Lin28A activates androgen receptor via regulation of c-myc and promotes malignancy of ER-/Her2+ breast cancer

Abstract

Having previously demonstrated the co-expression status of the Lin28A and androgen receptor (AR) in ER-/Her2+ breast cancer, we tested the hypothesis that Lin28A can activate AR and promotes growth of ER-/Her2+ breast cancer. The expression of Lin28A and AR were examined after Lin28A siRNA and Lin28A plasmid were transfected into ER-/Her2+ breast cancer cells. Chromatin immune-precipitation (ChIP) analysis and Luciferase Assays were used to evaluate the effect of Lin28A and c-myc on AR promoter activity. MTT assays, Boyden chamber invasion assays, colony formation assays and flow cytometry analysis were performed. ER-/Her2+ breast cancer cells which transfected with Lin28A siRNAs and Lin28A plasmid were injected into nude mice, and tumorigenesis was monitored. Our data showed that Lin28A can induced AR expression in ER-/Her2+ breast cancer cells. ChIP analysis showed that Lin28A stimulates the recruitment of c-Myc to the promoter of the AR gene. Lin28A enhanced growth ability, colonies ability, cells proliferation activities, invasive ability and inhibited cells apoptosis of ER-/Her2+ breast cancer cells. Lin28A high expression cells exhibited significantly higher tumorigenic ability in vivo. Our study demonstrates that Lin28A can activates androgen receptor via regulation of c-myc and promotes malignancy of ER-/Her2+ breast cancer. Our findings underline a novel role for Lin28A in breast cancer development and activation of the AR axis.

Keywords: Her2; Lin28A; androgen receptor; breast cancer; c-myc.

Figure 1. Lin28A increases AR and c-myc expression

Relative expression levels of Lin28A A. and AR B. were determined in MDA-MB-231, MDA-MB-453, and SK-BR-3 cells by qRT-PCR. Results are presented as relative fold change compared to expression levels in MDA-MB-231 cells. C. MDA-MB-453 cells were transfected with Lin28A siRNA#2 plasmid, and AR and c-myc mRNA levels were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in MDA-MB-453 cells transfected with control plasmid. Con, control. D. Western blot analysis showing the decrease in AR and c-myc protein expression in MDA-MB-453 cells transfected with Lin28A siRNA#2 plasmid. Actin is shown as loading control. E. SK-BR-3 cells were transfected with Lin28A plasmid, and AR and c-myc mRNA levels were analyzed by qRT-PCR. F. Western blot analysis showing the up-regulation of AR and c-myc protein expression in SK-BR-3 cells transfected with Lin28A.

Figure 2. Regulation of AR by Lin28A is mediated by regulation of c-myc

A. MDA-MB-453 cells were cotransfected with control or Lin28AsiRNA along with the pGL4-AR-Prom-Luc reporter. AR promoter activity was repressed when Lin28A was down-regulated. Data points represent the mean±S.D. of triplicate samples from two independent experiments. RLU: Relative Luciferase Units. B. SK-BR-3 cells were cotransfected with control or Lin28A along with the pGL4-AR-Prom-Luc reporter. AR promoter activity was enhanced in Lin28A-overexpressing cells. C. MDA-MB-453 cells were cotransfected with control or c-myc shRNA along with the pGL4-AR-Prom-Luc reporter. Luciferase assays showed that down-regulation of c-myc reduced transactivation of the AR promoter. D. SK-BR-3 cells were cotransfected with control or c-myc along with the pGL4-AR-Prom-Luc reporter. Overexpression of c-myc enhanced AR promoter activity. E. SK-BR-3 cells were transfected with Lin28A alone or Lin28A and c-myc shRNA together along with the pGL4-AR prom-Luc reporter. Repression of c-myc could overcome the promotion of AR promoter activity by Lin28A. F, G. Recruitment of c-myc to the c-myc binding site in the AR promoter was analyzed by ChIP assays. Overexpression of Lin28A enhanced the recruitment of c-myc to the AR promoter. Down-regulation of Lin28A reduced the recruitment of c-myc to the AR promoter.

Figure 3. Lin28A promotes growth, invasive and clonogenic ability of ER-/Her2+ breast cancer cells

A, B. MDA-MB-453 cells were transfected with empty vector or Lin28A siRNA#2 plasmid and SK-BR-3 cells were transfected with empty vector or Lin28A plasmid. Cells growth was monitored at 0, 24, 48, 72 hours. Expression of Lin28A enhanced growth rates of ER-/Her2+ breast cancer cells. Western blot analysis of a series of cell proliferation related molecules, including GRO-α and YB-1 were detected. C, D. Boyden chamber invasion assays showed that Lin28A increases invasiveness of ER-/Her2+ breast cancer cells. Inset: Representative images of invading cells. Western blot analysis of E-cadherin and Vimentin were detected. E, F. Lin28A-expressing ER-/Her2+ breast cancer cells exhibited higher clonogenic ability compared to control cells. Inset: Representative images of clonogenic cells. Data are presented as means±SD of three experiments performed in triplicate.

Figure 4. Lin28A played a critical role in the G1/S progression and apoptosis in ER-/Her2+ breast cancer cells

A. The flow cytometry analysis showed that the number of MDA-MB-453 cells in G1 phase increased, while the number of cells in S phase decreased in the Lin28A siRNA#2 group compared to the control group. Western blot analysis of a series of cell cycle-related molecules, including PCNA and cyclin E were detected. B. The number of SK-BR-3 cells in G1 phase decreased, while the number of cells in S phase increased in the Lin28A group compared to the control group. Western blot analysis of a series of cell cycle-related molecules, including PCNA and cyclin E were detected. C. The percentage of apoptotic cells was higher in the Lin28A siRNA#2 group compared to the control group in MDA-MB-453 cells. Western blotting was used to analyse the levels of the anti-apoptosis marker Bcl-2, pro-apoptotic marker procaspase-3 and cleaved caspase-3. D. The percentage of apoptotic cells was lower in the Lin28A group compared to the control group in SK-BR-3 cells. Western blotting was used to analyse the levels of the anti-apoptosis marker Bcl-2, pro-apoptotic marker procaspase-3 and cleaved caspase-3.

Figure 5. Lin28A promotes tumor growth of ER-/Her2+ breast cancer in vivo

We injected 2×10⁶/100ul Lin28A siRNA MDA-MB-453 cells or control cells and Lin28A SK-BR-3 cells or control cells into subcutaneous of female nude mice. A. Mice tumors which injected with Lin28A siRNA MDA-MB-453 cells exhibited significantly lower rates of incidence and growth of tumors compared to control cells(Left: tumor growth curve graph; Right: mice and tumor). B. Mice tumors injected with Lin28A SK-BR-3 cells exhibited significantly higher tumors growth compared to control cells(Left: tumor growth curve graph; Right: mice and tumor). C, D. Total RNA extracts from the tumors were examined for expression levels of Lin28A and AR. Lin28A was correlated positively with expression levels of AR in ER-/Her2+ breast tumor.

Figure 6. The expression of Lin28A, AR, and Ki67 in xenograft tumor was detected by using immunohistochemistry

Breast cancer cells were inoculated in the left inguinal mammary fat pad of syngeneic Balb/c mice, and the mice were divided into four groups. In mice tumors which injected with MDA-MB-453 cells, Lin28A siRNA group showed downregulation of Lin28A A., AR B. and Ki67 C. expression compared with the control group D, E, F. In mice tumors which injected with SK-BR-3 cells, Lin28A group induced upregulation of Lin28A G., AR H. and Ki67 I. expression compared to the control group J, K, L.

Figure 7. Hematoxylin-eosin staining of mice lungs A. and livers B