Trop2 is a potential biomarker for the promotion of EMT in human breast cancer
- Authors:
- Published online on: June 14, 2018 https://doi.org/10.3892/or.2018.6496
- Pages: 759-766
Abstract
Introduction
BC is the second leading cause of cancer-related deaths in women after lung cancer and it accounts for nearly 25% of all cancers in females (1,2). Approximately 1.4 million women worldwide are diagnosed with BC each year, and half a million BC patients succumb to the disease (3). BC, which is comprised of several different phenotypes, is a highly heterogeneous disease (4). According to studies (1,2), based on the analysis of the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67, BC is classified into five subtypes, including luminal A, luminal B, normal breast-like, HER2 and basal-like. One of these subtypes, triple negative breast cancer (TNBC), is defined by the lack of ER, PR and HER2 expression in tumor tissues, and it accounts for 15–23.8% of BC patients (5). Compared with the other subtypes, TNBC is frequently found in premenopausal women, particularly in young women (<50 years), who may have a higher grade and a higher rate of a cellular tumor antigen p53 mutation (6). TNBC is more prevalent in African-American women and more aggressive than other molecular subtypes of breast tumors (7). Although the risk and roles of ER-positive cancer are well defined, those for TNBC are not as well defined. Therefore, it is critical to understand gene expression and genetic variability in the etiology and pathogenesis of these types of BC.
Human trophoblast cell surface protein 2 (Trop2/ TACSTD2/M1S1/GA733-1), is a 36-kDa transmembrane protein that is expressed primarily in the membrane surface of epithelial cells (8). Trop2 has been revealed to affect tumor proliferation, metastasis and invasion as an oncogene, and it is overexpressed in various epithelial tumors, which may be associated with aggressive tumor effects (9–12). In our previous study, we found that Trop2 has an important role in regulating stem cells and inducing EMT of gastric cancer cells. However, little is known about the regulatory role of Trop2 in TNBC.
E-cadherin is a type of adhesion molecule that mainly exists in human and animal epithelium, and its main function is to maintain normal epithelial cell morphology and structural integrity (13–15). E-cadherin can regulate the close adhesion of epithelial cells and its downregulation is a key factor in epithelial neoplasm development (16). The aberrant structure of E-cadherin was found in tumor cells, and the adhesion ability of cells was decreased, which could easily lead to infiltration and distal metastasis of cells to peripheral tissues (17). E-cadherin has been considered to be an important component of intercellular adhesion and it is also a key factor in initiating EMT transformation (18,19). Decreased E-cadherin expression in cancers, which could induce the EMT phenomenon has been a popular research area in recent years (20). Many researchers have found that inhibiting E-cadherin expression could be related to the differentiation, invasion and metastasis of tumors (21,22).
In the present study, we examined the relationship between Trop2 and E-cadherin in BC tissues obtained from Chinese patients and compared the expression of two genes in the BC tissues with matched adjacent non-tumor tissues.
Materials and methods
Tissue sample and clinical information
We studied a cohort of 312 patients who were diagnosed with primary BC from January 2009 to December 2012. A total of 354 formalin-fixed, paraffin-embedded (FFPE) BC tissue samples were investigated, including BC tissues (n=295) and matched adjacent tissues (n=59). All of the tissue blocks were obtained from the Department of Pathology, Nanjing First Hospital. Clinical information about the tissues was collected, including age, location, tumor size, tumor-node-metastasis (TNM) stage, ER/PR/HER2 status, Ki-67 expression, histological grade, lymph node status, metastasis and overall survival (OS). In addition, 20 pairs of freshly frozen BC tissues were obtained from the Department of Pathology, Nanjing First Hospital. Written informed consent was obtained before the patients underwent surgery. All the results obtained from the study will have no effect on the patient's health and future treatment. The study protocol was approved by the Human Research Ethic Committee of Nanjing First Hospital.
qRT-PCR
We detected Trop2 and E-cadherin mRNA expression levels in 20 pairs of human BC tissues, and then we compared them with matched adjacent tissues. Cell RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then reverse-transcribed into cDNA using a PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Human GAPDH served as the internal control. The primers used in the study were asfollows: GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; Trop2 forward, 5′-TGTCCTGATGTGATATGTCTGAG-3′ and reverse, 5′-GGGTGAGAGTGGGTTGGG-3′; E-cadherin forward, 5′-GACGCGGACGATGATGTGAAC-3′ and reverse, 5′-TTGTACGTGGTGGGATTGAAG-3′ (GenScript Co., Ltd., Nanjing, China). qRT-PCR was performed on an ABI Prism 7500 HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) in 96-well plates. Relative expression levels were calculated as ratios normalized against those of GAPDH. Results were normalized to the respective internal controls. The Ct-value for each sample was calculated using the ΔΔCt method, and the results were expressed as 2−ΔΔCt.
Tissue microarray (TMA) construction and IHC
The TMAs were constructed at the Department of Pathology, Nanjing First Hospital, (Nanjing, China) using the Quick-Ray tissue system (Unitma, Co., Ltd., Seoul, Korea) manual. Core tissue biopsies (2 mm in diameter) were obtained from 60 individuals. FFPE blocks were made and then arranged in new recipient paraffin blocks. A total of 5 breast TMAs were created. Tissue sections were deparaffinized and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by incubation in 3% H2O2. Tissues were then placed in 0.01 M citrate buffer (pH 6.0), and heated in a microwave for antigen retrieval. Trop2 was detected using a monoclonal antibody rabbit anti-human Trop2 (dilution 1:100) (cat. no. ab227689; Abcam, Cambridge, UK), and a monoclonal antibody mouse anti-human E-cadherin (dilution 1:100; cat. no. sc-8426; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Reactions were detected with an EnVision™ peroxidase kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Tissues were then incubated in 3,3′-diaminobenzidine (Dako; Agilent Technologies, Inc.), counterstained with hematoxylin, dehydrated through graded alcohols, cleared in xylene, and coverslipped with permanent mounting media. Staining was quantified in all of the tissues without knowledge of clinical characteristics. Trop2 expression was scored using the semi-quantitative H-score method, which takes into account both the staining intensity and the percentage of cells at that intensity (8). The following staining intensity scores were used: 0 indicated no staining; 1+ indicated weak staining; 2+ indicated moderate staining; and 3+ indicated intense staining. The total number of cells at each intensity level was multiplied by the corresponding intensity score to yield an intensity percentage score. Final staining scores were then calculated by summing the four intensity percentage scores; the minimum possible final staining score was 0 (no staining) and the maximum possible score was 300 (100% of cells with 3+ staining intensity).
Cell lines and cell culture
The human BC cell lines MCF-7 and MDA-MB-231 and the human breast epithelial cell line MCF-10A were preserved in our laboratory. Cell lines were maintained at 37°C in RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin-streptomycin (all from Gibco; Thermo Fisher Scientific, Inc). A humidified atmosphere containing 5% CO2 was used to incubate the cells.
Protein extraction and western blot analysis
Intracellular proteins were extracted using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Inc.) based on the manufacturer's protocol. Protein concentrations were determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). The same amounts of protein were resolved by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 1 h and then incubated with primary antibodies (anti-Trop2 antibody or anti-E-cadherin antibody) overnight at 4°C. After washing three times with PBS-Tween-20 (PBST), the membrane was incubated with secondary antibodies, including HRP-goat anti-rabbit secondary antibody (cat. no. A12004-1; Amy Jet Scientific, Wuhan, China), HRP-goat anti-mouse secondary antibody (A12003-1; Epigentek, Farmingdale, NY, USA) at room temperature for 1 h. The membrane was subsequently washed with PBST and incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) for 5 min. The specific bands were exposed to the ChemiDoc XRS+ System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression of GAPDH was used as the internal control.
Statistical analysis
The SPSS 19.0 statistical software package (SPSS, Inc., Chicago, IL, USA) was utilized to analyze all of the statistics. Unpaired Student's t-test was performed to compare two groups. One-way ANOVA followed by Tukey's multiple comparison test was used when three or more groups were compared. χ2 tests were performed to evaluate whether the expression levels of Trop2 and E-cadherin were associated with clinicopathological parameters. Kaplan-Meier analysis was used to estimate cumulative patient survival. Survival curves were compared by log-rank test. P<0.05 was considered to indicate a statistically significant result.
Results
Trop2 and E-cadherin mRNA expression in BC tissues compared with matched adjacent tissues
To study the levels of Trop2 and E-cadherin mRNA, we used qRT-PCR in 20 pairs of BC tissues and matched adjacent tissues. Trop2 and E-cadherin mRNA expression levels in BC tissues were 1.55±0.78 and 0.70±0.38 fold higher than those in the matched adjacent tissues, respectively (P=0.018). Notably, we also found Trop2 and E-cadherin mRNA expression levels in the TNBC tissues (T6, 9, 11, 12, 15, 16, 19 and 20) were 2.76±0.77 (P=0.013) and 0.54±0.21 (P=0.010) fold higher than those in other types of BC (T1, 2, 3, 4, 5, 7, 8, 10, 13, 14, 17 and 18, respectively) (Fig. 1).
Trop2 and E-cadherin protein expression in BC tissues compared with matched adjacent tissues
We used an IHC assay to detect Trop2 and E-cadherin protein expression levels in BC tissues, and we found that Trop2 and E-cadherin were localized at the membrane and cytoplasm of the BC cells (Fig. 2). Trop2 was overexpressed in the BC tissues, while E-cadherin had a low expression in BC tissues. We defined high and low Trop2/E-cadherin expression levels based on OS in BC patients through the x-tile software program for TMA data analysis. For Trop2, the cut-off of 130 was selected, and a score of 0–130 was considered to be low or no expression, while 131–300 was considered to be a high expression. For E-cadherin, a score of 0–120 was considered to be low or no expression, while 121–300 was considered to be a high expression.
High Trop2 and high E-cadherin (T+E+), high Trop2 and low E-cadherin (T+E−), low Trop2 and high E-cadherin (T−E+), and low Trop2 and low E-cadherin (T−E−) expression was detected in the BC tissues, compared with those in the matched adjacent tissues. The levels of T+E+, T+E−, T−E+ and T−E− expression in the TNBC tissues were 7 (7.3%), 68 (70.8%), 16 (16.7%), and 5 (5.2%), while in the BC tissues, they were 22 (11.1%), 102 (51.3%), 61 (30.7%), and 14 (7.0%), and compared with those in matched adjacent tissues, they were 8 (13.6%), 13 (22.0%), 28 (47.5%), and 10 (16.9%), respectively (χ2=37.105, P<0.001) (Table I).
Association of Trop2 and E-cadherin expression with clinicopathological characteristics in BCs
Next, we investigated the relationship between Trop2/E-cadherin protein expression and clinicopathological variables in tissues of BC patients. The results indicated that the T+E− expression in BC was associated with lymph node status (χ2=36.688, P<0.001), metastasis (χ2=42.958, P<0.001), TNM stage (χ2=15.91, P<0.014), and TNBC (χ2=10.429, P<0.015). However, we did not detect a significant association between Trop2 and E-cadherin expression levels and tumor location, age and tumor size (Table II).
Table II.Association of the expression level of Trop2 and E-cadherin with clinicopathological characteristics in BC patients. |
Prognostic value of Trop2 and E-cadherin protein expression in BC
We also used univariate and multivariate analyses to examine the possible prognostic factors for BC. The results revealed that T+E− expression was significantly associated with a shorter survival in univariate analysis, along with lymph node status, metastasis, TNM stage and TNBC. Multivariate analysis further indicated that T+E− expression was associated with poor OS, as did lymph node status, metastasis and TNBC (Table III). Kaplan-Meier survival curves revealed that T+E− expression was associated with poor OS (Fig. 3). These results may help in our analysis of the 5-year survival rate of BC patients.
Table III.Univariate and multivariate analysis of prognostic markers for overall survival in breast cancer. |
Trop2 and E-cadherin mRNA and protein expression in BC cell lines
To better demonstrate the level of Trop2 and E-cadherin expression in BC, we assessed the protein level of Trop2 and E-cadherin in two types of BC cell lines (MCF-7 and MDA-MB-231) and the human breast epithelial cell line MCF-10A. Trop2 expression was the lowest and E-cadherin was the highest in the MCF-10A cells when compared with the BC cell lines. The MCF-7 and MDA-MB-231 cell lines exhibited increased Trop2 and decreased E-cadherin expression levels when compared with MCF-10A cells as determined by qRT-PCR and western blot assay (Fig. 4).
Discussion
The invasion and metastasis of tumors refers to the process of tumor cells being transferred to other tissues from the original site (23–25). EMT is a developmental process in which epithelial cells lose their phenotypes, such as E-cadherin and N-cadherin, and mesenchymal cells gain their phenotypes, such as vimentin and fibronectin. EMT is considered to be important in the invasive and metastasis progression of cancer (26). EMT promotes migration and invasion, accelerates stem cell properties, conduces immunosuppression, and deters apoptosis and senescence (20). Both Trop2 and E-cadherin have a great influence on EMT transformation.
In our previous study (unpublished data), we confirmed that Trop2 upregulated the expression of the mesenchymal phenotype and downregulated the epithelial phenotype, such as E-cadherin and N-cadherin. We also demonstrated that Trop2 was involved in β-catenin/TGF-β1-mediated EMT in gastric cancer. Lin et al (27) have revealed that Trop2 is highly expressed in BC patients and it is related to the expression level of cyclin D1. However, the potential role of Trop2 in the promotion of EMT in BC has not been fully studied.
In the present study, Trop2 mRNA was highly expressed in BC tissues while E-cadherin mRNA was decreased in BC tissues compared with those in matched para-carcinoma tissues, results which were similar to a previous study (20). Notably, we also found that the Trop2 and E-cadherin mRNA expression levels in the TNBC tissues were 2.76±0.77 and 0.54±0.21 fold higher than those in other types of BC. A similar result was found in protein expression. Our TMA results also revealed that the Trop2+/E-cadherin− expression was associated with lymph node status, metastasis, TNM stage and ER−/PR−/HER2−. Furthermore, a high Trop2/low E-cadherin expression predicted poor OS in BC patients. Lastly, we detected Trop2 and E-cadherin expression in BC cell lines (MCF-7 and MDA-MB-231) and the normal breast epithelial cell line MCF-10A, and we found that Trop2 had a higher expression while E-cadherin had a lower level compared with that in the normal breast epithelial cell line.
Different expression of Trop2/E-cadherin between cancer tissues and adjacent tissues indicate that high Trop2/low E-cadherin expression is connected with BC or even TNBC. Our findings revealed that Trop2 promoted the migration and invasion of BC cells by inducing the EMT phenomenon, resulting in lymph node involvement and distant metastasis which always indicates poor prognosis and short OS. The function of E-cadherin does the opposite in BC.
Overall, high Trop2 and low E-cadherin expression may predict poor prognosis and short survival in BC. Furthermore, Trop2 and E-cadherin could be considered as therapeutic targets. In the future, more studies should be performed to confirm the mechanism of Trop2 and E-cadherin in vivo and in vitro.
Acknowledgements
We thank all the patients enrolled in the study.
Funding
The present study was supported by grants from the ‘Six Talent Peaks Project’ in Jiangsu Province (no. WSN-068) and the National Natural Science Foundation of China (no. 81601618).
Availability of data and materials
The datasets used during the present study are available from the corresponding author upon reasonable request.
Authors' contributions
WBH and YCZ designed the study; WZ, XWK and XYZ acquired the data and drafted the article; LZJ, JSW and XBY analyzed and interpreted the data; ZDT, XLW, QL and BW revised the article critically for important intellectual content. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
The study protocal was approved by the Human Research Ethic Committee of Nanjing First Hospital. Written informed consent was obtained before the patients underwent surgery.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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