T4 MAb RIA, MP Biomedical, Irvine, CA, USA). Detection limits were 14 pg/mL and 0.76 μg/dL for TSH and free T4, respectively. The intra-assay variations were <14% and 5.7% for TSH, and free T4, respectively. All samples were measured in the same assay.
Total reduced thiol levels
Mammary tissue samples were homogenized in 5 mM Tris–HCl buffer (pH 7.4), containing 0.9% NaCl (w/v) and 1 mM EDTA, followed by centrifugation at 750 g for 10 min at 4°C. Total reduced thiols were determined by using DTNB immediately after sample homogenization. NTB2− formation was quantified in a spectrophotometer (Hitachi U-3300) by measuring the absorbance at 412 nm and was expressed as nmol of reduced DTNB per mg protein (Ellman 1959).
Western blot analysis
Total protein extracts were analyzed as previously described (Andrade-Lima et al. 2015). Quantification was performed using the Pierce BCA Protein Assay kit (Thermo Scientific). Fifty micrograms of total protein from tissue and cell extracts were separated on a 10% SDS polyacrylamide gel and blotted onto a nitrocellulose membrane. Membranes were blocked for 1 h in 5% (w/v) milk powder in PBS-Tween 20 (PBST), incubated overnight at 4°C with the primary antibody, washed, and incubated for 1 h with secondary antibody. The blots were developed using an ECL detection system (Immobilon Western Chemiluminescent HRP Substrate, Merck Millipore). Protein expression was detected using anti-TSHR (1:500, Santa Cruz Biotechnologies Inc. sc-53542); anti-HNE (1:1000, Abcam ab48506); anti-H2A.X (1:1000 dilution, Cell Signaling Technologies #2595); anti-γH2A.X (1:1000 dilution, Cell Signaling Technologies #2595); anti-p21 (1:800, Santa Cruz Biotechnology Inc. sc-397) and anti-β actin (1:1000 dilution, Sigma-Aldrich A5441) antibodies.
NADPH oxidase activity in mammary tissue
NOX activity was quantified in particulate fractions of mammary tissue by the Amplex red/horseradish peroxidase assay (Molecular Probes, Invitrogen). The tissues were homogenized in a buffer (pH 7.2, containing 0.25 M sucrose, 0.1 mM dithiothreitol, 1 mM EGTA, 50 mM sodium phosphate, 5 mg/mL aprotinin and 34.8 mg/mL phenylmethanesulfonyl fluoride (PMSF)) and centrifuged at 720 g for 15 min at 4°C. After that, the pellet was resuspended in 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2 mM MgCl2, 5 mg/mL aprotinin and 34.8 mg/mL PMSF, and the homogenate was centrifuged at 100,000 g for 35 min at 4°C, followed by a wash of the pellet at 100,000 g. Finally, the pellet was resuspended in 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2 mM MgCl2, 5 mg/mL aprotinin, and 34.8 mg/mL PMSF and stored at −80°C until H2O2 generation measurements, as follows. The microsomal fractions were incubated in 150 mM sodium phosphate buffer (pH 7.4) containing SOD (100 U/mL; Sigma), horseradish peroxidase (0.5 U/mL, Roche), Amplex red (50 μM; Molecular Probes), 1 mM EGTA, and 1 mM NADPH. The fluorescence was immediately measured in a microplate reader (Victor X4; PerkinElmer) at 30°C, using excitation at 530 nm and emission at 595 nm (Fortunato et al. 2010). The specific enzymatic activity was expressed as nanomoles H2O2 per hour per milligram of protein (nmol/h/mg). Protein concentration was determined by the Bradford assay (Bradford 1976).
Quantitative PCR (qPCR)
Total RNA was extracted using TRI Reagent® (Sigma-Aldrich), and 2 μg were used for RT with High Capacity cDNA RT Kit (Applied Biosystems). Quantitative real-time PCR was performed with HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) (Solis BioDyne®, USA). Data were analyzed using the 2−ΔΔCt method (Schmittgen & Livak 2008). GAPDH was used as the housekeeping control gene. Primers used are described in the Table 1.
Primers used for the real-time PCR assay.
| Gene | Species | Sequence | |
|---|---|---|---|
| NOX2 | Rattus norvegicus | Forward | CCA TTC ACA CCA TTG CAC ATC |
| Reverse | CGA GTC ACA GCC ACA TAC AG | ||
| NOX4 | R. norvegicus | Forward | TCC ATC AAG CCA AGA TTC TGA G |
| Reverse | GGT TTC CAG TCA TCC AGT AGA G | ||
| DUOX1 | R. norvegicus | Forward | GAT ACC CAA AGC TGT ACC TCG |
| Reverse | GTC CTT GTC ACC CAG ATG AAG | ||
| DUOX2 | R. norvegicus | Forward | TGC TCT CAA CCC CAA AGT G |
| Reverse | TCT CAA ACC AGT AGC GAT CAC | ||
| GAPDH | R. norvegicus | Forward | TGA TTC TAC CCA CGG CAA GT |
| Reverse | AGC ATC ACC CCA TTT GAT G | ||
| NOX2 | Homo sapiens | Forward | AGG AGT TTC AAG ATG CGT GG |
| Reverse | TTG AGA ATG GAT GCG AAG GG | ||
| NOX4 | H. sapiens | Forward | GCT GAC GTT GCA TGT TTC AG |
| Reverse | CGG GAG GGT GGG TAT CTA A | ||
| NOX5 | H. sapiens | Forward | TGT TCA TCT GCT CCA GTT CC |
| Reverse | ACA AGA TTC CAG GCA CCA G | ||
| DUOX1 | H. sapiens | Forward | AGG AGT GGC ATA AGT TTG AGG |
| Reverse | GCG TCA CCC AGA TGA AGT AG | ||
| DUOX2 | H. sapiens | Forward | GGA GAA CCC CAA TCT GAA CAG |
| Reverse | AGT CTG TAC GGG AGA ATT TGC | ||
| GAPDH | H. sapiens | Forward | ATC TTC CAG GAG CGA GAT CC |
| Reverse | ACC ACT GAC ACG TTG GCA GT | ||
Activity of antioxidant enzymes in mammary tissue
Mammary tissue samples (100 mg) were homogenized in 5 mM Tris–HCl buffer (pH 7.4), containing 0.9% NaCl (w/v) and 1 mM EDTA, followed by centrifugation at 750 g for 10 min at 4°C. The supernatant aliquots were stored at −80°C. Catalase activity was assayed following the method of Aebi and was expressed as catalase units per milligram of protein (U/mg) (Aebi 1984). Glutathione peroxidase (GPX) activity was assayed by measuring NADPH oxidation at 340 nm in the presence of an excess of glutathione reductase, reduced glutathione, and tert-butyl hydroperoxide, as substrates and expressed as nmol of oxidized NADPH per milligram of protein (nmol/mg) (Flohé & Günzler 1984). Total superoxide dismutase activity was determined by the reduction of cytochrome C at 550 nm (Crapo et al. 1977).
Cell culture
Non-tumor human mammary epithelial cell lineage MCF10A was maintained in phenol red-free DMEM medium containing 10% fetal bovine serum (Gibco®/Life Technologies), penicillin and streptomycin (2%), and amphotericin B (1 mg/mL) and supplemented with cholera toxin (100 ng/mL), EGF (20 ng/mL), insulin (10 μg/mL), and hydrocortisone (500 ng/mL). The cells were incubated with TSH at 10 and 20 mU/mL in medium for 24 and 72 h. In some experiments, the cells were incubated with 1 mM N-acetyl cysteine (NAC) 24 h before and concomitant to the treatment with TSH. After treatment, the cells were harvested for the assays.
Extracellular H2O2 generation
Extracellular H2O2 generation was quantified by the AmplexRed/ HRP assay. 1 × 105 cells in phenol red-free Hank’s Balanced Salt Solution (HBSS) were incubated with D-glucose (1 mg/mL), SOD (100 U/mL; Sigma), HRP (0.5 U/mL; Roche), and Amplex Red (50 μM; Molecular Probes) and the fluorescence was measured in a microplate reader (Victor X4; PerkinElmer) at 530 nm excitation and 595 nm emission. Data were calculated using standard calibration curves and were expressed as nmol H2O2/h/105 cells.
Intracellular and mitochondrial ROS levels
Cells were dissociated and incubated with 10 μM H2DCF-DA (Invitrogen®/Life Technologies) for 30 min or with 5 μM MitoSOX Red (Invitrogen/Life Technologies) for 10 min at 37°C. Mean fluorescence intensity was detected by flow cytometry using BD FACSCalibur™ (BD Biosciences). Excitation and emission settings were 495/520 nm for H2DCF-DA and 510/580 nm for MitoSOX Red.
Comet assay
1 × 105 cells were suspended in 0.5% low melting point agarose, and then homogenously spread onto two microscope slides pre-coated with 1.5% agarose, immediately covered with coverslips, and kept at 4ºC for 10 min. The solidified slides were immersed in cell lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10) at 4°C overnight. The slides were then placed horizontally in an electrophoresis chamber with cold alkaline buffer (300 mM NaOH, 1 mM EDTA, pH >13) for 20 min, and electrophoresis was performed for 25 min at 25 V and 300 mA. Then, slides were neutralized with neutralization buffer (0.4 M Tris, pH 7.5) and fixed with ice-cold 100% ethanol. Finally, the slides were stained with DAPI and imaged with a fluorescence microscope (EVOS M5000 Cell Imaging System, ThermoFisher Scientific) at a magnification of 200×. At least 100 comets per slide were scored, on duplicate slides, using the OpenComet v1.3.1 software. The results were plotted as percentage of tail DNA content (Tice et al. 2000).
Immunocytofluorescence staining
After treatment, cells were fixed with 4% paraformaldehyde at room temperature for 20 min, incubated in a 50 mM ammonium chloride solution for 30 min. Following the blocking with 5% PBS/BSA, the coverslips were incubated with anti-53BP1 primary antibody (rabbit pAb to 53PB1, Bethyl Laboratories Inc., A300-273A, 1:500 dilution) in 3% PBS/BSA solution overnight at 4oC. Then, they were incubated with secondary antibody with AlexaFluor™-488 (goat anti-rabbit IgG, Invitrogen by ThermoFisher Technologies). Nuclei were counterstained with 1 μM DAPI (Sigma-Aldrich). The microscopic images were taken using a confocal microscope Leica TCS SP5 (Leica Microsystems). Quantification of 53BP1 foci was determined with ImageJ software (NIH).
Cell viability
4 × 103 cells were seeded in 96-well plates for 24 h and then treated or not with freshly diluted TSH (10 and 20 mU/mL) for 24, 48 and 72 h. Subsequently, MTT (0.5 mg/mL) was added. After 4 h, MTT solution was aspirated and formazan crystals were diluted in 100 μL DMSO. Finally, the absorbance of formazan solution was measured at 570 nm in a microplate reader (Victor X4, PerkinElmer). In addition, cell viability was measured using the dye exclusion method with trypan blue (Strober 2015). Cells were trypsinized and washed with HBSS solution. Then, they were mixed with 1-part of 0.4% trypan blue and 1-part cell suspension (dilution of cells), allowing the mixture to incubate for ∼3 min at room temperature. A drop of the trypan blue/cell mixture was placed on a hemacytometer under an optical microscope. The unstained (viable) and stained (nonviable) cells were counted separately.
Sub-G1 and cell cycle analysis by flow cytometry
Sub-G1 and cell cycle analysis were performed as previously described (Andrade-Lima et al. 2015). MCF10A cells treated or not with TSH were trypsinized, washed with PBS, and fixed with chilled 70% ethanol for at least 24 h, at −20°C. Staining was performed at room temperature for 30 min in filtered PBS containing 200 μL propidium iodide (PI) solution (200 mg/mL RNase A, 20 mg/mL PI, and 0.1% Triton X-100 in PBS) and each sample was analyzed in a flow cytometer (BD FACSCalibur™/BD Biosciences). Flowing Software 2.5.1 was used to analyze sub-G1 and cell cycle phases.
TGCA analysis
By using the online database analysis tool The Cancer Genome Atlas (TCGA) from cBioPortal for Cancer Genomics (https://www.cbioportal.org/), we obtained the TSH receptor (TSHR) expression profile of different types of human breast cancers and adjacent normal tissues in the METABRIC database (Curtis et al. 2012, Pereira et al. 2016), containing 1904 patient samples, evaluated via RNA Seq and microarray (Fig. 1). We searched the database for the TSHR gene and its expression alterations in breast cancer using the filters of differential analysis (cancer vs normal) and cancer type (breast cancer). Students’ t-test was used to generate a P value. To obtain the most significant probes, we set the following parameters: the cut-off of P value and z-score were defined as 0.01 and 2.0, respectively.
Statistical analysis
All results were expressed as mean ± s.e.m. and were analyzed by one-way ANOVA followed by Dunnet's multiple comparison test. In the case of animal group comparisons, unpaired t-test was used. Statistical analyses were done using GraphPad Prism (version 5.01, GraphPad Software Inc.) and the minimum level of significance was set at P < 0.05.
Results
Animals body weight and hormonal measurements
Serum TSH levels of the Hypo group at 7 or 21 days were significantly higher than their respective controls. Moreover, serum TSH levels were two-fold higher in 21 days Hypo group when compared to 7 days Hypo group. Total T4 serum levels were lower in treated animals, both after 7 and 21 days. After 7 days of treatment, as expected, the body weight of the control animals increased (Δ = 21.78 ± 1.81; P < 0.05), while the body weight of Hypo group did not differ (Δ = 13.40 ± 2.182). After 21 days of treatment, body weight of control animals increased (Δ = 19.39 ± 6.9; P <0.05), while those of Hypo group decreased (Δ = −20.09 ± 8.5; P < 0.05), as expected. Thyroid weight increased two-fold in the treated group for the two periods of treatment in relation to their respective controls (Table 2).
Characteristics of female Wistar rats treated or not with MMI for 7 or 21 days.
| 7 days | 21 days | |||
|---|---|---|---|---|
| Control | Hypo | Control | Hypo | |
| Body weight (g) | Prior: 221.2 ± 5.9 | Prior: 244.8 ± 6.9 | Prior: 185.9 ± 8.5 | Prior: 204.1 ± 12.2 |
| Following: 243.0 ± 6.8* | Following: 258.2 ± 6.7 | Following: 205.3 ± 3.0* | Following: 184.0 ± 4.5* | |
| Relative thyroid weight (mg/100 g body weight) | 5.58 ± 0.25 | 11.48 ± 0.72*** | 5.54 ± 0.17 | 12.73 ± 0.42*** |
| TSH (pg/mL) | 1438 ± 121.8 | 9161 ± 828.4**** | 3116 ± 974.1 | 20,671 ± 1111**** |
| Total T4 (μg/dL) | 8.15 ± 0.26 | 4.63 ± 0.39*** | 7.18 ± 0.44 | 3.72 ± 0.19*** |
Data are shown as mean ± s.e.m. of 6–10 rats per group. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Effects of hypothyroidism on oxidative stress biomarkers of breast tissue
Thiol residues are found mainly in proteins and low molecular mass metabolites, such as GSH, and can be reversibly oxidized by ROS to nitrosothiols or sulfonic acids, decreasing their cellular levels (Winterbourn & Hampton 2008). Total reduced thiol levels were lower in 7-day Hypo group when compared to its control. However, there was no significant difference between groups after 21 days of treatment (Fig. 2A). Lipid oxidation produces 4-hydroxy-2-nonenal aldehyde (4-HNE) as one of its products. Lipid aldehyde is an electrophile and there is a high reactivity of 4-HNE to protein nucleophiles (i.e. Cys, His, and Lys) (Majima et al. 2002). We observed that after 7 days of treatment, there was an increase in proteins linked to 4-HNE (Fig. 2B and C). However, after 21 days, this difference was no longer found.
Effects of 7 and 21 days of hypothyroidism on oxidative stress biomarkers in breast tissue of female Wistar rats. (A) Total reduced thiol groups were evaluated by the reaction of thiols with DTNB and measured in a spectrophotometer at 412 nm. (B) Representative blots of 4-HNE immunostaining. (C) Proteins modified by 4-hydroxynonenal, a biomarker of lipid peroxidation, were analyzed by Western blot. The data were expressed as relative units. Data are shown as mean±s.e.m.. N = 6–10 per group. *P < 0.05; *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on oxidative stress biomarkers in breast tissue of female Wistar rats. (A) Total reduced thiol groups were evaluated by the reaction of thiols with DTNB and measured in a spectrophotometer at 412 nm. (B) Representative blots of 4-HNE immunostaining. (C) Proteins modified by 4-hydroxynonenal, a biomarker of lipid peroxidation, were analyzed by Western blot. The data were expressed as relative units. Data are shown as mean±s.e.m.. N = 6–10 per group. *P < 0.05; *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on oxidative stress biomarkers in breast tissue of female Wistar rats. (A) Total reduced thiol groups were evaluated by the reaction of thiols with DTNB and measured in a spectrophotometer at 412 nm. (B) Representative blots of 4-HNE immunostaining. (C) Proteins modified by 4-hydroxynonenal, a biomarker of lipid peroxidation, were analyzed by Western blot. The data were expressed as relative units. Data are shown as mean±s.e.m.. N = 6–10 per group. *P < 0.05; *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of hypothyroidism on the NADPH oxidase activity and mRNA levels in breast tissue
There was no significant difference in NADPH oxidase activity between control and Hypo groups after 7 and 21 days (Fig. 3A). On the other hand, calcium-dependent H2O2 production, which is related to the DUOX activity, had a significant decrease after 7 days of hypothyroidism compared to its control. However, there was no difference in DUOX activity between the 21-day control and Hypo groups (Fig. 3B). Significantly lower levels of NOX2 were found in the 7 and 21 days Hypo groups compared to their respective controls (Fig. 3C and D). No differences were found for NOX4 and DUOX1 mRNAs between groups (Fig. 3C and D). DUOX2 enzyme was less expressed after 7 days of hypothyroidism in comparison to control (Fig. 3C), thus suggesting that the reduction of calcium-dependent H2O2 generation found in this group might be attributed to the decreased DUOX2 expression. On the other hand, we did not observe any difference in DUOX2 mRNA levels between the groups at 21 days (Fig. 3D).
Effects of 7 and 21 days of hypothyroidism on the NADPH oxidase activity and mRNA levels from breast tissue of female Wistar rats. (A) Total and (B) calcium-dependent NOX activities were determined by Amplex Red/HRP assay in the breast tissue of rats. Relative mRNA expression of NOX2, NOX4, DUOX1 and DUOX2 enzymes evaluated by qPCR in breast tissue of rats treated with MMI for (C) 7 and (D) 21 days. The data were expressed as relative to the control, using the GAPDH as an internal control for normalization and presented as the mean ± s.e.m. N = 6–10 per group. * P < 0.05; ** P < 0.01.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on the NADPH oxidase activity and mRNA levels from breast tissue of female Wistar rats. (A) Total and (B) calcium-dependent NOX activities were determined by Amplex Red/HRP assay in the breast tissue of rats. Relative mRNA expression of NOX2, NOX4, DUOX1 and DUOX2 enzymes evaluated by qPCR in breast tissue of rats treated with MMI for (C) 7 and (D) 21 days. The data were expressed as relative to the control, using the GAPDH as an internal control for normalization and presented as the mean ± s.e.m. N = 6–10 per group. * P < 0.05; ** P < 0.01.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on the NADPH oxidase activity and mRNA levels from breast tissue of female Wistar rats. (A) Total and (B) calcium-dependent NOX activities were determined by Amplex Red/HRP assay in the breast tissue of rats. Relative mRNA expression of NOX2, NOX4, DUOX1 and DUOX2 enzymes evaluated by qPCR in breast tissue of rats treated with MMI for (C) 7 and (D) 21 days. The data were expressed as relative to the control, using the GAPDH as an internal control for normalization and presented as the mean ± s.e.m. N = 6–10 per group. * P < 0.05; ** P < 0.01.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of hypothyroidism on the activity of antioxidant enzymes in breast tissue
After 7 days of hypothyroidism, catalase and GPX activities were significantly lower in treated group in comparison to its control, but there was no significant difference between groups in SOD activity (Fig. 4). However, after 21 days, we observed an increase in catalase (Fig. 4A) and SOD (Fig. 4C) activities in the Hypo group compared to its control, while no differences were observed for GPX activity (Fig. 4B).
Effects of 7 and 21 days of hypothyroidism on superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase activities in breast tissue of female Wistar rats. Catalase (A), GPX (B) and SOD (C) activities were measured in tissue homogenate by specific spectrophotometric assays for each enzyme in breast tissue of rats. Results are presented as mean ± s.e.m. N = 6–10 per group. ** P < 0.01.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase activities in breast tissue of female Wistar rats. Catalase (A), GPX (B) and SOD (C) activities were measured in tissue homogenate by specific spectrophotometric assays for each enzyme in breast tissue of rats. Results are presented as mean ± s.e.m. N = 6–10 per group. ** P < 0.01.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase activities in breast tissue of female Wistar rats. Catalase (A), GPX (B) and SOD (C) activities were measured in tissue homogenate by specific spectrophotometric assays for each enzyme in breast tissue of rats. Results are presented as mean ± s.e.m. N = 6–10 per group. ** P < 0.01.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of hypothyroidism on DNA damage levels in breast tissue
ROS are unstable and extremely reactive molecules capable to react with other biomolecules nearby, including DNA. As shown in Fig. 2B, lipid peroxidation was higher after 7 days of hypothyroidism. Thus, we aimed to evaluate if the DNA molecule was also affected by the treatment. For this purpose, we analyzed the phosphorylation of histone H2A.X, a marker of DNA damage, and the expression of p21, a protein that regulates the transition from G1 to S phase of the cell cycle. This transition is particularly important in the study of cancer cells, since DNA replication occurs in the S phase, so there is a greater probability of genetic mutations caused by loss of genome integrity. In fact, we observed an increase in γ-H2A.X/ H2A.X ratio in the breast of 7 days Hypo group (Fig. 5B); however, no significant difference in the expression of p21 was observed (Fig. 5C). In the case of 21 days Hypo group, there was no change in γ-H2A.X/ H2A.X ratio or in p21 expression (Fig. 5A,B, and C).
Effects of 7 and 21 days of hypothyroidism on γH2A.X and p21 levels in breast tissue of Wistar rats. Western blot of total H2A.X, γH2A.X and p21. (A) Representative immunoblot of, γH2A.X and p21. (B) Ratio between the relative densities of γ H2A.X and total H2A.X of the breast tissue. (C) Relative density of p21 protein in the breast tissue of rats. Results presented as the mean ± s.e.m. N = 4–8 per group. *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on γH2A.X and p21 levels in breast tissue of Wistar rats. Western blot of total H2A.X, γH2A.X and p21. (A) Representative immunoblot of, γH2A.X and p21. (B) Ratio between the relative densities of γ H2A.X and total H2A.X of the breast tissue. (C) Relative density of p21 protein in the breast tissue of rats. Results presented as the mean ± s.e.m. N = 4–8 per group. *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of 7 and 21 days of hypothyroidism on γH2A.X and p21 levels in breast tissue of Wistar rats. Western blot of total H2A.X, γH2A.X and p21. (A) Representative immunoblot of, γH2A.X and p21. (B) Ratio between the relative densities of γ H2A.X and total H2A.X of the breast tissue. (C) Relative density of p21 protein in the breast tissue of rats. Results presented as the mean ± s.e.m. N = 4–8 per group. *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
TSHR expression on mammary cell lines and breast tissue
Our experimental model of hypothyroidism was characterized by lower levels of total T4 and higher levels of TSH. It has been shown that TSH can induce oxidative stress in the thyroid and other tissues (Dardano et al. 2006, Morshed et al. 2010, 2013, Weyemi et al. 2010, Gagnon et al. 2014). Thus, we hypothesize that TSH could be involved in the observed induction of oxidative stress after 7 days of MMI. In order to evaluate the presence of TSH receptors in the breast tissue, Western blot was performed in the mammary tissue of rats and in breast cell lines (Fig. 6). As positive controls, we used the follicular cell line of rat thyroid, PCCL3, and thyroid of control rats. We observed that both breast epithelial cell lines MCF10A (non-tumoral) and MCF7 (tumoral) express the TSH receptor (TSHR). In addition, the presence of TSH receptors was observed in murine breast tissue (Fig. 6).
TSH receptor expression in mammary cell lines and breast tissue. Western blot analysis of the TSH receptor in breast and thyroid cell lines and in rat breast and thyroid tissue homogenates. Lane 1: MCF10A cells; lane 2: MCF7 cells; lane 3: PCCL3 cells; lane 4: Rat mammary gland homogenate; lane 5: Rat thyroid homogenate. In all cases, 30 μg of protein were submitted to electrophoresis.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
TSH receptor expression in mammary cell lines and breast tissue. Western blot analysis of the TSH receptor in breast and thyroid cell lines and in rat breast and thyroid tissue homogenates. Lane 1: MCF10A cells; lane 2: MCF7 cells; lane 3: PCCL3 cells; lane 4: Rat mammary gland homogenate; lane 5: Rat thyroid homogenate. In all cases, 30 μg of protein were submitted to electrophoresis.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
TSH receptor expression in mammary cell lines and breast tissue. Western blot analysis of the TSH receptor in breast and thyroid cell lines and in rat breast and thyroid tissue homogenates. Lane 1: MCF10A cells; lane 2: MCF7 cells; lane 3: PCCL3 cells; lane 4: Rat mammary gland homogenate; lane 5: Rat thyroid homogenate. In all cases, 30 μg of protein were submitted to electrophoresis.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on extracellular and intracellular ROS production and mRNA expression of NADPH oxidases in MCF10A cells
Non-tumor mammary epithelial cells were incubated with 10 and 20 mU/mL of TSH, which correlates with serum TSH concentrations found in our experimental model of hypothyroidism. Both concentrations of TSH treatment (10 and 20 mU/mL) increased extracellular ROS production (Fig. 7A), but only the higher concentration of TSH was able to increase intracellular ROS generation (Fig. 7C and D) and mitochondrial superoxide production (Fig. 7E and F). Taking into consideration the Ca2+-dependent ROS generation (activity of the DUOX enzymes), there was also a significant increase in cells treated with TSH at 20 mU/mL at 24 h (Fig. 7G). Moreover, a significant increase in NOX2, NOX4, and DUOX2 mRNA levels was observed after TSH treatment (Fig. 7G). On the other hand, the treatment decreased DUOX1 mRNA levels (Fig. 7G). No differences between groups were observed for NOX5.
Effects of TSH after 24 h treatment on extracellular ROS production, NADPH oxidase expression, intracellular and mitochondrial ROS levels in MCF10A cells. (A, B) The extracellular generation of hydrogen peroxide was evaluated by AmplexRed/HRP method in MCF10A cells treated with TSH for 24 h with (B) and without (A) calcium. The results were expressed as nanomoles of hydrogen peroxide corrected for time and quantity of cells. The intracellular and mitochondrial levels of ROS were evaluated in MCF10A cells treated with different TSH concentrations for 24 h by flow cytometry using the CM-H2DCF-DA and mitoSOX™ probes, respectively. (C) Histograms of DCF data from flow cytometry. (D) Relative fluorescence unit results of DCF assay. (E) Histograms of MitoSOX™ data from flow cytometry. (F) Relative fluorescence unit results of MitoSOX™ assay. The results were presented as the mean ± s.e.m. in three independent experiments with three replicates each. (G) Expression of four enzymes of the NADPH oxidase family expressed in breast epithelial cells was assessed by qPCR. The data were expressed as relative to control and presented as the mean ± s.e.m. of two independent experiments with three replicates each. Significance level: *P < 0.05; ** P < 0.01; *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH after 24 h treatment on extracellular ROS production, NADPH oxidase expression, intracellular and mitochondrial ROS levels in MCF10A cells. (A, B) The extracellular generation of hydrogen peroxide was evaluated by AmplexRed/HRP method in MCF10A cells treated with TSH for 24 h with (B) and without (A) calcium. The results were expressed as nanomoles of hydrogen peroxide corrected for time and quantity of cells. The intracellular and mitochondrial levels of ROS were evaluated in MCF10A cells treated with different TSH concentrations for 24 h by flow cytometry using the CM-H2DCF-DA and mitoSOX™ probes, respectively. (C) Histograms of DCF data from flow cytometry. (D) Relative fluorescence unit results of DCF assay. (E) Histograms of MitoSOX™ data from flow cytometry. (F) Relative fluorescence unit results of MitoSOX™ assay. The results were presented as the mean ± s.e.m. in three independent experiments with three replicates each. (G) Expression of four enzymes of the NADPH oxidase family expressed in breast epithelial cells was assessed by qPCR. The data were expressed as relative to control and presented as the mean ± s.e.m. of two independent experiments with three replicates each. Significance level: *P < 0.05; ** P < 0.01; *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH after 24 h treatment on extracellular ROS production, NADPH oxidase expression, intracellular and mitochondrial ROS levels in MCF10A cells. (A, B) The extracellular generation of hydrogen peroxide was evaluated by AmplexRed/HRP method in MCF10A cells treated with TSH for 24 h with (B) and without (A) calcium. The results were expressed as nanomoles of hydrogen peroxide corrected for time and quantity of cells. The intracellular and mitochondrial levels of ROS were evaluated in MCF10A cells treated with different TSH concentrations for 24 h by flow cytometry using the CM-H2DCF-DA and mitoSOX™ probes, respectively. (C) Histograms of DCF data from flow cytometry. (D) Relative fluorescence unit results of DCF assay. (E) Histograms of MitoSOX™ data from flow cytometry. (F) Relative fluorescence unit results of MitoSOX™ assay. The results were presented as the mean ± s.e.m. in three independent experiments with three replicates each. (G) Expression of four enzymes of the NADPH oxidase family expressed in breast epithelial cells was assessed by qPCR. The data were expressed as relative to control and presented as the mean ± s.e.m. of two independent experiments with three replicates each. Significance level: *P < 0.05; ** P < 0.01; *** P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on DNA damage in MCF10A cells
Since ROS was increased by TSH in MCFA10 cells, we decided to evaluate its effect on DNA damage. After 24 h of treatment, TSH-induced a two-fold increase in DNA damage in mammary cells, which was also observed after 72 h (Fig. 8A). The response to DNA damage is a network of cellular pathways that detects signals and repairs DNA lesions, affecting the state of chromatin at the site of the damaged DNA. Proteins like 53BP1 are important regulators of the cellular response to the double-strand break that supports signaling and promotes the repair of damaged DNA (Sulli et al. 2012). We observed that treatment with 20 mU/mL TSH increased the number of 53BP1 focal points in MCF10A cells, confirming the results of comet assay (Fig. 8B and C). In order to verify the role of ROS on TSH-induced DNA damage, MCF10A cells were treated with TSH with or without NAC. While TSH treatment induced a two-fold increase in DNA damage, the concomitant incubation of TSH and NAC resulted in only 1.3-fold increase, but the results were not statistically different (Fig. 8D).
Effects of TSH on DNA damage in MCF10A cells. MCF10A cells were treated with TSH at 20 mU/mL for 24 h or 72 h to assess DNA damage through the comet assay (A) and the response to DNA damage through immunostaining of 53BP1 analyzed by immunocytofluorescence (B–C). Moreover, MCF10A cells were treated with TSH at 20 mU/mL for 24 h with or without 1 mM N-acetyl cysteine (NAC) to assess DNA damage through the comet assay (D). The graph data were presented as the mean ±s.e.m. in three independent experiments with two replicates each. Significance level: *P < 0.05; ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on DNA damage in MCF10A cells. MCF10A cells were treated with TSH at 20 mU/mL for 24 h or 72 h to assess DNA damage through the comet assay (A) and the response to DNA damage through immunostaining of 53BP1 analyzed by immunocytofluorescence (B–C). Moreover, MCF10A cells were treated with TSH at 20 mU/mL for 24 h with or without 1 mM N-acetyl cysteine (NAC) to assess DNA damage through the comet assay (D). The graph data were presented as the mean ±s.e.m. in three independent experiments with two replicates each. Significance level: *P < 0.05; ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on DNA damage in MCF10A cells. MCF10A cells were treated with TSH at 20 mU/mL for 24 h or 72 h to assess DNA damage through the comet assay (A) and the response to DNA damage through immunostaining of 53BP1 analyzed by immunocytofluorescence (B–C). Moreover, MCF10A cells were treated with TSH at 20 mU/mL for 24 h with or without 1 mM N-acetyl cysteine (NAC) to assess DNA damage through the comet assay (D). The graph data were presented as the mean ±s.e.m. in three independent experiments with two replicates each. Significance level: *P < 0.05; ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on cell viability and cell cycle in MCF10A cells
As shown in Fig. 9A, there were no significant differences in the viability between the control and treated groups by the MTT assay. Since MTT assay assesses cell viability through mitochondrial activity, which could be modulated by TSH treatment, we also used the trypan blue staining method. Likewise, no significant differences among groups were observed in the number of live cells (Fig. 9B) and the number of dead cells (Fig. 9C), corroborating the results of the MTT test. We also investigated the regulation of cell proliferation by quantifying the proportion of cells in each phase of the cell cycle. The analysis of cell cycle and G1 was performed by incorporating propidium iodide (PI) and visualized by flow cytometry. TSH treatment did not alter the cell cycle and the number of cells in sub-G1 24 h or 72 h after treatment (Fig. 9D,E,F, and G).
Effects of TSH on cell viability and cell cycle in MCF10A cells. MCF10A cells were treated with different TSH concentrations for 24, 48 and 72 h for cell viability evaluation assessed by the MTT assay and by trypan blue method. (A) Cell viability by the MTT assay. (B) The total number of living cells by trypan blue assay. (C) The total number of dead cells by trypan blue assay. MCF10A cells were treated with 20 mU/mL TSH for (D) 24 h and (E) 72 h and the cell cycle and sub-G1 analysis of the cells was evaluated by the incorporation of propidium iodide, measured by flow cytometry. Histograms of cell cycle data in (F) 24 h and (G) 72 h. Results are presented as the mean ±s.e.m. of three independent experiments with four to six replicates in the MTT assay, two independent experiments with three replicates each using the blue trypan method, and four independent experiments with three replicates for cell cycle analysis.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on cell viability and cell cycle in MCF10A cells. MCF10A cells were treated with different TSH concentrations for 24, 48 and 72 h for cell viability evaluation assessed by the MTT assay and by trypan blue method. (A) Cell viability by the MTT assay. (B) The total number of living cells by trypan blue assay. (C) The total number of dead cells by trypan blue assay. MCF10A cells were treated with 20 mU/mL TSH for (D) 24 h and (E) 72 h and the cell cycle and sub-G1 analysis of the cells was evaluated by the incorporation of propidium iodide, measured by flow cytometry. Histograms of cell cycle data in (F) 24 h and (G) 72 h. Results are presented as the mean ±s.e.m. of three independent experiments with four to six replicates in the MTT assay, two independent experiments with three replicates each using the blue trypan method, and four independent experiments with three replicates for cell cycle analysis.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Effects of TSH on cell viability and cell cycle in MCF10A cells. MCF10A cells were treated with different TSH concentrations for 24, 48 and 72 h for cell viability evaluation assessed by the MTT assay and by trypan blue method. (A) Cell viability by the MTT assay. (B) The total number of living cells by trypan blue assay. (C) The total number of dead cells by trypan blue assay. MCF10A cells were treated with 20 mU/mL TSH for (D) 24 h and (E) 72 h and the cell cycle and sub-G1 analysis of the cells was evaluated by the incorporation of propidium iodide, measured by flow cytometry. Histograms of cell cycle data in (F) 24 h and (G) 72 h. Results are presented as the mean ±s.e.m. of three independent experiments with four to six replicates in the MTT assay, two independent experiments with three replicates each using the blue trypan method, and four independent experiments with three replicates for cell cycle analysis.
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
TSH receptor mRNA levels in breast cancer tissues
Since TSH was able to increase ROS production and DNA damage, two events involved in the carcinogenic process, we decided to compare the expression of TSHR among 1904 samples of breast cancer patients of various types using the METABRIC database (Curtis et al. 2012, Pereira et al. 2016). Only 102 samples (5.4%) showed TSHR alterations, 9 of which were gene amplifications, 58 were related to increase in mRNA, and 35 samples to decrease in mRNA. No gene mutations were found. Most changes were increased in TSHR mRNA, mainly found in invasive ductal carcinoma (Fig. 10A). TSHR mRNA levels were present was detected in all types of breast cancer; however, the triple-negative claudin-low type had a higher level of expression compared to the other classifications (Fig. 10B).
TSHR expression profile in different types of breast cancer. (A) Mutation, copy number aberration (CNA) and changes in TSHR mRNA levels found in different types of cancer. (B) Expression of TSHR mRNA in different classifications of breast carcinoma. Results obtained from the METABRIC database (Curtis et al. 2012, Pereira et al. 2016).
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
TSHR expression profile in different types of breast cancer. (A) Mutation, copy number aberration (CNA) and changes in TSHR mRNA levels found in different types of cancer. (B) Expression of TSHR mRNA in different classifications of breast carcinoma. Results obtained from the METABRIC database (Curtis et al. 2012, Pereira et al. 2016).
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
TSHR expression profile in different types of breast cancer. (A) Mutation, copy number aberration (CNA) and changes in TSHR mRNA levels found in different types of cancer. (B) Expression of TSHR mRNA in different classifications of breast carcinoma. Results obtained from the METABRIC database (Curtis et al. 2012, Pereira et al. 2016).
Citation: Endocrine-Related Cancer 28, 7; 10.1530/ERC-21-0010
Discussion
In the current study, we investigated the effect of hypothyroidism on rat mammary tissue redox homeostasis and DNA damage, the role of TSH regulating these events in mammary cells, as well as the expression of TSH receptor in breast cancer using the METABRIC database.
We found that after 7 days of hypothyroidism, lipid peroxidation levels were higher than controls, while total reduced thiol group levels were lower. These results suggest that, in this time point, the mammary glands were under oxidative stress. Interestingly, the same effect was not observed after 21 days of treatment, in which no differences were found between groups, suggesting that mechanisms were activated to counteract the oxidative stress found in the beginning of the treatment.
Calcium-dependent hydrogen peroxide generation was reduced after 7 days of treatment with MMI. Methimazole has previously been shown to inhibit DUOX activity (Ferreira et al. 2003), and DUOX2 mRNA levels were lower in the MMI-treated group, which could explain the reduction in DUOX activity in this group. Moreover, NOX2 mRNA levels were lower after 7 and 21 days of treatment, while DUOX2 mRNA levels decreased only after 7 days. Thus, the oxidative stress found in the mammary gland of rats treated for 7 days with MMI could not be attributed to NOX family. It is important to note that other sources of ROS, such as mitochondria, could be affected by MMI treatment, but they were not evaluated in the present study.
Regarding antioxidant enzymes, we observed that after 7 days of treatment with MMI, catalase and GPX activities were decreased, while after a more prolonged time of hypothyroidism (21 days), catalase and SOD activities were higher when compared to their respective controls. Taken together, our results suggest that oxidative stress found in short treatment (7 days) might be due to the lower antioxidant capacity, while the redox balance was restored in the long term (21 days) thanks to the increased catalase and SOD activities. Thus, after 21 days of MMI treatment, biomolecules oxidation no longer occurred, probably due to an adaptive response that was responsible for the higher antioxidant activity observed in at this time point. Regarding DNA damage, γH2Ax/H2Ax ratio was higher after 7 days of treatment, but no differences were observed after 21 days, following the same pattern of oxidative damage. Thus, our findings suggest a possible relationship between the higher ROS availability and DNA damage in our model of experimental hypothyroidism.
The action of TSH on thyroid cells is well established, but its effects on other cell types are beginning to be elucidated. Interestingly, it has been shown that TSH can induce oxidative stress in the thyroid and other tissues. In a primary human follicular thyroid cell culture, TSH increased NOX4-derived ROS generation in a dose-dependent manner (Weyemi et al. 2010). In vitro studies with thyrocytes exposed to TSHR-stimulating antibodies present in Graves' disease increased intracellular ROS generation in a dose-dependent manner, with the participation of increased mitochondrial ROS generation (Morshed et al. 2010, 2013). The administration of recombinant TSH to 21 patients monitored for differentiated thyroid carcinoma for two consecutive days increased serum lipoperoxide levels and reduced the ferric reducing antioxidant power (Dardano et al. 2006). Moreover, an in vitro study with differentiated human adipocytes found that TSH was also able to increase the activity of NADPH oxidase (Gagnon et al. 2014). Thus, we hypothesized that TSH could be involved in the induction of oxidative stress and DNA damage in breast tissue. The first step was to identify the presence of the TSHR in the MCF10A cells and in the breast tissue, since data in the literature are scarce. The first identification of the TSHR in breast tissue dates back to 2005 when the group of Oh et al. demonstrated the expression of TSHR in breast carcinoma tissue and correlated this finding with the type of tumor pathology, demonstrating that the TSHR gene expression was higher in low-grade breast cancer (Oh et al. 2005). In 2012, Govindaraj et al. demonstrated the expression of TSHR in human normal breast tissue, which was lower than in breast tumor samples (Govindaraj et al. 2012). We observed in the present study that TSHR is expressed by non-tumoral epithelial mammary cell MCF10A, as well as by rat breast tissue and the cell line MCF7, thus confirming that this hormone can act in the breast.
We observed that the cells treated with TSH had a higher generation of extracellular, intracellular, and mitochondrial ROS. Moreover, NOX2, NOX4, and DUOX2 mRNA levels were higher after TSH treatment. NOX enzymes have ROS generation as their sole function, and their upregulation is associated with different phases of the carcinogenic process, which was demonstrated in several tumor types (Lim et al. 2005, Block & Gorin 2012, Weyemi et al. 2012, Roy et al. 2015). DUOX1 mRNA levels were lower in cells treated with TSH compared to control. Previous studies have shown a decrease in DUOX1 expression in lung cancers, liver, and breast cancer (Luxen et al. 2008, Ling et al. 2014, Fortunato et al. 2018). These studies suggest that the epigenetic inactivation of DUOX1 is an important factor in cancer tumorigenesis. We have demonstrated that the silencing of DUOX1 in MCF12A cells (a non-tumoral epithelial mammary cell line) increased cellular proliferation, decreased cell adhesion, and blunted the response to genotoxic stress (Fortunato et al. 2018). TSH treatment also led to increased levels of DNA damage, and higher levels of 53BP1 nuclear foci, which is involved in DNA damage response. Taking into account that the treatment with TSH increased the damage to DNA in cells, a cell cycle arrest was expected in response to this damage (Sulli et al. 2012). However, cell number, viability, and cell cycle were not affected by treatment, suggesting that TSH signaling was capable to bypass DNA damage response. The role of TSH in thyroid follicular cell to promote cell proliferation is well documented (García-Jiménez & Santisteban 2007, Milas et al. 2009). The higher DNA damage after 72 h of TSH treatment also suggests that DNA damage response was surpassed.
Finally, we evaluated the profile of TSHR expression in different classifications of breast carcinoma using the TGCA platform. We observed that only 5.4% of the studied samples presented some type of alteration in the TSHR mRNA levels, with the majority being an increase in the levels of mRNA, most associated with invasive ductal carcinoma. These results may be related to the characteristics of more invasive breast tumors found in patients and animals with hypothyroidism (Martínez-Iglesias et al. 2009, Franco et al. 2016). TSHR expression was similar among the various molecular classifications of the mammary tumors; however, we observed an increased TSHR expression in the claudin-low type. Several groups have characterized this tumor subtype and have shown that claudin-low tumors are responsible for 7–14% of all invasive breast cancers, are enriched in genes associated with epithelial to mesenchymal transition, immune cell infiltration, IFNγ activation, presence of stem cells or breast tumor initiator cells and typically demonstrate high levels of genomic instability (Perou 2010, Prat et al. 2010, Prat & Perou 2011, Dias et al. 2017).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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