Downregulated METTL14 Expression Correlates with Breast Cancer Tumor Grade and Molecular Classification

It is unclear whether the methyltransferase-like 14 (METTL14) protein promotes or suppresses cancer growth. We examined the association between METTL14 expression, cancer progression, and patient prognosis in a total of 398 breast cancer tissue specimens. Significantly fewer cancer tissue specimens compared with normal breast tissue expressed high levels of METTL14 (52.8% vs. 75.0%). METTL14 expression was negatively associated with tumor grade and positively associated with patient age, estrogen, and progesterone receptor status. High METTL14 expression was more common in luminal A and luminal B tissue (75.9% and 60.8%, respectively), compared with human epidermal growth factor receptor 2- (HER2-) enriched and triple-negative breast cancer (TNBC) samples (38.2% and 18.6%, respectively). In multiple logistic regression analysis, independent predictors of METTL14 expression in breast cancer included higher tumor grade (odds ratio (OR) = 0.494, 95% confidence interval (CI): 0.289–0.844; P = 0.010), TNBC subtype (OR = 0.109, 95% CI: 0.054–0.222; P < 0.001), and HER2-enriched subtype (OR = 0.298, 95% CI: 0.156–0.567; P < 0.001). No clear relationship was observed between patient prognosis and METTL14 expression. It appears that downregulated METTL14 expression in breast cancer is associated with tumor grade and molecular classification.


Introduction
Breast cancer is the leading cause of cancer-related death among women [1]. Breast cancer is a biologically diverse disease, reflected by different molecular subtypes that have different biological behaviors and clinical treatment options [2][3][4]. The different subtypes include luminal A and luminal B, human epidermal growth factor receptor 2-(HER2-) enriched, and triple-negative or basal-like; immunohistochemistry (IHC) can assess standard biomarkers (estrogen receptor (ER), progesterone receptor (PR), and HER2) as well as Ki-67 status (a marker of cell proliferation) [2,5]. Triple-negative breast cancer (TNBC) has a more aggressive clinical course and poorer prognosis than other types of breast cancer, and currently there is no effective treatment other than chemotherapy [6][7][8][9]. Thus, studying the molecules related to the occurrence and progression of breast cancer and their clinical significance is of great significance for guiding the treatment of breast cancer, especially TNBC.
The catalytic protein methyltransferase-like 14 (METTL14) participates in mediate cellular N 6 -methyladenosine (m 6 A) deposition [10] and is closely related to the occurrence and development of malignant tumors [11][12][13][14][15][16][17][18][19][20][21][22][23]. METTL14 plays a complex role in malignant tumors: low levels of METTL14 expression are found in hepatocellular carcinoma [13], colorectal cancer [14,16,17], bladder cancer [15], gastric cancer [18], childhood ETV6/RUNX1-positive acute lymphoblastic leukemia [19], and papillary thyroid carcinoma [20], where METTL14 functions as a tumor suppressor [13][14][15][16][17][18], whereas levels of METTL14 expression are high in pancreatic cancer [21,22] and acute myeloid leukemia [23], where this protein functions as a tumor promoter. In breast cancer, contrasting findings have been reported from analyses of the expression and function of METTL14. In one study, significantly decreased METTL14 expression was observed in patients with breast cancer compared with healthy controls, and overexpression of METTL14 inhibited breast cancer cell viability, colony formation, and migration [24]. Conversely, in another study, significantly upregulated METTL14 expression was observed in breast cancer tissue compared with normal tissue, and METTL14 overexpression enhanced the migration and invasion capacities of breast cancer cells [25], while other researchers have reported significantly upregulated levels of METTL14 and m 6 A in breast cancer cells and tissues, and the modulation of METTL14 by long noncoding RNA LINC00942 (LNC942) promoted breast cancer initiation and progression [26]. Thus, further research is needed to better understand the expression and function of METTL14 in breast cancer. This study performed an IHC analysis of METTL14 expression in breast cancer tissue samples obtained from 398 Chinese Han women, to clarify the expression of METTL14 in breast cancer and its clinicopathological and prognostic significance. Twenty-four samples of adjacent normal breast tissue were also obtained following surgical resection. A pathohistological diagnosis was made according to the breast tumor classification criteria of the World Health Organization [27]. Histological grading was based on the Scarff-Bloom-Richardson system [28]. According to ER, PR, HER2, and Ki-67 status, the samples were classified into 4 subtypes [2,5]: luminal A (ER + /PR + ½≥20%/HER2 -, Ki67 < 14%); luminal B, containing hormone receptor-positive cases that did not meet the conditions of luminal A; HER2-enriched (ER -, PR -, and HER2 + ); or TNBC (ER -, PR -, and HER2 -). Follow-up information was available for 228 breast cancer patients with a median follow-up time of 60 months (range, 6-70 months). The Medical Ethics Committee of the Affiliated Dongyang Hospital of Wenzhou Medical University approved this study. All study methods satisfied the relevant guidelines and regulations issued by the Affiliated Dongyang Hospital of Wenzhou Medical University.

Tissue Array Preparation.
We followed the methods described by Wang et al. [29]. In brief, the Quick-Ray ® UT-06 (Unitma Co., Ltd., Seoul, Korea) tissue microarray system and the Quick-Ray premade recipient block (UB-06) wax model were used to prepare tissue specimens (1 mm in diameter). Two representative sites from each breast cancer tissue sample were selected for sampling.

Assessment of Staining.
Each entire section was scanned and scored independently by 2 pathologists. The intensity of nuclear staining for METTL14 was assessed in breast tissue. Staining intensity was scored on a 4-point scale from 0 (negative) to 1 (weak), 2 (moderate), or 3 (strong). High METTL14 expression was defined as a staining intensity of positive invasive cancer cells of 2 or 3 [31]. A case was considered to be ER-or PR-positive if the percentage of positive invasive cancer cells (nuclear staining) was ≥1% [32]. HER2 status was determined by the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing in breast cancer [33].
2.5. Patient Follow-Up. We followed the methods of Wang et al. [29]. In brief, each patient was followed up postoperatively by telephone call and thereafter at 6 monthly hospital appointments; follow-up was discontinued in the event of the patient's death. A diagnosis of local breast cancer recurrence was made by clinical or histology results. Relapse-free survival (RFS) was defined as the time from surgery to relapse/metastasis; overall survival (OS) was the time from surgery to death (excluding non-tumor-related deaths).

Flow Diagram.
A flowchart of the study methodology is shown in Figure 1.

Statistical
Analysis. Statistical analyses were conducted using SPSS software version 19.0 (SPSS Inc., Chicago, IL, USA). Between-group differences were compared using Pearson's chi-square test for qualitative variables. The relative risk was expressed by odds ratios (ORs) and their 95% confidence intervals (CIs). The independent correlation factor of METTL14 expression in breast cancer was assessed by multiple logistic regression analysis. RFS and OS rates were estimated by the Kaplan-Meier method and compared using log-rank testing. P < 0:05 was considered to be statistically significant.  Figure 2); the between-group difference was significant (P = 0:034) ( Table 1). As shown in Table 2, high METTL14 expression was identified in 64.2% (174/271) of grade I-II tumor tissue and in 28.3% (36/127) of grade III tumor tissue; the betweengroup difference was highly significant (P < 0:001). In younger-aged patients (≤35 years) and in those whose tumors were ER-or PR-negative, rates of high METTL14 expression were 31.8%

No Association between METTL14 Expression and
Survival of Patients with Breast Cancer. When we examined the data for potential associations between levels of METTL14 expression and survival rates in a cohort of 228 breast cancer patients, 5-year RFS and OS rates were 79.8% and 88.6%, respectively. As shown in Figure 3, no clear asso-ciations were observed between METTL14 expression and survival. The 137 patients with high levels of METTL14 expression had a mean RFS of 54.0 months and an estimated 5-year RFS rate of 79.6%; corresponding values in the 91 patients whose tumors expressed low levels of METTL14 were 53.2 months and 80.2%, respectively (P = 0:955; Figure 3(a)). Mean OS was 57.9 months (with an estimated 5-year OS rate of 89.8%) in the patients with high levels of METTL14 expression and 57.0 months (with an estimated 5-year OS rate of 86.8%) in those with low levels of METTL14 (P = 0:478; Figure 3(b)).

Discussion
Several studies have reported that METTL14 plays a complex role in the occurrence and development of malignant tumors [11][12][13][14][15][16][17][18][19][20][21][22][23]. For example, in hepatocellular carcinoma, RFS is adversely affected by downregulated levels of METTL14 expression [13], while a reduction in m 6 A mRNA methylation caused by the METTL14 loss-of-function R298P mutation increases the proliferation and tumorigenicity of endometrial cancer cells [34]. In contrast, METTL14 is highly expressed in acute myeloid leukemia cells and promotes leukemogenesis via m 6 A modification [23]. Findings from three investigations into METTL14 expression and function in breast cancer are controversial, with one study showing that METTL14 acts as a tumor suppressor [24], while the other two show that METTL14 promotes cancer growth [25,26].
Our findings revealed that METTL14 expression was significantly downregulated in women with higher-grade tumors. Since such tumors are known to be more aggressive and to have a poor prognosis [28], this finding indicates that the downregulation of METTL14 expression may be closely related to highly invasive breast cancer and a poor prognosis. Our results also revealed significantly decreased levels of METTL14 expression in ER-and PR-negative disease, which was closely and negatively correlated with hormone receptor expression. Furthermore, evidence has shown that overexpression of METTL14 in the ER-and PR-negative breast cancer cell line MDA-MB-231 inhibits its proliferation and migration ability [24]. Since hormone receptor-negative breast cancer patients are unsuitable candidates for endocrine therapy, further research should examine whether these patients may benefit from the exogenous overexpression of METTL14. We also found in this study that METTL14 expression was significantly decreased in younger-aged women (≤35 years), who have been shown to have more aggressive breast cancer and a worse prognosis [35,36]. Our analysis showed that having higher-grade disease, the TNBC subtype or the HER2-enriched subtype is independently correlated with low METTL14 expression in breast cancer. These results suggest that METTL14 expression in breast cancer is correlated with tumor grade and molecular classification, although our survival analysis failed to reveal any association between METTL14 expression and survival. More research is needed to determine how METTL14 expression affects the prognosis of breast cancer patients. TNBC disease is hormone receptor-negative and HER2negative, is associated with a younger age at diagnosis, a higher tumor grade, a more aggressive clinical course, and poorer prognosis than other types of breast cancer, and is not amenable to endocrine therapy or HER2-targeted agents, such as trastuzumab [6][7][8][9]. All of these factors highlight the need for new treatment options for TNBC. Our findings showing that METTL14 expression was the lowest in TNBC compared with other subtypes of breast cancer suggests that it may be worth targeting this protein in the treatment of patients with TNBC.

Data Availability
All data generated or analyzed during this study are included in this published article.

Conflicts of Interest
The authors declare no conflicts of interest.