Abstract
Background/Aim: Thyroid hormones (THs) stimulate breast cancer (BC) cell proliferation. We hypothesized that these hormones and the proliferative marker thymidine kinase 1 (TK1) represent the initial and final steps of the proliferative pathway, respectively. Patients and Methods: We measured the serum levels of thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4), along with serum TK1 activity, in 144 newly diagnosed BC patients, and examined the associations between THs and proliferation in different BC receptor profiles. Results: TK1 activity did not correlate with TSH (r=0.06, p=0.473) or FT4 levels (r=0.04, p=0.665), but did correlate with FT3 levels (r=0.28, p=0.001). Elevated FT3 (>6.0 pmol/l) predicted increased TK1 activity (>140 Du/l) after adjusting for age (odds ratio 4.1, p=0.014). We also found a significant association of the combined elevation of FT3 and TK1, assumed as a surrogate marker of accomplished proliferative signal, with triple-negative (TN) profile (p=0.003). The rates of combined FT3 and TK1 elevation in TN and ER-positive profiles were 20.0% and 1.8%, respectively (p=0.005). Conclusion: FT3 may be involved in proliferative signaling, as measured by TK1 activity, predominately in TN breast cancer.
Thyroid hormones (THs) are involved in the regulation of mammary cell growth, maturation, and differentiation and can potentially affect the growth and spread of breast cancer (BC) cells (1, 2). The thyroid gland produces two main forms of THs: T4 (3,5,3’,5’-tetraiodothyronine) and T3 (3,3’,5-triiodothyronine). Specific iodothyronine deiodinases modify the prohormone T4 to the natural T3 hormone. The hypothalamic–pituitary–thyroid axis regulates blood concentrations of THs, and the association of THs and thyroid-stimulating hormone (TSH) is regulated by a negative feedback loop (3).
In studies examining the influence of THs on cells, these hormones have been shown to be associated with proliferative processes (4-6). T3 stimulated MCF-7 proliferation in a dose-dependent manner and induced the activation of an estrogen response element–mediated gene expression (6). In the estrogen-responsive cell line T47-D, T3 regulates cell cycle progression and proliferation, causing hyper-phosphorylation of the retinoblastoma (Rb) tumor suppressor protein (7). Sequestered from the RB-E2F complex, the transcription factor E2F activates genes involved in DNA synthesis and cell proliferation (8-11). One of these genes is thymidine kinase 1 (TK1), which encodes a key enzyme in the salvage pathway for deoxythymidine monophosphate synthesis. Its activity is highest at the G1-S translation checkpoint in proliferating normal and tumor cells. Dividing cells release TK1 during mitotic exit, a process that is mediated by the ubiquitin system (12). In a previous study, we showed that serum TK1 activity can be assessed with a new high-resolution assay (13).
Both normal and malignant proliferating cells release TK1 into the circulation, and serum TK1 activity reflects the overall condition (proliferative background) of each individual (13). TK1 activity within a malignant tumor and its contribution to serum TK1 activity are unknown; however, the drastic decrease in TK1 after surgery suggests that the tumor is the main source of the analyte (14). The strong coupling of TK1 expression to the cell cycle and proliferation provides the rationale for investigating TK1 as a marker of the effect of different activators of proliferative signaling. The hypothesis that THs and TK1 represent the initial and final elements of the proliferative pathway could be useful for evaluating the relationships between these parameters in BC patients. The aim of this study was to investigate whether TSH, FT3, and FT4 are related to cell proliferation in terms of serum TK1 activity in patients with BC.
Patients and Methods
Our institutional ethical review board approved this study (380-23.04.04), which included 168 female patients who underwent surgery for BC between May 2004 and December 2008, and were followed at Hadassah University Hospital. All patients provided signed informed consent. Patients with a prior history of malignancy, known metastatic BC, pure ductal carcinoma in situ, or pre-operative neoadjuvant chemotherapy or hormonal therapy were excluded, as were patients who were taking levothyroxine (n=23) as a thyroid supplement.
Serum samples were obtained from all patients preoperatively, aliquoted, and stored at –80°C until analysis. Tests for TSH, FT3, and FT4 were performed using the commercially available IMMULITE kits, which are solid-phase, two-site chemiluminescent immunometric assays (Immulite, Siemens Healthcare Diagnostics, Lianberis, United Kingdom).
To measure serum TK1 activity, we used a colorimetric enzyme-linked immunosorbent assay kit (DiviTum; Biovica International AB, Uppsala, Sweden), as described previously (13). TK1 levels were expressed in DiviTum units/l (Du/l). The cut-off level for TK1 was 140.0 Du/l, based on the 95th percentile in a reference group of 149 healthy women (13).
Statistical analysis. We evaluated TSH, FT3, and FT4 concentrations and TK1 activity as both continuous and categorical variables. The distribution of these variables was asymmetric, so we used the Mann–Whitney test to compare numeric variables. Fisher’s exact test was used for categorical variables. Potential correlations between numerical variables were evaluated by the Pearson product-moment (Pearson r) or by Spearman’s rank correlation coefficient (Spearman r), as appropriate. Logistic regression was used for the prediction of increases above the cutoff TK1 activity. Statistical calculations were performed using SPSS, version 17 for Windows (SPSS Inc., Chicago, IL, USA). A value of p<0.05 was considered significant.
Results
This study included 144 women with a histological diagnosis of invasive BC. Age ranged between 24 to 88 [median 56, interquartile range (IQR) 46-65]. Invasive ductal carcinoma was diagnosed in 127 (88.2%), and 17 patients (11.8%) had other histological types. Eighty patients (55.6%) had tumors ≤2.0 cm, and 64 patients (44.4%) had tumors >2 cm. Histologic tumor grade was I/II in 58 patients (40.3%), and 76 (52.8%) had grade III breast cancer. Sixty-two (43.1%) were lymph node-positive, and 82 were node-negative (56.9%).
TSH, FT4, FT3, and TK1 by age. Among the 144 patients, age did not correlate with TSH (r=–0.01, p=0.930), FT4 (r=– 0.04, p=0.605), or TK1 (r=–0.09, p=0.277). Only FT3 levels negatively weakly correlated with age (r=–0.28, p=0.001). The median serum levels of FT3 were higher in women below 50 years of age compared to those above (median, IQR: 5.24, 4.58-5.76 vs. 4.67, 4.11-5.49 pmol/l, p=0.022). TK1 did not correlated with either FT4 (r=0.04, p=0.665) or TSH (r=0.06, p=0.473), but FT3 and TK1 significantly correlated (r=0.28, p=0.001).
Distribution of TK1 by FT3 levels. The distribution of TK1 by FT3 levels in BC patients is shown in Figure 1. There was no significant change in TK1 activity with an increase in FT3 up to 6.0 pmol/l (p=0.786). The median TK1 activity in the upper interval of FT3 levels (>6.0 pmol/l) was significantly higher compared to that at each of the two lower intervals: ≤4.0 pmol/l and >4.0-6.0 pmol/l, respectively (p=0.023 and p=0.019, Mann–Whitney U-test).
Distribution of TK1 activity by FT3 levels in patients with breast cancer. FT3: Free triiodothyronine; TK1: thymidine kinase 1; IQR: interquartile range. Each boxplot shows the median (thin line), quartiles (box ends), and extreme values (whiskers) within the category shown. p-Values were derived from Mann–Whitney U-test.
TK1 activity in patients with FT3 above 6.0 pmol/l was significantly higher than that in patients with FT3 below [median (IQR): 57.1 (38.0-156.3) vs. 31.7 (18.0-64.8) Du/l, p=0.012]. Based on these data, 6.0 pmol/L was defined as the threshold for detection of patients with increased TK1 activity (>140.0 Du/l). In the total cohort, FT3 levels >6.0 pmol/l predicted increased serum TK1 activity after adjustment for age [odds ratio (OR)=4.1 (95% confidence interval (CI) =1.3–13.0, p=0.014].
Association of elevated FT3 and TK1 and their combination with histopathologic characteristics. There was no association of elevated FT3 and TK1 or their combination (Table I) with tumor stage (17.5% vs. 12.5%, p=0.488; 11.3% vs. 10.9%, p=1.000, and 5.0% vs. 3.1%, p=0.693), tumor grade (17.5% vs. 14.5%, p=0.640; 7.0% vs. 14.5%, p=0.268, and 1.8% vs. 6.6%, p=0.237), and nodal status (11.3% vs. 18.3%, p=0.350; 9.7% vs. 12.2%, p=0.790 and 1.6% vs. 6.1, p=0.236).
Association of elevated FT3 and TK1 and their combination with histopathological characteristics in BC.
Elevated TK1 (>140.0 Du/l) was more often found in patients with ER-negative and TN tumors (Table I), than in ER-positive (22.6% vs. 8.0%, p=0.046) and non-TN tumors (25.0% vs. 8.9%, p=0.049), respectively. Elevated FT3 (>6.00 pmol/l) showed very low prevalence in patients with Her2/neu-positive compared to Her2/neu-negative tumors (3.2% vs. 18.6%, p=0.046).
In the whole cohort of 144 patients, six cases (4.2%) had combined elevation of FT3 levels and TK1 activity. Four of six patients were ER-negative/PR-negative/Her2neu-negative and remaining two patients had ER-positive/PR-positive/Her2neu-negative tumors.
The rate of cases with combined elevation of FT3 levels and TK1 activity was higher in ER-negative and TN cases compared to ER-positive (12.9% vs. 1.8%, p=0.020) and non-TN cases (20.0% vs. 1.6%, p=0.003), respectively. The rate of combined elevation of FT3 and TK1 in ER-positive and TN profiles comprised 1.8% and 20.0%, respectively (p=0.005). A significant correlation between FT3 and TK1 was found in TN (r=0.61, p=0.008), but not in ER-positive BC (r=0.17, p=0.069) (Figure 2).
Scatterplot showing correlation between FT3 levels and TK1 activity in triple-negative (A) and ER-positive (B) breast cancer. FT3: Free triiodothyronine; TK1: thymidine kinase 1.
Discussion
In preclinical studies involving different BC lines, THs have demonstrated growth-promoting effects (4-6). However, little clinical data are available confirming the association of THs with proliferative activity in patients with BC.
Tumor cell proliferation is one of the most important characteristics of tumor aggressiveness and is a commonly used variable in evaluating tumor progression (15). One way to assess proliferation is measurement of the enzyme TK1, which is involved in DNA synthesis. The expression of TK1 peaks in the S-phase of the cell cycle and is regulated by the transcription factor E2F (8-11). Although TK1 activity is measured in serum, it primarily reflects proliferative activity of tumors (14). In this study, we evaluated potential correlations of serum TK1 activity with TSH, FT4, and FT3 levels.
We found no association of serum FT4 and TSH levels with the proliferative background in patients with BC. These two hormones did not correlate with TK1. Mourouzis et al. (16) have also reported a lack of correlation of FT4 and TSH with proliferation activity, based on measuring the KI67 index in patients newly diagnosed with BC.
A significant positive correlation was found between FT3 and TK1. Analysis of the distribution of TK1 among three intervals of FT3 revealed significantly higher TK1 activity in the upper interval (>6.0 pmol/l) compared with the two lower intervals: ≤4.0 pmol/l and >4.0-6.0 pmol/l. Median TK1 activity in patients with FT3 above 6.0 pmol/l was significantly higher compared to those below (57.1 vs. 31.7 Du/l, p=0.012).
In accordance with our previous data (17), serum FT3 significantly reversely correlated with age. Higher median FT3 level was detected in younger (below 50 years) compared to elder patients (5.24 vs. 4.67 pmol/l, p=0.022). Based on logistic regression, elevated FT3 predicted an increased proliferative background after adjustment for age, with an OR of 4.1. This finding is the first to show a positive association between FT3 and proliferative activity (TK1) in patients with BC.
Further, we analyzed the distribution of combined elevation of FT3 and TK1, assumed a surrogate marker of accomplished proliferative signal in BC patients. In the whole cohort, the combined elevation was found in six patients (4.2%). These cases were unevenly distributed among different hormone receptor profiles. The prevalence of the combined elevation of FT3 and TK1 in TN profile was more than ten times higher than in ER-positive profile (20.0% vs. 1.8%, p=0.003). These findings are consistent with stronger correlation between FT3 and TK1 observed in TN than in ER positive profiles (r=0.61 and r=0.17, respectively), suggesting that FT3 stimulates proliferative activity predominately in TN BC.
In studies examining the influence of T3 on cells, this hormone has shown associations with proliferative processes (6, 7). Earlier, Dinda et al. (7) reported that T3 mimics the effects of estrogen in BC cell lines. Like estrogen, T3 could regulate cell cycle progression, causing hyperphosphorylation of Rb by a mechanism involving ER mediated pathways. Acting at the plasma membrane by a non-genomic mechanism via integrin αvβ3, T3 triggers the MAPK–dependent phosphorylation of ER (18, 19). In ER-positive human lung cancer cells, T3 induces ERK1/2 activation, as well as ER-alpha phosphorylation, proliferating-cell nuclear antigen expression, and hormone-dependent thymidine uptake by tumor cells (20). Thymidine uptake intensity in this study most likely reflected the activity of thymidine kinase. In our study combined elevation of FT3 and TK1 was, however, rarely observed in ER-positive BC (1.8%) and more often in TN BC (20.0%).
Different signaling pathways could play a role in the activation of proliferation in TN BC. Abnormally activated MAPK, PI3K/AKT/mTOR and Wnt/b-catenin signaling pathways provide ER-negative cells with the ability to proliferate (21-23). Recently, Silva et al. (24) reported that at physiological doses T3 upregulated the expression of transforming growth factor α (TGFα) independent of MAPK/ERK pathway activation in MCF7 cells. An activation of phosphoinositide 3-kinase (PI3K) was necessary for T3 to modulate TGFα expression (25). In primary BC tissue and BC cell lines T3 upregulated TGFα mRNA expression more efficiently than 17β-estradiol (26). T3 also upregulated the expression of amphiregulin (27). TGFα and amphiregulin are known ligands of EGFR, activating EGFR signaling pathways for cell proliferation. Mitogenic signaling via most pathways is bound to the transcription factor E2F (28). It can be hypothesized that FT3 in TN BC drives cell proliferation via the EGFR pathway and subsequent activation of TK1, downstream to this pathway. A more detailed investigation of the signaling from T3 to TK1 in hormone receptor negative BC is warranted.
In summary, elevated FT3 in patients with BC was significantly associated with increased TK1 activity, suggesting an increased proliferative background in these patients. This new evidence could provide a plausible explanation for the role of this TH as a stimulator of proliferative processes in BC. The relationship between FT3 and TK1 was dependent on hormone receptor expression. The strongest association of elevated FT3 levels with high TK1 activity was detected in patients with TN BC. The prevalence of combined elevation of FT3 and TK1 assumed as a surrogate marker of accomplished proliferative signal, in TN profile was 20.0%, while in ER-positive profile only 1.8%. This pattern is of important physiological and clinical relevance because it demonstrates the involvement of FT3 in proliferative signaling predominately in patients with TN BC, and suggests its possible detrimental effect in these patients.
Acknowledgements
The Authors thank Dr. Mario Baras of the Hebrew University, Hadassah School of Public Health, for his advice on statistical analysis.
Footnotes
↵* These Authors contributed equally to the present study.
Authors’ Contributions
Conception and design: BN, TA, TP, LK. Acquisition of data: TA, BN, EC, TP, LK, OM. Analysis and interpretation of data: BN, TA, TP, LK, AM. Histological examination of the breast: BM. Writing, review, and/or revision of the manuscript: BN, TA, TP, LK.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
Funding
This work was partly funded by Biovica/Ronnerbol (Uppsala, Sweden).
- Received December 6, 2020.
- Revision received December 22, 2020.
- Accepted December 23, 2020.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
