GTSE1 is involved in breast cancer progression in p53 mutation-dependent manner
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With the rapid development of the high throughput detection techniques, tumor-related Omics data has become an important source for studying the mechanism of tumor progression including breast cancer, one of the major malignancies worldwide. A previous study has shown that the G2 and S phase-expressed-1 (GTSE1) can act as an oncogene in several human cancers. However, its functional roles in breast cancer remain elusive.
In this study, we analyzed breast cancer data downloaded from The Cancer Genome Atlas (TCGA) databases and other online database including the Oncomine, bc-GenExMiner and PROGgeneV2 database to identify the molecules contributing to the progression of breast cancer. The GTSE1 expression levels were investigated using qRT-PCR, immunoblotting and IHC. The biological function of GTSE1 in the growth, migration and invasion of breast cancer was examined in MDA-MB-231, MDA-MB-468 and MCF7 cell lines. The in vitro cell proliferative, migratory and invasive abilities were evaluated by MTS, colony formation and transwell assay, respectively. The role of GTSE1 in the growth and metastasis of breast cancer were revealed by in vivo investigation using BALB/c nude mice.
We showed that the expression level of GTSE1 was upregulated in breast cancer specimens and cell lines, especially in triple negative breast cancer (TNBC) and p53 mutated breast cancer cell lines. Importantly, high GTSE1 expression was positively correlated with histological grade and poor survival. We demonstrated that GTSE1 could promote breast cancer cell growth by activating the AKT pathway and enhance metastasis by regulating the Epithelial-Mesenchymal transition (EMT) pathway. Furthermore, it could cause multidrug resistance in breast cancer cells. Interestingly, we found that GTSE1 could regulate the p53 function to alter the cell cycle distribution dependent on the mutation state of p53.
Our results reveal that GTSE1 played a key role in the progression of breast cancer, indicating that GTSE1 could serve as a novel biomarker to aid in the assessment of the prognosis of breast cancer.
KeywordsBreast cancer GTSE1 p53 Cell cycle Prognosis
American Type Culture Collection
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
False discovery rate
G2 and S phase-expressed-1
Hematoxylin and eosin
Nottingham prognostic index
Polymerase chain reaction
Small interfering RNA
The Cancer Genome Atlas
Triple negative breast cancer
Breast cancer is one of the major malignancies worldwide [1, 2]. There are standardized therapies accessible for the Her2 and luminal subtypes, but for TNBC there are no standard treatment methods owing to its heterogeneity. [3, 4, 5]. Progress has been made in the treatment of breast cancer in recent decades, however its recurrence and metastasis remain the most common reasons for treatment failure . In order to eliminate the bottleneck of breast cancer treatment, intensive research has been carried out in this field, but the molecular driving factors of tumor progression are still unclear . Therefore, identifying the key molecular events associated with the malignant transformation of breast cancer cells and tumor progression is of great significance as it would allow the development of new solutions for the diagnosis, treatment and improvement of the patients’ survival.
The rapid development of high-throughput detection techniques has enabled the accumulation of tumor-related Omics data which are of great significance to study the mechanism of tumor progression [8, 9]. The management and mining of large quantities of biological data have become important in cancer research . The TCGA database provides a large amount of tumor public data, which is widely used in tumor research, providing useful information for the discovery of new tumor biological indicators and drug targets [11, 12]. By taking advantages of bioinformatics, molecules related to the histological classification, recurrence and metastasis of breast cancer were screened to find out potential therapeutic targets which could benefit to the survival of breast cancer patients . By analyzing public database, GTSE1 attracted our attention.
GTSE1 is located in chromosome 22q13.2-q13.3, which is expressed specifically during the G2 and S phases of the cell cycle . A previous study has identified that the protein GTSE1 is mainly located in the cytoplasm and is associated with the activity of cytoplasmic tubulin and microtubules during mitosis . It is a key regulator for chromosomal movement and spindle integrity during mitosis . In addition, GTSE1 can act as a negative regulatory factor of p53 by accumulating in the nucleus and binding to p53 protein to transport it out of the nucleus for further degradation [17, 18]. Furthermore, overexpression of GTSE1 can lead to a delay in the transition of the G2 to M phase . Further studies have shown that the expression level of GTSE1 was upregulated in different human cancers, while its high expression was not associated with lung cancer prognosis . After treatment with cisplatin, GTSE1 was upregulated in myeloma cells, which showed a mechanism for clinical-acquired drug resistance . GTSE1 was found to be overexpressed in HCC and could serve as a marker for poor prognosis as apart from reducing the sensitivity of HCC cells to 5-FU, it could also promote the malignant biological behavior of HCC by improving its proliferative and metastatic ability and upregulating the expression of EMT makers [22, 23]. In addition, it conferred to cisplatin resistance by the inhibition of p53 apoptotic signaling in gastric cancer cells . GTSE1 was shown to be a regulated cytoskeletal protein required to promote cell migration . Previous studies have also reported that GTSE1 was identified as a novel YAP/TAZ-TEAD4 regulatory protein that could promote metastasis in breast cancer cells when it is overexpressed, especially in TNBC [26, 27]. However, up to now, there has been no detailed study regarding the functional role of GTSE1 in breast cancer.
In the present study, we showed that GTSE1 was associated with worse outcome and malignant phenotype including enhanced abilities of tumor proliferation and metastasis in breast cancer. Moreover, GTSE1 contributed to multidrug resistance in breast cancer cells. Intriguingly, GTSE1 could regulate the p53 function to alter the cell cycle distribution, dependent on the mutational status of p53. Taken together, we brought light on the function of GTSE1 and showed its potential significance as a novel biomarker for assessing the breast cancer progression.
TCGA data analysis and bioinformatics analysis
RNA-Seq data of normal breast and breast cancer tissues were downloaded from TCGA and analyzed to find genes that were significantly upregulated in breast tumors by using the EdgeR method. The candidate genes were identified by the following conditions: (1) the genes had to be significantly upregulated in samples of breast cancer as compared to samples from normal breast tissue, (false discovery rate [FDR] < 5%); (2) the expression difference should be at least two of fold change; (3) the direction of gene expression had to be inversely and significantly associated with survival (P < 0.05). Survival analyses of the candidate genes were performed using the two online databases namely bc-GenExMiner and PROGgeneV2, to determine the prognostic significance of the mRNA expression level of GTSE1, and to perform further bioinformatics analysis.
Cell lines and cell culture
The HCC38, MDA-MB-468, MDA-MB-231, MCF7, MCF10A cells were purchased from the American Type Culture Collection (ATCC). HCC38 cells were cultured in RPMI 1640 medium (GIBCO, C11875500BT) supplemented with 10% fetal bovine serum (FBS). MCF10A cells were cultured in RPMI 1640 medium supplemented with horse serum (5%), insulin (10μg/mL) and epidermal growth factor (20 ng/mL), hydrocortisone (0.5μg/mL). MCF7, MDA-MB-231 and MDA-MB-468 cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO, C11995500BT) supplemented with 10% FBS at 37 °C and 5% CO2,100 units per mL penicillin (MDbio, P003-10 g), and 100 mg/mL streptomycin (MDbio, S007-25 g).
Quantitative real-time polymerase chain reaction
Trizol reagent (Invitrogen) was used to extract total RNA from cultured cell lines, which was then applied to reverse transcription using a cDNA Synthesis Kit (ThermoFisher, EP0441). qRT PCR was conducted using a UNICONTM qPCR SYBR® Green Master Mix (YEASEN, 11198ES08). Expression data were standardized to the reference gene GAPDH in order to control the variability in expression levels and calculated as 2- [(CT of candidate genes) - (CT of GAPDH)], where CT represents the threshold cycle for each transcript. The average for each gene and sample was calculated and the experiments were independently repeated three times. The primers used are listed in the Additional file 1: Table S1.
Western blotting was performed according to the standard protocol. The primary antibodies, including GTSE1(Proteintech, 21,319–1-AP), Ki67(Proteintech, 27,309–1-AP),mutant p53(Abcam, ab32049), p53(Proteintech, 10,442–1-AP),Cyclin D1(Proteintech, 60,186–1-Ig),Cyclin E1(Proteintech, 11,554–1-AP), p21(Proteintech,60,214–1-Ig), E-Cadherin (Proteintech, 20,874–1-AP), Desmoplakin (Proteintech,25,318–1-AP), N-Cadherin (Proteintech,22,018–1-AP), Vimentin (Proteintech, 10,366–1-AP), Nanog (Proteintech, 14,295–1-AP), ABCG2(CST, 42078S), TAZ (CST, 4883S), YAP (CST, 14074S), ERK1/2(Affinity, AF0155), phospho-ERK1/2(Affinity, AF1015), AKT (CST, 2938S), Phospho-Akt (Thr308) (CST, 13038S), Phospho-Akt (Ser473) (CST, X4060S), FoxC2(CST, 12974S),Slug (CST, 9585 T),Twist1(CST, 46702S),Snail (Proteintech, MG-3879 T) and GAPDH (Proteintech, 60,004–1-Ig) were used at a dilution of 1:1000.
We obtained 16 non-cancerous breast tissues and 37 primary breast cancer tissues from the Department of Breast Carcinoma, at the Sun Yat-sen University Cancer Center (Guangzhou, P. R China) to investigate the mRNA expression levels of GTSE1. A total of 154 paraffin-embedded primary breast cancer tissues were obtained from patients with informed consent who underwent surgery for primary breast cancer at the Sun Yat-Sen University Cancer Center between 2003.04 and 2011.12, with the approval of the Institutional Clinical Ethics Review Board of Sun Yat-Sen University Cancer Center (IRB Approval Number GZR2019–101). The immuno-histochemical (IHC) score was composed of a score for the percentage of positive tumor cells and the staining intensity grade. The percentage of positive cells were classified as follows: 0–5% of stained cells = 0, 6–25% = 1, 26–50% = 2, 50–75% = 3 and 76–100% = 4. The intensity score of positive cells contained the cytoplasmic and nucleus staining intensity. The staining intensity was classified into the following four grades: no staining = 0, weak staining = 1, moderate staining = 2 and strong staining = 3. Then multiplied positive proportion with intensity to produce a total score which varied from 0 to 12. We then used the follow-up data of 154 breast cancer cases (TNBC, n = 90, non-TNBC, n = 64) for further survival analysis after immunohistochemical (IHC) staining for GTSE1.
Small interfering RNA transfection
siRNAs and the negative control small interfering RNA (NC) were purchased from GenePharma (sequences shown in Additional file 1: Table S2). Transient transfections were performed in an antibiotic-free medium using the Lipofectamine RNAiMAX Reagent (Invitrogen, 13,778,150) following the manufacturer’s protocol. The GTSE1 silencing was performed by transfecting the siRNAs in MDA-MB-231 and MDA-MB-468 cell lines for 48 h.
Lentivirus transfection and transduction
To establish GTSE1 knockdown stable cell lines, the human GTSE1 ‘SureSilencing shRNA’ plasmids were purchased from GeneCopoeia (sequences shown in Additional file 1: Table S3). Lentiviruses were produced by co-transfecting 293 T cells with the GTSE1 shRNA or negative control shRNA plasmid along with packaging plasmids using Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia, LT003). After transfection for 48 h, infectious lentivirus was harvested through a 0.45-filter (Millipore, SG079) before using them to infect the MDA-MB-231 cells. Then the infected cells were screened with 1 μg/mL of puromycin (MPbio, 219,453,925) for 7 days. To produce MDA-MB-231 and MCF7 cells with stable overexpression of GTSE1(sequences shown in Additional file 1: Table S4), the full-length human GTSE1 was cloned into a lentiviral vector. The efficiency of the knockdown and overexpression of GTSE1 was determined by Immunoblotting.
MTS assay, and colony formation assay
Cell growth curves were plotted using the GraphPad software based on the data obtained from the MTS assays, which used the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, G3581) to detect the cancer cells viability and growth. In brief, a concentration of 1000 cells/200 μL of mixture medium was seeded into a 96-well plate (jet, TCP011096) and cultured under normal conditions for 7 days and tested MTS every day. At each corresponding time points after seeding, we incubated cells with 200 μL of the mixture of MTS and DMEM (1:10) for 2.5 h, and adjusted the absorbance to 490 nm to detect its viability with a microplate research reader (Bio-Tek EPOCH2, America). In order to perform the colony formation assay, 1000 cells/2 mL were seeded into a 6-well plate (jet, TCP011006) and the cultured medium was changed twice a week. Two weeks later, the colony number were calculated after washing these cells with 1X phosphate-buffered saline (MP, 92810307) solution, fixing them with methanol for 20 min, and staining with 2% crystal violet for 30 min. These experiments were independently repeated three times.
Analysis of cell motility
Cell motility was determined by using the transwell and wound healing assays. Migration assays were performed using chambers without Matrigel (Falcon, 353,097), and invasion assays were conducted with the Matrigel Invasion Chamber (Corning, 354,480) based on the manufacturer’s protocol. Briefly, cells in 200 μL of serum free medium were seeded to the upper chamber and allowed to migrate or invade to the other side of the membrane. After 48 h, the chambers were stained with crystal violet. Images were captured from each membrane using the ImageJ software to count for the metastatic cell numbers. In migration assay, 30,000 number of cells were seeded to the upper chamber and the chamber was not covered with Matrigel. While in invasion assay, 50,000 number of cells were seeded to the upper chamber and the chamber was covered with Matrigel. After 48 h, the chambers of transient transfection groups (siNC and siGTSE1) were stained with crystal violet, and the stable cell line groups (vector and GTSE1) were stained after 30 h.The mean number obtained from three independent assays under each experimental condition was used in the final analysis. The wound healing assay was conducted using a 200 uL pipette tip to scratch the cell layer 24 h after seeding, and the wound healing rate was recorded after 0, 24, 36, 48 and 72 h. All experiments were independently repeated three times.
Sphere culture assay
A concentration of 1000 cells/well were seeded on 6-well low-attachment culture plates (Corning, 3471) in order to perform the tumor sphere assay. Cells were cultured in serum-free DMEM/F12 medium (GIBCO, C11330500BT) supplemented with 20 ng/ml of EGF (epidermal growth factor) (NovoProtein, C029-B) and 20 ng/mL of bFGF (basic fibroblast growth factor) (NovoProtein, C046-A). The number of spheres was counted using the Image J software. Three independent experiments were performed.
Cell apoptosis and cell cycle assay
Cell apoptosis and cell cycle were respectively detected by the Annexin V FITC Apop Dtec Kit (BD, 556547) and a cell cycle staining kit (BestBio, BB-4104-3), in accordance to the manufacturer’s manual. Briefly, the cells were grown to 70% confluency, after 48 h of incubation with or without drug (Taxol, 5-fluorouracil, and Adriamycin), the cells were collected and processed for analysis. The cell samples were then analyzed using a flow cytometer ACEA NovoCyte equipped with the FlowJo software (version 0.7).
Xenograft tumor model
All animal experiments were conducted according to the instructions approved by the Research Animal Resource Center of Sun Yat-Sen University (Approval number L102042018030E). Three-week-old female BALB/c mice were purchased from the Sun Yat-sen University Laboratory Animal Center and maintained under standard conditions. Tumor cells of MDA-MB-231 scramble and shGTSE1 groups (2 × 106 cells/tumor in 100 μl DMEM) were suspended in 100 μl DMEM containing 50% Matrigel (Corning, 356,237) and injected into their left and right axillary areas. The mice were observed three times per week for apparent tumor formation. The experiment was finished at 22 days after tumor cell inoculation. For lung metastasis experiment, the tumor cells of MDA-MB-231 scramble and shGTSE1 groups (1 × 106 cells/tumor in 100 μl DMEM) were intravenously injected through the tail vein of the mice. Lung metastatic nodules were calculated after 5 weeks when the mice died of cachexia. Lungs were excised for hematoxylin and eosin staining. In addition, six-week-old female BALB/c mice were used to perform situ breast pad injection. Tumor cells of MCF7 vector and GTSE1-overexpression groups (7 × 106 cells/tumor in 100 μl DMEM) were suspended in 100 μl DMEM containing 50% Matrigel and injected into the left and right breast pad areas. The mice received estradiol supplementation (0.4 mg/kg) every 7 days for 35 days after cell injection, and were observed and palpated for tumor appearance. Tumor growth was measured weekly using calipers. Tumor volume was determined using the standard formula: L*W2*0.52, where L and W refered to the longest and shortest diameters, respectively.
Senescence-associated β-gal assay
Senescence-associated β-gal assay was carried out to determine the cells’ senescence state using an SA-β-gal staining kit (Roche, 11,828,673,001) according to introductions. Briefly, the cells were washed with PBS (1X), fixed with 2% formaldehyde and 0.2% glutaraldehyde for 10 min after removing medium, the cells were washed with PBS twice and incubated with fresh SA-β-gal stain solution, which was then kept overnight at 37 °C without CO2. The cells were then photographed using a microscope (Nikon Eclipse 80i), and the percentage of senescence cell was counted by counting three random fields.
Statistical analyses were conducted using the GraphPad Prism version 7.0 (USA, GraphPad Software) and the SPSS software version 22.0 for Windows (IBM, USA). The relationship between GTSE1 expression and clinicopathologic status was analyzed using the chi-square test. The correlations between the GTSE1 expression to OS and RFS were analyzed using the Kaplan–Meier survival and log-rank test. Data were analyzed using the Student’s t-test or ANOVA methods. P-value of less than 0.05 was considered as statistically significant for all tests.
Identification of GTSE1 in breast cancer progression based on the analysis of the online databases
p53 mutation is correlated with the high expression of GTSE1
GTSE1 is closely related to differentiation and prognosis of breast cancer
GTSE1 promotes breast cancer cell growth by activating AKT pathway
GTSE1 promoted breast cancer metastasis by regulating EMT
GTSE1 can cause multidrug resistance in breast cancer cells
GTSE1 regulates p53 function to alter cell cycle distribution dependent on the mutational status of p53
Breast cancer is a heterogeneous malignancy, with multiple epigenetic and genetic alterations, and involving complex molecular signaling pathways [45, 46, 47]. In this study, we found that both the GTSE1 mRNA and protein expression levels are increased in breast cancer cell lines and tissues, especially in TNBC and p53 mutant breast cancer cells. Furthermore, its expression level was positively correlated with Ki67, highlighting its important role in human breast cancer progression. The expression level of GTSE1 protein in breast cancer tissues was related to differentiation and reduced overall survival time and recurrence free survival time of patients. Taken together, our study demonstrated that GTSE1 could serve as a novel and potential marker for the prognosis of breast cancer.
Our functional studies demonstrated that GTSE1 could promote the growth of breast cancer cells, which is in line with the data on HCC . In addition, our data demonstrated that GTSE1 may affect the AKT pathway to facilitate breast cancer cells growth. Moreover, our data also indicated that GTSE1 could enhance breast cancer cells metastasis by regulating EMT, which is in line with the data on HCC . Inhibiting AKT pathway has no effect on cell metastasis, which may be due to the compensation of other tumor signaling pathways. It has also been reported that the up-regulated expression of GTSE1 in gastric cancer cells has a contribution to cisplatin resistance . Importantly, our data implicated that GTSE1 downregulation contributed to an increase in sensitivity to chemotherapy-induced apoptosis after treatments with Taxol, 5-fluorouracil and Adriamycin, while the overexpression of GTSE1 exhibited opposing effects. Since GTSE1 could promote the proliferative ability of breast cancer cells, we found that it had no function on breast cancer cells spontaneous apoptosis but affected the cell cycle distribution. GTSE1 is necessary for chromosome arrangement and progression to mitosis . Silencing GTSE1 significantly increased the percentage of S phase owing to the inhibition of mitosis resulting to an S phase arrest in breast cancer cells, meanwhile, the overexpression of GTSE1 increased the percentage of cells in the G2 peak, indicating that overexpression of GTSE1 delayed the M-to-G2 phase transition of breast cancer cells, which is consistent to previous study . However, since GTSE1 affects breast cancer differentiation without affecting self-renewal ability, we assumed that GTSE1 may regulate the differentiation of breast cancer cells by influencing other biological processes such as EMT and cell cycle.
It has been reported that GTSE1 is one of the target genes of p53, which may also serve as a negative regulator of p53. Previous studies have demonstrated that GTSE1 may influence the cell cycle distribution by translocating p53 from the nucleus to the cytoplasm for degradation, and can downregulate p53 protein level . P21 is one of the most important downstream target genes of p53 . P21 protein is known as the cell cycle inhibitory protein with the most extensive kinase inhibition activity, which binds to the Cyclin-CDK complex and inhibits its activation. By inhibiting the activation of CyclinD1-CDK4/6 and CyclinE1-CDK2, p21 can prevent the phosphorylation of Rb protein and the release of transcriptional factors E2F, causing cell-cycle arrest and affecting the distribution of cell cycle [50, 51, 52]. Our findings revealed that a novel GTSE1 function in regulating cell cycle distribution. GTSE1 has no effect on a mutant p53 homozygote but had an effect on the wild-type p53 homozygote and mutant p53 heterozygote. In cells expressing the mutant p53 heterozygote, the role of GTSE1 in regulating the p53 function was mainly dependent on the wild-type portion of the p53 mutant heterozygote. We hypothesized that GTSE1 regulates the p53 function to alter the cell cycle distribution mainly by the following three ways. First, in MCF7 cells, GTSE1 played a role in a p53-dependent manner, which interacted with the wild-type p53 protein to form a complex in the nucleus and transported the p53 to the cytoplasm for degradation resulting to p53 downregulation, which in return downregulated the p21 protein level meanwhile upregulated CyclinD1 and CyclinE1 protein levels. Second, in MDA-MB-231 cells, GTSE1 bonded with the wild-type part protein of p53 mutant heterozygotes to modulate the downstream protein levels, playing a role in a p53-partial-dependent manner. Third, in MDA-MB-468 cells, GTSE1 plays a role in a p53-independent manner, which does not bind to the mutant p53 protein and had no function on it as well as its downstream proteins. Interestingly, Bublik et al found that GTSE1 protects p21 from proteasome-dependent degradation in a p53-independent mechanism, and p21 protein levels altered in accordance with GTSE1 expression . Since GTSE1 is an oncogene and p21 is a tumor suppressor gene, our research showed that the variation trend of GTSE1 was opposite to that of p21, and GTSE1 regulates p21 protein level dependent on the mutation state of p53 and such regulation of p21 levels depend on the changes of the cell cycle.
In summary, GTSE1 have effects on regulation of breast cancer growth and metastasis. Moreover, GTSE1 confers multi-drug resistance in breast cancer cells, and can regulate p53 function to alter cell cycle distribution dependent on p53 mutational status.
This work was supported by National Natural Science Foundation of China 81773162, National Natural Science Foundation of China 81572901, National Natural Science Foundation of China 31170151, Natural Science Foundation of Guangdong Province, China 2017A030313866, Science and Technology Planning Project of Guangdong Province, China 2014A020209024, Science and Technology Planning Project of Guangdong Province, China2014B020212017.
Availability of data and materials
For all manuscripts, especially those containing data from the Sun Yat-sen University Cancer Center (SYSUCC) it is recommended that the authors to deposit the data in the Research Data Deposit (RDD) at the following website: http://www.researchdata.org.cn/
FL and Y-JX carried out most of the experimental work and the analysis of data; FL was responsible for drafting the manuscript, B-JH provided scientific and administrative oversight for the conduct of the research and revised the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All human tissue samples were obtained with patient consent and the approval of the Institutional Clinical Ethics Review Board at Sun Yat-Sen University Cancer Center. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Sun Yat-Sen University Cancer Center.
Consent for publication
The authors declare that they have no competing interests.
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