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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 7  |  Page : 1597-1602

MALAT1 and BACH1 are prognostic biomarkers for triple-negative breast cancer


Department of Breast Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, China

Date of Submission23-Apr-2019
Date of Decision12-Sep-2019
Date of Acceptance11-Oct-2019
Date of Web Publication13-Jan-2020

Correspondence Address:
Dr. Feng Liu
Hengyang Medical School of University of South China
China
Dr. Xiangsheng Xiao
Department of Breast Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_282_19

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 > Abstract 


Aims: The purpose of this study was to investigate the potential correlation between metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the transcription factor BTB and CNC homology 1 (BACH1) and their clinicopathological significance in triple-negative breast cancer (TNBC).
Subjects and Methods: MALAT1 and BACH1 were detected by immunohistochemistry using TNBC tissue microarrays of 240 patients. The association between MALAT1 and BACH1 expression levels was statistically analyzed. Moreover, the prognostic roles as well as clinical and pathological significance of MALAT1 and BACH1 expression in TNBC were determined.
Statistical Analysis Used: Two-tailed Pearson correlation was used to examine the correlation of BACH1 and MALA1 expression. Comparisons of clinicopathological variables between different BACH1 and MALA1 expression groups were performed using χ2 tests. Overall survival (OS) and disease-free survival (DFS) curves were plotted with the Kaplan-Meier method and the differences in OS and DFS between three groups were compared by the log-rank test. Multiple comparisons were performed using χ2 tests for subsequent individual group comparisons.
Results: MALAT1 and BACH1 expression was significantly correlated with tumor-node-metastasis stage, distant metastasis, pathological stage, and survival outcomes of patients. Patients with high MALAT1 and BACH1 expression exhibited shorter overall survival and disease-free survival.
Conclusions: These findings provide further insight into the expression pattern of MALAT1 and BACH1 in TNBC and suggest them as prognostic biomarkers for TNBC.

Keywords: BTB and CNC homology 1, metastasis-associated lung adenocarcinoma transcript 1, prognosis, triple-negative breast cancer


How to cite this article:
Ou X, Gao G, Bazhabayi M, Zhang K, Liu F, Xiao X. MALAT1 and BACH1 are prognostic biomarkers for triple-negative breast cancer. J Can Res Ther 2019;15:1597-602

How to cite this URL:
Ou X, Gao G, Bazhabayi M, Zhang K, Liu F, Xiao X. MALAT1 and BACH1 are prognostic biomarkers for triple-negative breast cancer. J Can Res Ther [serial online] 2019 [cited 2020 Jan 22];15:1597-602. Available from: http://www.cancerjournal.net/text.asp?2019/15/7/1597/275550




 > Introduction Top


Among females, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. A status report on the global burden of cancer estimated 2,088,849 (11.6%) new cases of breast cancer and 626,679 (6.6%) breast cancer deaths among women in 2018.[1] Triple-negative breast cancer (TNBC) tumors lack estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression [2] and are found in 10%–20% of patients with newly diagnosed breast cancer. Patients with TNBC exhibit a dismal prognosis.[3] Moreover, effective diagnostic and prognostic markers for TNBC are lacking.

Metabolic activity can regulate tumor growth.[4] BTB and CNC homology1 (BACH1) is a heme-binding transcription factor and a member of the Cap 'n' Collar/basic region leucine zipper factor (CNC-bZip) family.[5] BACH1 can reprogram metabolic pathways by targeting mitochondrial metabolism.[4],[6] In addition, BACH1 expression was upregulated in the tumors of patients with TNBC.[7],[8]

Long noncoding RNAs (lncRNAs) can regulate survival, proliferation, invasion, metastasis, and angiogenesis of cancer cells.[9] Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched abundant transcript 2, is the most abundant (~3000 copies/cell) nuclear-retained lncRNA.[10],[11] Previously, MALAT1 was considered an important marker for metastatic lung cancer. However, several studies have demonstrated that MALAT1 is associated with progression and metastasis of various cancers, including breast cancer.[12] Moreover, it has been demonstrated that lysine-specific demethylase 5B (KDM5B) can upregulate MALAT1 expression in TNBC cells, and subsequently increase tumor invasion and clonogenic potential.[13] Furthermore, high MALAT1 expression has been shown to correlate with poor prognosis in patients with breast cancer.[14],[15]

In this study, we have investigated the correlation between the expression of BACH1 and MALAT1 and their clinicopathological significance in TNBC. We have also investigated the prognostic roles of BACH1 and MALAT1 in patients with breast cancer. Our study provides evidence that BACH1 and MALAT1 can serve as prognostic factors and potential therapeutic targets in TNBC.


 > Subjects and Methods Top


Ethics statement

All procedures performed in this study involving human participants were approved by the Ethics Committee of Sun Yat-Sen University Cancer Center and were in accordance with the 1964 Helsinki Declaration and its later amendments. Written informed consent about the researchable use of the clinical data was obtained from each participant patient. All patient data were anonymous and deidentified prior to analysis. This study was conceived and presented according to the reporting recommendations for tumor marker prognostic studies guidelines.

Patients and specimens for tissue microarray

A total of 240 female patients who were histopathologically diagnosed with TNBC at Sun Yat-Sen University Cancer Center were included in this study. TNBC tissue specimens were collected by surgery, formalin-fixed and paraffin-embedded using standard techniques, and preserved at the Department of Specimen and Resource in Sun Yat-Sen University Cancer Center. Only those patients who underwent therapeutic surgical treatment (conservative or radical surgery with axillary evaluation or mastectomy) were recruited in this study. The exclusion criteria included: male patients, inflammatory breast cancer, bilateral carcinoma, and history of malignant tumor. In addition, all of the patients included in this study had not received chemotherapy or radiation therapy previously; their complete clinical and pathological information, including age, menopause status, tumor size, lymph node status, stage, and distant metastasis, were available and can be reviewed. The histological grade of the tumor was classified based on the tumor-node-metastasis (TNM) staging system (American Joint Committee on Cancer Classification). Follow-up data were obtained by reviewing clinical records and by contacting the patient by telephone. The dates of death and relapse were used to estimate overall survival (OS) and disease-free survival (DFS), respectively. Tissue microarrays (TMAs) were constructed as follows: briefly, histological slides were retrieved and reviewed, and representative tumor areas were selected for TMA construction. The presence of carcinoma in the tumor core was used as an inclusion criterion. We compared both hematoxylin and eosin-stained and immunostained TMA sections with the corresponding full-section slides to assess representativeness and heterogeneity of staining.

Immunohistochemical analysis

Immunohistochemistry (IHC) was performed on the 240 paraffin-embedded TNBC tissue sections. Briefly, the slides were deparafinized, rehydrated, and treated with a 90% methanol/3% H2O2 solution for 10 min at room temperature to block endogenous peroxidase. Thereafter, the slides were soaked in sodium citrate buffer (10 mM sodium citrate; 0.05% Tween 20, pH 6.0) at 96°C for 5 min for antigen retrieval. The slides were incubated overnight at 4°C with the following antibodies after blocking with BSA: anti-BACH1 antibody (dilution 1:100, Santa Cruz Biotechnology, Europe) and anti-MALAT1 antibody (dilution 1:500, Caiyou, Shanghai, China). The slides were subsequently incubated at room temperature with a biotinylated secondary antibody for 10 min, and finally with Horseradish Peroxidase (HRP)-streptavidin for 10 min. The results were identified as positive and negative after DAB staining.

Statistical analysis

All statistical analyses were performed using SPSS 25.0 statistical package (SPSS Inc., Chicago, IL, USA). A two-tailed Pearson correlation was used to examine the correlation between BACH1 expression and MALA1 expression. The clinicopathological variables between different BACH1 and MALA1 expression groups were compared using Chi-square tests. OS and DFS curves were plotted using the Kaplan–Meier method and the differences in OS and DFS between these groups were compared using the log-rank test. Multiple comparisons were performed using Chi-square tests for subsequent individual group comparisons. All results were statistically significant at P < 0.05.


 > Results Top


Metastasis-associated lung adenocarcinoma transcript 1 and BTB and CNC homology 1 expression is upregulated in triple-negative breast cancer

The mRNA expression of MALAT1 and BACH1 was assessed by IHC using TNBC TMAs of 240 patients [Figure 1]. MALAT1 and BACH1 were both overexpressed in TNBC tissues. The characteristics of the patients with overexpressed MALAT1 and BACH1 are summarized in [Table 1]. This result further indicates that LDHA and AMPK were expressed synchronously in TNBC tissues.
Figure 1: The expression of metastasis-associated lung adenocarcinoma transcript 1 and BTB and CNC homology 1 was detected by immunohistochemistry using breast cancer tissue microarrays of 240 patients. Representative Immunohistochemistry images of two staining outcomes (negative and positive) of metastasis-associated lung adenocarcinoma transcript 1 and BTB and CNC homology 1 expression under two multiples of the microscope (×400 and ×100) were showed

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Table 1: Patient characteristics (n=240)

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Coexpression of metastasis-associated lung adenocarcinoma transcript 1 and BTB and CNC homology 1 is correlated with clinicopathological parameters

Next, we explored the potential clinicopathological implications of altered MALAT1 and BACH1 expression. The clinical tissue specimens of 240 breast cancer patients were divided into multiple groups based on the expression scores of MALAT1 and BACH1. Of these, 65 (27%) overexpressed MALAT1 and BACH1 [Table 2]. The results showed that MALAT1 and BACH1 expression is correlated with tumor size, Lymph node metastasis (LNMET), and TNM stage [P = 0.001, 0.000, and 0.002, respectively; [Table 2]. Multiple comparisons were performed to further investigate the differences. The results showed that compared with both negative groups, coexpression of MALAT1 and BACH1 was correlated with tumor size, LNMET, and TNM stage [P = 0.000, 0.000, and 0.001, respectively; [Table 3]. Patients overexpressing MALAT1 and BACH1 exhibited large tumor size, lymph node metastasis, and advanced TNM stage, suggesting that MALAT1 and BACH1 may play critical roles in carcinogenesis and progression of TNBC.
Table 2: Clinicopathological variables and LDHA and AMPK expression in 240 breast cancer patients

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Table 3: Multiple comparisons of clinicopathological variables between different expression groups

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Coexpression of metastasis-associated lung adenocarcinoma transcript 1 and BTB and CNC homology 1 is correlated with poor clinical outcome of patients with breast cancer

To analyze the significance of MALAT1 and BACH1 expression for the clinical prognosis of TNBC, Kaplan–Meier survival analysis was performed using OS [Figure 2]a and DFS [Figure 2]b. The results showed that patients overexpressing MALAT1 and BACH1 exhibit shorter OS and DFS than those with downregulated expression of MALAT1 and BACH1 [P < 0.0001 for both OS and DFS; [Figure 2]a and [Figure 2]b. These results indicated that coexpression of LDHA and AMPK is significantly associated with shorter survival of TNBC patients.
Figure 2: Coexpression of metastasis-associated lung adenocarcinoma transcript 1 and BTB and CNC homology 1 were correlated with poor clinical outcomes of breast cancer. (a) Overall survival curves for 240 studied patients. (b) Disease-free survival curves for 240 studied patients

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 > Discussion Top


LncRNAs are a class of noncoding RNAs of more than 200 nucleotides but lack protein-coding potential. They are emerging as a new class of indispensable molecules for the development and promotion of malignant diseases.[16] Therefore, it is reasonable that abnormal expression of lncRNAs results in dysregulation of normal biological and pathological processes of disease.[17] From a clinical perspective, lncRNA can serve as a potential therapeutic target, especially for TNBC because of the constant failure of chemotherapy and other conventional treatment options.[2],[18] MALAT1 is one of the earliest identified lncRNAs. Several studies have shown that MALAT1 plays a pivotal role in the development and progression of various cancers [10],[19] and promotes proliferation, migration, metastasis, and blood-tumor-barrier permeability of tumor cells.[20] High expression of MALAT1 is associated with increased tumor size and advanced stage of breast cancer,[21] as well as poor outcome,[22] especially in TNBC, suggesting that MALAT1 could be an important prognostic factor and a therapeutic target in TNBC.

BACH1 is a transcriptional factor that forms a heterodimer with the small Maf family proteins. BACH1 can repress Maf recognition element expression in vivo.[5],[23] According to recent studies, BACH1 is primarily involved in the physiological regulation of oxidative stress, heme oxidation, and senescence.[24] Alvarez et al. (2011) have predicted that BACH1 might be a regulator of the prostate cancer marker ACPP; although, this hypothesis has not been experimentally verified. Furthermore, Han et al. (2019) found that BACH1 could promote EMT gene expression by recruiting HMGA2 in epithelial ovarian cancer cells. HMGA2 plays a role in BACH1-induced EMT of epithelial ovarian tumor cells.[8] Yun et al. have indicated that BACH1 can upregulate metastatic genes such as CXCR4 and MMP1 and promote bone metastasis of breast cancer.[25],[26] These evidence indicate that BACH1 could function as cancer development and migration factor and is often associated with poor outcomes.[26],[27] Hence, in this study, we attempted to explore the feasibility of BACH1 as a prognostic factor for TNBC.

In this study, we investigated the expression pattern of BACH1 and MALAT1 in TNBC. We found that BACH1 and MALAT1 expression was synchronously upregulated in TNBC tissues. These results indicated that BACH1 and MALAT1 could be used as biomarkers for TNBC. Next, we explored the potential clinicopathological implications of altered LDHA and AMPK expression. We found that patients with upregulated BACH1 and MALAT1 expression exhibited large tumors, lymph node metastasis, and advanced TNM stage. Moreover, the patients with upregulated BACH1 and MALAT1 expression were associated with shorter OS and DFS. Our results are consistent with those reported previously, suggesting that BACH1 and MALAT1 together play critical roles in breast cancer. Hence, BACH1 and MALAT1 could be important prognostic factors and promising therapeutic targets in TNBC.

In summary, this study demonstrated that BACH1 and MALAT1 are upregulated synchronously in patients with TNBC who had poor clinical outcomes. Our results indicate that BACH1 and MALAT1 could be important prognostic factors and potential therapeutic targets in TNBC. The simultaneous detection and targeting of BACH1 and MALAT1 should be performed in the clinical management of TNBC.


 > Conclusion Top


In summary, this study demonstrated that BACH1 and MALAT1 were up-regulated synchronously in TNBC, which was associated with poor clinical outcomes. Our findings provide significant evidence that BACH1 and MALAT1 could be prognostic factors and potential therapeutic targets in TNBC. Detecting and targeting BACH1 and MALAT1 at the same time should be recommended in clinical management of TNBC.

Financial support and sponsorship

This research was supported by the National Natural Science Foundation of China (81372133) and the Natural Science Foundation of Hunan Province, China (2019JJ40258).

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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