Abstract
Endocrine therapies, which inhibit estrogen receptor signaling, are the most common and effective treatments for estrogen receptoralpha-positive breast cancer. However, the utility of these agents is limited by the frequent development of resistance, and the precise mechanisms underlying endocrine therapy resistance remain incompletely understood. Here, we demonstrate that peptidyl-prolyl isomerase Pin1 is an important determinant of resistance to tamoxifen and show that Pin1 increases E2F-4- and Egr-1-driven expression of LC-3 as a result of an increased interaction with and phosphorylation of MEK1/2. In human tamoxifen-resistant breast cancer, our results show a significant correlation between Pin1 overexpression and high levels of LC-3. Promoter activity as well as expression levels of Pin1 were drastically higher in tamoxifen-resistant MCF7 cells than control MCF7 cells, as were levels of LC-3 mRNA and protein, an autophagy marker. Pin1(-/-) mouse embryonic fibroblasts showed lower 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MEK1/2 phosphorylation than Pin1(+/+) mouse embryonic fibroblasts. Silencing of Pin1 expression inhibited TPA-induced MEK1/2 phosphorylation in MCF7 cells. Moreover, PD98059, a specific inhibitor of MEK1/2, and juglone, a potent Pin1 inhibitor, significantly suppressed the TPA-induced expression of E2F-4 as well as Egr-1 transcription factors, which control LC-3 gene expression. Importantly, 4-hydroxy tamoxifen, when used in combination with silencing of Pin1 or LC-3, increased cleaved poly(ADP-ribose) polymerase and DNA fragmentation to inhibit cologenic growth of MCF7 cells. We therefore link the Pin1-MEK pathway and LC-3-mediated tamoxifen resistance and show the therapeutic potential of Pin1 in the treatment of tamoxifen-resistant breast cancer.
Highlights
Tors (ERs),3 and after surgery, they can be treated with hormonal therapy alone, in the absence of more toxic chemotherapy, resulting in a relatively favorable prognosis [2]
To further examine whether Pin1 or LC-3 facilitates the development of antiestrogen resistance, we did a drug selection in which MCF7 cells were exposed to small, incremental increases of 4-OH tamoxifen
The stepwise drug selection was continued until the MCF7 cell population could sustain viability and proliferate when challenged with 3.0 M 4-OH tamoxifen
Summary
Reagents and Antibodies—Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimal essential medium, L-glutamine, gentamicin, and FBS were purchased from Invitrogen. DMEM containing 10% charcoal/dextran-treated FBS and 3 M 4-OH tamoxifen were used to culture the tamoxifen-resistant MCF7 breast cancer cells (TAMR-MCF7), which were provided by Dr Keon Wook Kang (College of Pharmacy, Chosun University). Detection of Apoptosis—An apoptosis assay was performed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling, and apoptosis was detected with an in situ cell death detection kit (Roche Applied Science) according to the manufacturer’s instructions. Cells were stained with terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end-labeling solution and incubated at 37 °C for 2 h, washed twice with phosphate-buffered saline, and mounted with crystal mount reagent for 4 h in the dark. Cells were transfected with siRNA-sc or -Pin, incubated for 48 h, and treated with 4-OH tamoxifen. Data from cell viability, promoter activity, RT-PCR, or soft agar assay were statistically analyzed using unpaired t test, and p values Ͻ0.05 were considered to be significant.
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