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Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells.

Li L, Qi L, Liang Z, Song W, Liu Y, Wang Y, Sun B, Zhang B, Cao W - Int. J. Mol. Med. (2015)

Bottom Line: Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts.In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion.In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, P.R. China.

ABSTRACT
Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

No MeSH data available.


Related in: MedlinePlus

Transforming growth factor-β1 (TGF-β1) induces CD44 and EGFR expression and co-localization. (A) Analysis of the expression levels of epidermal growth factor receptor (EGFR) and CD44 by western blot analysis in A549 and MCF-7 cells, in the absence or presence of TGF-β1. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in A549 and MCF-7 cells, which were stimulated with or without TGF-β1 for 24 h. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was performed using the A549 and MCF-7 cells. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.
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f3-ijmm-36-01-0113: Transforming growth factor-β1 (TGF-β1) induces CD44 and EGFR expression and co-localization. (A) Analysis of the expression levels of epidermal growth factor receptor (EGFR) and CD44 by western blot analysis in A549 and MCF-7 cells, in the absence or presence of TGF-β1. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in A549 and MCF-7 cells, which were stimulated with or without TGF-β1 for 24 h. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was performed using the A549 and MCF-7 cells. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.

Mentions: In accordance with the results of RT-PCR (Fig. 1C), western blot analysis also demonstrated that EGFR cell surface expression levels increased following stimulation of the A549 and MCF-7 cells with TGF-β1 (Fig. 3A). We found that the CD44 expression levels were also increased. Moreover, the expression levels and cellular localization of EGFR and CD44 following stimulation with TGF-β1 were assessed by confocal laser scanning microscopy using the A549 and MCF-7 cells. As shown in Fig. 3B, without TGF-β1 stimulation, the EGFR (green) and CD44 (red) proteins were weakly expressed around the cytomembrane. However, following stimulation with TGF-β1, both the EGFR and CD44 expression levels were upregulated and showed an obvious co-localization, with visible yellow signals (merged image) (Fig. 3B).


Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells.

Li L, Qi L, Liang Z, Song W, Liu Y, Wang Y, Sun B, Zhang B, Cao W - Int. J. Mol. Med. (2015)

Transforming growth factor-β1 (TGF-β1) induces CD44 and EGFR expression and co-localization. (A) Analysis of the expression levels of epidermal growth factor receptor (EGFR) and CD44 by western blot analysis in A549 and MCF-7 cells, in the absence or presence of TGF-β1. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in A549 and MCF-7 cells, which were stimulated with or without TGF-β1 for 24 h. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was performed using the A549 and MCF-7 cells. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494581&req=5

f3-ijmm-36-01-0113: Transforming growth factor-β1 (TGF-β1) induces CD44 and EGFR expression and co-localization. (A) Analysis of the expression levels of epidermal growth factor receptor (EGFR) and CD44 by western blot analysis in A549 and MCF-7 cells, in the absence or presence of TGF-β1. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in A549 and MCF-7 cells, which were stimulated with or without TGF-β1 for 24 h. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was performed using the A549 and MCF-7 cells. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.
Mentions: In accordance with the results of RT-PCR (Fig. 1C), western blot analysis also demonstrated that EGFR cell surface expression levels increased following stimulation of the A549 and MCF-7 cells with TGF-β1 (Fig. 3A). We found that the CD44 expression levels were also increased. Moreover, the expression levels and cellular localization of EGFR and CD44 following stimulation with TGF-β1 were assessed by confocal laser scanning microscopy using the A549 and MCF-7 cells. As shown in Fig. 3B, without TGF-β1 stimulation, the EGFR (green) and CD44 (red) proteins were weakly expressed around the cytomembrane. However, following stimulation with TGF-β1, both the EGFR and CD44 expression levels were upregulated and showed an obvious co-localization, with visible yellow signals (merged image) (Fig. 3B).

Bottom Line: Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts.In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion.In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, P.R. China.

ABSTRACT
Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

No MeSH data available.


Related in: MedlinePlus