TTK predicts triple positive breast cancer prognosis and regulates tumor proliferation and invasion.

This study was conducted to investigate the expression of the spindle assembly checkpoint kinase tyrosine/threonine kinase (TTK) in triple-positive breast cancer (TPBC) and its effect on TPBC cells. We analyzed the status of TTK in 69 TPBC samples using immunohistochemistry. The correlation between TTK and clinicopathological parameters was analyzed using a chi-squared test. The prognostic value of TTK was evaluated using Kaplan-Meier survival curves. We analyzed the role of TTK in the invasion and proliferation of TPBC cells in vitro and in vivo. The mean age of the 69 patients with TPBC enrolled in this study was 53 years (range: 29-86 years). TTK expression was positively correlated with tumor size (P = 0.034), p53 status (P = 0.023), TNM stage (P = 0.023), and Ki-67 index (P < 0.001). The Kaplan-Meier curves revealed that TTK expression was correlated with poor disease-free survival (P = 0.001) and overall survival (P = 0.050). Multivariate proportional hazard regression analyses showed that TTK and TNM staging were significant independent predictors of disease-free survival (P = 0.007 and P = 0.034, respectively). Additionally, TTK knockdown inhibited the invasion and proliferation of the BT474 TPBC cell line. The findings of this study indicate that TTK overexpression is associated with cancer progression and prognosis in patients with TPBC, whereas TTK knockdown inhibits the invasion and proliferation of TPBC cells. Thus, TTK might serve as a prognostic marker for TPBC.


Background
Understanding breast cancer biology is important in terms of both therapy management and assessment of patient outcomes. Breast cancer has been classi ed into different subtypes based on the expression of estrogen receptors (ERs), progesterone receptors, and human epidermal growth factor receptor 2 (HER2). Recently, a distinct subcategory of HER2-enrichedbreast cancer with positive hormone receptors has been identi ed (HER2-positive breast cancer). This subtype exhibits high levels of hormone receptor expression and is referred to as "triple positive" breast cancer (TPBC). In ER + luminal tumors, preclinical evidence suggests that strong crosstalk occurs between the ER and HER2 signaling pathways and that activation of the ER pathway leads to resistance to anti-HER2 therapy; furthermore, from a genomic/transcriptomic perspective, ER+/HER2 + tumors may be much more common than ER−/HER2 + tumors and have fewer tumor-in ltrating lymphocytes and lower PD-L1 expression [1,2]. Current treatment guidelines, however, do not propose distinct management strategies for ER+/HER2 + and ER−/HER2 + breast cancer [3,4].
TPBC responds distinctly to conventional treatment [5]. This raises the question of whether it is necessary to tailor TPBC therapy to individuals.
The spindle assembly checkpoint is a signaling cascade that prevents chromosome missegregation by arresting mitosis until all chromosomes are properly attached to the mitotic spindle [6]. As the core spindle assembly checkpoint kinase, tyrosine/threonine kinase (TTK) is a dual-speci city kinase that can phosphorylate serine/threonine and tyrosine residues [7]. TTK is critical for the recruitment of spindle assembly checkpoint proteins to unattached kinetochores, mitotic checkpoint complex formation, and mitotic arrest [8]. Inhibition of TTK activity causes cells to prematurely exit mitosis with unattached chromosomes, resulting in severe chromosome missegregation, aneuploidy, and eventually cell death [9][10][11][12]. Overexpression of mitotic checkpoint genes leads to chromosomal instability in cancer cells [13][14][15][16].
Previous studies have shown that TTK is overexpressed in breast cancer cells, particularly in the HER2 + and triple negative breast cancer (TNBC) subtypes [13,14,17,18]. However, whether TTK is also a prognostic factor in TPBC remains unclear. In this study, we analyzed the expression of TTK in TPBC and further analyzed the biological function of TTK in the progression of TPBC.

TPBC tissue and cell line
We retrospectively collected data and tissue specimens from 69 patients with TPBC who had undergone tumor surgical resection at the Second Hospital of Jilin University between January 2009 and January 2013. Patients administered preoperative neoadjuvant chemotherapy or hormonal treatment were excluded from this study. The 69 patients had been diagnosed with invasive carcinoma of no special type. The last follow-up was conducted in April 2018 (mean follow-up duration: 44.2 months, range: 1.67-72.10 months).
Clinicopathological data were retrieved from patient medical records. These parameters included patient age at initial diagnosis, histological subtype, tumor size, lymph node metastasis, and clinical outcome.
The study protocol was approved by the Institutional Ethics Committee of the Second Hospital of Jilin University, Jilin, China (IRB approval number: 2020008). The need for written informed consent was waived because of the retrospective nature of the study.
Two-micrometer-thick sections were prepared from para n blocks of breast cancer tissues retrieved from the Pathology Department, Second Hospital of Jilin University, for immunohistochemistry and hematoxylin and eosin staining. The latter was used to con rm the diagnoses, and the results were reviewed by Min Yao and Yunhe Gao. Tumor histological grades were assessed using the Nottingham grading system [19].
A human breast carcinoma cell line, BT474, which is a TPBC cell line, was purchased from the American Type Culture Collection (Manassas, VA, USA). The cell line was maintained in RPMI-1640 supplemented with 10% fetal bovine serum at 37°C in a humidi ed atmosphere with 5% CO 2 .

Review and scoring of immunostained tissue sections
The immunostained tissue sections were independently reviewed and scored by two investigators to determine the immunostaining percentage and staining intensity, as described previously [20]. A numerical nal expression score (FS) was calculated for each tissue sample by multiplying the staining intensity (I) score (0, negative; 1, weak; 2, moderate; 3, strong staining) by the percentage (P) of positively stained cells (FS = P × I). Final expression score, therefore, ranged from 0 to 300. Thereafter, each case was scored as positive or negative using the median nal expression score as the cutoff value for statistical analysis [21]. MiaoLingBio, Changchun, China), which also expressed green uorescent protein, was constructed and transfected into BT474cells. A functional, nontargeting shRNA, and an empty vector were used as controls.

Cell proliferation and colony formation experiments
Quantitative reverse transcription-PCR (qRT-PCR) Total RNA was isolated from 5 × 10 6 cells by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA concentration and purity were analyzed in duplicate samples by using a NanoDropND-2000 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA). cDNA was synthesized from quali ed RNA by using an RT-PCR reverse transcription kit (TransGen Biotechnology LLC), and 1 µg of total RNA was reverse-transcribed into cDNA under the following conditions: 25°C for 10 min, 42°C for 30 min, and 85°C for 5 s, as per the manufacturer's recommendations. The cDNA was stored at − 20°C until use. PCR was performed using a PCR kit (TransGen Biotechnology LLC), and the PCR products were electrophoresed on 1.5% agarose gels. qPCR was conducted with either Taq-Man or SYBR Green PCR reagents on an ABI Prism 7300 detection system (all from Applied Biosystems, Foster City, CA, USA). The reaction program was as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 30 s and 55°C for 20 s, and 72°C for 15 s. GAPDH served as an internal control, and relative mRNA levels were calculated using the 2 −ΔΔCt method [22]. The

Transwell invasion assay
The transfected cells were inoculated into the upper chamber of a Transwell chamber (Corning, Inc., Corning, NY, USA) at 1×10 5 cells/mL, and RPMI-1640 medium supplemented with 30% fetal bovine serum was added to the lower chamber. The cells were cultured in 5%CO 2 and 100% humidity and at 37°C constant temperature for 24 h. The chamber was removed, and the cells were rinsed with PBS and xed in absolute ethanol for 40 min. After crystal violet staining, the cells that did not pass through the upper chamber were wiped off using a cotton swab, and cells that had passed through the upper chamber were counted under an inverted microscope. Finally, invasive cells on the lower surface of the membrane were counted using a microscope. Five elds of view were randomly selected to detect changes in invasion ability. The experiment was repeated three times.

Animal studies
Fourteen female BALB/c nude mice (6 weeks old) were purchased from SPF Biotechnology (Beijing, China) and randomized into two groups. All animal experiments were conducted in accordance with the institutional guidelines and approved by the Experimental Animal Ethical Committee of Jilin University.
Cells were injected subcutaneously into the right buttock of each mouse at 1 × 10 6 cells per 0.1 mL PBS.
All mice were maintained in a standardized barrier system environment at the Animal Experiment Center of the Basic Medical College of Jilin. After 4 weeks of observation, the mice were euthanized. The xenograft tumors were removed and xed in 10% formalin. The tumors were measured using a Vernier caliper, and the volume was calculated using the following formula: V = (width 2 × length)/2 [23]. The xenograft tumors were examined macroscopically and subjected to hematoxylin and eosin staining and immunohistochemistry.

Statistical analyses
All statistical analyses were performed using SPSS v. 21.0 (SPSS, Inc, Chicago, IL, USA). Continuous variables were evaluated using the Mann-Whitney U test, whereas categorical variables were analyzed with the chi-squared test. Furthermore, signi cant variables (P < 0.05) from the univariate analyses were included in multivariate analyses. A multivariate Cox proportional hazards model was used to obtain hazard ratios for survival and to identify factors affecting disease-free survival (DFS) and overall survival (OS). Kaplan-Meier survival curves and log-rank tests were used to evaluate DFS and OS. Experiments were repeated at least three times. P < 0.05 was considered to indicate statistically signi cant differences.

Patient characteristics
The median age of the69 patients with invasive breast cancer included in this study was 53 years (range: 29-86 years). According to the seventh edition of the UICC cancer staging system grouping criteria, 17.4% (12/69), 43.5% (30/69), and 39.1% (27/69) of the TPBC patients had stage I, II, and III disease, respectively. All patients exhibited invasive breast ductal carcinoma and underwent modi ed radical mastectomy. Among the patients with available adjuvant treatment information, 97.1% (67/69) were administered chemotherapy (two ceased treatment because of an allergic reaction) and 33.3% (23/69) were treated with radiation.
Associations between TTK expression and clinicopathological parameters in patients with breast cancer Page 7/20 The associations between TTK expression and clinicopathological parameters were analyzed using Pearson's chi-squared test (Table 1). TTK positivity was observed in 39.1% (27/69) of TPBC tissues (Fig. 1a). TTK expression was positively associated with tumor size (P = 0.034), p53 mutation (P = 0.023), TNM stage (P = 0.023), and Ki-67 expression (P < 0.001). The larger tumor size and higher Ki-67 expression under TTK overexpression suggest that TPBC is more proliferative and has higher vitality than TTK-negative TPBC. Furthermore, the p53 mutation and advanced TNM stages associated with TTK overexpression indicate that TPBC has more aggressive clinicopathological features than other forms of breast cancer.

Association between TTK expression and patient survival
Kaplan-Meier curves and a log-rank test were used to analyze OS and DFS after strati cation by TTK expression. TTK expression was signi cantly correlated with DFS (P = 0.001, Fig. 1b) and OS (P = 0.050, Fig. 1b).

TTK knockdown inhibits TPBC cell proliferation
To investigate the potential role of TTK in TPBC cells, we designed and tested TTK-targeting shRNAs and identi ed two shRNAs that effectively knocked down TTK expression in BT474 cells (Figs. 2a and 2b). The CCK-8 assay showed that the survival rate of BT474 cells was signi cantly reduced after TTK knockdown (shTTK group vs. control group, P < 0.001; Fig. 3a). Furthermore, colony formation ability experiments and an EdU assay were performed to mimic the ability of cells to form multicellular clones from individual tumor cells (Figs. 3b and 3c). The number of BT474 cells in the shTTK group was remarkably lower than that in the control group (P < 0.05 and P < 0.05). Furthermore, in the shTTK group, CCND1, CCND2, CDK4, CDK6, and p21 expression levels were signi cantly lower than those in the control group (Fig. 3d). These ndings suggest that downregulation of TTK can signi cantly inhibit proliferation of the BT474 TPBC cell line and reduce colony formation. After transfection with TTK-targeting shRNA, AKT expression was signi cantly lower in BT474 cells than in control cells (Fig. 3d).
Epithelial-mesenchymal transition (EMT), an invasive phenotype of cancer cells, plays a key role in tumor metastasis. Western blot analysis revealed that the expression of EMT proteins (E-cadherin, vimentin, N-cadherin, and Snail) was signi cantly altered by TTK knockdown. The expression of Ecadherin was signi cantly higher, and that of vimentin, N-cadherin, and Snail protein was signi cantly lower, in the shTTK group than in the control group (Fig. 4a). The Transwell assay revealed the effects of TTK knockdown on the invasiveness ofBT474 cells. There were signi cantly fewer invading cells in the shTTK group than in the control group (P = 0.005) (Fig. 4b). These results suggest that TTK enhances the invasiveness of BT474 cells by regulating EMT.
TTK knockdown inhibits tumor growth in a breast cancer xenograft model TTK suppression was examined in nude mice bearing established control and shTTK BT474 xenograft tumors. TTK suppression inhibited tumor growth in the shTTK group compared to that in the control group in this xenograft model (Fig. 5a). Moreover, based on the tumor tissue immunohistochemistry results, TTK, AKT, and Ki-67 expression was suppressed in the shTTK group relative to that in the control group (Figs. 5b and5c); this is consistent with the in vitro results. These results indicate that TTK enhanced tumor growth in vivo, possibly via the AKT pathway.

Discussion
In this study, we found that TTK expression enhanced the proliferation and metastasis of BT474 TPBC cells. Furthermore, unlike in TNBC, TTK overexpression was associated with worse prognosis in patients with TPBC. Encouraging results have been obtained using a dual blockade strategy comprising anti-HER2 agents and hormonal therapy [24]. Patients with the HER2-enriched molecular subtype of breast cancer and those at high risk of relapse are typically administered adjuvant trastuzumab as the anti-HER2 agent, along with chemotherapy and endocrine therapy.
Consistent with our results, AI-Ejeh et al. [17] revealed that TTK protein levels were elevated in aggressive tumors, leading to poor survival. Most previous TNBC studies focused on aberrant TTK expression in TNBCs, an event that is signi cantly associated with an elevated risk of relapse and docetaxel resistance [17,18,25,26]. Based on these ndings, we hypothesized that aberrant TTK expression facilitates the progression of TPBC. Accordingly, we found that TTK expression was signi cantly higher in TPBCs with a poor prognosis than in those with a good prognosis. Assessing TTK expression augments the prognostic information provided by the traditional prognostic indicators ER, progesterone receptor, and HER2. This is the rst study to evaluate the effect of TTK expression on the survival of a consecutive TPBC cohort.
To further investigate the functional role of TTK in the progression of TPBC, we used a lentivirus to achieve TTK knockdown in BT474 cells. TTK knockdown inhibited the proliferation, colony formation, and invasiveness of TPBC cells, both in vivo and in vitro. Additionally, TTK knockdown may affect the expression of proteins associated with proliferation and invasiveness. Recent research has indicated that TTK promotes cell migration and EMT by enhancing the expression of dihydropyrimidinase-like 3 (DPYSL3), thus promoting snail-regulated EMT, and reinforcing the metastatic potential and ultimately tumor metastasis in TPBC. TTK and DPYSL3 upregulation was negatively correlated with clinical outcome in patients with lung cancer [27]. Importantly, our ndings revealed that TTK protein expression was positively correlated with tumor size, TNM stage, p53 mutation status, and the Ki-67 index. Consistent with our ndings, previous studies have shown that TTK is expressed signi cantly more in breast cancers expressing mutant p53 than in those expressing wild-type p53, independent of the breast cancer subtype [28]. In patients with breast cancer, TTK overexpression is associated with increased breast cancer grade and aggressiveness, possibly because it promotes the proliferation or survival of cancer cells [25].
The AKT signaling pathway can exert proliferative effects by targeting downstream transcription factors. TTK promotes cell proliferation and invasiveness by activating the AKT/mTOR and MDM2/p53 signaling pathways [29]. Our ndings demonstrate that reducing TTK expression can inhibit the activation of AKT expression and proliferation of breast cancer cells. Many clinical trials [9-11, 13, 30-32] have assessed the effects of TTK inhibitors in TNBC treatment and showed that TTK inhibitors may be effective as second-line therapy for TPBC, particularly for resistant patients; our ndings are consistent with these results. Additional large cohort studies are required to validate the positive effects of anti-TTK therapy.
In conclusion, TTK upregulation was closely associated with TPBC progression and poor prognosis. Thus, TTK may serve as a valuable prognostic marker and therapeutic target for TPBC.