Research ArticleClinical Studies

Bromodomain-containing Protein 4 Is a Favourable Prognostic Factor in Breast Cancer Patients

CHIHO SUZUKI, AKIMITSU YAMADA, SHOKO ADACHI, HIDETAKA SHIMA, KUMIKO KIDA, MASANORI OSHI, SADATOSHI SUGAE, SHINYA YAMAMOTO, KAZUTAKA NARUI, MIKIKO TANABE, KAZUAKI TAKABE, YASUSHI ICHIKAWA, TAKASHI ISHIKAWA and ITARU ENDO
Anticancer Research July 2021, 41 (7) 3597-3606; DOI: https://doi.org/10.21873/anticanres.15148

Abstract

Aim: To evaluate the association between bromodomain-containing protein 4 (BRD4) expression and clinicopathological factors and prognosis in human breast cancer specimens. Patients and Methods: We used tissue microarrays constructed from samples of patients (n=183) who underwent surgery. We validated the association between BRD4 expression and prognosis in solid tumours, including breast cancer, using The Cancer Genome Atlas (TCGA) database. Results: Immunohistochemical staining showed that BRD4 was widely distributed in breast cancer tissues. BRD4 was strongly expressed in 19.7% of patients but BRD4 staining intensity was not correlated with other clinicopathological factors. Most importantly, patients with a strong BRD4 expression had a significantly longer disease-specific survival than those with a weak BRD4 expression (100.0% vs. 91.3% at 5 years, p=0.027). mRNA expression analysis showed similar results (91.2% vs. 80.2% at 6 years, p=0.047). Conclusion: Strong BRD4 expression was associated with a significantly better prognosis in breast cancer tumours.

Key Words:

Breast cancer is the most common malignancy in women worldwide. In recent years, there has been a significant advancement in subtype-based systemic therapeutic methods and strategies for breast cancer treatment (1). Although the mortality rate is still high owing to disease metastases and treatment-resistant recurrences, recent advances have resulted in the improvement of survival outcomes.

Cancer stem cells (CSCs) comprise a small population of cells within a tumour and have characteristics similar to those of normal stem cells, in that they can self-renew and differentiate (2). Owing to these characteristics, CSCs are considered to be involved in the metastasis and recurrence after initial treatment, chemotherapy, and/or radiation therapy (3). In breast cancer, ESA+, CD44+/CD24–, and ALDH1A1+ are known CSC markers (4). We previously reported that patients with ALDH1A1-positive breast cancer have poor prognosis and are less likely to achieve pathological complete response with preoperative chemotherapy (5).

In a previous study, we used network analysis and identified 10 genes, namely, SIRT2, PCAF, LXR, BRD4, SMAD4, Pitx3, RARα, MUC1, HASH1, and C/EBPβ, that are believed to directly control ALDH1 expression (6). In this study, we focused on one of these genes, bromodomain-containing protein 4 (BRD4), which has multiple functions as an epigenetic factor. BRD4 is a member of the bromo- and extra-terminal domain (BET) protein family that contains two tandem bromodomains (BDI and BDII) and an extra-terminal domain (7). It interacts with the hyperacetylated histone region and acts at both the transcription initiation and elongation steps, promoting gene transcription (8-10). BRD4 binds to the acetylation region of histones, cooperates with RNA polymerase and acts as an enhancer to promote transcription of various genes. Among them, cancer-related genes such as Myc, FOSCL, and RUNX2 are thought to promote tumour growth (8, 11, 12).

There are several reports on BRD4 in haematological and solid tumours, including breast cancer, and it is being developed as a new target for cancer treatment (13, 14). Several in vitro and in vivo studies have also shown that inhibition of BRD4 reduces the tumour size in breast cancer (15, 16). However, the association between BRD4 and clinicopathological factors in breast cancer remains unclear. Therefore, in this study, we aimed to evaluate the association between BRD4 expression and the clinicopathological factors and prognosis in human breast cancer specimens.

Patients and Methods

Patient sample collection. Tissue samples were obtained from 183 patients who underwent surgery without presurgical treatment at Yokohama City University Medical Center, between April 2006 and December 2008. The median follow-up period was 120 months (5-54 months). This study was approved by the Institutional Review Board of Yokohama City University (190700023) and informed consent was obtained from all individual participants included in the study. The primary outcomes of this study were disease-free survival (DFS) and disease-specific survival (DSS). The DFS interval was defined as the period from diagnosis to the relapse of breast cancer or metastasis, and any cause of death; DSS was defined as the period from the diagnosis to breast cancer-related death.

To quantify the expression of BRD4 protein, we used tissue microarrays (TMAs) constructed from formalin-fixed, paraffin-embedded tumour blocks obtained from the surgical specimens of primary breast cancer, as previously described (5, 17). Two board-certified pathologists performed the pathological diagnosis and immunohistochemical (IHC) evaluation in a blinded manner. Oestrogen receptor (ER) and progesterone receptor (PgR) status were evaluated using the Allred score (18). Human epidermal growth factor receptor 2 (HER2) status was examined using the Dako HercepTest™ (K5204; Dako, Santa Clara, CA, USA) or PathVysion™ (Abbott, Abbott Park, IL, USA). HER2 positivity (overexpression or amplification) was scored according to the American Society of Clinical Oncology/College of American Pathologists guidelines (19). IHC staining was performed to examine BRD4 protein expression and localisation by using a monoclonal anti-human BRD4 [EPR5150(2), Abcam, Cambridge, UK]. The slides were incubated at 95°C for 40 min and deparaffinised in xylene four times for 5 min, followed by 5 min each in 95, 90 and 70% ethanol and washed twice for 1 min in distilled water. Antigen retrieval was performed by autoclaving the slides in citrate buffer (pH 6.0) at 121°C. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide/methanol solution for 15 minutes. The tissue sections were incubated with the primary antibody at 4°C for 8 h and with the secondary antibody for 30 min. 3,3-Diaminobenzidine (DAB; Dako) was used as a chromogenic substrate. The slides were lightly counterstained with Mayer’s haematoxylin. Immunohistochemically stained sections were examined for BRD4 expression and quantified using the Allred scoring method (18) by a pathologist who had a subspecialty training in breast pathology. The score consisted of two components: 1) the average intensity of BRD4 staining (negative: 0; weak: 1; medium: 2; and strong: 3) and 2) the percentage of BRD4-stained nuclei (none: 0; <1%: 1; 1%-10%: 2; 11%-33%: 3; 34%-66%: 4; and 67%-100%: 5). The sum of the two component scores formed the overall score with a possible value of 0 or 2-8. We evaluated the average intensity of BRD4 staining, and a cut-off score of 2 was determined by assessing the nuclear BRD4 expression levels. We evaluated the association between BRD4 expression and clinicopathological findings.

Data of The Cancer Genome Atlas cohort. Expression levels of the tumour gene in patients with breast (n=1082), pancreatic (n=176), gastric (n=375), lung (n=510), and ovarian (n=300) cancers in The Cancer Genome Atlas (TCGA) database were obtained from cBioPortal (20). DSS was defined as the period from the diagnosis to breast, pancreatic, gastric, lung, or ovarian cancer-related death. For breast cancer, we selected female patients whose BRD4 gene expression data (n =1069) were available. To evaluate the DFS and DSS, we used data from the curated and filtered pan-Cancer Clinical Data Resource of survival endpoints for TCGA cases(21). To categorise the patients into two groups based on their tumour BRD4 expression, we used the intra-cohort median value of gene expression.

Statistical analysis. The data were analysed using the Statistical Package for the Social Sciences (SPSS) Statistics v.24 software (IBM SPSS Statistics for Windows, Version 24.0. IBM Corp., Armonk, NY, USA) and R software (version 4.0.1). SPSS was used for data analysis of patient data by TMA, and R was used for TCGA data analysis. We used the Mann-Whitney U-test was used for the association of BRD4 expression with patient age and tumor size, and Fisher’s exact test was used for the association with other biomarkers and clinicopathological factors. Survival data were evaluated using the Kaplan–Meier method and log-rank test. For survival analyses in R, by group comparison of the survival times, the log-rank test, and Cox proportional hazards regression models, the survival package was used. For the boxplot of BRD4 mRNA expression, we used Mann-Whitney U and Fisher’s exact test to compare two groups, and Kruskal-Wallis test to compare multiple groups. Statistical significance was determined at p<0.05.

Results

BRD4 expression in human breast cancer. IHC staining was performed on TMAs to examine the differential expression of BRD4. The results are shown in Figure 1. BRD4 was expressed in the nucleus of most tumour cells but not in stromal cells. In all cases, BRD4-positive cells accounted for one-third or more of the tumour cells, and the proportion score was 4 or more in all cases. As the staining intensity was different for each case, cases with an intensity score of 2 or less were considered the weak group (147 cases) and those with a score of 3 were considered the strong group (36 cases).

Figure 1.
Figure 1.

Immunohistochemical staining of bromodomain-containing protein 4 (BRD4) in human breast cancer specimens. Representative images according to the intensity scoring (IS) are shown. (a) Weak (IS=1), (b) Intermediate, and (c) Strong (IS=3). Scale bar=100 μm.

Association of BRD4 expression with clinicopathological factors. Patient characteristics and histopathological data are shown in Table I. The median age was 58 (30-69) years, and 81.4% of the patients (n=149) were ER-positive, 9.3% (n=17) were HER2-positive, and 11.4% (n=21) were triple-negative (TN: ER-negative, PgR-negative, and HER2-negative). We compared the weak and strong groups. The association of BRD4 expression with clinicopathological factors is shown in Table II. There were no significant differences between the two groups in terms of age, hormone status, operative procedure, adjuvant therapy, or axillary lymph node metastasis.

View this table:
Table I.

Patient characteristics.

View this table:
Table II.

Correlation between BRD4 expression and clinicopathological factors

Survival analyses according to BRD4 expression. Survival data according to the BRD4 expression are shown in Figure 2. The median period of observation was 120 months. Although not significantly different, the strong group had a lower rate of breast cancer recurrence than the weak group (18.4% vs. 8.3%, p=0.182) and no breast cancer-related deaths (13.6% vs. 0.0%, p=0.041). The strong group tended to have a higher rate of DFS (96.9% vs. 86.5% at 5 years, p=0.207, Figure 2a) and significantly better DSS (100.0% vs. 91.3% at 5 years, p=0.027, Figure 2b) than the weak group.

Figure 2.
Figure 2.

Survival analysis according to bromodomain-containing protein 4 (BRD4) expression in patients with breast cancer. (a) Kaplan–Meier disease-free survival (DFS) and (b) disease-specific survival (DSS) in patients with strong BRD4 expression [intensity score (IS)=3; red] in comparison to those with weak BRD4 expression (IS=1, 2; blue). Breast cancer patients with high BRD4 expression with an IS of 3 have a significantly longer DSS and better prognosis than those with an IS of 2 or less.

Prognostic analysis using the TCGA cohort. Next, we validated the clinical relevance of BRD4 using TCGA datasets. There was no significant difference in DFS between the groups with high and low expression of BRD4 (78.4% vs. 77.1 % at 6 years, p=0.854, Figure 3a); however, the DSS was significantly more prolonged and better in the high expression group compared to the low expression group (91.2% vs. 80.2% at 6 years, p=0.047, Figure 3b), which was consistent with the results of our IHC analysis. The association between BRD4 expression and the clinicopathological factors in the TCGA cohort is shown in Table III. In this cohort, 54.3% (n=581) of the cases were ER-positive/HER2-negative, 16.5% (n=176) were HER2-positive and 14.2% (n=152) were triple-negative breast cancer (TNBC). A significantly greater number of TNBC cases (18.5% vs. 11.2%, p=0.003) and fewer lymph node metastatic cases (47.6% vs. 53.6%, p=0.026) were observed in the BRD4-high expression group than in the BRD4-low expression group. The expression of BRD4 mRNA was significantly higher in TNBC than in the other subtypes (Figure 4). Nonetheless, the subtype-based analysis showed no significant difference in prognosis between the high and low BRD4 expression groups in any of the subtypes (data not shown).

Figure 3.
Figure 3.

Survival analysis according to bromodomain-containing protein 4 (BRD4) expression in The Cancer Genome Atlas (TCGA) cohort. (a) Kaplan–Meier disease-free survival (DFS) and disease-specific survival (DSS) (b) in patients with high BRD4 expression (red) compared with those with low BRD4 expression (blue) in the TCGA cohort. Classification by mRNA; median cut-off values are used.

View this table:
Table III.

Correlation between BRD4 expression and clinicopathological factors in the TCGA cohort.

Figure 4.
Figure 4.

Boxplots of BRD4 mRNA expression according to (a) subtype, (b) American Joint Committee on Cancer (AJCC) stage and (c) histologic grade of tumor. All plots are of Tukey type and boxes represent the median with inter-quartile range. p-Values were calculated with Mann-Whitney U and Kruskal-Wallis test.

To analyse the association between BRD4 expression and prognosis in solid tumours, we used the data on breast, pancreatic, gastric, lung and ovarian cancers. A significantly prolonged DSS was observed in breast cancer with high BRD4 expression [hazard ratio (HR)=0.57, 95% confidence interval (CI): 0.36-0.91, p=0.019], whereas a high BRD4 expression was associated with a poor prognosis in ovarian cancer (HR=1.28, 95% CI=1.02-1.61, p=0.034). There were no significant differences between lung and gastric cancers; however, a tendency for high BRD4 expression leading to poor prognosis was observed (Figure 5).

Figure 5.
Figure 5.

Significance of tumour bromodomain-containing protein 4 (BRD4) expression for survival in various solid cancers. The association of gene expression (as continuous variable) with disease-specific survival is examined with univariate Cox proportional hazards regression for five types of cancers in The Cancer Genome Atlas cohort. The hazard ratio (HR) for mortality associated with high gene expression and its 95% confidence interval (CI) are plotted. Wald test p-values are shown.

Discussion

We evaluated the association between BRD4 expression determined by IHC staining, clinicopathological features, and clinical outcomes in breast cancer patients who underwent surgery. We found that the BRD4 protein was universally observed in breast cancer cases; however, the intensity of its expression varied between cases. Breast cancer with strong expression of BRD4 [intensity score (IS)=3 points] was associated with a favourable outcome. We validated the association between BRD4 expression and breast cancer prognosis using TCGA cohort data and found that the results were consistent with our protein analysis. Although the cut-off values for protein and mRNA expression were different, we would like to emphasise that the two analyses produced the same result, such that breast cancers with high BRD4 expression had a better prognosis than those with low BRD4 expression. Notably, BRD4 expression was associated with poor prognosis in other solid tumours, including ovarian, lung, and gastric cancers.

BRD4 promotes oncogene transcription (22-24). It has also been suggested that BRD4 is associated with cancer cell migration and invasion through epithelial-mesenchymal transition (EMT). In breast cancer cells, inhibition of BRD4 rapidly reduces the expression of Snail, a potent EMT transcription factor (EMT-TF) (25); hence, we hypothesised that BRD4 expression was related to poor prognosis in breast cancer. In a previous study, breast cancers with strong BRD4 expression were shown to have a large tumour size and a high Ki-67 index, as well as a high number of premenopausal patients (26). Moreover, it has been reported that breast cancers with high BRD4 expression have a poor prognosis (27). The discordance between these studies and our study may stem from differences in probes used for BRD4 detection and sample size of the database. In the current study, BRD4 expression did not correlate with poor prognostic factors, such as the tumour stage and grade. Contrary to our hypothesis, BRD4 expression was identified as a favourable prognostic factor in breast cancer and the results were validated in the TCGA cohort. Notably, BRD4 in other solid tumours was associated with poor prognosis, suggesting that BRD4 might have a different function in breast cancer from that in other solid tumours.

In this study, there was no significant difference in the recurrence rate of breast cancer according to BRD4 expression; however, no breast cancer-related death was observed in patients with high BRD4 expression. Thus, we speculate that BRD4 may modify the nature of metastatic tumours, reducing their invasiveness, decreasing the tumour grade and making them chemosensitive. Activation of BRD4 reduces both the invasiveness and mobility of highly metastatic cell lines without affecting cell proliferation rates. Nevertheless, the activation of BRD4 has been shown to result in a significant decrease in tumour proliferation and metastatic capacity in in vivo experiments (28). Furthermore, BRD4 mediates resistance to neoplastic transformation in normal cells by altering genome-wide binding patterns, leading to the inhibition of oncogenic dedifferentiation (29).

BRD4 plays a dual role in cancer, suggesting that it exerts either antitumor or protumor functions, depending on the intracellular context. Wu et al. showed that BRD4 has two isoforms, BRD4-L and BRD4-S, which exert contrasting functions in breast cancer (30, 31); BRD4-L has an extended disordered proline-rich region and a P-TEFβ interaction domain, which are absent in BRD4-S (7). BRD4-S deficiency suppresses the proliferation, invasion, and migration of breast cancer cells. Ectopic BRD4-S expression in the mammary gland significantly promotes primary tumour growth and lung metastasis, while ectopic BRD4-L expression suppresses tumour growth in vivo (30). The antibody used in this study could not distinguish between the two isoforms. However, the same study reported that BRD4-L accounts for a large proportion of the isoforms, which is consistent with our findings.

Inhibitors against BETs, including BRD4, have received much attention as potential therapeutic agents for cancer (15, 32-35). However, this study showed that BRD4 has contrasting functions in breast and other cancers. Furthermore, the detailed functions of BRD4 in breast cancer proliferation and prognosis remain unclear and controversial. Therefore, for the safe and appropriate use of BET inhibitors, further investigations regarding the signalling pathways and complementary effects of BRD4 are warranted.

Nonetheless, this study has several limitations. First, as this study used TMA without presurgical treatment from a single institution, there is a selection bias. We had some patients who were ER-positive/HER2-negative, and a few other subtypes. Second, it was a retrospective study with a small number of patients. Third, because the BRD4 antibody used in this study could not distinguish between the different BRD4 isoforms, we were unable to address the association between the expression of BRD4-S/L isoforms and breast cancer prognosis. Similarly, for the TCGA cohort, we could not distinguish between the BRD4 isoforms in breast and other cancers. Nonetheless, this study suggests that the prognostic function of BRD4 may differ across malignancies, and further analysis of the distribution and functions of BRD4 isoforms is warranted.

In conclusion, BRD4 was widely distributed in human breast cancer specimens; nonetheless, its expression varied between cases. Breast cancer with a high BRD4 expression had a better prognosis than that with a low BRD4 expression; however, this was not noted in other solid tumours. Further investigations are warranted to further examine the antitumour function of BRD4.

Acknowledgements

This work was supported by the National Institutes of Health (NIH) grant R01CA160688 to K.T. We would like to thank Editage (www.editage.com) for English language editing.

Footnotes

  • Authors’ Contributions

    All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Conception and design of study: C.S, A.Y, T.I; Acquisition of data: C.S, S.A, H.S, M.T; Analysis and/or interpretation of data: K.K, M.O, S.S ; Drafting the manuscript: C.S, A.Y, S.Y, K.N; Revising the manuscript critically for important intellectual content: K.T, Y.I, T.I, I,E.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest.

  • Received May 11, 2021.
  • Revision received June 6, 2021.
  • Accepted June 7, 2021.

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Bromodomain-containing Protein 4 Is a Favourable Prognostic Factor in Breast Cancer Patients
CHIHO SUZUKI, AKIMITSU YAMADA, SHOKO ADACHI, HIDETAKA SHIMA, KUMIKO KIDA, MASANORI OSHI, SADATOSHI SUGAE, SHINYA YAMAMOTO, KAZUTAKA NARUI, MIKIKO TANABE, KAZUAKI TAKABE, YASUSHI ICHIKAWA, TAKASHI ISHIKAWA, ITARU ENDO
Anticancer Research Jul 2021, 41 (7) 3597-3606; DOI: 10.21873/anticanres.15148
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