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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

Inhibition of hyaluronan (HA) by 4-methylumbelliferone (4-MU) abolishes the transforming growth factor-β1 (TGF-β1)-induced CD44/epidermal growth factor receptor (EGFR) expression and co-localization. (A) The mRNA expression levels of different HAS subtypes were determined by RT-PCR following stimulation of the A549 and MCF-7 cells with TGF-β1 or TGF-β1 and 4-MU. Changes in mRNA expression levels were then detected. Western blot analysis revealed that CD44 and EGFR protein expression levels were altered following stimulation with TGF-β1 or TGF-β1 plus 4-MU. β-actin was used as a loading control. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in the A549 and MCF-7 cells, that were stimulated with TGF-β1 or TGF-β1 plus 4-MU. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was carried out using the A549 and MCF-7 cells following stimulation with TGF-β1 or TGF-β1 plus 4-MU. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.
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f4-ijmm-36-01-0113: Inhibition of hyaluronan (HA) by 4-methylumbelliferone (4-MU) abolishes the transforming growth factor-β1 (TGF-β1)-induced CD44/epidermal growth factor receptor (EGFR) expression and co-localization. (A) The mRNA expression levels of different HAS subtypes were determined by RT-PCR following stimulation of the A549 and MCF-7 cells with TGF-β1 or TGF-β1 and 4-MU. Changes in mRNA expression levels were then detected. Western blot analysis revealed that CD44 and EGFR protein expression levels were altered following stimulation with TGF-β1 or TGF-β1 plus 4-MU. β-actin was used as a loading control. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in the A549 and MCF-7 cells, that were stimulated with TGF-β1 or TGF-β1 plus 4-MU. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was carried out using the A549 and MCF-7 cells following stimulation with TGF-β1 or TGF-β1 plus 4-MU. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.

Mentions: Hyaluronan synthesis and degradation are regulated by HAS (15). We found that stimulation with TGF-β1 increased both HAS2 and HAS3 expression at the mRNA level (Fig. 4A), suggesting that HAS2 and HAS3 are involved in the TGF-β1-mediated effects on HA production. Moreover, the upregulation of HAS2 was more obvious than that of HAS3 in the A549 and MCF-7 cells, whereas the mRNA expression levels of HAS1 were not altered (Fig. 4A). We then treated the A549 and MCF-7 cells with the HAS inhibitor, 4-MU, and measured the mRNA expression levels of HAS2 and HAS3. The results revealed that the expression levels were significantly downregulated (Fig. 4A). Western blot analysis (Fig. 4A) and immunofluorescence staining (Fig. 4B) also demonstrated that 4-MU suppressed the expression of CD44 and EGFR, decreasing their expression to levels similar to those of the cells not stimulated with TGF-β1 and disrupted the CD44-EGFR co-localization induced by TGF-β1 (Fig. 4A and B). Additionally, 4-MU inhibited the interaction between CD44 and EGFR, even in the presence of TGF-β1 (Fig. 4C).


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)

Inhibition of hyaluronan (HA) by 4-methylumbelliferone (4-MU) abolishes the transforming growth factor-β1 (TGF-β1)-induced CD44/epidermal growth factor receptor (EGFR) expression and co-localization. (A) The mRNA expression levels of different HAS subtypes were determined by RT-PCR following stimulation of the A549 and MCF-7 cells with TGF-β1 or TGF-β1 and 4-MU. Changes in mRNA expression levels were then detected. Western blot analysis revealed that CD44 and EGFR protein expression levels were altered following stimulation with TGF-β1 or TGF-β1 plus 4-MU. β-actin was used as a loading control. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in the A549 and MCF-7 cells, that were stimulated with TGF-β1 or TGF-β1 plus 4-MU. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was carried out using the A549 and MCF-7 cells following stimulation with TGF-β1 or TGF-β1 plus 4-MU. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494581&req=5

f4-ijmm-36-01-0113: Inhibition of hyaluronan (HA) by 4-methylumbelliferone (4-MU) abolishes the transforming growth factor-β1 (TGF-β1)-induced CD44/epidermal growth factor receptor (EGFR) expression and co-localization. (A) The mRNA expression levels of different HAS subtypes were determined by RT-PCR following stimulation of the A549 and MCF-7 cells with TGF-β1 or TGF-β1 and 4-MU. Changes in mRNA expression levels were then detected. Western blot analysis revealed that CD44 and EGFR protein expression levels were altered following stimulation with TGF-β1 or TGF-β1 plus 4-MU. β-actin was used as a loading control. (B) Immunofluorescence staining was conducted to detect EGFR (green) and CD44 (red) expression in the A549 and MCF-7 cells, that were stimulated with TGF-β1 or TGF-β1 plus 4-MU. The nuclei were visualized with 4′,6-diamidino-2-phenylindole staining (DAPI, blue). Images were captured at ×200 magnification. (C) Co-IP analysis was carried out using the A549 and MCF-7 cells following stimulation with TGF-β1 or TGF-β1 plus 4-MU. EGFR proteins were immunoprecipitated using an antibody against CD44. IgG was used as a negative control.
Mentions: Hyaluronan synthesis and degradation are regulated by HAS (15). We found that stimulation with TGF-β1 increased both HAS2 and HAS3 expression at the mRNA level (Fig. 4A), suggesting that HAS2 and HAS3 are involved in the TGF-β1-mediated effects on HA production. Moreover, the upregulation of HAS2 was more obvious than that of HAS3 in the A549 and MCF-7 cells, whereas the mRNA expression levels of HAS1 were not altered (Fig. 4A). We then treated the A549 and MCF-7 cells with the HAS inhibitor, 4-MU, and measured the mRNA expression levels of HAS2 and HAS3. The results revealed that the expression levels were significantly downregulated (Fig. 4A). Western blot analysis (Fig. 4A) and immunofluorescence staining (Fig. 4B) also demonstrated that 4-MU suppressed the expression of CD44 and EGFR, decreasing their expression to levels similar to those of the cells not stimulated with TGF-β1 and disrupted the CD44-EGFR co-localization induced by TGF-β1 (Fig. 4A and B). Additionally, 4-MU inhibited the interaction between CD44 and EGFR, even in the presence of TGF-β1 (Fig. 4C).

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