Comparison of the expression levels of lysine‑specific demethylase 1 and survival outcomes between triple‑negative and non‑triple‑negative breast cancer
- Authors:
- Published online on: December 8, 2020 https://doi.org/10.3892/ol.2020.12363
- Article Number: 102
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Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Breast cancer is a heterogeneous disease, that may be subclassified into triple-negative breast cancer (TNBC) and non-triple-negative breast cancer (NTNBC). Compared with NTNBC, TNBC has unique clinicopathological characteristics, such as higher risk of recurrence, larger tumor size, lymph node metastasis and poor prognosis, and represents a major health concern (1). TNBC accounts for 15–20% of breast cancers and is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) (2). Compared with the other subtypes of breast cancer, TNBC has a relatively early onset and a higher degree of malignancy (3). In addition, patients with TNBC have a worse prognosis compared with other breast cancer subtypes, mainly because TNBC has no specific targets; therefore, the hormone receptors or HER2 cannot be targeted with therapy as in other subtypes (4). Although the molecular alterations in TNBC have been widely investigated, identifying the mechanisms that regulate the initiation and progression of TNBC may provide further insight into the development and progression of TNBC.
Lysine-specific demethylase 1 (LSD1), also referred to as KDM1A and AOF2, was the first histone demethylase to be discovered (5). LSD1 encodes a nuclear protein containing a SWIRM domain, a FAD-binding motif and an amine oxidase domain. The protein is a component of several histone deacetylase complexes and may silence genes by functioning as a histone demethylase. Notably, alternative splicing results in multiple transcript variants. The expression levels of LSD1 in certain subtypes of breast cancer have been investigated and LSD1 expression has been found to be frequently upregulated in several human malignancies, including breast (6), prostate (7), lung (8) and colon (9) cancer, neuroblastoma (10) and hepatocellular cancer (11). Notably, Lim et al (12) reported a significant positive association between LSD1 upregulation and a negative ER status. Another previous study identified an inverse correlation between high LSD1 expression levels and a low PR status (6). Recently, Cao et al (13) demonstrated that the overexpression of LSD1 promoted breast cancer cell proliferation, migration and invasion. In addition, it has been suggested that the ability of LSD1 to promote breast cancer growth and pulmonary metastasis may involve the resistance to immune checkpoint blockade (14). However, the expression and significance of LSD1 in breast cancer, particularly in the most aggressive subtype, TNBC, remain unclear.
The present study aimed to systematically investigate the expression levels of LSD1 in normal breast tissue, TNBC and NTNBC tissues using immunohistochemical staining, and analyze the potential association between LSD1 expression levels and clinicopathological characteristics of breast cancer.
Materials and methods
Bioinformatics analysis
The expression profile of LSD1 across various types of human cancer was examined through the Broad Institute FireBrowse portal (http://firebrowse.org). On the homepage, ‘LSD1’ was typed into the search box and ‘View Expression Profile’ was selected. The boxplots produced plotted the expression levels of the target gene, with red bars representing tumor samples and blue bars representing normal samples.
The mRNA expression levels of LSD1 in breast cancer tissues were compared with their matched normal tissues using The Cancer Genome Atlas (TCGA) datasets in the Oncomine database (http://www.oncomine.org). The thresholds used to obtain the most significant probes of the queried gene for each microarray dataset included a 2-fold difference in expression levels between the cancer and normal tissues and P<1×10-4. For each gene, the mRNA expression levels in three independent datasets were analyzed. The prognostic values of LSD1 in breast cancer were analyzed using the Kaplan-Meier plotter (http://kmplot.com/analysis) and the survival rates of patients with high and low expression levels of LSD1 were illustrated using a Kaplan-Meier survival plot.
Tissue specimens
The participants in the present study were all newly diagnosed patients with breast cancer who were treated at the Department of Breast Surgery of The Affiliated People's Hospital of Jiangsu University between December 2010 and October 2016. Samples were collected from 238 patients with breast cancer, including 112 TNBC and 126 NTNBC tissues. In addition, 80 normal tissues adjacent to the TNBC and 93 normal tissues adjacent to NTNBC were collected. The pathological data of all patients were complete and included tumor size, age, lymph node metastasis, clinical stage and histological type. All patients were regularly followed up; the follow-ups mainly occurred via phone or partly using an online platform. The patients survival, living conditions and presence of any abnormal symptoms were assessed until the follow-up deadline, which was set as June 2019. No subjects were lost during the follow-up period. All the tissue specimens were collected after obtaining informed patient consent and the use of the breast cancer specimens was approved by The Affiliated People's Hospital of Jiangsu University Institutional Review Board.
Immunohistochemical analysis
Immunohistochemistry (IHC) was performed to determine LSD1 expression levels in the tissues. The anti-LSD1 antibody (1:400; cat. no. 2184S) was purchased from Cell Signaling Technology, Inc.; a rabbit two-step detection kit and DAB color development kit were purchased from Beijing Zhongshan Jinqiao Biotechnology, and hematoxylin and water-soluble mounting tablets were purchased from Boster Biological Technology. All tissue samples for the experiment were provided by The People's Hospital of Jiangsu University and tissue chips were prepared with the assistance of Shanghai Changzheng Hospital. Each experiment used a known positive tissue section as the positive control and PBS solution instead of the primary antibody as the negative control.
IHC scoring
The interpretation of the results was performed using a double-blind reading under the guidance of a pathology expert. The lower part that was brownish yellow referred to the immunoreactive score, which was based on the comprehensive evaluation of the degree of cellular staining and the percentage of positive cells. In total, ≥10 fields of view were visualized under a high magnification and 100 tumors were viewed in each field of view. The percentage of positive cells was counted and the following scoring system was used: 0 points (≤5%), 1 point (5–25%), 2 points (25–50%), 3 points (25–75%) and 4 points (≥75%). The staining intensity was scored as follows: 0 (no staining), 1 (weakly stained), 2 (moderately stained) or 3 (strongly stained). The LSD1 immunostaining score was calculated as (positive percentage score) × (staining intensity score). In this experiment, a score of ≥4 points was considered as positive.
Statistical analysis
Statistical analysis was performed using SPSS 25.0 software (IBM Corp.). The association between the expression levels of LSD1 and clinicopathological characteristics was analyzed using a χ2 test or Fisher exact probability method. The log-rank test and Kaplan-Meier method were also used and the survival analysis was depicted graphically. Following the univariate analysis, variables with P<0.05 were used for subsequent multivariate analysis based on the Cox proportional hazards model. P<0.05 was considered to indicate a statistically significant difference.
Results
Upregulated LSD1 expression is not associated with a poor prognosis in breast cancer
The gene expression levels of LSD1 were analyzed in 37 cases of human cancer using TCGA database. The columns in Fig. 1A represent the accurate quantification of the gene and isoform expression levels from the RNA-Seq data. The results revealed that LSD1 expression levels were upregulated in almost all cancer tissues compared with their respective matched normal tissues. The expression levels of LSD1 were the highest in testicular germ cell tumor and the lowest in cholangiocarcinoma. Notably, the LSD1 gene exhibited a similar expression pattern in breast cancer (Fig. 1A). The Oncomine database analysis comparing the cancer tissues with normal tissues also revealed that the mRNA expression levels of LSD1 were significantly upregulated in breast cancer tissues compared with the corresponding normal tissues in three independent analyses (Fig. 1B-D). The results of the Kaplan-Meier analysis revealed no significant association between the expression levels of LSD1 and the overall survival rate of patients with breast cancer (P=0.31; Fig. 1E).
LSD1 expression is upregulated in TNBC and NTNBC tissues
To further investigate the results obtained through the bioinformatics analysis, the protein expression levels of LSD1 in breast cancer tissues were also investigated. For this analysis, samples from 238 patients with breast cancer (including 112 TNBC and 126 NTNBC cases) who were diagnosed between 2010 and 2016 were used; >95% of the tumors were ≤5 cm, ~35% of the patients had lymph node metastasis and ~15% of the patients were diagnosed at an advanced TNM stage (stage III/IV). The protein expression levels of LSD1 in both the TNBC and NTNBC subtypes were significantly upregulated compared with those in adjacent normal tissues (P<0.001; Table I). Subsequently, the proportion of LSD1 expression in breast tumors was further determined. IHC staining revealed that LSD1 was localized mainly to the cell nucleus in both breast cancer subtypes (Fig. 2). Positive staining for LSD1 expression was recorded in 90 (40%) of the 231 breast cancer samples; specifically, in 42 (40%) of the 105 TNBCs and 48 (38%) of the 126 NTNBCs (Fig. 2). There were no significant differences in the LSD1 expression levels between the TNBC and NTNBC samples (P>0.05; Table SI).
Association between LSD1 expression and clinicopathological characteristics
The present study further analyzed the association between LSD1 expression levels and the clinicopathological characteristics of breast tumors, including age, tumor size, lymph node metastasis status and clinical stage. The expression levels of LSD1 in breast cancer were not significantly associated with any of the individual clinical indicators (P>0.05; Table II). However, in TNBC, the LSD1 expression levels were significantly associated with age (P=0.019) and TNM stage (P=0.031), but not with tumor size or lymph node metastasis (P>0.05; Table II). Positive staining for LSD1 was detected in 48.9% of the patients aged >55 years and in 44.9% of patients with TNM stage I+II disease. In addition, positive staining for LSD1 was also detected in 33.8% of patients aged <55 years and in 12.5% of patients with TNM stages III+IV disease. In NTNBC, the expression levels of LSD1 were not significantly associated with any of the clinicopathological indicators (P>0.05; Table II). Taken together, these results suggest that LSD1 expression levels may be associated with patient age and TNM stage in TNBC.
Association between LSD1 expression and patient prognosis
To determine the role of LSD1 and the clinicopathological indicators in predicting breast cancer outcomes, the postoperative survival in patients with breast cancer was tracked and analyzed. Patients were classified into LSD1-positive and LSD1-negative expression groups according to the IHC results. For breast cancer, the mass size (4 cm), lymph node metastasis (N) and TNM stage were significantly inversely associated with patient survival. The survival period of the patients with a large tumor diameter, late clinical stage and lymph node metastasis was significantly decreased compared with that of patients with smaller tumors, diagnosed at an earlier clinical stage and without metastasis to the lymph nodes (Table III). The χ2 value of the log-rank test revealed that the cumulative survival difference between tumor size (4 cm) was the most significant, followed by TNM stage and N stage. In TNBC, the tumor size (4 cm) and N stage were significantly inversely associated with patient survival (Table III). The cumulative survival rate difference in the N stage was the most significant, followed by tumor size. In NTNBC, the tumor size (4 cm) and TNM stage were significantly inversely associated with patient survival (Table III). The cumulative survival rate difference in the TNM stage was the most significant, followed by tumor size. Taken together, these data suggested that tumor metastasis, but not LSD1 expression levels, may be a major factor associated with mortality in patients with TNBC and NTNBC.
 | Table III.Univariate analysis of the association between LSD1 expression and clinicopathological variables in patients with breast cancer (log-rank test). |
Cox regression analysis was subsequently used to calculate various prognostic parameters for the survival of patients with TNBC and NTNBC. Univariate analysis identified four prognostic factors: TNM stage (I+II vs. III+IV), N stage (N0 vs. N1-3), LSD1 expression levels (negative or positive) and tumor size (≤4 vs. >4 cm). However, upon performing multivariate Cox proportional hazard regression, increased LSD1 expression levels were not identified as a significant independent predictor of poor survival in patients with breast cancer (P>0.05; Table IV), which was consistent with the results of the bioinformatics analysis. Taken together, these data suggested that LSD1 expression levels may not be inversely associated with a poor prognosis in breast cancer and, in fact, the best predictor of poor prognosis in TNBC may be the N stage (P<0.05; Table IV).
Discussion
LSD1 is a member of the monoaminoxidase enzyme family, which play an important role in controlling gene expression through histone modifications (15). Consistent with the perceived role of LSD1 in cell proliferation, overexpression of LSD1 has been reported in a diverse range of human tumors, including breast cancer (6). For example, Serce et al (6) reported upregulated expression levels of LSD1 in invasive ductal breast cancer. The expression levels of LSD1 were also found to increase with progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (6). Similarly, a study by Scoumanne and Chen (16), which analyzed the role of LSD1 in the human malignant breast cancer cell line MCF7, discovered that downregulation of LSD1 expression reduced the number of proliferating breast cancer cells. However, the expression levels of LSD1 in TNBC and NTNBC have not been analyzed to date. Therefore, to the best of our knowledge, the present study was the first to systematically analyze LSD1 expression levels in TNBC and NTNBC.
From the clinical data, it may be concluded that the survival of patients with TNBC is poor. The poor prognosis of TNBC may be due to its biological characteristics, such as younger age at onset, higher rate of breast cancer family history, larger tumor size, more advanced clinical stage at diagnosis, higher rate of lymph node metastasis, higher histological grade, earlier recurrence and metastasis, and resistance to endocrine and targeted therapy. The present study revealed that LSD1 expression levels were upregulated in ~40% (90/231) of breast cancer cases. Importantly, LSD1 was found to be upregulated in both TNBC and NTNBC. The expression levels of LSD1 also appeared to be similar between older and younger patients with breast cancer. In addition, the expression levels of LSD1 were not significantly associated with poor prognosis or the cumulative survival rate of postoperative patients with breast cancer. Bioinformatics analysis also revealed that LSD1 expression levels were not significantly associated with the prognosis of breast cancer. However, Nagasawa et al (17) concluded that the upregulation of LSD1 was a poor prognostic factor in breast cancer, particularly the basal-like subtype of invasive breast cancer. This inconsistency may be due to the insufficient data on LSD1 expression levels obtained via IHC staining in the present study, which may lead to differences in the LSD1 prognostic impact. In the present study, LSD1 expression levels were found to be closely associated with the breast tumor size and distant metastasis (TNM stage) in TNBC. Lim et al (12) demonstrated that the overexpression of LSD1 in breast cancer was significantly positively correlated with the absence of the ER. In addition, the knockdown of LSD1 by small interfering RNA induced the regulation of multiple proliferation-related genes, including p21, ERBB2 and CNA2, thereby inhibiting the proliferation of breast cancer cells (12). However, the specific mechanisms underlying the association between LSD1 and cancer development have not been fully elucidated. Upregulated expression levels of LSD1 have been reported to be a hallmark of breast cancer cells (18). However, according to the data obtained in the present study, LSD1 was not found to play an important role in breast cancer progression or metastasis. Thus, LSD1 may be a secondary factor associated with breast cancer-related mortality.
LSD1 may be involved in the carcinogenesis and progression of breast cancer. It has been reported that the CtBP/LSD1/COREST complex interacts with ZNF516 to participate in the epithelial-to-mesenchymal transition (EMT) process, and inhibits the proliferative and invasive ability of breast cancer cells (19). LSD1 was also discovered in another study to regulate the EGFR signaling pathway and affect EMT (11), thereby inhibiting breast cancer cell invasion. LSD1 appears to serve a role in regulating the expression of oncogenic proteins in breast cancer cells (20), and the upregulation of LSD1 may be an early tumor-promoting event in breast cancer. Thus, LSD1 may be involved in the occurrence and development of TNBC, in addition to NTNBC, which may hold therapeutic promise. The combination of a LSD1 inhibitor (21), pargyline (22) and the HDAC inhibitor SAHA (vorinostat) (23) significantly inhibited the growth and apoptosis of TNBC cells. Therefore, to further elucidate the specific mechanism underlying the role of LSD1 in breast cancer in vivo and in vitro, future studies should explore the association between LSD1 and breast cancer cell proliferation, migration and invasion.
In conclusion, the findings of the present study indicated that LSD1 expression levels may be upregulated in breast cancer and the expression levels of LSD1 may be associated with clinical stage in TNBC. However, the detection of LSD1 was not found to be a marker for the early diagnosis of breast cancer or a potential target for early treatment strategies. The results of the present study may shed some light on the complex epigenetic regulatory mechanisms of breast cancer, which may help identify novel therapeutic targets.
Supplementary Material
Supporting Data
Acknowledgements
The authors would like to thank Dr Miao Chen (Department of Pathology, The Affiliated People's Hospital of Jiangsu University, Zhenjiang), for reviewing the clinicopathological data in this manuscript.
Funding
The present study was supported by grants from the National Natural Science Foundation of China (grant no. 81170573) and the Key Research and Development Program of Jiangsu Province (grant no. BE2020678).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors contributions
GS and QL conceived and designed the study; KZ performed the experiments; KZ and YL wrote the manuscript; YL, TH, AS, ML and WB were involved in the conception of the study. All authors 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. All authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate
Human breast tissue specimens were preserved in the Breast Cancer Tissue Bank at The Affiliated People's Hospital of Jiangsu University (Zhenjiang, China). All the tissue specimens for this study were collected after obtaining informed patient consent and the use of the breast cancer specimens was approved by The Affiliated People's Hospital of Jiangsu University Institutional Review Board.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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