In the study, we firstly evaluated AGR2 expression in BRCA. Generally, AGR2 upregulates in non-basal-like BRCA compared to normal tissues, which is consistent with previous studies18,19. We also found that AGR2 level was the lowest in basal-like BRCA and was even lower than in normal tissues. Consistently, Jilong Guo et al. performed immunohistochemistry and revealed that AGR2 was significantly upregulated in non-TNBC tissues than TNBC tissues, and was also significantly downregulated in TNBC than normal tissues20. Basal-like breast cancer, also known as triple-negative breast cancer (TNBC), lacks estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) amplification. Compared with other subtypes, the TNBC subtype has poorer prognosis, higher mortality and the highest probability of recurrence after treatment. Its poor outcome is due to the lack of available targeted therapies, and its intrinsically aggressive nature21. We also found that AGR2 expression was higher in mucinous breast cancer (MBC) than invasive ductal cancer (IDC) and invasive lobular cancer (ILC). As a rare histological cancer type, MBC exhibits lower malignancy and a comparatively better prognosis. A study showed that patients with MBC had a significantly better outcome than patients with IDC22. Plus, lower AGR2 level was found in SBR3 group and NPI3 group. It appears that AGR2 expressions are lower in more invasive tumors, though Wang Z suggested that increased AGR2 contributes to tumor growth and invasion and metastasis formation23.
To understand the reasons behind AGR2 overexpression in BRCA, we explored the relationship between AGR2 mRNA expression and CNV or DNA methylation. Because of changes in gene sequence location, CNV can affect gene expression, increase disease susceptibility and aggravate disease progression24. Changes in DNA methylation have an impact on regulating gene expression during cancer progression and metastasis25. Hence, we planned to explore what may be the potential reason behind changes of AGR2 mRNA expression in BRCA at the DNA level. Our study found that AGR2 copy number was significantly increased in BRCA, and the main type of AGR2 alteration was amplification. However, AGR2 CNV was not associated with mRNA expression or survive. But we found AGR2 mRNA expression was negatively associated with DNA methylation status, lower methylation status indicating higher mRNA level. HYE YOUN SUNG et al analyzed AGR2 mRNA expression and DNA methylation data in metastasized ovarian tumor in the mice and showed mRNA overexpression and hypomethylation at CpG sites in its promoter region26. Furthermore, they revealed that treatment with 5-aza-2'-deoxycytidine, a kind of the DNA methyltransferase inhibitor, could reduce the level of CpG methylation in the AGR2 promoter and increase the level of AGR2 expression. We carried out further exploration and found AGR2 methylation status were lower in BRCA than normal tissues, and it showed strong negative correlation between AGR2 methylation and mRNA expression at probe cg25343204, where also showed significant association with survival, lower methylation status showing better outcome. Thus, for the first time, we propose that upregulation of AGR2 mRNA in BRCA may be caused by DNA methylation rather than CNV.
The prognostic value of AGR2 remains largely inconclusive. A study demonstrated that higher level of AGR2 was related to less aggressive tumors and better prognosis in epithelial ovarian carcinoma27, which is in line with Ames and colleagues who found absence of AGR2 was associated with increased malignancy and tumor progression, and relapse in endometrioid and muinous subtypes28. AGR2 overexpression was found to contributed to the poor OS in lung cancer29, whereas another study showed that AGR2 overexpression did not show any prongnostic value in non-small-cell lung cancer30. Fritzsche FR et.al found that for patients with AGR2-expressing breast carcinomas, significantly longer OS became apparent than those without AGR2-expression19. However, other reports suggested that high AGR2 expression was related to poor outcome in BRCA31–33. In our study, no significant prognostic values of AGR2 were found according to GEPIA2 database. Interestingly, in subtype analysis, for patients with basal-like BRCA, higher AGR2 level showed better outcome according to TCGAPortal and bc-GenExMiner v4.4 databases. Combining data from our expression analysis of AGR2, it seems like that AGR2 plays a protective role in BRCA due to its higher level in less aggressive tumors such as Luminal subtype and MBC BRCA.
Many findings are in favor of the hypothesis that AGR2 is an oncogene and can promote tumor cell survival, migration, and invasion; drug resistance; angiogenesis; and metastasis2,4,23,34−36. However, Shao-bo Tian and colleagues performed a systematic review and meta-analysis about the prognostic value of AGR2 expression in solid tumors, and speculated that AGR2 might act as a tumor suppressor in some tumor types, which needed further verifications37. Hence, we performed functional analysis of AGR2 for better understanding. GO and KEGG pathway enrichment analyses of AGR2 associated genes in BRCA were performed by using GSEA. Go term analysis indicated that these genes were mainly enriched in chromosome segregation (BP), DNA conformation change (BP), chromosomal region (CC), and GABA receptor activity (MF). KEGG pathway analysis demonstrated that “ribosome biogenesis in eukaryotes” was the most significant signaling pathway, and genes enriched in this pathway were negatively associated with AGR2. Ribosomes play important roles in translating information contained in mRNAs into functional proteins, the final step in the genetic programme38. Ribosome biogenesis can increase the size of nucleolar organizing regions (NORs) which have long been used as a marker of tumor cell proliferation that negatively correlates with patient survival39. Richard Y. et al. indicated that the increasing of ribosomal content in epithelial cell fates may enhance their metastatic potential in breast cancer40. Studies also demonstrated that drugs inhibiting ribosome biogenesis might provide a feasible therapeutic method for cancer treatment41. Moreover, Varsha Prakash et al. demonstrated that upregulation of ribosome biogenesis could fuel the execution of Epithelial-to-Mesenchymal Transition (EMT), which is a migratory cellular program connected with development and tumor metastasis, and inhibition of rRNA synthesis in vivo turns primary tumors into a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative (a fundamental constituent of the EMT-promoting mammalian target of rapamycin complex 2 ) phenotype and reduced metastasis42. Lucia Sommerova et al. described that loss of intracellular AGR2 in cell lines caused a variation from epithelial to mesenchymal phenotype and establishment of intracellular AGR2 expression launched EMT43. However, other studies suggested that extracellular AGR2 could interrupt adherens junctions, disrupt basal laminin and activate fibroblast-associated cancer invasion, promoting epithelial morphogenesis and tumorigenesis44. To date, no researches about correlation between AGR2 and ribosome biogenesis have been reported. For the first time, we speculate that AGR2 may be a potential regulator of ribosome biogenesis in BRCA. In our opinion, the function of AGR2 should be explored according to different ER status and its different locations: intracellular or extracellular.
There were still limitations to the current study. Firstly, all the results were validated by using web tools, meaning a lack of in vitro and in vivo experiments. Secondly, all the researches should be performed in different subtypes respectively, due to results may be affected by ER, PR, or Her2 status.