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Epigenetic silencing of tropomyosin alters transforming growth factor beta control of cell invasion and metastasis
Breast Cancer Research volume 7, Article number: P4.15 (2005)
Transforming growth factor beta (TGF-β) is a potent tumor suppressor but it can also enhance tumor metastasis by inducing epithelial to mesenchymal transition, cell migration, and changes in tumor microenvironment. The mechanisms underlying the metastatic switch in TGF-β function are not well understood. We have recently reported that TGF-β regulates tropomyosin-based actin microfilament fibers , which are essential for cell proliferation, morphology and motility . Smads and p38 MAPK mediate induction of tropomyosin and formation of stable actin microfilament fibers (stress fibers), thereby reducing cell motility. Tropomyosin (TM) is a dimeric coil-coiled protein that binds along actin microfilaments forming a head-to-tail polymer. TM stabilizes microfilaments and protects them from the depolymerizing action of gelsolin and cofilin. Importantly, TGF-β induction of stress fibers inversely correlated with metastatic behavior of tumor cells. The metastastic breast cancer MDA-MB-231 cell line with active TGF-β signaling did not express TM isoforms encoded by the TPM1 gene. DNA demethylating agent increased TPM1 expression. We hypothesized that DNA methylation may suppress TPM1 and tropomyosin-based actin fibers, thereby reducing TGF-β control of tumor cell invasion and metastasis. The goals are to define the mechanisms underlying loss of tropomyosin expression and changes in TGF-β tumor suppressor function.
RT-PCR and immunoblotting analysis of expression tropomyosin isoforms encoded by TPM1 and TPM2 genes in a panel of normal epithelial (MCF10A, NMuMG) and carcinoma (MCF7, MDA-MB-231, MDA-MB-435, A549, SW620, SW480) cell lines. DNA methylation of the TPM1 promoter was analyzed by bisulfite sequencing in normal and cancer breast cell lines. Cell migration/invasion was studied using transwell and wound-healing assays. Actin filaments and focal adhesions were studied by immunofluorescence. The role of TPM1 was studied using inducible Tet-Off MDA-MB-231 cell lines.
Both TPM1 and TPM2 genes were expressed in normal and non-metastatic tumor cell lines. In metastatic breast and colon tumor cell lines, however, TPM1 expression was significantly reduced or absent, whereas TPM2 was expressed at low levels. Treatment of metastatic cell lines (MDA-MB-231, MDA-MB-435, SW620) with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) increased TPM1 expression with little 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 using the 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.
Tropomyosin-based stress fibers are essential for TGF-β control of cell motility and invasion. Epigenetic suppression of TPM1 may alter tumor suppressor function of TGF-β and contribute to the acquisition of metastatic phenotype.
Bakin AV, Safina A, Rinehart C, Daroqui C, Darbary H, Helfman DM: A critical role of tropomyosins in TGF-β regulation of the actin cytoskeleton and cell motility in epithelial cells. Mol Biol Cell. 2004, 15: 4682-4694. 10.1091/mbc.E04-04-0353.
Pawlak G, Helfman DM: Cytoskeletal changes in cell transformation and tumorigenesis. Curr Opin Genet Dev. 2001, 11: 41-47. 10.1016/S0959-437X(00)00154-4.
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Bakin, A., Varga, A., Zheng, Q. et al. Epigenetic silencing of tropomyosin alters transforming growth factor beta control of cell invasion and metastasis. Breast Cancer Res 7, P4.15 (2005) doi:10.1186/bcr1145
- Stress Fiber
- Bisulfite Sequencing
- Tumor Suppressor Function
- Metastatic Cell Line
- Tropomyosin Isoforms