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Radiation promotes malignant phenotypes through SRC in breast cancer cells.

Kim RK, Cui YH, Yoo KC, Kim IG, Lee M, Choi YH, Suh Y, Lee SJ - Cancer Sci. (2014)

Bottom Line: However, the molecular mechanisms underlying radiation-induced cancer progression remain obscure.Importantly, radiation-activated SRC induced SLUG expression and caused epithelial-mesenchymal cell transition through phosphatidylinositol 3-kinase/protein kinase B and p38 MAPK signaling.In addition, downregulation of SRC also abolished radiation-acquired resistance of breast cancer cells to anticancer agents such as cisplatin, etoposide, paclitaxel, and IR.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, Korea.

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Irradiation promotes epithelial–mesenchymal transition (EMT) through activation of SRC in breast cancer cells. (a) Kinase assay for SFK proteins (SRC, LYN, FYN, and LCK) using enolase as a substrate in MCF7 breast cancer cells after exposure to fractionated radiation. (b) Migration and invasion assay in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA (si-Cont) prior to irradiation. (c, d) Western blot analysis (c) and immunocytochemistry (d) for EMT markers E-cadherin, N-cadherin, and vimentin in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (e, f) Western blot analysis for EMT transcription factors SLUG, SNAIL, ZEB1, and TWIST (e), and immunocytochemistry for EMT transcription factor SLUG (f) in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (g, h) Migration and invasion assay in MCF7 (g) and SKBR3 (h) cancer cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. (i) Western blot analysis for E-cadherin and N-cadherin in MCF7 cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. *P < 0.05; **P < 0.01. Cont, control; IP, Immunoprecipitation.
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fig02: Irradiation promotes epithelial–mesenchymal transition (EMT) through activation of SRC in breast cancer cells. (a) Kinase assay for SFK proteins (SRC, LYN, FYN, and LCK) using enolase as a substrate in MCF7 breast cancer cells after exposure to fractionated radiation. (b) Migration and invasion assay in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA (si-Cont) prior to irradiation. (c, d) Western blot analysis (c) and immunocytochemistry (d) for EMT markers E-cadherin, N-cadherin, and vimentin in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (e, f) Western blot analysis for EMT transcription factors SLUG, SNAIL, ZEB1, and TWIST (e), and immunocytochemistry for EMT transcription factor SLUG (f) in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (g, h) Migration and invasion assay in MCF7 (g) and SKBR3 (h) cancer cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. (i) Western blot analysis for E-cadherin and N-cadherin in MCF7 cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. *P < 0.05; **P < 0.01. Cont, control; IP, Immunoprecipitation.

Mentions: To investigate the molecular mechanisms underlying radiation-induced EMT in breast cancer cells, we examined the activation status of SFKs. Of note, we observed that irradiation specifically increases the phosphorylation of SRC kinase protein among SFKs (Fig.2a). As irradiation promoted the activation of SRC, we next examined whether SRC activation is responsible for radiation-triggered EMT in breast cancer cells. To this end, we analyzed the migratory and invasive behavior of MCF7 cells that are pretreated with siRNA targeting SRC and then irradiated. Of note, when SRC is downregulated by treatment with siRNA, radiation effects on migration and invasion were inhibited in MCF7 cancer cells (Fig.2b). In parallel with these results, siRNA-mediated downregulation of SRC also attenuated radiation-induced EMT markers (Fig.2c,d). Downregulation of SRC recovered the expression levels of E-cadherin, N-cadherin, and vimentin in irradiated cancer cells to the levels of non-irradiated cells. In agreement, downregulation of SRC blocked the radiation-induced SLUG expression, whereas other EMT regulators were not altered (Fig.2e,f). To further confirm that radiation-activated SRC contributes to EMT, we overexpressed SRC in breast cancer cells and analyzed the migratory and invasive properties as well as EMT markers. As phosphorylation of Try527 inactivates SRC through the interaction of p-Tyr527 with the SH2 domain, we also used a mutant form of SRC Y527F that is constitutively active. Overexpression of either WT SRC or mutant form Y527F enhanced the migratory and invasive properties of MCF7 breast cancer cells (Figs2g,h, S2a). In agreement, SRC overexpression also increased N-cadherin and decreased E-cadherin, although the effect was weak compared to the effect of SRC downregulation (Fig.2i). Collectively, these results suggest that radiation triggers the EMT program through activation of SRC in breast cancer cells.


Radiation promotes malignant phenotypes through SRC in breast cancer cells.

Kim RK, Cui YH, Yoo KC, Kim IG, Lee M, Choi YH, Suh Y, Lee SJ - Cancer Sci. (2014)

Irradiation promotes epithelial–mesenchymal transition (EMT) through activation of SRC in breast cancer cells. (a) Kinase assay for SFK proteins (SRC, LYN, FYN, and LCK) using enolase as a substrate in MCF7 breast cancer cells after exposure to fractionated radiation. (b) Migration and invasion assay in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA (si-Cont) prior to irradiation. (c, d) Western blot analysis (c) and immunocytochemistry (d) for EMT markers E-cadherin, N-cadherin, and vimentin in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (e, f) Western blot analysis for EMT transcription factors SLUG, SNAIL, ZEB1, and TWIST (e), and immunocytochemistry for EMT transcription factor SLUG (f) in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (g, h) Migration and invasion assay in MCF7 (g) and SKBR3 (h) cancer cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. (i) Western blot analysis for E-cadherin and N-cadherin in MCF7 cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. *P < 0.05; **P < 0.01. Cont, control; IP, Immunoprecipitation.
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fig02: Irradiation promotes epithelial–mesenchymal transition (EMT) through activation of SRC in breast cancer cells. (a) Kinase assay for SFK proteins (SRC, LYN, FYN, and LCK) using enolase as a substrate in MCF7 breast cancer cells after exposure to fractionated radiation. (b) Migration and invasion assay in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA (si-Cont) prior to irradiation. (c, d) Western blot analysis (c) and immunocytochemistry (d) for EMT markers E-cadherin, N-cadherin, and vimentin in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (e, f) Western blot analysis for EMT transcription factors SLUG, SNAIL, ZEB1, and TWIST (e), and immunocytochemistry for EMT transcription factor SLUG (f) in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (g, h) Migration and invasion assay in MCF7 (g) and SKBR3 (h) cancer cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. (i) Western blot analysis for E-cadherin and N-cadherin in MCF7 cells transfected with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. *P < 0.05; **P < 0.01. Cont, control; IP, Immunoprecipitation.
Mentions: To investigate the molecular mechanisms underlying radiation-induced EMT in breast cancer cells, we examined the activation status of SFKs. Of note, we observed that irradiation specifically increases the phosphorylation of SRC kinase protein among SFKs (Fig.2a). As irradiation promoted the activation of SRC, we next examined whether SRC activation is responsible for radiation-triggered EMT in breast cancer cells. To this end, we analyzed the migratory and invasive behavior of MCF7 cells that are pretreated with siRNA targeting SRC and then irradiated. Of note, when SRC is downregulated by treatment with siRNA, radiation effects on migration and invasion were inhibited in MCF7 cancer cells (Fig.2b). In parallel with these results, siRNA-mediated downregulation of SRC also attenuated radiation-induced EMT markers (Fig.2c,d). Downregulation of SRC recovered the expression levels of E-cadherin, N-cadherin, and vimentin in irradiated cancer cells to the levels of non-irradiated cells. In agreement, downregulation of SRC blocked the radiation-induced SLUG expression, whereas other EMT regulators were not altered (Fig.2e,f). To further confirm that radiation-activated SRC contributes to EMT, we overexpressed SRC in breast cancer cells and analyzed the migratory and invasive properties as well as EMT markers. As phosphorylation of Try527 inactivates SRC through the interaction of p-Tyr527 with the SH2 domain, we also used a mutant form of SRC Y527F that is constitutively active. Overexpression of either WT SRC or mutant form Y527F enhanced the migratory and invasive properties of MCF7 breast cancer cells (Figs2g,h, S2a). In agreement, SRC overexpression also increased N-cadherin and decreased E-cadherin, although the effect was weak compared to the effect of SRC downregulation (Fig.2i). Collectively, these results suggest that radiation triggers the EMT program through activation of SRC in breast cancer cells.

Bottom Line: However, the molecular mechanisms underlying radiation-induced cancer progression remain obscure.Importantly, radiation-activated SRC induced SLUG expression and caused epithelial-mesenchymal cell transition through phosphatidylinositol 3-kinase/protein kinase B and p38 MAPK signaling.In addition, downregulation of SRC also abolished radiation-acquired resistance of breast cancer cells to anticancer agents such as cisplatin, etoposide, paclitaxel, and IR.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, Korea.

Show MeSH
Related in: MedlinePlus