miR-452 Reverses Abnormal Glycosylation Modification of ERα and Estrogen Resistance in TNBC (Triple-Negative Breast Cancer) Through Targeting UGT1A1

Abstract

Background: The breast epithelial cells in patients with triple-negative breast cancer (TNBC) actually have specific estrogen receptor (ER) expression, and the abnormal glycosylation of UGT1A1 in TNBC cells resulted in abnormal expression and function of ERα through regulating the modification of ERα. Therefore, our study targets the role of UGT1A1 expression, then glycosylation modification of ERα (estrogen receptor α) and estrogen resistance in development of TNBC. Methods: The differential expression of mRNA and miRNA in TNBC tissues was tested. Luciferase activity was analyzed in TNBC cells treated with miR-452. Moreover, the human mammary gland and TNBC cell lines were dealt with estrogen and miR-452 or its inhibitors, then proliferation ability was further determined. Moreover, the role of interaction between UGT1A1 and ERα in the glycosylation modification of ERα and UGT activity, and metabolism of estrogen were assessed. The effects of miR-452 on TNBC by improving abnormal glycosylation modification of ERα by targeting UGT1A1 and estrogen resistance were studied in vitro and in vivo. Results: The expression level of UGT1A1 in TNBC tumor tissues was higher than its matched para-tumorous tissues, but the miR-452 expression was opposite. The glycosylation modification site of ERα expressed in TNBC cells was different from that of normal mammary epithelial cells. The estrogen 17β-estradiol (E2) significantly promoted mitotic entry of TNBC cells. The interaction between UGT1A1 and ERα affected the expression level of each other, as well as the UGT enzyme activity and proliferation of TNBC cells. UGT1A1 induced production of intracellular estrogens and TNBC proliferation, but it could be reversed by overexpression of ERα. Upregulation of ERα caused the downregulation of UGT1A1 and marked decrease of intracellular estrogen products, and then suppressed TNBC proliferation. Moreover, UGT1A1 was the target gene of miR-452; miR-452 antagomir restrained TNBC xenograft. Conclusion: Our results demonstrated that estrogen was a positive factor in the proliferation of TNBC cells at onset of mitosis through accentuating the expression and enzyme activity of UGT1A1. However, miR-452 targeted to UGT1A1, then regulated glycosylation modification of ERα, estrogen metabolism, and TNBC development associated with estrogen resistance.

Keywords: UGT); estrogen receptor (ER); estrogen resistance; glycosylation modification; triple-negative breast cancer (TNBC); uridine diphosphate glucuronyl transferase (UDPGT.

Figure 1

Low expression of ERα and high expression of UGT1A1 in TNBC. To explore the expression of UGT1A1 in TNBC, immunohistochemistry staining was conducted. The results indicated a high expression level of UGT1A1 in tumorous tissues of patients with TNBC (A). We found that UGT1A1 was present in the nucleus, cytoplasm, and cell surface of para-tumorous tissues at a low expression level (B). The immunohistochemical staining was used to conduct the determination of ERα expression in TNBC tissues and their paired normal control tissues. Consequently, the positive stains of ERα were observed in the normal mammary gland tissues with the moderate level (C). However, in TNBC tissues, the low expression of ERα was visible in endothelium (D).

Figure 2

The expression of UGT1A1 in tissues of patients with TNBC. Moreover, the expression of UGT1A1 in tumorous tissues of patients with TNBC was much more than in the matched para-tumorous tissues.

Figure 3

Post-translational glycosylation modification of ERα induced by UGT1A1. The ERα protein expression in normal human breast epithelial cells has a modification site at the 10th amino acid with the O-GalNAc glycosylation (A), which was in accord with the bio-information analysis based on the online software of Uniprot (https://www.uniprot.org/) (B). However, the expression of ERα protein was present on the surface of TNBC cells with a low expression level, whereas the glycosylation site at the 10th amino acid site was UDP-GlcNAc glycosylation modification (C).

Figure 4

Estrogen upregulated the expression of UGT1A1 and its enzyme activity in TNBC cell line MDA-MB-231, and promoted cell proliferation. In this experiment, the cell line MDA-MB-231 was treated with 17β-estradiol (E2) in the presence of concentration gradients. Our results demonstrated that the 17β-estradiol (E2) could strongly promote the expression of UGT1A1 in a dose-dependent manner, but lightly upregulate the ERα expression (A). Moreover, the enzyme activity of UGT1A1 was determined, and these results indicated the presence of concentration gradients, too. Our results demonstrated that the E2 induced forcefully the UGT1A1 activity in a dose-dependent manner (B). Furthermore, the cell mitosis was released after using the block with double thymidine to synchronize cell at the G1/S phase, then the synthesis of DNA was evaluated with BrdU. Incorporating BrdU into the control, the mitotic cells treated with E2 increased obviously than control in a dose-dependent manner (C). Our results indicated that the expression and enzyme activity of UGT1A1 upregulated by E2 may control the DNA synthesis, and then transition of G1/S boundary or mitosis in TNBC cells. The flow cytometry and stain of phospho-H3 was additionally used to confirm the role of UGT1A1 expression and enzyme activity in mitotic entry of TNBC cells (D). Estrogen 17β-estradiol (E2) significantly promoted mitotic entry of TNBC cells. In summary, these results sustained powerfully the estrogen as a positive factor in the proliferation of TNBC cells at onset of mitosis through accentuating the expression and enzyme activity of UGT1A1. **P < 0.01, ***P < 0.001.

Figure 5

The interaction between UGT1A1 and ERα in TNBC cells. Moreover, the CO-IP assay was conducted to verify the interaction between UGT1A1 and ERα in TNBC cells (A). Furthermore, upregulating expression of ERα induced remarkably the reduction of UGT1A1 expression (B), and then the UGT enzyme activity also notably decreased in a time-dependent manner (D). At the same time, overexpression of ERα inhibited dramatically the proliferation of MDA-MB-231 cells in a time-dependent manner (F). Besides, upregulating expression of UGT1A1 caused prominently the decrease of ERα expression (C), and the UGT enzyme activity significantly increased in a time-dependent manner, too (E). Moreover, overexpression of UGT1A1 promoted markedly the proliferation of MDA-MB-231 cells in a time-dependent manner (G). *P < 0.05, **P < 0.01.

Figure 6

UGT1A1 induced production of intracellular estrogens. At first, the intracellular estrogen products in human mammary gland cell line MCF-12A, human breast cancer cell line MCF-7, and triple-negative breast cancer (TNBC) subtype cell line MDA-MB-231 were determined, respectively. In MDA-MB-231 cells, the productions of E1, E2, and E1S were all remarkably greater than that in MCF-12A and MCF-7. However, there was no significant difference between MCF-12A and MCF-7 (A). Moreover, the expression levels of ERα and UGT1A1 were detected in these cell lines (B). The results showed that the expression of UGT1A1 in MDA-MB-231 was much greater than MCF-12A and MCF-7, but the ERα expression in MDA-MB-231 was lower than MCF-12A and MCF-7. Meanwhile, the expression of UGT1A1 in MCF-7 was slightly more than MCF-12A, but there was no significant difference. Besides, the expression level of ERα in MCF-7 was slightly lower than MCF-12A, but there was no significant difference (B). In cell line MCF-12A, overexpression of UGT1A1 downregulated the expression of ERα (C), and then resulted in an obvious increase of intracellular estrogen products (D). Meanwhile, Ad-UGT1A1 promoted markedly the cell proliferation of MCF-12A (E). Besides, overexpression of ERα was induced in cell line MDA-MB-231, which caused the downregulation of UGT1A1 (F) and marked decrease of intracellular estrogen products (G). Meanwhile, Ad-ERα significantly reduced cell proliferation of MDA-MB-231 (H). **P < 0.01.

Figure 7

The expression level of miR-452 in tissues of TNBC. The expression level of miR-452 in non-tumorous, para-tumorous, and tumorous tissues of patients with TNBC were detected. The result showed that the relative expression level of miR-452 in tumor was remarkably lower than that of para-tumorous and non-tumorous tissues. However, there was no significant difference between non-tumorous and para-tumorous tissues. **P < 0.01, ***P < 0.001.

Figure 8

miR-452 inhibited UGT1A1 expression and its bio-functions in TNBC. Further experiments were conducted using luciferase reporter gene system to identify whether miR-452 could straightly bind with 3′UTRs of UGT1A1 mRNA. The data indicated 3′UTR luciferase activities of UGT1A1 significantly downregulated in TNBC treated with miR-452 (A). However, it did not show an obvious change in the group of UGT1A1 mutation (B). Moreover, miR-452 did not bind straight to 3′UTRs of ERα (C). Besides, miR-452 restrained UGT1A1 protein expression level and promoted ERα protein expression in TNBC cell line MDA-MB-231 (D). Moreover, miR-452 inhibitors could promote expression of UGT1A1, but suppress the ERα protein expression level, respectively (D). Furthermore, miR-452 induced a remarkable decrease in intracellular estrogen products in cell line MDA-MB-231 (E). Meanwhile, the inhibitors of miR-452 significantly reduced cell proliferation of MDA-MB-231 (F). **P < 0.01.

Figure 9

miR-452 antagomir suppressed TNBC xenograft. To detect anti-tumor activity induced by miR-452 antagomir in vivo, human TNBC xenografts were established with MDA-MB-231 cell line. The xenografts of control groups grew rapidly, but in the experiment group, which was treated with antagomir of miR-452, they grew slowly in vivo (A). Moreover, the weight of tumors in the experiment mice (treated with antagomir of miR-452) was significantly less than that of the control group (B,C). At the time of end, the average tumor volume of control mice was much larger than that of the experiment group, and the difference between the two groups had statistical significance (C). As we can see, the results revealed that compared with the control group, treatment with antagomir of miR-452 downregulated the expression of UGT1A1 with statistical significance and promoted protein expression of ERα in xenografts (D). *P < 0.05, **P < 0.01.