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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2024 Nov 26;150(12):512. doi: 10.1007/s00432-024-06021-9

TTK promotes HER2 + breast cancer cell migration, apoptosis, and resistance to targeted therapy by modulating the Akt/mTOR axis

Shaolin Zhang 1,3, Hua Ding 2, Yongfen Deng 4, Yu Ren 1,2, Fulin Zhou 1,5, Qian Zhang 1, Shu Liu 1,2,✉
PMCID: PMC11599621  PMID: 39589549

Abstract

Background

HER2 + breast cancer is a malignant neoplasm with a high degree of aggressiveness and therapeutic challenge. In recent years, studies have indicated a strong correlation between TTK and various tumors, though its role in HER2 + BRCA remains unclear.

Objectives

Studying the biological function of the TTK gene in HER2 + BRCA and its resistance to targeted therapy it provides new ideas for targeted drug research.

Methods

TTK was knocked down by small interfering RNA transfection, and its biological function in HER2 + BRCA cells was verified, and its mechanism of action was verified by RT-PCR and Western blot.

Results

The study demonstrated that TTK promoted cell proliferation and migration by activating the Akt/mTOR pathway in HER2 + breast cancer and enhanced the drug sensitivity of BRCA cell lines SKBR3 and BT474 to pyrotinib, in addition, knockdown of TTK induced apoptosis and arrested cells in G1 phase.

Conclusion

Which implies that TTK is an oncogene in HER2 + BRCA and is a valuable research target.

Keywords: HER2 + breast cancer, Igration, Apoptosis, Drug resistance, Target

Introduction

Currently, the most common malignant tumor in women is BRCA(Siegel et al. 2023), with 2.26 million new cases as early as 2020 (Sung et al. 2021), which is increasing year by year and is also the leading cause of cancer deaths in women (J. Qi et al. 2023a, b). The primary cause of mortality in most patients is the progression, recurrence, and metastasis of the disease following treatment. BRCA originates from breast epithelial cells and can be divided into four subtypes according to molecular typing: LuminalA, LuminalB, HER2 + , and TNBC (Yeo and Guan 2017). HER2 + BRCA accounts for 15–20% of all breast cancer cases (Curtis et al. 2012); HER2 + BRCA is characterized by rapid growth, aggressiveness, and high mortality. Over half of patients have a survival rate of only five years, with resistance to chemotherapy being a critical factor contributing to disease recurrence, metastasis, and poor prognosis (Slamon et al. 1987). Pyrotinib is an oral, small-molecule tyrosine kinase inhibitor that has been approved as a first-line drug for HER2 + BRCA in China (Qi et al. 2023a, b) and has achieved outstanding clinical results. In phase III clinical study, XU found that erlotinib can significantly improve the survival rate of patients with metastatic HER2 + BRCA who have received trastuzumab and taxane chemotherapy (Xu et al. 2021) and yet with clinical use. Resistance to chemotherapy drugs is still unavoidable, resulting in poor disease treatment outcomes (Jiang et al. 2023). TTK is a bispecific protein kinase that acts as a key regulator during cell mitosis (Pachis and Kops 2018), which mainly maintains centrosome amplification in HER2 + BRCA (Lee et al. 2014), and the expansion and accumulation of phosphorylation of centrosomes also promote the occurrence and development of tumors. Centrosome amplification occurred in more than 20% of patients with HER2 + BRCA, particularly in patients with HER2 + ER-PR- and luminal B subtypes (Guo et al. 2007; Schneeweiss et al. 2003), and TTK expression was negatively correlated with the overall survival rate of patients in HER2 + BRCA (Lee and Gollahon 2013). HER2 overexpression is an oncogenic driver of BRCA progression, inducing cell proliferation by activating downstream signals, promoting the Ras mitogen-activated protein kinase (MAPK) pathway, or inhibiting cell death via the phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway (Pattanayak et al. 2022; Patel et al. 2020). Hence, there is a notable correlation between centrosome amplification and the pathogenesis of HER2 + BRCA, which further confirms that the mutation of the TTK gene may be a key factor in the progression and drug resistance of HER2 + BRCA. In studies of CDK4/6i inhibitor resistance, resistance is associated with mutations in the TTK gene, which lead to excessive mitotic errors, DNA damage, and cell death, thereby increasing drug sensitivity (Soria-Bretones et al. 2022). Through the analysis of bio-information GSEA technology, we found that the Akt/mTOR signaling pathway was significantly related to TTK and determined that this pathway was associated with drug resistance in tumors (Hua et al. 2021, 2019; Dey et al. 2017). Therefore, we hypothesize that TTK may effectively address pyrotinib resistance in HER2 + BRCA by activating the Akt/mTOR pathway and that TTK is expected to be a valuable target for the clinical treatment of HER2 + BRCA.

Materials and methods

Cell culture

In this study, four strains of BRCA cells and one strain of human normal breast cells MCF-10A were employed. Purchased two strains of SKBR3 and BT474 cells in the Chinese Academy of Sciences and MCF-10A, MCF-7, and HCC1806 cells were kindly donated by the Translational Research Center of Guizhou Medical University. All cells were grown in Proxel's medium (DMEM, 1640) containing 10% fetal bovine serum (FBS) and penicillin–streptomycin solution at 37 °C and 5% CO2.

Clinical specimens and immunohistochemistry

The clinical specimen tissues were from the Department of General Surgery, Dejiang County People's Hospital, and the tumor tissues were from two HER2 + and HER2- BRCA patients and adjacent tissues, respectively. The patient and histopathologist agree to use this clinicopathological tissue. The use of this sample was reviewed and approved by the Ethics Committee of Dejiang County People's Hospital, and the principles of the Declaration of Helsinki were also followed. Each patient enrolled in this study has provided informed consent by signing a patient consent form. The process adheres to a specific protocol, including steps such as deparaffinization, dehydration, inhibition of endogenous peroxidase and non-specific antigens, antigen retrieval, incubation with primary antibodies, staining, and blocking. The experimental tissue sections are then examined using an inverted microscope.

Transient transfection

Ten thousand cells per well were plated in a six-well plate, and after 12 h, the cells were adherent, siRNA and overexpression plasmid were introduced into the cells with liposome Lipo3000 to obtain a temporarily silenced TTK cell line, and 12 h after transfection with fresh complete medium for follow-up experiments.

Quantitative real-time PCR

Extract the total RNA of the cells with an RNA kit, then reverse transcribe and amplify the cDNA. A quantitative polymerase chain reaction was performed on cDNA using primers ordered by Guizhou Weiboxin Biological Company. Finally, the BioRad CFX96 System was adopted to detect genetic changes through computational data.

Western blot analysis and antibodies

Cell total protein extraction, pure protein ultrasonic distraction collection, centrifugation, supernatant extraction, the addition of reagent ratio: lysate: protease inhibitor: phosphatase inhibitor = 100:1:1, BCA protein analysis kit quantification. The protein was separated by SDS–polyacrylamide gel electrophoresis. It is subsequently moved to a 0.45 mm membrane and incubated with the desired primary antibody for 24 h at 4 °C. The Te antibodies used in this experiment included TTK, mTOR, AKT, E Cadherin, N Cadherin, Vimentin, β-Actin, p-mTOR, p-AKT, Cleaved-casp3, and Cleaved-casp7. Following the protein is fully bound to the primary antibody, it is imaged in a chemical DocXRS + molecular imager using a chemiluminescence kit to unpack the communication of the protein, and the ImageJ software is processed with gray values.

The viability of CCK-8 cells and the IC50 value of the drug were measured

Cell viability is measured with the CCK-8 kit and resistance to pyrotinib, and the inhibition rate and IC50 value of pyrotinib before and after TTK knockout were verified. Prepare a 96-well plate, take the experimental cell digestion and off-center suspension, count 5000 cells by cell tallying, dilute with 100 μL of culture medium, seed each well, and continue to incubate in the incubator for 24 h. After watching the merisis of the cells, ten μL of pyrotinib at distinct consistency was mixed in every well and put in the incubator for 24 h. Remove the 96-well plate and add ten μL of CCK-8 reagent to each well in the dark. Wrap the 96-well plate with aluminum foil and continue incubating for four hours. Remove the 96-well plank, open the cap, and determine the OD value with an enzyme marker at 450 nm. GraphPad Prism 9.5 processes the data.

Wound healing test

Six-well plates were plated first, cells were adherent for eight hours for transfection, and when the cells were cultured to 90% concentration, a straight line was drawn on the cell monolayer with a ten μl pipette tip. The wound width was measured at 0 h and 48 h, respectively. The rate of cell migration was determined by calculating wound closure, normalized against the NC group. Wound healing trials were performed three times, and GraphPad Prism 9.5 processed data.

Plate cloning

200 SKBR3 and BT474 were added to each well of the six-well plate, and after three weeks of continuous culture, they were washed twice with PBS. The cells in each well are fixed with 3 ml of methanol for 30 min and twice with PBS. Cells are stained with 1% crystal violet for 30 min, washed off with slow water, and subsequently air dry. The data were then observed and counted under the microscope and processed with GraphPad Prism 9.5.

Flow cytometry was used to detect cell cycle and apoptosis

Cell Cycle Assay Kits are used; take 1 ml of cell suspension (1 × 10^6/mL), fix it with 70% cold ethanol 500ul for two hours, then wash twice in PBS, add 500ul PI/RNase staining solution, protect from light for 60 min at room temperature, and detect on the machine, the wavelength is 488 nm; The trial was conducted three times. Six-well plates were plated with approximately 2 × 10^6 cells per well using the Annexin V-FITC Apoptosis Assay Kit, and cells were resuspended with 100 ul of 1XAnnexin V Binding Buffer. Then, add 2.5 μl of Annexin V/FITC Reagent and 2.5ul of PI Reagent to each EP tube, mix well, and incubate for 20 min at room temperature. Add 400 μL of 1X Annexin V Binding Buffer to resuspend the cells, and proceed with immediate testing. The experiment was repeated three times.

Animal modeling

A mouse model of xenografts for breast cell carcinoma was established; SKBR3 and SKBR3 cells were directly injected into the left and right lower abdomen of nude mice, respectively, and approximately 10^7 cells were inoculated into each nude mouse, and the merisis of tumors was watched, and record the size of the tumor with vernier calipers once every three days. Following 30 days, the tumors were dislodged, photographed, and weighed, and tumor information was gathered for statistical analysis. The Animal Center and the Use Committee of Guizhou Medical University approved all animal experimental protocols.

Bioinformatics analysis

The databases employed in the study include Cancer Genome Atlas (TCGA), Breast Cancer Integrated Platform Data BCIP (http://www.omicsnet.org/bcancer/), UALCAN (http://ualcan.path.uab.edu/); The KEGG and GO pathways of TTK were enriched by Sangerbox technology to obtain the pathways, cell cycle and apoptosis-related to their expression.

Statistical analysis

The collected experimental data were analyzed by GraphPad Prism 9.5 software. One-way ANOVA was used for comparison between multiple groups, and LSD t test was used for pairwise comparison between groups, and on the condition that p < 0.05, the difference was significant or extremely significant. All experiments were performed with n ≥ 3.

Results

TTK overexpression in HER2 + breast cancer is strongly associated with tumor progression and prognosis

At the beginning of the experimental design, bioinformatics analysis was performed using the TCGA database, Sangerbox (Fig. 1A), UALCAN (Fig. 1B, C), and BCIP database, and it was determined that the expression level of TTK in human BRCA pathological tissues was much higher than that in healthy breast tissues (Figs. 2, 3, 4, 5 and 6). High expression of TTK significantly reduced OS, DS, and DFS in BRCA patients, and the prognosis was generally poor (Fig. 1D, E, F).

Fig. 1.

Fig. 1

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The expression of TTK in breast cancer was analyzed by relevant databases. A The expression of TTK in different cancer types was analyzed by Sangerbox. B, C The UALCAN (http://ualcan.path.uab.edu/) database was used to analyze the relationship between TTK expression and tumor stage in normal people and breast cancer patients. D–F The association between TTK gene and survival in HER2 + breast (OS,DS,DFS) was analyzed through the BCIP (http://www.omicsnet.org/bcancer/) database. G HER2 + , HER2- and pathological tissues of adjacent tissues were used for HE staining and immunohistochemistry to understand the expression of TTK. H–I PCR and WB were used to verify the expression of TTK in normal breast cells and different subtypes of breast cancer cells.J It is the quantitative calculation statistics of the gray value of the WB band. (*p < 0.05,**p < 0.01,***p < 0.001,****p < 0.0001

Fig. 2.

Fig. 2

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The sensitivity of SKBR3 and BT474 to pyrotinib was affected by the Akt/mTOR signaling pathway. A, B, D, E After the knockout of TTK gene in SKBR3 and BT474 cells was detected by DCCK-8 assay, the survival rate of pyrotinib was reduced and the IC50 value was reduced. C, F It was stained with 1% crystal violet and the morphological changes of the nuclei under different drug concentrations were observed. G BT474 breast cancer cells, which are more sensitive to pyrotinib, were cultured with the IC50 value of pyrotinib as the starting concentration, and the protein was raised once a month for four months, and the expression of TTK gene increased with the occurrence of drug resistance in the cells. H Quantitative statistics on the gray value of the WB band. I The schematic diagram shows that pyrotinib affects patients with HER2 + breast cancer and the mechanism of action related to TTK genes. (**p < 0.01)

Fig. 3.

Fig. 3

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TTK promotes the horizontal and vertical migration of SKBR3 and BT474 cells through the Akt/mTOR signaling pathway. A Wound healing experiment: Silencing of TTK gene reduced the horizontal migration ability of SKBR3 and BT474 cells (scale bar = 100um). B Histograms were used to count the horizontal migration distance of cells. C The effect of TTK on the vertical migration of SKBR3 and BT474 cells was investigated by transwell migration assay without Matrigel. Cell counts were counted after staining with 1% crystal violet, scale bar = 1 mm. D Count the number of cells migrating in the vertical direction with a histogram. E, F PCR and WB experiments showed that TTK gene migration in HER2 + breast cancer cells was also related to EMT. G The gray value of the WB strip was quantified. (**p < 0.01,***p < 0.001)

Fig.4.

Fig.4

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TTK affects the phosphorylation of SKBR3 and BT474 cells through the Akt/mTOR signaling pathway. A–C The pathways, cell cycle, apoptosis and other pathways, cell cycles, and apoptosis related to TTK genes were analyzed through the Sangerbox database. D, E SKBR3 and BT474 cells were transfected with siRNA, and verified by PCR and WB. F It is a quantification of the gray value of the WB strip. G The relevant pathway was verified by WB, and TTK gene knockout was closely related to the phosphorylation of SKBR3 and BT474 in tumor cells. H Rapamycin inhibitor inhibition of the Akt/mTOR signaling pathway, followed by transfection of the TTK overexpression plasmid with the reverse transcription signaling pathway for reply verification. (**p < 0.01,***p < 0.001)

Fig. 5.

Fig. 5

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TTK affects the cell cycle and apoptosis of SKBR3 and BT474 cells through the Akt/mTOR signaling pathway. A After knocking out the TTK gene in SKBR3 and BT474 cells by flow cytometry, the two cells mainly acted on the G1 phase of the cell cycle. B Histograms are used to represent changes in proportion at each stage of the cell cycle. C Flow cytometry was used to detect the increase of apoptosis after knocking out TTK gene in SKBR3 and BT474 cells. D The histogram was used to represent the proportion of apoptosis before and after TTK gene knockout in the two cells. E The apoptotic proteins caspase-3 and caspase-7 were detected by WB assay, and apoptosis increased after knockout of TTK gene. F The gray value of the WB strip was quantified. (**p < 0.01,***p < 0.001)

Fig. 6.

Fig. 6

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TTK promotes HER2 + breast cancer cell proliferation through the Akt/mTOR signaling pathway. A, B The TTK gene was knocked out by sh transfection and verified by PCR and WB. C The gray value of the WB strip was quantified. D Plate cloning experiments were used to verify that the cell proliferation ability was weakened after TTK gene knockout. Stained with 1% crystal violet and counted. E Histograms were used to count the cell proliferation before and after knockout of TTK gene. F SKBR3 and BT474 cells were transfected with shRNA, and their cell proliferation was counted. G The subcutaneous tumorigenesis experiment of nude mice was used to verify that the proliferation ability of tumor cells was significantly weakened after TTK gene knockout. H HE staining and immunohistochemistry were performed on the subcutaneous tumor tissues of nude mice, and the TTK gene was observed After knockout, the tumor proliferation ability is significantly reduced. (*p < 0.05, **p < 0.01, ***p < 0.001)

TTK is overexpressed in breast cancer cell lines and pathological tissue specimens

To gain insight into the biological function of TTK in BRCA, first, we assessed TTK expression in both BRCA cell lines and pathological tissue samples. In comparison to normal breast epithelial MCF-10A cells, TTK was found to be upregulated in breast cancer cell lines SKBR3, BT474, MCF-7, and HCC1806 (Fig. 1I). In addition, we collected HER2 + , HER2- pathological tissues, and adjacent tissues for HE staining and immunohistochemistry, and the results showed that the expression of TTK in HER2 + and HER2- BRCA patients was significantly higher than that in adjacent tissues (Fig. 1G). Through the above studies, it can be further implied that TTK is up-regulated in BRCA cell lines and pathological tissues, which may be associated with the progression of BRCA.

TTK down-regulated in BT474 and SKBR3 BRCA cells

To further understand the function of TTK in BRCA cells, we transiently transfected BT474 and SKBR3 cells with NC or siRNA and confirmed TTK knockout by qPCR and western blotting (Fig. 4D, E). The results demonstrate that both siRNA significantly reduced the mRNA and protein levels of TTK, and then we verified the function of TTK silencing on BRCA cell proliferation, migration, and drug sensitivity (Fig. 2B, E). Through colony formation experiments, we found that the TTK silencing group formed smaller as well as fewer colonies than the control group (Fig. 6D). In the Transwell and wound healing assays, TTK-silencing significantly reduced the migration of breast cancer cells compared with wild-type and negative control cells, and in addition, the IC50 value of TTK-silenced BT474 and SKBR3 cell lines for pyrotinib was substantially reduced, and the inhibition rate of the drug on cells was significantly increased, further indicating that TTK knockout raised the sensitivity of HER2 + BRCA cells to drugs. Simultaneously, crystal violet staining experiments indicated that nuclear fission occurred in the TTK silencing group at a small drug concentration. It is further clarified that TTK is closely related to the sensitivity or resistance of cells to drugs, and TTK may lead to drug resistance in BRCA cells. Based on the aforementioned findings, we also conducted a subcutaneous tumorigenesis experiment in nude mice. SKBR3 cells were transfected with either NC or shRNA-TTK, followed by subcutaneous tumorigenesis in the nude mice model.

TTK induces HER2 + breast cancer cell cycle arrest in G1 stage

When enriching the pathway through bio-information GSEA technology, it was determined that TTK was closely related to the cell cycle, and the mechanism of chemotherapy resistance in HER2 + BRCA was closely associated with the regulation of the cell cycle. To further understand the key role of TTK in cell cycle regulation, we adopted flow cytometry to measure the cycle distribution of two cells before and after TTK knockout, and we determined that the percentage of cells in the G1 phase was larger than that in the control group (Fig. 5A, B), but the number of cells in the G2/S phase was significantly reduced. Further analysis suggests that the silencing of TTK predominantly induces cell cycle arrest in the G1 phase. Previous studies have found that silencing of TTK enhances the drug susceptibility of HER2 + BRCA cells to pyrotinib, and these outcomes suggest that TTK has an essential role in chemoresistance in HER2 + BRCA.

TTK facilitates the migration and apoptosis of HER2 + BRCA cells by activating the Akt/mTOR pathway

To further discuss the framework of action of TTK in HER2 + BRCA, which affects cell biology function. First, we enriched the related pathways of TTK through GSEA technology in Sangerbox and enriched many pathways related to TTK, among which the Akt/mTOR pathway score was relatively high, and subsequently we detected the correlation between TTK and Akt/mTOR pathway by qPCR and WB and discovered that the expression level of Akt and mTOR did not change significantly following silencing TTK, while the phosphorylated proteins p-Akt and p-mTOR decreased significantly. In alignment with our prior research, the development and progression of breast neoplasms are linked to protein phosphorylation. Additionally, through GSEA technology, we discovered that TTK is closely associated with apoptosis. Subsequently, we employed western blot analysis to confirm the influence of TTK on apoptosis markers and potential signaling pathways in HER2 + BRCA. Down-regulation of the TTK gene significantly up-regulated caspases 7,3, while phosphorylated proteins p-Akt and p-mTOR were sensibly decreased, further suggesting that TTK plays an essential role in the anti-apoptotic process of HER2 + BRCA cells (Fig. 5C). In addition, Experimental proof has found that the TTK gene is obviously bound up with centrosome amplification in breast cancer and frequently has the effect of promoting tumor cell invasion, migration, and proliferation, and it is currently speculated that TTK can promote the migration of HER2 + BRCA cells promotes cell migration in BRCA cells (Fig. 3A, C).

TTK promotes resistance to pyrotinib-targeting drugs in HER2 + BRCA cells by activating the Akt/mTOR pathway

At present, pyrotinib is a first-line drug for advanced HER2 + BRCA, which can significantly improve the survival rate of patients with advanced HER2 + BRCA. Due to the characteristics of HER2 + BRCA with strong aggressiveness, poor prognosis, and easy recurrence, and erlotinib resistance occurs in clinical medication, we speculate that TTK affects the resistance of HER2 + BRCA to pyrotinib, we raised two HER2 + breast cancer cell lines BT474 and SKBR3, transiently transfected BT474 and SKBR3 cells with NC and siRNA-TTK, and acted with distinct concentrations of pyrotinib for 24 h. The CCK-8 experiment was employed to test the cell livability and IC50 value of the drug, and the cell property and IC50 quantity of the TTK silencing group were significantly reduced, and research results revealed that the semi-inhibitory attention of pyrotinib in BT474/SiRNA-TTK cells was 1.494 μg/ml, while that in the control group was 2.716 μg/ml. The semi-inhibitory concentration is 13.87 μg/ml in SKBR3/SiRNA-TTK cells and 29.18 μg/ml in NC SKBR3 cells (Fig. 2B, E). The results demonstrated that silencing TTK decreased cell practicality and increased drug sensitivity. At the same time, BT474 cells were used to culture for resistance, cultured with increasing drug concentration for four months, and then WB was adopted to verify that TTK expression also increased with the dosing concentration, and these results illustrated that TTK gene enhanced the resistance of HER2 + BRCA cells to pyrrhotine (Fig. 2G). The study determined that EMT is cyneoplasm cell migration and drug resistance, and we hypothesized that TTK resistance to pyrotinib might also impact EMT. Consequently, we assessed the relationship between TTK and EMT using qPCR and Western blot analysis. Our findings revealed a decrease in N-cadherin and Vimentin levels following TTK silencing, while there was a significant increase in E-cadherin. It was further suggested that pyrotinib resistance in HER2 + breast cancer was also associated with EMT (Fig. 3E, F).

Discussion

HER2 + targeted breast cancer has the characteristics of rapid growth, strong invasiveness, easy distant metastasis, and poor prognosis; in recent years, it has gradually drawn the attraction of researchers. HER2 is a part of the EGFR kindred and has tyrosine kinase capability. Besides, HER2 overexpression significantly affects the prognosis and survival of patients (Chi et al. 2022). In recent years, the main drugs targeting HER2 are monoclonal antibodies and tyrosine kinase inhibitors. The persistent use of medications has led to drug resistance emerging as one of the primary factors contributing to unfavorable clinical outcomes.

TTK is a bispecific protein kinase that acts as a key regulatory factor in mitosis, mainly maintaining genome integrity (Musacchio 2015; London and Biggins 2014; Santaguida and Amon 2015). When TTK is overexpressed, centrosome amplification and chromosomal instability will occur, which may result in tumorigenesis. Research studies have confirmed that Overexpression of TTK is related to the onset of a variety of cancerous tumors, column as follows: glioblastoma (Kessler et al. 2020), BRCA (Tang et al. 2019), lung cancer (Zheng et al. 2019), HCC (Choi et al. 2017), COAD and other malignant tumors. Tang's study found that TTK expression is related to the histological grade of BC, and continuous reduction of TTK can substantially reduce the survival and growth of BRCA cells (Tang et al. 2019). In the process, in cancer research, it was determined that high TTK expression is associated with the aggressive subtype of tumors and treatment resistance (Lee et al. 2014; Maire et al. 2013; Daniel et al. 2011; Sugimoto et al. 2017; Maia et al. 2015).

In this experiment, we detected the action of TTK in HER2 BRCA, especially its resistance to pyrotinib chemotherapy in HER2 + BRCA, and determined that TTK expression enhanced the proliferation, migration, drug sensitivity, and reduced cell apoptosis of HER2 breast cancer cells BT474 and SKBR3. Through bioinformatics analysis, we discovered that the demonstration of TTK in HER2 + BRCA was significantly higher than that in HER2- BRCA and normal tissues, and TTK significantly affected the survival rate and prognosis of patients. Increased TTK expression correlates with decreased OS, DS, and DFS values among patients; this finding aligns with the observed expression of TTK in TPBC and its impact on TPBC cells (Gao et al. 2022). At the same time, this result has also been verified in triple-negative breast cancer. The (King et al. 2018) study confirmed that drug inhibition or silencing of the TTK gene can reverse the epithelial-mesenchymal transition of TNBC cells. In the (Maia et al. 2015) TNBC mouse model, the combination of TTK inhibitor and paclitaxel can slow down the merisis of BRCA tumors in mice, thereby prolonging survival of mice. To further understand whether TTK is related to targeted drug resistance in HER2 breast cancer, we used CCK-8 experiments and determined that following silencing TTK with siRNA, the inhibition rate of pyrotinib on BT474 and SKBR3 cells was substantially increased, while the IC50 value was significantly reduced, further confirming that silencing TTK enhanced the drug susceptibility of HER2 BRCA cells to pyrotinib, and TTK may affect the resistance to pyrotinib in HER2 BRCA; to further confirm how TTK affects counteraction in HER2 BRCA, in vitro drug experiments proved that pyrotinib inhibited the proliferation, Invasion, migration and apoptosis of HER2 BRCA cells; induced cell block in the G1 phase, and downregulated the expression of p-p65, p-Akt and FOXC1 (Collins et al. 2021). Furthermore, the mechanism of action of pyrotinib is that it executes its powerful anti-tumor effect primarily by restraining the sensitization of Ras/MAPK or PI3K/AKT signaling passages (Wu et al. 2023). Lauring's study also confirmed that the phosphoinositide-3-kinase-AKT-mTOR approach is a key objective for targeted therapy of BRCA (Lauring et al. 2013). Through flow cytometry experiments, we found that after silencing TTK, cell apoptosis mainly occurred during the G1 period of the cell cycle, which is consistent with the clinical drug action cycle of pyrotinib. To further verify the framework of the function of TTK in HER2 BRCA, we enriched the Akt/mTOR signaling pathway through bioinformatics enrichment analysis and detected the correlation between TTK and the Akt/mTOR pathway through qPCR and WB. We discovered that after silencing TTK, the reflection of Akt and mTOR did not change significantly, while the phosphorylated proteins p-Akt and p-mTOR reduced obviously. Protein phosphorylation is related to tumor occurrence and progression, which further verified that TTK mainly promotes HER2 BRCA resistance to pyrotinib by activating the Akt/mTOR pathway. In gastric cancer, TTK was also found to promote cell ion and apoptosis by starting the AKT/mTOR signaling access (Huang et al. 2020). To further confirm how TTK regulates the Akt/mTOR pathway to play a crucial role in HER2 BRCA? Studies have confirmed that Akt phosphorylation substrates have multiple effects on tumor cell proliferation, migration, differentiation, apoptosis, DNA damage repair, and metabolism (Lauring et al. 2013); first, Akt phosphorylation can relieve the inhibition of mTORC1, thereby raising the function of mTORC1 kinase; and the mTORC2 complex acts upstream of Akt and plays a key role in regulating Akt phosphorylation; on the contrary, mTOR is a key downstream target of the PI3K/AKT signaling access, which is abnormally activated in BRCA and is related to cell multiplication, apoptosis, satis and metabolism; usually mTOR inhibits AKT phosphorylation activation by inhibiting mTORC1 and mTORC2 through inhibitors, thereby enhancing its anti-tumor activity (Lauring et al. 2013; Basu et al. 2015). We used the mTOR inhibitor rapamycin to inhibit the Akt/mTOR signaling pathway and then reverse transcribed with the TTK overexpression plasmid. Interestingly, the TTK overexpression plasmid can substantially reverse transcribe p-Akt and p-mTOR; further responses verified that TTK acts through the Akt/mTOR signaling passage, which is in accord with the results of the previously expected experiments. The aforementioned study suggests that TTK could serve as a promising therapeutic target in the clinical management of HER2-associated advanced BRCA. Considering the limited research currently available regarding TTK in the context of HER2-related BRCA, there is a need for more comprehensive investigations into its functional roles.

Conclusion

In summary, TTK plays a crucial role in the progression of HER2 + BRCA by modulating the Akt/mTOR axis to promote cell migration, inhibit apoptosis, and induce resistance to targeted therapy, which provides a Novel target and idea for the healing strategy of HER2 + BRCA and is expected to lay the foundation for the advancement of more effective therapy options in the future. Nonetheless, further research is still needed to fully uncover the complexity and potential regulatory mechanisms of TTK, as well as to explore targeted interventions to ameliorate the prognosis of sufferers with HER2 + BRCA.

Abbreviations

TTK

Threonine tyrosine kinase

BRCA

Breast cancer

TNBC

Triple-negative breast cancer

TPBC

Triple-positivity breast cancer

TCGA

The Cancer Genome Atlas

UALCAN

The University of Alabama at Birmingham Cancer

BCIP

Breast Cancer Integrative Platform

OS

Overall survival

DS

Disease specific survival

DFS

Disease free survival

Author contributions

ShaoLinZhang,HuaDing and YongFenDeng contributed equally to this work.Z.D and D.L were mainly responsible for writing manuscript texts.Z.R and Z were mainly responsible for collecting pictures.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82060480) ,Doctoral research start-up Fund of Guizhou Medical University (Gyfybsky-2021-42, Gyfybsky-2023-19) and QianKeHe Science Foundation - [2024] Youth Project 281.

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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