OCT-4: a novel estrogen receptor-α collaborator that promotes tamoxifen resistance in breast cancer cells

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Abstract

Tamoxifen has shown great success in the treatment of breast cancer; however, long-term treatment can lead to acquired tamoxifen (TOT) resistance and relapse. TOT classically antagonizes estradiol (E2) -dependent breast cancer cell growth, but exerts partial agonist/antagonist behavior on gene expression. Although both E2 and TOT treatment of breast cancer cells results in recruitment of the estrogen receptor (ER) to common and distinct genomic sites, the mechanisms and proteins underlying TOT preferential recruitment of the ER remains poorly defined. To this end, we performed in silico motif-enrichment analyses within the ER-binding peaks in response to E2 or TOT, to identify factors that would specifically recruit ER to genomic binding sites in the presence of TOT as compared to E2. Intriguingly, we found Nkx3-1 and Oct-transcription factor homodimer motifs to be enriched in TOT preferential binding sites and confirmed the critical role of Oct-3/4 (aka Oct-4) in directing ER recruitment to TOT preferential genomic binding sites, by chromatin immunoprecipitation (ChIP) analyses. Further investigation revealed Oct-4 expression to be basally repressed by Nkx3-1 in MCF-7 cells and TOT treatment appeared to elevate Nkx3-1 degradation through a p38MAPK-dependent phosphorylation of the E3 ligase, Skp2 at serine-64 residue, as observed by quantitative mass-spectrometry analyses. Consistently, Oct-4 upon induction by phospho-Ser64-Skp2-mediated proteasomal degradation of Nkx3-1, participated in ER transcriptional complexes along with p38MAPK and Skp2 in a tamoxifen-dependent manner leading to TOT-dependent gene activation and cell proliferation of the TOT-resistant MCF-7-tamr breast cancer cells. Notably, Oct-4 levels were highly elevated in MCF-7-tamr cells, and appeared critical for their TOT sensitivity in cell proliferation assays. Furthermore, overexpression of Oct-4 enhanced tumor growth in the presence of tamoxifen in mice in vivo. Collectively, our work presents a novel mechanism for tamoxifen-specific gene activation by ER, secondary to its TOT preferential recruitment to genomic sites by specific activation of Oct-4, a phenomenon that appears to underlie tamoxifen resistance in breast cancer cells and in xenograft tumor models, and could be useful in designing therapeutic interventions to improve treatment outcome.

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Abbreviations

CoIP:

Co-immunoprecipitation

Ub:

ubiquitin

E2:

estradiol

TOT:

tamoxifen

ER:

estrogen receptor

Skp2:

S-phase Kinase-associated Protein-2

p38MAPK:

p38-Mitogen Activated Protein Kinase

NFATc4:

Nuclear Factor Activated T-cell 4

siRNA:

short interfering RNA

Ad:

Adenovirus

ERE:

estrogen response element.

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Acknowledgements

We thank Dr Peter Yau, Biotechnology Core, UIUC, for contributions to the mass-spectrometry analyses. This work was supported by grants from the NIH 5RO1 CA18119 (BSK), The Breast Cancer Research Foundation (BSK) and Juvenile Diabetes Research Foundation (GW). SB conceived of, designed and executed the study, performed experiments, data analyses and interpretation and wrote the manuscript. JDS conceived the study, performed computational analysis and contributed to experimental data. GW and SJ contributed to experimental data. BSK conceived and designed the study, performed data analyses and interpretation and wrote the manuscript.

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Correspondence to B S Katzenellenbogen.

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