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Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-β

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Abstract

Loss of actin stress fibers has been associated with cell transformation and metastasis. TGF-β induction of stress fibers in epithelial cells requires high molecular weight tropomyosins encoded by TPM1 and TPM2 genes. Here, we investigated the mechanism underlying the failure of TGF-β to induce stress fibers and inhibit cell migration in metastatic cells. RT–PCR analysis in carcinoma cell lines revealed a significant reduction in TPM1 transcripts in metastatic MDA-MB-231, MDA-MB-435 and SW620 cell lines. Treatment of these cells with demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) increased mRNA levels of TPM1 with no effect on TPM2. Importantly, 5-aza-dC treatment of MDA-MB-231 cells restored TGF-β induction of TPM1 and formation of stress fibers. Forced expression of TPM1 by using Tet-Off system increased stress fibers in MDA-MB-231 cells and reduced cell migration. A potential CpG island spanning the TPM1 proximal promoter, exon 1, and the beginning of intron 1 was identified. Bisulfite sequencing showed significant cytosine methylation in metastatic cell lines that correlated with a reduced expression of TPM1. Together these results suggest that epigenetic suppression of TPM1 may alter TGF-β tumor suppressor function and contribute to metastatic properties of tumor cells.

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Accession codes

Accessions

GenBank/EMBL/DDBJ

Abbreviations

TGF-β:

transforming growth factor beta

MAPK:

mitogen-activated protein kinase

TPM:

tropomyosin

5-aza-dC:

5-aza-2′-deoxycytidine

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Acknowledgements

We thank Bert Vogelstein for providing reagents, Erika VanDette for assistance with immunofluorescence studies; Dominic Smiraglia for aid in initial design of methylation primers; Andrew Leppard for assistance with graphics in Figure 5; Michael Higgins and John Cowell for stimulating discussions. This work was supported by PHS grant R01 CA95263 and USAMRMC grant DAMD17-02-01-0602 (to A.V.B.).

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  1. Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, 14263, NY, USA

    Andrea E Varga, Nina V Stourman, Qiao Zheng, Alfiya F Safina, Lei Quan, Xiurong Li, Khalid Sossey-Alaoui & Andrei V Bakin

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  1. Andrea E Varga
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  2. Nina V Stourman
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  3. Qiao Zheng
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  4. Alfiya F Safina
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Correspondence to Andrei V Bakin.

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Varga, A., Stourman, N., Zheng, Q. et al. Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-β. Oncogene 24, 5043–5052 (2005). https://doi.org/10.1038/sj.onc.1208688

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