Background
Breast carcinoma (BRCA) is one of the most common malignant neoplasms in humans and has a high cancer-related morbidity in females, ranking 6th in mortality for females [
1]. BRCA is a highly heterogeneous neoplasm [
2], and the mechanism underlying its initiation and development remains unclear. Therefore, the identification of biomarkers for early diagnosis, prognosis judgment and treatment of BRCA is urgently needed. Ubiquitin-conjugating enzyme E2C (UBE2C), a crucial part of the ubiquitin-conjugating enzyme complex, is involved in the ubiquitin–proteasome system. The ubiquitin–proteasome pathway is one of the main pathways of protein degradation in eukaryotes and serves as an important component in the post-translational modification of proteins. The process of ubiquitination is associated with many biological processes [
3‐
6]. In recent studies, the dysregulation of the ubiquitination process has been discovered to play essential roles in the occurrence and progression of cancers, and ubiquitination has, thus, become a new therapeutic target for cancer [
7‐
9]. Previous studies have reported overexpression of UBE2C in some cancers such as colorectal carcinoma [
10], pancreatic carcinoma [
11], cervical cancer [
12], bladder carcinoma [
13], esophageal squamous cell carcinoma [
9] and lung cancer, etc. [
14]. Particularly, cancers with a high degree of malignancy, low differentiation and high metastatic tendency usually present with higher UBE2C expression and poor patient survival [
3]. Although several studies have confirmed UBE2C overexpression in BRCA and the prognostic significance of UBE2C in BRCA [
8,
15‐
17], the specific role and molecular mechanism of UBE2C expression in BRCA is unclear. Therefore, in this study, we employed immunohistochemistry (IHC) and bioinformatics analysis guided by public databases containing data on gene expression in cancer to detect UBE2C expression in BRCA and adjacent tissues. Moreover, we investigated the clinico-pathological significance of UBE2C expression in BRCA and endeavored to elucidate the molecular mechanism underlying it.
Discussion
Breast carcinoma is one of the most common malignant neoplasms that pose a serious threat to women’s life and health. It is of great importance to delve into the pathogenesis of BRCA and identify molecular biomarkers for BRCA with high sensitivity and specificity. UBE2C, as a crucial member of the ubiquitin-conjugating enzyme family (E2), plays a pivotal role in the ubiquitin–proteasome proteolytic (UPP) pathway. Disorder of the UPP pathway initiates abnormal degradation of proteins encoded by some oncogenes and tumor suppressor genes, subsequently leading to abnormal accumulation of these proteins in the body. Therefore, the UPP system is closely related to the occurrence and progression of cancers [
28,
29]. With respect to the relationship between UBE2C and BRCA, Parris et al. observed higher expression of UBE2C in invasive BRCA tissues than in normal breast tissues [
15]. The prognostic value of UBE2C has also been validated to be significant in high-risk early BRCA and node-positive BRCA samples [
16,
17]. In the research conducted by Rawat et al., the suppression of UBE2C could inhibit growth of BRCA cells and sensitized breast cancer cells to radiation, doxorubicin, tamoxifen and letrozole [
30]. Despite the above findings, the exact function and molecular basis of UBE2C in BRCA has remained elusive. We have, for the first time, comprehensively investigated the protein expression and gene alteration of UBE2C in BRCA as well as the clinico-pathological significance of UBE2C in BRCA using a combination of immunohistochemistry (IHC) and excavation of gene expression data from public databases.
To test the hypothesis that UBE2C exerts a carcinogenic influence on BRCA, we first detected UBE2C expression in BRCA and matched normal tissues by using IHC. The results from our immunohistochemistry experiments showed that highly positive UBE2C expression in BRCA tissues contrasted sharply with the negative UBE2C expression in adjacent tissues. In addition, the expression of UBE2C was positively correlated with histological grade, tumor size, clinical stage and lymph node metastasis of BRCA. In vitro experiment in previous articles have also proved that UBE2C could enhance the proliferative, viability and invasive capacity through MTT, colony formation assay and invasion assays in lung cancer and prostate cancer [
31,
32], which increased the reliability of our result that UBE2C exerted an oncogenic influence on breast cancer cells. There was also a difference between UBE2C expression in different molecular subtypes of breast cancer, among which the positive rate of UBE2C expression in HER2-overexpression BRCA was the highest, while the positive expression rate of UBE2C was the lowest in Luminal A BRCA. In the analysis of the relationship between UBE2C expression and other common biomarkers for BRCA, UBE2C expression was positively correlated with the expression of P53, HER2 and Ki-67. Conversely, UBE2C expression was negatively correlated with ER and PR expression. Research conducted by Pan et al. confirmed the overexpression of UBE2C in BRCA tissues as well as the significant correlation between UBE2C expression and the degree of cell differentiation, molecular types, and Ki-67 or HER2 expression [
33]. Similarly, Berlingier et al. also reported that the expression of UBE2C in BRCA tissues was significantly higher than that in normal breast tissues, and UBE2C expression was positively correlated with Ki-67 and HER2 expression (P < 0.05) [
34], which agreed with our results. With regard to the prognostic value of UBE2C expression in BRCA, a study from Psyrri et al. demonstrated that BRCA patients with high UBE2C mRNA expression experienced worse overall survival [
16]. Furthermore, a Cox multivariate regression analysis from the study of Psyrri et al. suggested that UBE2C mRNA expression was an independent prognostic factor for BRCA patients [
16], which was in accordance with the results from the Kaplan–Meier survival analysis for BRCA patients with different UBE2C expression in Oncolnc. Nevertheless, the Kaplan–Meier survival analysis and multivariate Cox hazard regression analysis in our study from IHC data yielded contradictory results that there was no statistically significant difference between UBE2C expression and the survival of BRCA patients. We considered that the limited patient samples and short follow-up time might contribute to the contradictory results. There were only eight patients that died during the follow-up time before April 16, 2016, which might have had a significant impact on the results. Further studies with larger patient cohorts and longer follow-up time were necessary to assess the prognostic significance of UBE2C on survival of BRCA patients.
Apart from the IHC data, UBE2C gene expression data from public databases including the HPA database, Oncomine and Firebrowse provided a consistent result showing UBE2C overexpression in BRCA tissues, which supported the IHC results. Specifically, we demonstrated an increasing significance in the difference between UBE2C expression in BRCA tissues and normal tissues with the enhanced malignancy of the same type of BRCA using data from the Oncomine database. We speculated that UBE2C expression was indicative of the malignant progression of BRCA. Moreover, the result that UBE2C expression in ductal carcinoma was obviously higher than most other types of breast cancer suggested that UBE2C could be used as a potential marker for distinguishing ductal carcinoma from other types of breast cancers.
To facilitate a comprehensive understanding of the mechanism of UBE2C expression in BRCA, we investigated gene alteration of UBE2C in BRCA with available data from cBioPortal. From the BRCA cases with UBE2C gene alteration, amplification and mRNA up-regulation comprised the major types of gene alteration, which might be triggered by the aberrant expression of upstream molecules that moderate the expression of target mRNAs. MicroRNA (miRNA), a short non-coding RNA 21–24 nucleotides in length that silences target mRNA expression through complete or incomplete binding to the 3′-untranslated region (3′-UTR) of the target mRNAs [
35‐
43], is an typical example of a molecule that regulates downstream mRNA expression. Recently, accumulating studies have noted that miRNAs were engaged in a series of biological processes including proliferation, diversification, metastasis, and apoptosis in the development of cancer [
44‐
52]. Several studies have also revealed that miRNAs interact with UBE2C to affect the progression of cancers. In gastric cancer, miRNA-17/20 promoted gastric cancer cell growth via targeting UBE2C [
53]. In BRCA, the study of Han et al. identified UBE2C as a direct and functional target of miR-196a which can upregulate the expression of UBE2C [
54]. Meanwhile, we obtained a list of miRNAs including miR-671, miR-615, miR-20a, miR-17 and miR-196a through the gene-miRNA targets function of miRWalk 2.0, which were predicted to regulate the expression of UBE2C. Thus, we hypothesized that UBE2C might serve as a substrate of specific miRNAs that stimulate the overexpression of UBE2C and exert an oncogenic influence on BRCA. Future studies were needed to investigate the role for microRNAs in regulation of UBE2C expression. To trace the origins of the UBE2C gene alteration, we explored the interactions between UBE2C and the top 50 frequently altered neighboring genes. From the gene network, some genes such as ASB7, ARIH2, KLHL20, KLHL9, ASB13, HERC2 and MGRN1 can be seen to form a complex with UBE2C; some other genes, including TRIM11, PSMD12, WWP2, SMD3 and PSMD8, directly interact with UBE2C. We hypothesized that the gene alteration of UBE2C might be explained by the abnormal activities of these genes, and these genes may also exert carcinogenic effects in BRCA through cooperating with UBE2C. Further studies are warranted to validate the interaction between UBE2C and these genes in the tumorigenesis of BRCA.
Authors’ contributions
CHM: performed most of the experiments as well as statistical analysis and supervised the progression of research. LG: analyzed and interpreted data and drafted the manuscript. XFZ, KLW, JJZ, GC and ZBF: participated in sample collection and provided information from database. All authors read and approved the final manuscript.