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. 2018 Oct 16;37(1):254.
doi: 10.1186/s13046-018-0928-7.

AFF3 upregulation mediates tamoxifen resistance in breast cancers

Yawei Shi  1 , Yang Zhao  2 , Yunjian Zhang  1 , NiJiati AiErken  3 , Nan Shao  1 , Runyi Ye  1 , Ying Lin  4 , Shenming Wang  5
Affiliations

AFF3 upregulation mediates tamoxifen resistance in breast cancers

Yawei Shi et al. J Exp Clin Cancer Res. .

Abstract

Background: Although tamoxifen is a highly effective drug for treating estrogen receptor-positive (ER+) breast cancer, nearly all patients with metastasis with initially responsive tumors eventually relapse, and die from acquired drug resistance. Unfortunately, few molecular mediators of tamoxifen resistance have been described. Here, we describe AFF3 (AF4/FMR2 family member 3), which encodes a nuclear protein with transactivation potential that confers tamoxifen resistance and enables estrogen-independent growth.

Methods: We investigated AFF3 expression in breast cancer cells and in clinical breast cancer specimens with western blot and Real-time PCR. We also examined the effects of AFF3 knockdown and overexpression on breast cancer cells using luciferase, tetrazolium, colony formation, and anchorage-independent growth assays in vitro and with nude mouse xenografting in vivo.

Results: AFF3 was overexpressed in tamoxifen-resistant tumors. AFF3 overexpression in breast cancer cells resulted in tamoxifen resistance, whereas RNA interference-mediated gene knockdown reversed this phenotype. Furthermore, AFF3 upregulation led to estrogen-independent growth in the xenograft assays. Mechanistic investigations revealed that AFF3 overexpression activated the ER signaling pathway and transcriptionally upregulated a subset of ER-regulated genes. Clinical analysis showed that increased AFF3 expression in ER+ breast tumors was associated with worse overall survival.

Conclusions: These studies establish AFF3 as a key mediator of estrogen-independent growth and tamoxifen resistance and as a potential novel diagnostic and therapeutic target.

Keywords: AFF3; Breast cancer; Estrogen receptor-positive; Resistance; Tamoxifen.

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Conflict of interest statement

Ethics approval and consent to participate

For the use of clinical tissues for research purposes, the prior consent of the patients and approval from the Institutional Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University were obtained. All animal experiments were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Sun Yat-sen University.

Consent for publication

All authors approved of the manuscript and consented to its publication.

Competing interests

The authors declare that have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
AFF3 upregulation in TamR breast cancers. a Venn diagrams showing the overlapping proteins upregulated across two models (P < 0.05). b Western blotting performed on whole cell lysates demonstrating increased AFF3 protein levels in the TamR cell lines. GAPDH antibody was used as the loading control. c Western blotting of AFF3 expression in breast cancer tissues compared with noncancerous breast tissues
Fig. 2
Fig. 2
AFF3 overexpression leads to tamoxifen resistance. a Whole cell lysates were harvested from cell lines and used for western blotting. GAPDH was used as the loading control. b and c MTT assay (b) and colony formation assay (c) of MCF-7 and T47D cells, derived TamR cells and AFF3 overexpressed clones seeded in assay medium and exposed to vehicle (ethanol) and 1 μM 4-OH-tamoxifen for 7 days
Fig. 3
Fig. 3
AFF3 knockdown reverses tamoxifen resistance. a Whole cell lysates were harvested from cell lines and used for western blotting. GAPDH was used as the loading control. b and c MTT assay (b) and colony formation assay (c) of MCF-7 and T47D derived TamR cells, and their AFF3 knockdown clones seeded in assay medium and exposed to vehicle (ethanol) and 1 μM 4-OH-tamoxifen for 7 days
Fig. 4
Fig. 4
AFF3 overexpression leads to a TamR phenotype in vivo. a Soft agar growth analysis of MCF-7 and T47D cells with AFF3 overexpression or knockdown (left) and MCF-7 and T47D colony quantification (right). *P < 0.05. b-d In vivo tumorigenesis assay of MCF-7-TamR#1 cells with AFF3 knockdown. b Representative images of the tumors in each group; c growth curves; d tumor weight
Fig. 5
Fig. 5
AFF3 overexpression activates the ER signaling pathway and increases the expression of ER-regulated genes. a Luciferase assay indicating the trans-activity of ER in MCF-7 and T47D cells with AFF3 overexpression or knockdown. *P < 0.05. Bars represent the mean ± SD of three independent experiments. b Real-time PCR analysis of mRNA levels of the ER-related genes in MCF-7 and T47D cells with AFF3 overexpression or knockdown
Fig. 6
Fig. 6
Primary luminal breast cancers with AFF3 overexpression have worse prognosis. a Immunohistochemical staining of AFF3 expression in breast cancer tissues. b and c Kaplan–Meier survival curves of the relapse-free survival (b) and overall survival (c) of patients with breast cancer with high or low AFF3 protein levels
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References

    1. Ginsburg O, Bray F, Coleman MP, Vanderpuye V, Eniu A, Kotha SR, Sarker M, Huong TT, Allemani C, Dvaladze A, et al. The global burden of women’s cancers: a grand challenge in global health. Lancet. 2017;389(10071):847–860. doi: 10.1016/S0140-6736(16)31392-7. - DOI - PMC - PubMed
    1. Vuong D, Simpson PT, Green B, Cummings MC, Lakhani SR. Molecular classification of breast cancer. Virchows Arch. 2014;465(1):1–14. doi: 10.1007/s00428-014-1593-7. - DOI - PubMed
    1. Ignatiadis M, Sotiriou C. Luminal breast cancer: from biology to treatment. Nat Rev Clin Oncol. 2013;10(9):494–506. doi: 10.1038/nrclinonc.2013.124. - DOI - PubMed
    1. Jordan VC. Selective estrogen receptor modulation: concept and consequences in cancer. Cancer Cell. 2004;5(3):207–213. doi: 10.1016/S1535-6108(04)00059-5. - DOI - PubMed
    1. Ring A, Dowsett M. Mechanisms of tamoxifen resistance. Endocr Relat Cancer. 2004;11(4):643–658. doi: 10.1677/erc.1.00776. - DOI - PubMed