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A multi-omics approach to uncover estrogen receptor (ER) and activator protein 1 (AP-1) signaling networks in breast cancer

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posted on 2024-09-03, 03:41 authored by Huan He

Estrogen receptor (ER) binds to DNA indirectly through other transcription factors (e.g. AP-1) to modulate gene expression, which is a tethering mechanism. The ER/AP-1 crosstalk plays an important role in tamoxifen therapy resistance. However, the overlap in DNA binding profiles of ER and AP-1 transcription factors at genome-wide level has not been described. Moreover, AP-1 plays a pivotal role in various cellular processes in breast cancer. The transcriptional activity of AP-1 is controlled by coregulators, thereby regulating the expression of specific genes. Understanding protein-protein interactions is fundamental to the mechanism of AP-1 signaling. In addition, ERα is one of the key biomarkers for diagnosis and endocrine therapy of breast cancer. However, ERα status is not considered to be a perfect marker for responsiveness to anti-estrogens. It has been shown that ERβ may act as a tumor suppressor and could be a therapeutic target for breast cancer, however the functions of ERβ in this setting remain to be further explored. The use of multi-functional genomic technologies to identify cistrome, transcriptome and proteome of ER or AP-1 has resulted in comprehensive deciphering of the role of the ER and AP-1 in breast cancer, which also provides information for developing novel therapeutic strategies for breast cancer.

In Paper I, we investigated the genome-wide assessment of c-Jun, a potent member of AP-1 family, and ERα cistrome and transcriptome in ERα-positive breast cancer cells. Our findings demonstrate the genome-wide co-localization of ERα and c-Jun binding regions and suggest that ERα tethering to AP-1 is a global mechanism for gene transcription regulated by ERα. In addition, the results confirm that the sensitivity of ERα-positive breast cancer cells to tamoxifen therapy is reduced by c-Jun overexpression. Moreover, it is shown that expression of transforming growth factor β induced (TGFBI) protein is associated with poor outcomes of ERα-positive breast cancer patients receiving endocrine therapy and thus as a candidate gene that may cause tamoxifen resistance through ERα and AP-1 crosstalk.

In Paper II, we elucidated the first Fra-1 associated interactome in triple-negative breast cancer (TNBC) cells using Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins (RIME) approach, showing that the most enriched Fra-1 interacting protein was DDX5. The cistrome and transcriptome of DDX5 extensively overlapped with that of Fra-1, which is highly associated with the TNBC cell growth. Furthermore, we found that DDX5 acts as a transcriptional coactivator for Fra-1, enhancing Fra-1-dependent TNBC cell proliferation through increasing the transcriptional activity of Fra-1. We also showed that higher expression level of DDX5 protein was detected in triple-negative basal-like tumors compared with that in non-basal-like ones. In addition, the direct target gene set of DDX5 can predict poor clinical outcome of breast cancer patients.

In Paper III, we generated a novel breast cancer cell model with overexpression of ERβ in the absence of ERα. We used CRISPR/Cas9 system to knock out ERα in MCF7 breast cancer cells with stable Tet-Off-inducible ERβ expression. We found that only ERβ-expressing MCF7 cells displayed a significant reduction in cell proliferation in response to E2 compared with vehicle, conversely, only ERα-expressing MCF7 cells displayed an increased cell proliferation upon E2 treatment. The RNA-seq results indicated that ERβ could modulate specific gene expression profile different from that of ERα. Furthermore, functional enrichment analysis showed that the two ER isoforms regulate cell proliferation in opposite direction; ERβ is significantly involved in the biological process “negative regulation of cell proliferation”.

In conclusion, the studies presented in the thesis contribute to comprehensive understanding of the mechanism of ER and AP-1 signaling in breast cancer. We characterized two molecules, TGFBI and DDX5, in breast cancer, suggesting that they could be the candidates of therapeutic targets. We also provided evidences that ERα and ERβ have opposite effects on E2-dependent breast cancer cell proliferation by regulating distinct gene sets.

List of scientific papers

I. He H, Sinha I, Fan R, Haldosen LA, Yan F, Zhao C, Dahlman-Wright K. c-Jun/AP-1 overexpression reprograms ERalpha signaling related to tamoxifen response in ERalpha-positive breast cancer. Oncogene. 2018 May;37(19):2586-2600.
https://doi.org/10.1038/s41388-018-0165-8

II. He H, Song D, Sinha I, Hessling B, Li X, Haldosen LA, Zhao C. Endogenous interaction profiling identifies DDX5 as an oncogenic coactivator of transcription factor Fra-1. Oncogene. 2019 Jul:38(28):5725-5738.
https://doi.org/10.1038/s41388-019-0824-4

III. He H, Song D, Sinha I, Haldosen LA, Zhao C. ER𝛼 and ERβ exert differential regulation of gene expression in MCF7 cells. [Manuscript]

History

Defence date

2019-10-18

Department

  • Department of Medicine, Huddinge

Publisher/Institution

Karolinska Institutet

Main supervisor

Zhao, Chunyan

Co-supervisors

Dahlman-Wright, Karin; Lehtiö, Janne

Publication year

2019

Thesis type

  • Doctoral thesis

ISBN

978-91-7831-542-0

Number of supporting papers

3

Language

  • eng

Original publication date

2019-09-27

Author name in thesis

He, Huan

Original department name

Department of Biosciences and Nutrition

Place of publication

Stockholm

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