Breast cancer is an aggressive malignancy tumor in females, the common metastasis locations in clinical included the lung, bone, and brain, leading to approximately 522,000 deaths yearly [1]. Until now, the specific mechanism of BC is still unclear. Currently, the causes of BC were considered to beinvolvedin a variety of events, including genetics and epigenetics modification, especially for epigenetic change, such as DNA promoter methylation, gene mutation and deletion in tumorigenesis. In the past decades, DNA methylation has been demonstrated to be a promising early diagnostic biomarker for BC, however, useful markers in practice have not been completely identified.
Zinc finger protein is an important family of transcription factors and the majority of human zinc finger proteins contain the KRAB domains, which has been proved to act as a transcriptional repressor by interacting with KAP1 and subsequently recruiting histone-modifying proteins [20, 23, 24]. ZNF132, a member of the zinc finger protein family, was only reported to be downregulated by promoter methylation in ECSC and PC[25, 26]. However, its diagnostic and prognostic values have not been elucidated in BC until now. The present study, to the best of our knowledge, is the first one to systematically explore the clinical significance of ZNF132 in BC.
In agreement with the study of ZNF132 in ECSC and PC, our results indicated that ZNF132 had a significantly lower expression in BC tissues than adjacent normal tissues both in mRNA and protein level, which implied that ZNF132 might serve as a tumor suppressor in BC. Without a doubt, the larger samples need to be collected to provide more powerful evidence to verify the role of ZNF132 in BC. In addition, the ROC curve revealed that ZNF132 displayed a significant diagnostic value for BC (AUC =0.887, P <0.001). Significantly, lower ZNF132 expression was correlated with the worse prognosis of BC. Consequently, ZNF132 might be served as a promiing diagnostic and prognostic marker for BC. However, as demonstrated in Table 2 and Table 3, univariate and multivariate analysis did not provide independent prognostic information for ZNF132 expression. The possible reasons we summarized are as follows: a. A series of mixed factors were involved in the prognosis of BC, the common factors were only listed in the present study. b. Samples in our study based on the TCGA database were not enough to clarify the correlation between ZNF132 expression and prognostic value, so large-scale prospective study would be necessary to confirm the value of ZNF132 in future. Moreover, analysis from Kaplan-Meier plotter revealed that low ZNF132 expression was significantly associated with a shorter RFS for patients of BC, but not with OS. Therefore, we speculated that ZNF132 expression can detect BC recurrence. Owing to tumor heterogeneous characteristics, BC has usually been classified several molecular subgroups: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2), normal, and basal-like based on immunohistochemical evaluation of estrogen receptor (ER), progesterone receptor (PR), HER2 and proliferation marker Ki-67. Analysis based on TCGA database revealed that downregulated ZNF132 was significantly correlated with the malignant phenotype of BC, including positive HER2 status, larger tumor sizes, distant metastasis and advanced clinical stage, which suggested that ZNF132 might inhibit the progression of BC by inhibiting the growth, invasion, and metastasis of tumor cells. Simultaneously, ZNF132 expression was positively associated with ER and PR status, further investigation is called for to identify whether combined detection of ZNF132 together with some of these other molecules would be valuable in improving prognosis assessment.
To recognize the potential mechanisms of ZNF132 in BC, the first investigation from cBioPortal showed that approximately 6% BC patients exhibited ZNF132 alterations, among which, the mRNA downregulation was the predominant type of alteration, which could contribute to the downregulation of ZNF132 in BC. In addition, the analysis based on GSEA demonstrated that ZNF132 participated in a variety of important biological processes and pathways. Significantly, ZNF132 expression was negatively correlated with CCNE1 and ENO1. Cyclin E1 (CCNE1), belongs to the highly conserved cyclin family, forms a complex with CDK2, whose activity is required for cell cycle G1/S transition. CCNE1 has been reported to upregulated in various human cancer, including breast[27], bladder[28], and ovarian[29], by mediating premature S-phase entry, ineffective DNA replication, and genomic instability. Alpha-enolase (ENO1), as a prominent glycolytic enzyme, was upregulated in multiple cancers and its overexpression was involved in tumor cell proliferation and metastasis, such as glioma[30], gastric[31], pancreatic[32], colorectal[33], BC[34]. So we speculated that ZNF132 might inhibit the progression of BC by regulating the expression of ENO1 and CCNE1.
Aberrant promoter methylation can permanently inactivate tumor-associated genes, particularly tumor suppressor genes. In the present study, we found that the ZNF132 methylation level was higher in BC tissue than that in normal tissues.. Besides, 3 DNA methyltransferases were also overexpressed in the ZNF132low group. Simultaneously, methylated modification of ZNF132 was detected in 6 BC cell lines and 5-Aza-dC treatment could restore the mRNA and protein levels of ZNF132 in some cell lines, including MDA-MB-231, MCF7, and HCC1937. However, 5-Aza-dC could not restore the expression of ZNF132 between the mRNA and protein levels in the T47D cell. Besides, the MDA-MB-453 cell did not restore ZNF132 expression at the protein level. Moreover, an opposite trend was obtained in DU4475. The most likely reason is that the ZNF132 gene may undergo other epigenetic modifications, including transcription and post-transcriptional regulation. Finally, methylation analysis in 10 CpG island sites, including cg169294963, cg11618529, cg07878486, cg00868383, cg24366702, cg00547077, cg13877915, cg03735888, cg12042659, and cg19776201, indicated that these sites were hypermethylated in BC sample, suggesting that DNA methylation in these sites might inactivate ZNF132 gene transcription. Importantly, clinical samples analysis provided us a strong evidence between promoter methylation status and expression of ZNF132 in BC. Therefore, ZNF132 hypermethylation may act as an independent risk factor in BC patients.