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Twist activates miR-22 to suppress estrogen receptor alpha in breast cancer.

ABSTRACT: TWIST1 (Twist) is a basic helix-loop-helix transcription factor that is overexpressed in many cancers and promotes tumor cell invasion, metastasis, and recurrence. In this study, we demonstrate that Twist upregulates expression of microRNA 22 (miR-22) which, in turn, downregulates estrogen receptor alpha (ER) expression in breast cancer. Initial analysis of miR-22 and Twist expression in a panel of breast cancer cell lines showed a direct correlation between Twist and miR-22 levels with miR-22 being highly expressed in ER negative cell lines. Overexpressing Twist caused increased miR-22 levels while downregulating it led to decreased miR-22 expression. To characterize the upstream promoter region of miR-22, we utilized rapid amplification of cDNA ends and identified the transcription start site and the putative promoter region of miR-22. Mechanistically, we determined that Twist, in combination with HDAC1 and DNMT3B, transcriptionally upregulates miR-22 expression by binding to E-boxes in the proximal miR-22 promoter. We also established that miR-22 causes an increase in growth in 3D but not 2D cultures. Importantly, we observed a direct correlation between increased breast cancer grade and Twist and miR-22 expression. We also identified two potential miR-22 binding sites in the 3'-UTR region of ER and confirmed by promoter assays that miR-22 regulates ER expression by binding to both target sites. These results reveal a novel pathway of ER suppression by Twist through miR-22 activation that could potentially promote the ER negative phenotype in breast cancers.

SUBMITTER: Vesuna F

PROVIDER: S-EPMC9128495 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Twist activates miR-22 to suppress estrogen receptor alpha in breast cancer.

Vesuna Farhad F Lisok Ala A van Diest Paul P Raman Venu V

Molecular and cellular biochemistry 20210213 6

TWIST1 (Twist) is a basic helix-loop-helix transcription factor that is overexpressed in many cancers and promotes tumor cell invasion, metastasis, and recurrence. In this study, we demonstrate that Twist upregulates expression of microRNA 22 (miR-22) which, in turn, downregulates estrogen receptor alpha (ER) expression in breast cancer. Initial analysis of miR-22 and Twist expression in a panel of breast cancer cell lines showed a direct correlation between Twist and miR-22 levels with miR-22 b ...[more]

PMID: 33582945

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Project description:The role of estrogen receptor-? (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer.

Project description:Breast cancer resistance to the antiestrogens tamoxifen (OHT) and fulvestrant is accompanied by alterations in both estrogen-dependent and estrogen-independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either OHT or fulvestrant. We found that apigenin can function as both an estrogen and an antiestrogen in a dose-dependent manner. At low concentrations (1 mumol/L), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (>10 mumol/L), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either OHT or fulvestrant showed synergistic, growth-inhibitory effects on both antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or an antiestrogen, effects of the drug on estrogen receptor-alpha (ERalpha); transactivation activity, mobility, stability, and ERalpha-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERalpha and its coactivator amplified in breast cancer-1. However, higher doses (>10 mumol/L) of apigenin inhibited ERalpha mobility (as determined by fluorescence recovery after photobleaching assays), down-regulated ERalpha and amplified in breast cancer-1 expression levels, and inhibited multiple protein kinases, including p38, protein kinase A, mitogen-activated protein kinase, and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and a protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERalpha-dependent and ERalpha-independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.

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Twist activates miR-22 to suppress estrogen receptor alpha in breast cancer.

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Twist activates miR-22 to suppress estrogen receptor alpha in breast cancer.

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