Abstract
Background/Aim: DYRK2 is a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase induces degradation of telomerase reverse transcriptase (TERT). The expression of both proteins in breast cancer were investigated as predictors of recurrence. Patients and Methods: Two hundred and twenty-one patients with early breast cancer treated at our institute between 2000 and 2009, were included. We used immunohistochemical analyses to measure the expression of DYRK2 and TERT and correlated it with clinicopathological factors and survival. Results: DYRK2 and TERT were positive in 58 (26%) and 86 (39%) of 221 patients, respectively. There was no correlation between DYRK2 and TERT expression and clinicopathological factors. Better disease-free survival was observed in the DYRK2-positive group (p=0.032), and poorer disease-free survival was noted in the TERT-positive group (p=0.023). The DYRK2-positive TERT-negative group exhibited significantly better disease-free survival than the other groups (p=0.006). Conclusion: The combination of DYRK2 and TERT may be a powerful tool to stratify breast cancer patients.
Breast cancer is a common malignancy among women worldwide. Approximately 25-30% of breast cancer patients without lymph node metastases will develop distant metastases within ten years of surgery (1). Several genetic characteristics have been reported to be useful for predicting the prognosis and selecting appropriate therapies (2). Gene profiling has been established as a tool for predicting clinical outcomes (3).
Telomerase is a DNA polymerase that maintains the length of telomeres at the end of chromosomes. Its activity is relatively high in stem cells and is down-regulated in normal somatic cells (4). In cancer cells, telomerase activation leads to disordered cell proliferation and immortalization (5). This is due to the activation of telomerase either by promoting the synthesis of telomerase RNA template (TERC) or the transcription of telomerase reverse transcriptase (TERT), two subunits of telomerase (6). It was recently reported that TERT is phosphorylated by the dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase gene (DYRK2) and targeted for degradation by the ubiquitin-proteasome system, which results in the suppression of telomerase activity (7). DYRK2 was reported as a member of a conserved family of dual specificity tyrosine phosphorylation-regulated kinases that autophosphorylates tyrosine residues but functions as a serine/threonine kinase for its substrates (8). Previous reports have demonstrated that DYRK2 regulates p53 to induce apoptosis in response to DNA damage (9). However, the roles of DYRK2 have not been well analyzed and remain unclear. We previously reported the role of DYRK2 as a favorable prognostic marker in non-small cell lung cancer (NSCLC), and found that bronchiolo-alveolar carcinoma, which has a better prognosis than other adenocarcinoma subtypes, highly expressed this protein (10). DYRK2 is also down-regulated in ductal carcinoma of the breast (11). Furthermore, we previously reported that DYRK2 expression may be a prognostic factor in early-stage breast cancer and a new predictor of recurrence in patients with node-negative early-stage breast cancer (12). However, the function of this protein in breast cancer remains unclear.
In this study, we hypothesized that in high-DYRK2-expressing breast cancer, TERT protein expression is suppressed, thereby reducing telomerase activity, and improving prognosis. The expression of both proteins was investigated, and their combination was evaluated as a predictor of recurrence.
Patients and Methods
The study included 221 samples from breast cancer patients who underwent surgery at Fukuoka University Hospital between January 2000 and September 2009. The samples were diagnosed histologically as primary breast cancer by hematoxylin and eosin (H&E) staining. Patients who received radiotherapy or chemotherapy before surgery were excluded. The institutional ethical committee of Fukuoka University approved this retrospective study (H21-05-010) and waived the need for patient consent.
Immunohistochemical analysis. As previously reported (12), 4-μm sections were prepared for tissue slides. Antigen retrieval was performed at 121°C for 10 min for DYRK2 and for 15 min for TERT in an autoclave with citrate buffer (pH 6.0) after deparaffinization. To block nonspecific binding, 10% goat serum (Nichirei Tokyo, Japan) was used. Staining with polyclonal anti-DYRK2 antibody (AP7534a; Abgent, San Diego, CA, USA) and Polyclonal anti-TERT antibody (C-20; Santa Cruz, Princeton, NJ, USA) as a diluent at 1:50 was performed overnight at 4°C. After reacting with 3% hydrogen peroxide for 20 min at room temperature, polymer anti-rabbit (goat) antibody (K4002; Dako, Glostrup, Denmark) against DYRK2 and polymeric anti-goat antibody against TERT (Nichirei Tokyo, Japan) was applied and incubated for 30 min at room temperature. Negative controls were incubated without the primary antibody.
IHC staining was evaluated as follows: 0, no staining or faint cytoplasmic staining in less than 10% of tumor cells; 1+, faint cytoplasmic staining in more than 10% of tumor cells; 2+, weak or moderate cytoplasmic staining in more than 10% of tumor cells; and 3+, more than 10% of tumor cells having strong cytoplasmic staining. As we previously reported a DYRK2 IHC staining study, 0 or 1+ staining intensity was considered DYRK2-negative and 2+ or 3+ staining was considered positive. TERT was evaluated in the same manner. For evaluation of the reliability, two independent assessors estimated the staining positivity of two serial sections.
Statistical analysis. All statistical analyses were performed using SPSS 14.0 (SPSS Japan Inc., Tokyo, Japan). Different variables of the tumors and normal tissues were analyzed with the chi-square test or Fisher’s exact test. Disease-free survival (DFS) and overall survival (OS) were analyzed using the Kaplan–Meier method and evaluated by the log-rank test. Significant differences were accepted at p<0.05. Confounding factors were evaluated by Cox regression hazards models.
Results
The relationship between clinicopathological characteristics and DYRK2 and TERT expression. We investigated the correlation between the clinicopathological characteristics of breast cancer and DYRK2 and TERT expression. DYRK2 and TERT expression was observed in 58 (26%) and 86 (39%) patients, respectively. The expression patterns of DYRK2 and TERT are shown in Figure 1. Although DYRK2-positive samples exhibited strong granular staining in the cytoplasm of breast cancer cells from the resected specimen of invasive ductal carcinoma, TERT was positive in the nucleus. On the other hand, although normal stromal cells were negative for both proteins, ductal epithelial cells exhibited weak expression of DYRK2, but not TERT (data not shown). No correlation was found between DYRK2 and TERT expression and clinicopathological factors, including Her2 and Ki-67 (Table I). However, there was a significant correlation between TERT and DYRK2 expression (Table II). Although DYRK2 expression led to the down-regulation of TERT in 22 samples (10%), TERT was expressed in 50 samples (23%) in the absence of DYRK2.
Immunohistochemical staining of DYRK2 and TERT protein expression in breast cancer. The cytoplasm of invasive ductal carcinoma cells was strongly stained (A, DYRK2 ×100). Nuclear staining of TERT protein is observed in ductal carcinoma cells (C ×200). Negative staining of invasive ductal cancer (B, DYRK2; D TERT ×200).
Patient characteristics.
Correlation between DYRK2 and TERT expression in breast cancer samples.
Prognostic value of DYRK2 and TERT. We evaluated the potential role of these proteins in predicting prognosis. DFS and OS according to DYRK2 are shown in Figure 2. The DYRK2-positive group had a better DFS than the DYRK2-negative group (p=0.032), but there was no significant difference in OS. As shown in Figure 3, TERT-positive breast cancer had a poorer DFS than TERT-negative breast cancer (p=0.023), but there was no significant difference in OS. We divided samples into four groups according to different combinations of protein expression: group I, DYRK2 (+) and TERT (–); group II, DYRK2 (+) and TERT (+); group III, DYRK2 (–) and TERT (–); group IV, DYRK2 (–) and TERT (+). Comparison of survival in these four groups is shown in Figure 4. The median follow-up period was 63 months. The 10-year DFS rate was 100% in the DYRK2 (+) and TERT (–) group (n=22), 86.3% in the DYRK2 (+) and TERT (+) group (n=36), 68.4% in the DYRK2 (–) and TERT (–) group (n=113), and 66.3% in the DYRK2 (–) and TERT (+) group (n=50, p=0.006). The DYRK2 (+) and TERT (–) group had a better DFS than the other three groups (p=0.001), but there was no difference in OS among the groups.
Disease-free survival (DFS) and overall survival (OS) according to DYRK2 protein expression. The 10-year DFS was 94.3% in the DYRK2-positive group (n=58) and 68.1% in the negative group (n=163, p=0.032). OS was not significantly different between the groups.
DFS and OS according to TERT protein expression. The 10-year DFS was 79.2% in the DYRK2-positive group (n=86) and 68.2% in the negative group (n=135, p=0.023). OS was not significantly different between the groups. DFS: Disease-free survival; OS: overall survival.
Four groups regarding combinations of DYRK2 and TERT protein expression: Group I, DYRK2 (+) and TERT (–); group II, DYRK2 (+) and TERT (+); group III, DYRK2 (–) and TERT (–); group IV, DYRK2 (–) and TERT (+). The 10-year DFS rate was 100% in the DYRK2 (+) and TERT (–) group (n=22), 86.3% in the DYRK2 (+) and TERT (+) group (n=36), 68.4% in the DYRK2 (–) and TERT (–) group (n=113), and 66.3% in the DYRK2 (–) and TERT (+) group (n=50, p=0.006). DFS: Disease-free survival.
Ten-year OS did not significantly differ among the four groups because of the low incidence of death in the follow-up period (Figure 2, Figure 3 and Figure 4). T factor, estrogen receptor status, progesterone receptor status, chemotherapy, hormone therapy, DYRK2 expression, TERT expression, and the combination of both proteins were demonstrated to have prognostic significance by univariate analysis (Table III); however, multivariate Cox regression analysis revealed only the combination of DYRK2 and TERT expression to have the potential for a significant prognostic impact on DFS (Table III). Hormone therapy was a prognostic factor for OS by univariate and multivariate analyses (Table IV).
Univariate and multivariate Cox proportional hazards model (DFS).
Univariate and multivariate Cox proportional hazards model (OS).
Discussion
In this study, we evaluated the prognostic potential of DYRK2 and TERT in early-stage breast cancer. DYRK2 is a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase that phosphorylates both Ser/Thr and Tyr (8). The DYRK family has been implicated in regulating processes such as cell proliferation, cytokinesis, and cell differentiation (13). In addition to these activities, DYRK2 has effector kinetic potential for Ser46 of p53, leading to apoptosis in response to DNA damage (3). As the knockout of DYRK2 attenuates adriamycin (ADR)-induced apoptosis, DYRK2 plays a key role in p53-induced apoptosis (9). Furthermore, DYRK2 can induce apoptosis in a p53-independent manner (9).
As previously reported, DYRK2 is down-regulated in breast, colon, and prostate cancers (11). Moreover, down-regulated DYRK2 is involved in breast cancer invasion by increasing Snail protein, which is a zinc finger protein, leading to suppression of E-cadherin promoter via the epithelial-mesenchymal transition (EMT) (14). As EMT-mediated tumor development leads to metastasis, early recurrence may occur in patients with down-regulated DYRK2.
We have not evaluated the expression of Snail protein or E-cadherin, but we focused on TERT expression, which is also one of the target proteins of DYRK2. TERT is an essential protein for the maintenance of telomere length (7). Telomerase is activated in cancer cells to lead to cellular immortality by TERT over-expression (15). However, it is degraded by the proteasome ubiquitin pathway after DYRK2-induced phosphorylation (7). According to our hypothesis, DYRK2 over-expression followed by TERT down-regulation may lead to a better prognosis in patients with early breast cancer. On the other hand, DYRK2 down-regulation followed by TERT over-expression may lead to a poorer prognosis.
We investigated the relationship between DYRK2 and TERT expression and prognosis and found a significant correlation with DFS by univariate analysis. However, these proteins alone do not have prognostic potential because of several confounding factors. We evaluated the significance of both proteins in combination to predict recurrence. As these proteins were closely correlated, their combination was shown to significantly predict recurrence. If DYRK2 over-expression leads to sufficient TERT degradation, TERT down-regulation with DYRK2 expression may reduce telomerase activity and be a powerful prognostic factor in breast cancer. However, DYRK2 over-expression will not always lead to TERT down-regulation. On the other hand, the combination of TERT over-expression without DYRK2 expression leads to a poorer prognosis. Taken together, the different functions of DYRK2 may alter the prognosis of breast cancer and underlie the development of recurrence despite DYRK2 over-expression.
This study has the following limitations: it was a retrospective analysis at a single institution and analysed a relatively small number of patients. Longer follow-up and an increased number of patients may help confirm our results.
In conclusion, this study demonstrated that patients with DYRK2-positive and TERT-negative breast cancer have a better DFS than patients with DYRK2-negative and/or TERT-positive tumors. The combination of DYRK2 and TERT may be a powerful tool to stratify breast cancer patients. Further investigation of DYRK2 and other target proteins may provide new insight into this possibility.
Acknowledgements
The Authors appreciate the technical support of Ms. Yoko Miyanari.
Footnotes
Authors’ Contributions
Data acquisition (MT, YY, YE, YN, and SO), data analysis (RM), statistical analysis (SY), manuscript preparation (MT), manuscript editing (SY), and manuscript review (KN, AI, and TS).
Conflicts of Interest
There are no conflicts of interest regarding this study.
- Received January 26, 2022.
- Revision received February 15, 2022.
- Accepted February 16, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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