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Inhibitory mechanism of FAT4 gene expression in response to actin dynamics during Src-induced carcinogenesis.

Ito T, Taniguchi H, Fukagai K, Okamuro S, Kobayashi A - PLoS ONE (2015)

Bottom Line: Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression.Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression.Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Genetic Code, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, Japan.

ABSTRACT
Oncogenic transformation is characterized by morphological changes resulting from alterations in actin dynamics and adhesive activities. Emerging evidence suggests that the protocadherin FAT4 acts as a tumor suppressor in humans, and reduced FAT4 gene expression has been reported in breast and lung cancers and melanoma. However, the mechanism controlling FAT4 gene expression is poorly understood. In this study, we show that transient activation of the Src oncoprotein represses FAT4 mRNA expression through actin depolymerization in the immortalized normal human mammary epithelial cell line MCF-10A. Src activation causes actin depolymerization via the MEK/Erk/Cofilin cascade. The MEK inhibitor U0126 blocks the inhibitory effect of Src on FAT4 mRNA expression and Src-induced actin depolymerization. To determine whether actin dynamics act on the regulation of FAT4 mRNA expression, we treated MCF-10A cells with the ROCK inhibitor Y-27632. Y-27632 treatment decreased FAT4 mRNA expression. This suppressive effect was blocked by siRNA-mediated knockdown of Cofilin1. Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression. Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression. Additionally, we show that siRNA-mediated FAT4 knockdown induced the activity of the Hippo effector YAP/TAZ in MCF-10A cells. Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.

No MeSH data available.


Related in: MedlinePlus

The MEK/Erk pathway is indispensable for both FAT4 gene repression and Cofilin-mediated actin depolymerization by Src.A. Western blotting for phosphorylated and total Erk1/2 (P-Erk1/2 and Erk1/2, respectively) in MCF-10A v-Src:ER cells. Serum-starved cells (16 h) were cultured in the presence or absence of the MEK inhibitor U0126 for 1 h (30 μM) and then treated with 1 μM TAM for 1 h. B. Staining for F-actin (Phalloidin) and nuclei (DAPI) in TAM-treated cells (24 h) after pretreatment with 30 μM U0126 for 1 h. White bars, 50 μm. C. Western blotting for the indicated proteins in TAM-treated cells after pretreatment with U0126. D. RT-qPCR analyses of FAT4 mRNA expression levels in TAM-treated cells after pretreatment with U0126 (mean ± SD, n = 3). E. RT-qPCR analyses of FAT4 mRNA expression levels in MDA-MB-231 cells treated with 20 μM U0126 for 24 h after pretreatment with control or FAT4 siRNA (siControl and siFAT4, respectively, 20 nM) (mean ± SD, n = 3). F. WST-1 Assay in U0126-treated MDA-MB-231 cells (48 h) after pretreatment with siFAT4 for 24 h (mean ± SD, n = 5, * indicates P < 0.05).
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pone.0118336.g003: The MEK/Erk pathway is indispensable for both FAT4 gene repression and Cofilin-mediated actin depolymerization by Src.A. Western blotting for phosphorylated and total Erk1/2 (P-Erk1/2 and Erk1/2, respectively) in MCF-10A v-Src:ER cells. Serum-starved cells (16 h) were cultured in the presence or absence of the MEK inhibitor U0126 for 1 h (30 μM) and then treated with 1 μM TAM for 1 h. B. Staining for F-actin (Phalloidin) and nuclei (DAPI) in TAM-treated cells (24 h) after pretreatment with 30 μM U0126 for 1 h. White bars, 50 μm. C. Western blotting for the indicated proteins in TAM-treated cells after pretreatment with U0126. D. RT-qPCR analyses of FAT4 mRNA expression levels in TAM-treated cells after pretreatment with U0126 (mean ± SD, n = 3). E. RT-qPCR analyses of FAT4 mRNA expression levels in MDA-MB-231 cells treated with 20 μM U0126 for 24 h after pretreatment with control or FAT4 siRNA (siControl and siFAT4, respectively, 20 nM) (mean ± SD, n = 3). F. WST-1 Assay in U0126-treated MDA-MB-231 cells (48 h) after pretreatment with siFAT4 for 24 h (mean ± SD, n = 5, * indicates P < 0.05).

Mentions: Src has been shown to elicit Cofilin-mediated disruption of the actin cytoskeleton through the MEK/Erk pathway [5]. The function of Cofilin is activated via its dephosphorylation [32]. Thus, we examined whether Src can disrupt the actin cytoskeleton through dephosphorylation of Cofilin in MCF-10A v-Src:ER cells. Indeed, phalloidin staining and Western blot analysis demonstrated that Src can depolymerize the actin cytoskeleton and dephosphorylate Cofilin (Fig. 2). We further examined the involvement of the MEK/Erk pathway in Src-mediated modulation of the actin cytoskeleton. Treatment of MCF-10A v-Src:ER cells with the MEK inhibitor U0126 completely inhibited Src-mediated phosphorylation of Erk1/2, a major target of MEK1/2 (Fig. 3A). Under this condition, U0126 significantly inhibited both Src-induced actin dynamics (Fig. 3B) and the dephosphorylation of Cofilin (Fig. 3C). Therefore, these results indicate that Src induces Cofilin-mediated actin depolymerization through the MEK/Erk pathway in MCF-10A cells.


Inhibitory mechanism of FAT4 gene expression in response to actin dynamics during Src-induced carcinogenesis.

Ito T, Taniguchi H, Fukagai K, Okamuro S, Kobayashi A - PLoS ONE (2015)

The MEK/Erk pathway is indispensable for both FAT4 gene repression and Cofilin-mediated actin depolymerization by Src.A. Western blotting for phosphorylated and total Erk1/2 (P-Erk1/2 and Erk1/2, respectively) in MCF-10A v-Src:ER cells. Serum-starved cells (16 h) were cultured in the presence or absence of the MEK inhibitor U0126 for 1 h (30 μM) and then treated with 1 μM TAM for 1 h. B. Staining for F-actin (Phalloidin) and nuclei (DAPI) in TAM-treated cells (24 h) after pretreatment with 30 μM U0126 for 1 h. White bars, 50 μm. C. Western blotting for the indicated proteins in TAM-treated cells after pretreatment with U0126. D. RT-qPCR analyses of FAT4 mRNA expression levels in TAM-treated cells after pretreatment with U0126 (mean ± SD, n = 3). E. RT-qPCR analyses of FAT4 mRNA expression levels in MDA-MB-231 cells treated with 20 μM U0126 for 24 h after pretreatment with control or FAT4 siRNA (siControl and siFAT4, respectively, 20 nM) (mean ± SD, n = 3). F. WST-1 Assay in U0126-treated MDA-MB-231 cells (48 h) after pretreatment with siFAT4 for 24 h (mean ± SD, n = 5, * indicates P < 0.05).
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Related In: Results  -  Collection

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pone.0118336.g003: The MEK/Erk pathway is indispensable for both FAT4 gene repression and Cofilin-mediated actin depolymerization by Src.A. Western blotting for phosphorylated and total Erk1/2 (P-Erk1/2 and Erk1/2, respectively) in MCF-10A v-Src:ER cells. Serum-starved cells (16 h) were cultured in the presence or absence of the MEK inhibitor U0126 for 1 h (30 μM) and then treated with 1 μM TAM for 1 h. B. Staining for F-actin (Phalloidin) and nuclei (DAPI) in TAM-treated cells (24 h) after pretreatment with 30 μM U0126 for 1 h. White bars, 50 μm. C. Western blotting for the indicated proteins in TAM-treated cells after pretreatment with U0126. D. RT-qPCR analyses of FAT4 mRNA expression levels in TAM-treated cells after pretreatment with U0126 (mean ± SD, n = 3). E. RT-qPCR analyses of FAT4 mRNA expression levels in MDA-MB-231 cells treated with 20 μM U0126 for 24 h after pretreatment with control or FAT4 siRNA (siControl and siFAT4, respectively, 20 nM) (mean ± SD, n = 3). F. WST-1 Assay in U0126-treated MDA-MB-231 cells (48 h) after pretreatment with siFAT4 for 24 h (mean ± SD, n = 5, * indicates P < 0.05).
Mentions: Src has been shown to elicit Cofilin-mediated disruption of the actin cytoskeleton through the MEK/Erk pathway [5]. The function of Cofilin is activated via its dephosphorylation [32]. Thus, we examined whether Src can disrupt the actin cytoskeleton through dephosphorylation of Cofilin in MCF-10A v-Src:ER cells. Indeed, phalloidin staining and Western blot analysis demonstrated that Src can depolymerize the actin cytoskeleton and dephosphorylate Cofilin (Fig. 2). We further examined the involvement of the MEK/Erk pathway in Src-mediated modulation of the actin cytoskeleton. Treatment of MCF-10A v-Src:ER cells with the MEK inhibitor U0126 completely inhibited Src-mediated phosphorylation of Erk1/2, a major target of MEK1/2 (Fig. 3A). Under this condition, U0126 significantly inhibited both Src-induced actin dynamics (Fig. 3B) and the dephosphorylation of Cofilin (Fig. 3C). Therefore, these results indicate that Src induces Cofilin-mediated actin depolymerization through the MEK/Erk pathway in MCF-10A cells.

Bottom Line: Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression.Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression.Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Genetic Code, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, Japan.

ABSTRACT
Oncogenic transformation is characterized by morphological changes resulting from alterations in actin dynamics and adhesive activities. Emerging evidence suggests that the protocadherin FAT4 acts as a tumor suppressor in humans, and reduced FAT4 gene expression has been reported in breast and lung cancers and melanoma. However, the mechanism controlling FAT4 gene expression is poorly understood. In this study, we show that transient activation of the Src oncoprotein represses FAT4 mRNA expression through actin depolymerization in the immortalized normal human mammary epithelial cell line MCF-10A. Src activation causes actin depolymerization via the MEK/Erk/Cofilin cascade. The MEK inhibitor U0126 blocks the inhibitory effect of Src on FAT4 mRNA expression and Src-induced actin depolymerization. To determine whether actin dynamics act on the regulation of FAT4 mRNA expression, we treated MCF-10A cells with the ROCK inhibitor Y-27632. Y-27632 treatment decreased FAT4 mRNA expression. This suppressive effect was blocked by siRNA-mediated knockdown of Cofilin1. Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression. Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression. Additionally, we show that siRNA-mediated FAT4 knockdown induced the activity of the Hippo effector YAP/TAZ in MCF-10A cells. Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.

No MeSH data available.


Related in: MedlinePlus