The formation of L-isoaspartateyl, D-isoaspartateyl, and D-aspartic acid generally results in structurally nonfunctional peptides or proteins[14, 15]. As a protein repair enzyme, the main function of PCMT1 are to initiate the conversion of spontaneously isomerized aspartyl residues to their normal configuration[16]. Recent decade, most studies of PCMT1 focused on exploring its functions in protein repair[16], synaptic transmission[17], cellular matrix interactions[18–20] and lifespan[21, 22].
Recently, it has been found that PCMT1 is involved in tumor development and progression, and might be a potential biomarker for diagnosis or prognosis of several tumors. For example, PCMT1 expression was significantly higher in hepatocellular carcinoma, bladder cancer, advanced-stage lung adenocarcinoma and metastatic ovarian cancer, and higher PCMT1 expression predicted poor prognosis of above malignant tumors[6, 7, 11, 23]. Up to now, although it has showed that PCMT1 mRNA was highly expressed in BC, and the survival of BC patient with higher expression of PCMT1 was significantly poorer than those with lower expression[8–10]. However, no clinical information regarding the relationship between PCMT1 protein expression and clinicopathological characteristics of BC patients has been reported. In our study, based on the TCGA-BRCA data set, we explored the prognostic value of PCMT1 gene in BC. The results showed that high expression of PCMT1 gene was significantly correlated with shorter OS, DSS and PFS of BC patients, which is consistent with the previous studies[8–10]. Furthermore, the results of immunohistochemical assay indicated that PCMT1 protein expression was only obviously correlated with progesterone receptor expression of BC patients, but higher PCMT1 protein expression was an independent unfavorable prognostic factor for BC patients. Therefore, either at the mRNA or the protein level, PCMT1 high-expression might serve as a potential unfavorable prognostic factor for BC.
As for the biological function of PCMT1 in tumors, a previous study showed that the reduced motility of cells on aged type-I collagen could be restored by PIMT-treated repaired collagen by up to 72%[24]. PIMT could suppress the activity of p53 through methylation of its asparagine residues 29 and 30 and enhancing the p53-HDM2 interaction, which reduced gene expression dependent on p53 and protected cancer cells from apoptosis[10]. The latest research showed that PCMT1 promoted the migration and invasion ability of glioblastoma cells and plays a critical role in the development of glioblastoma[25, 26]. Through binding with LAMB3, PCMT1 activated integrin-FAK-Src signaling to enhance ovarian cancer cell adhesion, migration, invasion and metastasis formation[11]. PCMT1 also participated in bladder cancer cell migration and invasion by regulating epithelial-mesenchymal transition-related genes[6]. However, the study of Yamashita et al indicated that inhibition of PIMT expression under endoplasmic reticulum stress facilitated cell EMT process and invasion in some cell types of lung adenocarcinoma[27]. These studies showed that PCMT1 is important for the occurrence and development of a variety of malignant tumors. In addition, Ryu et al reported that PCMT1 and phosphorylated ERK were activated and expressed during the cell EMT process, and synergistically enhanced the migration and invasion of MDA-MB-231 cell[5]. In order to further explore the functions of PCMT1 in BC, we performed loss-of-function experiments in MCF-7 cell, and found that PCMT1 knockout cells displayed a clear inhibition in growth, migration and invasion. In the other hand, EMT process is a developmental program that enables epithelial cells drop their epithelial morphology and gain a mesenchymal phenotype, characterized by the reduction of epithelial protein such as E-cadherin, and the increase of mesenchymal protein such as N-cadherin and vimentin. Snail and β-catenin are key transcription factors in the regulation of EMT process[28]. In addition, Cyclin D1 is mostly known for the role played in the nucleus, as regulator of cell cycle progression[29]. Bcl2 has also been shown to inhibit cell cycle entry by decelerating the accumulation of E2F1, a critical inducer of cell cycle entry, and acting through mechanisms independent of Rb but dependent upon p130 and p27, all of which are negative regulators of cell cycle[30]. In order to explore the molecular mechanisms by which PCMT1 contributes to MCF-7 cell motility and growth, we performed western blot to detect the protein level of EMT and cell cycle-associated molecules. The results showed that E-cadherin, N-cadherin, Snail, β-catenin, Cyclin D1 and Bcl2 expression could be regulated by PCMT1, revealing that PCMT1 possessed a critical function in the biochemical regulation of cell cycle and EMT process, and further influnced the cell growth and motility. In addition, the AKT/GSK3β/mTOR is a signaling pathway involved in cell cycle, EMT process, glucose metabolism and DNA repair, and to play an important role in cell survival, invasion, migration and apoptosis[13, 31–33]. In this study, we also observed that the activity of AKT/mTOR signaling pathway was inhibited, while GSK3β was strengthened in PCMT1 knockout cell. This may explain the significant inhibition of cell growth and motility, as well as the expression of EMT and cell cycle-associated protein by PCMT1 knockout, which required to be tested further.