Role of Brf1 interaction with ERα, and significance of its overexpression, in human breast cancer

Abstract

TFIIB-related factor 1 (Brf1) modulates the transcription of RNA Pol III genes (polymerase-dependent genes). Upregulation of Pol III genes enhances tRNA and 5S RNA production and increases the translational capacity of cells to promote cell transformation and tumor development. However, the significance of Brf1 overexpression in human breast cancer (HBC) remains to be investigated. Here, we investigate whether Brf1 expression is increased in the samples of HBC, and we explore its molecular mechanism and the significance of Brf1 expression in HBC. Two hundred and eighteen samples of HBC were collected to determine Brf1 expression by cytological and molecular biological approaches. We utilized colocalization, coimmunoprecipitation, and chromatin immunoprecipitation methods to explore the interaction of Brf1 with estrogen receptor alpha (ERα). We determined how Brf1 and ERα modulate Pol III genes. The results indicated that Brf1 is overexpressed in most cases of HBC, which is associated with an ER-positive status. The survival period of the cases with high Brf1 expression is significantly longer than those with low levels of Brf1 after hormone treatment. ERα mediates Brf1 expression. Brf1 and ERα are colocalized in the nucleus. These results indicate an interaction between Brf1 and ERα, which synergistically regulates the transcription of Pol III genes. Inhibition of ERα by its siRNA or tamoxifen reduces cellular levels of Brf1 and Pol III gene expression and decreases the rate of colony formation of breast cancer cells. Together, these studies demonstrate that Brf1 is a good biomarker for the diagnosis and prognosis of HBC. This interaction of Brf1 with ERα and Brf1 itself are potential therapeutic targets for this disease.

Keywords: Brf1; ERα; Pol III genes; breast cancer; survival period.

Figure 1

Brf1 IHC staining of samples of HBC. (A); Brf1 staining. (A1,A3) IHC staining of Brf1 of HBC tumor tissues; (A2,A4) H&E staining of HBC tumor tissues. (A1,A2) 100× magnification; (A3,A4) 1000× magnification. A representative Brf1 staining of HCC samples. (B); Comparison of Brf1 staining in tumor foci or para‐can tissue of HBC. (B1,B3): Strong staining signals of Brf1 expression are seen in tumor foci of HBC; (B2,B4): Weak signals of Brf1 staining are detected in para‐can tissue of HBC. (B1,B3) 100× magnification; (B2,B4) 1000× magnification.

Figure 2

Comparison of Brf1 expression in tumor foci and ANT. (A): Brf1 staining. The levels of Brf1 expression were detected in four breast adenocarcinoma lesions (A, upper panel) and their paired ANT (A, lower panel). Brf1 expression was increased in the four breast adenocarcinoma lesions, compared to their matched no cancerous tissues. Magnification, 100×. (B): Staining intensity of Brf1 in the breast adenocarcinoma tumor tissues. In terms of the staining intensity of Brf1, the cases were divided into four groups: negative staining, weak staining, moderate staining, and strong staining from left side to right side (B, upper panel). Magnification, up: 100×; down: 400×.

Figure 3

Kaplan–Meier survival curve and log‐rank test analysis of the association between Brf1 expression and HBC patient survival. Brf1 expression of 218 HBC cases was determined by pathological analysis and IHC staining. (A) Total patients; (B) ER low expression or negative subgroup and (C) non‐triple‐negative breast cancer subgroup. n = number of patients in the subgroup; M = median survival in months of the subgroup. The group of high Brf1 expression or with ER+ status or non‐TNBC group display longer survival period. P‐values were calculated by log‐rank test.

Figure 4

Repression of ERα decreases expression of Brf1. (A): Expression of Brf1 and ERα in breast adenocarcinoma. Western blots show that the expression levels of Brf1 and ERα protein in four breast adenocarcinoma tumor tissues (T) are markedly higher than those of their paired ANTs. β‐Actin was used as a loading control. (B–C): ERα siRNA decreased the induction of ERα and Brf1 caused by ethanol. MCF‐7 cells were transfected with mismatch RNA (mm RNA) as a control RNA or ERα siRNA for 48 h and treated with ethanol as described previously (Zhang et al., 2013). The cell lysates and total RNA were extracted from these cells to determine protein levels of ERα, Brf1, and β‐actin by western blot (B and C, right panels). mRNA levels of ERα and Brf1 were measured by RT‐PCR (B,C, left panels). (D): ERα occupancy of Brf1 promoter: Chromatin was extracted from ethanol‐treated MCF‐7 cells to carry out ChIP assay with ERα or histone H3 antibodies, respectively. H3 is used as a control. These results indicate that ERα modulates Brf1 expression.

Figure 5

Colocalization and Interaction between Brf1 and ERα. Colocalization: Brf1 (red) and ER‐α (green) of the human breast adenocarcinoma tumor tissues were determined by immunofluorescence staining (A). The results indicate that both Brf1 and ERα are localized in nucleus of the tumor cells. Merging picture clearly shows that Brf1 and ERα reveal colocalization in nucleus of HBC biopsy (B). Magnification, up: 200×; down: 1000×. Interaction between Brf1 and ERα: MCF‐7 cells were treated with ethanol to extract cell lysates and to perform immunoprecipitation with Brf1 and ERα antibodies, respectively. Western blot analysis indicates that Brf1 antibody is able to put down ERα protein (C), whereas the antibody of ERα can also precipitate Brf1 protein (D). The input samples at C and D were from the ethanol‐treated cells. The results reveal the interaction of Brf1 with ERα in ER+ breast cancer cells.

Figure 6

ERα modulates Pol III gene transcription. (A‐D): MCF‐7 cells were transfected with mismatch (mm) RNA, ERα siRNA, or Brf1 siRNA for 48 h, respectively. The cells were treated as described above. The amounts of pre‐tRNA^{L eu} (A,C) and 5S rRNA (B,D) were measured by RT‐qPCR. Repression of ERα (A‐B) or Brf1 (C–D) by their siRNA decreases Pol III gene transcription. The fold changes are calculated by normalizing to the amount of GAPDH mRNA. (E–F) ChIP assay: MCF‐7 cells were treated as described above in Fig. 4. The results indicate that ERα occupies the promoter of tRNA ^leu and 5S rRNA. The bars represent mean ± SE of at least three independent determinations.

Figure 7

Brf1 and Pol III genes are mediated by Tam. (A–D): MCF‐7 cells were treated with 12.5 μm Tam (tamoxifen). The cells were treated as described above. The levels of Brf1 protein were measured by western blot (B). RT‐qPCR was performed to determine the amounts of Brf1 mRNA (A) and pre‐tRNA^{L eu} (C) and 5S rRNA transcription (D). The fold changes were calculated by normalizing to the amount of GAPDH mRNA. The bars represent mean ± SE of at least three independent determinations. (E–F): Soft agar assay: MCF‐7 cells were transfected with mm RNA, ERα siRNA, or Brf1 siRNA for 48 h, respectively. The cells were seeded in 6× well plates and treated with ethanol (25 mm) and Tam (12.5 μm) as previously described (Zhong et al., 2014). The cells were analyzed for colony formation in soft agar. Colonies were counted at 2–3 weeks after plating. Values are the means ± SE (n ≥ 3). *P < 0.05 as indicated.

Figure 8

Schematic illustration of Brf1 and ERα mediating Pol III gene transcription. Stimulus induces activation of JNK1 to increase cellular levels of Brf1 and ERα. Tam represses Brf1 expression and reduces ERα activity. The interaction of Brf1 with ERα in turn upregulates Pol III gene transcription to promote cell proliferation and transformation, eventually resulting in breast cancer development.