Background

Breast cancer has been the leading cause of cancer death in females for the past decade [1,2,3]. In 2020, female breast cancer has surpassed lung cancer as the leading cause of global cancer incidence, representing 11.7% of all cancer cases, which further increased the treatment burden [3]. The current cure rates of breast cancer are highly dependent on the molecular subtype of the tumor and the stage at diagnosis, which, in some cases, do not result in satisfactory clinical outcomes [4]. Therefore, the search for new biomarkers with therapeutic purpose is still needed to assist in the clinical management of breast cancer.

Epigenetic changes such as DNA methylation, histone modification and microRNAs (miRNAs) play a crucial role in tumorigenesis and cancer progression [5,6,7]. It has been found that the level of many histone modification markers was associated with the prognosis of human cancers [8,9,10,11]. Among these markers, histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 27 trimethylation (H3K27me3) were the hallmarks of repressive marker [12], and H3K9me3 was a key histone modification marker associated with the prognosis of many cancers [13,14,15,16], while one study found no association between the level of H3K9me3 and breast cancer survival [17]. However, cellular experiments revealed that H3K9me3 decreased during breast cancer transformation [18]. Moreover, a recent study found that decreased H3K9me3 level was associated with the upregulation of many oncogenes which were related to breast cancer prognosis [19].

We further noticed that the level of H3K9me3 was closely associated with estrogen receptor (ER) status (negative and positive) in breast cancer and the role of H3K9me3 depended on ER [20, 21], suggesting that the association of H3K9me3 with prognosis may be related to ER status. In addition, a recent update in the ASCO/CAP guideline recommends defining ER expression levels: negative, low level (1–10%) and high level (> 10%) depending on the proportion of positive tumor nuclei [22]. It has been found that ER-low positive patients revealed more similar clinicopathologic profiles to ER-negative rather than ER-high positive patients [23, 24]. It would be interesting to further examine the role of different expression levels of ER (low level and high level) on the association between H3K9me3 and the prognosis.

In the present study, therefore, we aimed to examine the prognostic value of H3K9me3 on breast cancer by ER status (negative vs. positive) and the different ER expression levels (ER-low positive vs. ER-high positive).

Results

Low level of H3K9me3 in breast cancer tissues

Of 917 women included in the analysis, almost all (99.0%) of them were pathologically diagnosed with invasive ductal carcinoma (IDC). The level of H3K9me3 in adjacent tissues was available in 633 patients, and the median (P25, P75) in tumor tissues [160.0 (62.5, 255.0)] was significantly lower than that in adjacent tissues [255.0 (210.0, 270.0)] (p < 0.001) (Table 1). When stratified by ER status, the level of H3K9me3 in tumor tissues was still lower than that in adjacent tissues for both ER-negative [210.0 (117.5.0, 255.0) vs 255.0 (180.0, 270.0), p < 0.001] and ER-positive [150.0 (56.7., 240.0) vs 255.0 (210.0, 270.0), p < 0.001] tumors.

Table 1 Level of H3K9me3 in tumor and adjacent tissues by ER status
Full size table

Demographic and clinicopathological characteristics and the associations with H3K9me3 in tumor tissues

The median age at diagnosis was 48 years (interquartile range: 41–56) among 917 eligible women and more than half (57.6%) of the women were premenopausal. The majority of the women were diagnosed with low histological grade (grade I/II: 73.3%), early clinical stage (stage I/II: 71.5%), ER-positive (73.1%), PR-positive (72.1%), or HER2-negative (66.8%) (Table 2). Univariate analysis showed that tumor size, nodal status, clinical stage and ER status were associated with OS and PFS (Additional file 1: Table S1).

Table 2 Demographic and clinicopathological characteristics and the associations with H3K9me3 [N (%)]
Full size table

The optimal cut-off value of H3K9me3 H-score was 240.0 according to the X-tile plot (Additional file 1: Fig. S2), and 667 (72.7%) patients had the H-score ≤ 240.0. Patients with the H-score ≤ 240.0 were more likely to have grade I/II, nodal positive, ER-positive, PR-positive and HER2-negative tumors than the subjects with H-score > 240.0 (Table 2). No marked differences in the level of H3K9me3 were observed between different age, menopausal status, tumor size and clinical stage.

Association of H3K9me3 with breast cancer prognosis

Of the 917 eligible women, 127 died and 203 experienced disease progression with a median follow-up time of 85.2 months (interquartile range: 59.0–121.8). Five-year OS rate and PFS rate were 92.0% and 84.9%, respectively. In univariate analysis, no significant association was found between the level of H3K9me3 and breast cancer OS, while significant association (HR = 0.67, 95% CI 0.47–0.95) between a better breast cancer PFS and the high level of H3K9me3 was observed (Table 3). In multivariate analysis, a similar pattern of association was observed (HR = 0.70, 95% CI 0.49–0.99 for PFS).

Table 3 Univariate and multivariate association of H3K9me3 with breast cancer prognosis
Full size table

Statistical interaction between ER status and H3K9me3

The results of stratified analysis by ER status showed that the prognostic value of H3K9me3 on breast cancer was significant only among patients with ER-positive tumors (Table 4). The high level of H3K9me3 was associated with a better breast cancer OS (HR = 0.43, 95% CI 0.21–0.86) and PFS (HR = 0.49, 95% CI 0.29–0.81) in ER-positive patients. Moreover, the interaction between the level of H3K9me3 and ER status on the prognosis was significant (Pinteraction = 0.011 for OS; Pinteraction = 0.022 for PFS).

Table 4 Associations of H3K9me3 with ER-negative and ER-positive breast cancer prognosis
Full size table

For patients with ER-positive tumors, stratified analysis by ER-low and ER-high positive tumors was conducted. Notably, the prognostic value of H3K9me3 was significant among patients with ER-high positive tumors (HR = 0.34, 95% CI 0.13–0.85 for OS; HR = 0.47, 95% CI 0.26–0.86 for PFS) (Table 5). However, no significant association was found among patients with ER-low positive tumors (HR = 0.64, 95% CI 0.21–1.91 for OS; HR = 0.53, 95% CI 0.20–1.38 for PFS).

Table 5 Associations of H3K9me3 with ER-low and ER-high positive breast cancer prognosis
Full size table

Discussion

In this study, we found that the level of H3K9me3 in breast cancer tissues was significantly lower than that in adjacent tissues. Moreover, the low level of H3K9me3 was associated with a poor breast cancer OS and PFS in ER-positive patients, while no significant association was found in ER-negative patients, and the interaction between H3K9me3 and ER on the prognosis was significant.

In consistent with our study, previous studies also found that the level of H3K9me3 was low in breast cancer tissues [18, 25]. The level of H3K9me3 in cells depended on histone lysine methyltransferases or the opposing demethylases, and various studies have shown that the demethylases KDM4A/JMJD2A, KDM4B/JMJD2B and/or KDM4C/JMJD2C are overexpressed in breast cancer [26]. In the present study, the decreased H3K9me3 level was observed for both ER-negative and ER-positive tumors when compared with the adjacent tissues, while the level of H3K9me3 in ER-positive tumors was lower than that in ER-negative tumors. It has been found that the histone demethylase KDM4B/JMJD2B is regulated by ERα [27, 28], which may explain that H3K9me3 level was lower in ER-positive tumors. In addition, we also showed that the low level of H3K9me3 was associated with low histological grade, nodal positive, PR-positive and HER2 negative tumors, while underlying mechanisms of these associations needs to be further explored.

Many cellular experiments revealed that a decreased level of H3K9me3 was associated with the overexpression of oncogenes; interestingly, some of these oncogenes were regulated by ER [19, 25, 27, 29]. These findings supported our result that the low level of H3K9me3 was associated with a poor prognosis and this association was significant only in ER-positive (particularly ER-high positive) patients but not in ER-negative patients. In addition, a previous population study found no significant association between H3K9me3 and breast cancer prognosis, which may be attributed to the high proportion (41.4%) of ER-negative patients [17].

Currently, epigenetic therapies are promising agents for overcoming clinical resistance to conventional treatments in breast cancer, but still not widely used [30, 31], and the further studies were needed. Our findings showed that the higher level of H3K9me3 was associated with a better prognosis of ER-positive (particularly ER-high positive) breast cancer. Combined with the related reports, inhibitors of H3K9me3 demethylase could be used for the treatment of ER-positive breast cancer [32, 33]. Interestingly, the H3K9me3 demethylase KDM4B/JMJD2B is regulated by ERα [27, 28], suggesting that the KDM4B/JMJD2B inhibitor would be more effective to improve the survival of ER-positive breast cancer patients.

Our study has several limitations that need to be taken into consideration. First, the IHC staining of H3K9me3 was evaluated by only one pathologist, which may lead to misclassification bias. However, the pathologist’s evaluation criteria are consistent; the relative relationship between all samples is almost unaffected. Therefore, even if the IHC staining was misclassified, the misclassification bias is likely to be non-differential and underestimate the true association. Second, only patients with tumor > 1 cm were included, which may lead to selective bias. However, the prognosis of patients with tumor ≤ 1 cm was excellent, even with less treatment [34]; thus, it is acceptable to select the patients with tumor > 1 cm as the study population. Third, the sample size of ER-low positive patients was small; the future study with larger sample size was more valuable. Finally, we didn not collect the information of treatment which was associated with the outcomes. However, since the treatment was determined according to the clinicopathological characteristics, and adjustment of these characteristics in the analysis was able to largely control the confounding effects of the treatment.

Conclusions

In conclusion, this study firstly demonstrated that there was an interaction between H3K9me3 and ER on breast cancer prognosis and the prognostic value of H3K9me3 was significant among only ER-positive tumors, particularly ER-high positive tumors, but not ER-negative and ER-low positive tumors. Our study highlights H3K9me3 as a prognostic marker in ER-positive breast cancer and more investigations are expected to prove that H3K9me3 is a therapeutic target for ER-positive breast cancer.

Methods

Study population

A total of 1062 female patients with pathologically diagnosed primary invasive breast cancer and > 1 cm of tumor size in diameter between January 2008 and December 2015 were recruited from the Cancer Center of Sun Yat-sen University in Guangzhou, China. Patients with metastatic tumor and missing information of age at diagnosis, ER status and the level of H3K9me3 in tumor tissues (N = 129) were excluded (Additional file 1: Fig. S1). Of 933 eligible women, 917 (98.3%) were successfully followed up until Dec 31, 2021 and then were included in the statistical analysis. This study was approved by the Ethics Committee of the School of Public Health at Sun Yat-sen University. Informed consent was obtained from each participant.

Baseline data collection

Information on demographic and clinicopathologic characteristics was collected at diagnosis from patients’ medical records, including age, menopausal status, clinical stage, histological grade, ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, etc. The definition of ER, PR and HER2 status was described in detail previously, and ER-positive was defined as ≥ 1% of tumor cell nuclei that are immunoreactive [35]. In the present study, furthermore, ER-low positive was defined as 1–10% of tumor cell nuclei that are immunoreactive; ER-high positive was defined as > 10% of tumor cell nuclei that are immunoreactive.

Tissue microarray and immunohistochemistry

The level of H3K9me3 was evaluated with tissue microarrays (TMAs) by immunohistochemistry (IHC). TMAs were constructed as previously described [36]. The TMAs were baked at 60 °C for 2 h and then dewaxed with xylene and ethanol. Then antigen retrieval was accomplished using EDTA (PH 9.0) in super-pressure kettle and endogenous peroxide was blocked using 3% H2O2. After the preparations, slides were incubated in rabbit monoclonal to H3K9me3- Chip Grade (ab8898, diluted 1:1000, Abcam) overnight at 4 °C and then labeled with the EnVision Detection System (Peroxidase/DAB, Rabbit/Mouse) (Dako K5007). Then slides were developed by diaminobenzidine (DAB) and counterstained by hematoxylin. These slides were finally dehydrated and mounted.

IHC-stained sections were digitally imaged using Pannoramic Scanner and CaseViewer software. IHC staining was analyzed by an experienced pathologist and scored for staining intensity (0-no staining, 1-weak, 2-moderate and 3-strong) and percentage of tumor cell staining (0–100). IHC scoring was done by H-score which was calculated by multiplying the staining intensity by the percentage of positive cells. Thus, the minimal H-score was 0, whereas the maximum H-score was 300. To avoid the observation variability, the mean value of duplicate scores was adapted for further analysis.

Follow-up and outcomes

Patients were followed up by phone calls or out-patient visits every 3 months in the first year, every 6 months in the second and third year after diagnosis and annually thereafter. Outcomes of interest were overall survival (OS) and progression-free survival (PFS). OS was defined as the time from diagnosis to death and PFS was the time from diagnosis to disease progression including recurrence, metastasis and death. The deaths were confirmed by calling the first-degree relatives of the patients and searching the Death Registration Reporting Information System of Guangzhou Center for Disease Control and Prevention. Survival status was censored at the latest follow-up date or Dec 31, 2021.

Statistical analysis

Wilcoxon signed rank test was used to compare the level of H3K9me3 between tumor tissues and adjacent tissues. Next, the H3K9me3 H-score was treated as binary variables. The optimal cut-off value was determined by the minimum P value from log-rank chi-square statistics based on PFS using the X-tile 3.6.1 software (Yale University, New Haven, CT, USA) [37]. Chi-square test was used to test the associations of H3K9me3 level with age, menopausal status, histological grade, tumor size, nodal status, clinical stage and expression of ER, PR and HER2. Kaplan–Meier method was used to estimate the 5-year survival. Cox proportional hazard model was used to estimate Hazard ratios (HRs) and their 95% confidence intervals (CIs) for the associations between various prognostic variables and the survival (OS and PFS). Multiplicative scale was used to estimate the interaction between H3K9me3 level and ER status on breast cancer prognosis.