Sperm‑associated antigen 5 is a potential biomarker for poor prognosis in breast cancer
- Authors:
- Published online on: November 19, 2018 https://doi.org/10.3892/ol.2018.9729
- Pages: 1146-1152
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Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Breast cancer (BC) is one of the most common cancer types in women. Approximately 1.68 million women are diagnosed with BC worldwide each year and it ranks as the leading cause of cancer-associated cases of mortality in women (1). Although China is a country with a lower incidence of BC, incidence and mortality still ranks the highest for BC in female malignant cancer types (2–4). BC is a heterogeneous disease that exhibits high sensitivity to chemotherapeutic treatment; approximately 50% of patients with advanced breast cancer benefit from combined chemotherapy, whereas the other half experience only the adverse effects from chemotherapy drugs (5). Therefore, it is important to further refine BC molecular classification to more precisely guide treatment decisions. There are four BC molecular subtypes, including luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpression and triple-negative (TN) (6). Significant differences have been identified in the prognosis and treatment efficacy among patients with different molecular subtypes of BC (7). Although the existing molecular subtyping provides an improved basis for the treatment and prognosis of patients, certain patients with heterogeneous BC remain insensitive to clinical treatment (8).
Sperm-associated antigen 5 (SPAG5), first identified in 2001, is also known as mitotic spindle-associated protein 5 (9–11). SPAG5 is involved in the formation of spindles during the cell cycle and may be closely associated with the function and movement of spindles during mitosis (12,13). Certain studies have also reported that SPAG5 may inhibit cell apoptosis through the inhibition of mammalian target of rapamycin complex 1 (14). In 2016, Abdel-Fatah et al (15) revealed that SPAG5 is an amplified gene in BC and may have clinical utility as a biomarker in estrogen receptor (ER)-negative BC, however this study did not examine the association between SPAG5 and the molecular classification of BC.
The current study analyzed SPAG5 expression in Chinese patients with primary BC and compared it with the expression level in matched adjacent nontumor tissues. The association between SPAG5 expression, clinical characteristics, ER/progesterone receptor (PR)/HER2/Ki-67 expression and overall survival (OS) in patients with BC was also investigated.
Materials and methods
Human tissue specimens and patient clinical information
A total of 379 formalin-fixed, paraffin-embedded (FFPE) breast tissue samples were collected from 159 female patients with an age range of 24–87 years. This included 159 cancer tissue samples, 159 matched adjacent nontumor tissue samples and 61 benign tumor tissue samples. All tissue blocks were obtained from the Department of Pathology at the Affiliated Hospital of Nantong University (Jiangsu, China) between July 2003 and March 2010. Medical records for tissue donor patients included information on age, histological grade, ER expression, PR expression, HER2 expression, Ki-67 expression, tumor size, lymph node metastasis and tumor-node-metastasis (TNM) stage. No patients received treatment, including radiation therapy, chemotherapy or immunotherapy, prior to surgical resection. OS was defined as the period from initial diagnosis of BC via biopsy until mortality. Information on patients who were alive at the last follow-up date was removed from the analysis. Disease-free survival was defined as the period from surgical treatment until disease recurrence. In addition, a total of 35 freshly frozen BC tumor tissues and matching adjacent nontumor tissues were obtained from a subset of the 159 patients at the Affiliated Hospital of Nantong University. Written informed consent was obtained from all patients involved in the current study. The Human Research Ethics Committee of the Affiliated Hospital of Nantong University approved the study protocol.
Tissue microarrays (TMAs) and immunohistochemistry (IHC)
TMAs were generated at the Department of Pathology, Affiliated Hospital of Nantong University, using a Quick Ray manual tissue microarrayer system (cat. no. UT06; Unitma Co., Ltd., Seoul, Korea). Specifically, core tissue biopsies (2 mm in diameter) were obtained from 159 individuals. FFPE blocks were made and then arranged in new recipient paraffin blocks (cat. no. 62595-08; Quick-Ray™ Recipient Block; Head Biotechnology Co., Ltd., Beijing, China), according to the manufacturer's protocol. A total of five breast TMAs were made. Sections (4 µm) were cut and placed on super frost-charged glass microscope slides to generate TMA slides. Tissue sections were deparaffinized and rehydrated through a graded series of alcohols. Endogenous peroxidase activity was blocked by incubation in 3% H2O2. Tissues were placed in 0.01 M citrate buffer at pH 6.0 and heated to boiling in a microwave for antigen retrieval. SPAG5 was detected using a polyclonal rabbit anti-human SPAG5 antibody for 2 h at room temperature (1:1,000; cat. no. HPA022008; Atlas Antibodies, Bromma, Sweeden). Reactions were detected using an Envision™ peroxidase kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Tissues were then incubated in 0.05% 3,3′-diaminobenzidine (Dako, Agilent Technologies, Inc.) for 30 sec at room temperature, counterstained with 0.45% hematoxylin for 3 min, dehydrated through a graded series of alcohols, cleared in xylene and coverslipped with permanent mounting media at room temperature.
Staining was quantified in all tissues without knowledge of clinical characteristics. SPAG5 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 (10,11). 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 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).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed to determine the SPAG5 mRNA expression level in 35 pairs of human BC tissue and matched adjacent nontumor tissue. Tissue samples were snap-frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Total RNA was extracted from frozen samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse transcribed into cDNA using a PrimeScript™ RT reagent kit (Clontech Laboratories, Inc., Mountainview, CA, USA), according to the manufacturer's protocol. Human β-actin served as the internal control for determining SPAG5 mRNA levels. The following primers were used for qPCR: human β-actin forward, 5′-TGGAGAAAATCTGGCACCAC-3′ and reverse, 5′-GATGATGCCTCGTTCTAC-3′; and SPAG5 forward, 5′-CATCTCACAGTGGGATAACTAATAAAC-3′ and reverse, 5′-CAGGGATAGGTGAAGCAAGGATA-3′ (GenScript, Nanjing, Jiangsu China). qPCR was performed using an ABI PRISM 7500HT Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in 96-well plates. The final volume for each reaction was 20 µl, which included 2 µl of cDNA template (corresponding to ~40 ng of retro-transcribed total RNA), the primers (20 nmol/l each) and 2X SYBR-Green PCR Master mixture (10 µl). Thermocycling conditions were as follows: Following an initial 2 min at 50°C to allow AmpErase-UNG activity and 10 min at 95°C, samples were cycled 40 times at 95°C for 15 sec and 58°C for 1 min. All experiments were performed in triplicate. mRNA levels were quantified using the 2−ΔΔCq method (16) and normalized to respective internal controls.
Statistical analysis
All statistical analyses were performed using the SPSS 18.0 statistical software package (SPSS Inc., Chicago, IL, USA). For statistical analysis, the continuous SPAG5 expression data from IHC were first converted into dichotic data (low vs. high) using specific cutoff values, which were selected based on significant differences in OS using the X-tile software program (www.tissuearray.org/rimmlab) (14). Statistical analysis of the RT-qPCR data was performed using an unpaired Student's t-test when two groups were compared. χ2 test was used to identify statistical differences between groups. Cumulative patient survival was estimated using the Kaplan-Meier method and a log-rank test was used to compare the survival curves. A Cox proportional hazards model was used to calculate univariate and multivariate hazard ratios (HRs) for the variables. P<0.05 was considered to indicate a statistically significant difference.
Results
SPAG5 mRNA is highly expressed in BC tissues
The expression levels of SPAG5 mRNA in BC tissues and matched adjacent nontumor tissues were examined using RT-qPCR. The SPAG5 mRNA expression level was 3.41±0.41-fold higher in BC tissues compared with matched adjacent nontumor tissues (P<0.001; Fig. 1A). In addition, it was identified that the SPAG5 mRNA expression level was 1.81±0.31-fold higher in TNBC tissues compared with other BC molecular subtypes (P<0.001; Fig. 1B).
SPAG5 protein is highly expressed in BC tissues
SPAG5 protein expression was demonstrated to be localized to the cytoplasm using IHC (Fig. 2). Furthermore, IHC revealed that SPAG5 protein was highly expressed in BC tissues. Using X-tile software to analyze TMA data, SPAG5 expression was classified as high or low according to OS for patients with BC. The cutoff value was selected as 130; a score of 0–130 was considered low or no expression and a score of 131–300 was considered high expression. High SPAG5 expression was detected more frequently in BC tissues (116/159, 72.96%) compared with benign tumor tissues (2/61, 3.28%) or matched adjacent nontumor tissues (0/159, 0.00%; Table I).
High SPAG5 expression is associated with clinicopathological characteristics in BC
The association between SPAG5 protein expression and clinicopathologic characteristics in BC tissues was investigated. Results indicated that high expression of SPAG5 in BC was associated with histological grade (χ2=9.965, P=0.007), ER expression (χ2=10.940, P<0.001), Ki-67 expression (χ2=21.799, P<0.001), tumor size (χ2=15.913, P=0.001), lymph node status (χ2=18.519, P<0.001), TNM stage (χ2=11.511, P=0.003) and TNBC subtype (χ2=10.429 P=0.015). However, no significant association was identified between SPAG5 expression and age, PR expression or HER2 expression (Table II).
Table II.Association of SPAG5 protein expression levels with clinicopathological characteristics in patients with breast cancer. |
High expression of SPAG5 protein is associated with a poor prognosis in BC
Lastly, the current study investigated the association between SPAG5 expression and prognostic factors in BC through univariate and multivariate analyses. High SPAG5 expression level was associated with poor OS in univariate analysis [HR=1.956; 95% confidence interval (CI)=1.040–2.009; P<0.001). OS was also identified to be associated with other prognostic markers, including TNM stage (HR=10.92; 95% CI=4.109–18.723; P<0.001), lymph node status (HR=2.192; 95% CI=1.528–2.428; P<0.001) and TNBC (HR=3.892; 95% CI=2.829–5.628; P<0.001). Multivariate analysis revealed that only high SPAG5 expression, molecular classification and TNM stage were associated with poor OS (Table III). Similar results were identified using Kaplan-Meier survival analysis; this indicated that SPAG5 high expression and the TNBC-positive subtype were significantly associated with poor OS (Fig. 3).
Table III.Univariate and multivariate analysis of prognostic markers for overall survival in breast cancer. |
Discussion
BC is regarded as a heterogeneous disease that can be classified into several molecular subtypes with prognostic significance (15). According to ER, PR, HER2 and Ki-67 expression status, BC can be categorized into four molecular subtypes, which can be used to predict outcome and contribute to the management of BC (7). TNBC typically predicts a poor prognosis and is characterized by no ER and PR expression, and no overexpression of HER2 (17). The majority of patients with TNBC have an aggressive tumor phenotype and exhibit a partial response to chemotherapy (17). Due to the aforementioned reasons, new targets are required for TNBC therapy.
SPAG5 serves an important role in cell meiosis and spermatid morphogenesis (10,18). Certain studies have indicated that knockdown of SPAG5 expression in cervical cancer cells can inhibit cell proliferation and induce cell apoptosis, thereby decreasing the ability of cancer cells to invade (19,20). According to the aforementioned studies, SPAG5 is currently considered to serve a role in promoting tumor cell growth. Cornen et al (21) reported that SPAG5 expression may be associated with the molecular classification of BC and is a potential oncogene in luminal BC.
Using RT-qPCR, the current study identified SPAG5 mRNA overexpression in fresh BC tumor tissues compared with matched adjacent nontumor tissues, particularly in TNBC tissues. A monoclonal antibody that recognizes an epitope in the SPAG5 protein was then used to demonstrate that SPAG5 was highly expressed in BC tissues and was associated with histological grade, ER expression, Ki-67 expression, lymph node status, TNM stage and TNBC subtype. Previously, studies have demonstrated that SPAG5 is highly expressed in BC tissues and is associated with tumor classification, p53 mutation and the amplification of HER2 (6). The current study also revealed that overexpression of SPAG5 predicted poor OS in patients with BC. This is similar to findings made by Abdel-Fatah et al (15), who reported that higher expression of SPAG5 was associated with a poor prognosis in patients with BC. Abdel-Fatah et al (15) reported that the role of SPAG5 could replace Ki67 as another proliferation factor. The current study also identified that SPAG5 protein expression was associated with Ki67 expression. In contrast to the study by Abdel-Fatah et al (15), the current study analyzed the association between SPAG5 protein expression and TNBC type, which may provide a new standard for the diagnosis of TNBC type in the future.
In conclusion, the current study indicated that high expression of SPAG5 may be associated with a poor prognosis in patients with BC, which may lead to the development of useful and effective targeted drugs for the treatment of BC.
Acknowledgments
Not applicable.
Funding
The current study was supported by the Science and Technology Development Fund of Nanjing Medical University (grant no. 2017NJMUZD107).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
XZ and QT designed the study; LJ, YS acquired the data and drafted the article; LJ, XW, LX and XZ analyzed and interpreted the data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study has been approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University (Nantong, China). Written informed consent was obtained from all patients involved in the current study.
Patient consent for publication
Written informed consent was obtained from all patients involved in the current study.
Competing interests
The authors declare that they have no competing interests.
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