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Aquaporin3 is required for FGF-2-induced migration of human breast cancers.

Cao XC, Zhang WR, Cao WF, Liu BW, Zhang F, Zhao HM, Meng R, Zhang L, Niu RF, Hao XS, Zhang B - PLoS ONE (2013)

Bottom Line: The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration.The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Breast Cancer Prevention and Treatment, Cancer Institute and Hospital, Tianjin, China.

ABSTRACT

Purpose: The aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers.

Methods: Potential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO4. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression.

Results: FGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO4, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.

Conclusions: AQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.

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FGF-2 induces AKT and ERK1/2 phosphorylation.For AKT and ERK1/2 phosphorylation, cells were treated with FGF-2 (10 ng/ml) and harvested at (2,5, 15, 30, 60 min). Phospho-AKT (p-AKT) and phospho-ERK1/2 (p-ERK1/2) were analyzed by Western blot and normalized to total AKT(t-AKT) and total-ERK1/2 (t-ERK1/2), respectively. The results represented mean ± SEM for triplicate experiments.
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pone-0056735-g008: FGF-2 induces AKT and ERK1/2 phosphorylation.For AKT and ERK1/2 phosphorylation, cells were treated with FGF-2 (10 ng/ml) and harvested at (2,5, 15, 30, 60 min). Phospho-AKT (p-AKT) and phospho-ERK1/2 (p-ERK1/2) were analyzed by Western blot and normalized to total AKT(t-AKT) and total-ERK1/2 (t-ERK1/2), respectively. The results represented mean ± SEM for triplicate experiments.

Mentions: To further investigate the signal pathway of FGF-2-induced AQP3 expression and cell migration, additional experiments were conducted using kinase inhibitors. As expected, FGF-2 induced AKT and ERK1/2 phosphorylation (Figs. 8A and 8D). ERK1/2 phosphorylation reached a peak at 5 min after FGF-2 treatment; AKT phosphorylation peaked as early as 2 min; the phosphorylation of AKT and ERK1/2 remained sustained for 1 h (Figs. 8B, 8C, 8E and 8F). Western blot analyses showed that pretreatment of PI3K inhibitor LY294002 or MEK1/2 inhibitor PD98059 inhibited, but not fully blocked FGF-2-induced AQP3 expression (Figs. 9A and 9C). In MDA-MB-231 cells, AQP3 expression was decreased from 1.74-(FGF-2alone) to 0.87-fold by LY294002 and 0.51-fold by PD98059. In Bcap-37 cells, AQP3 expression was reduced from 2.89-(FGF-2 alone) to 1.11-fold by LY294002 and 0.71-fold by PD98059. Likewise, FGF-2-induced cell migration was also reduced (Figs. 10A and 10C). In MDA-MB-231 cells, the average gap was increased from 0.32-(FGF-2 alone) to 0.50-fold by LY294002 and0.58-fold by PD98059. In Bcap-37 cells, the gap was increased from 0.27-(FGF-2alone) to 0.45-fold by LY294002 and 0.55-fold by PD98059 (Figs. 10B and 10D).


Aquaporin3 is required for FGF-2-induced migration of human breast cancers.

Cao XC, Zhang WR, Cao WF, Liu BW, Zhang F, Zhao HM, Meng R, Zhang L, Niu RF, Hao XS, Zhang B - PLoS ONE (2013)

FGF-2 induces AKT and ERK1/2 phosphorylation.For AKT and ERK1/2 phosphorylation, cells were treated with FGF-2 (10 ng/ml) and harvested at (2,5, 15, 30, 60 min). Phospho-AKT (p-AKT) and phospho-ERK1/2 (p-ERK1/2) were analyzed by Western blot and normalized to total AKT(t-AKT) and total-ERK1/2 (t-ERK1/2), respectively. The results represented mean ± SEM for triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585269&req=5

pone-0056735-g008: FGF-2 induces AKT and ERK1/2 phosphorylation.For AKT and ERK1/2 phosphorylation, cells were treated with FGF-2 (10 ng/ml) and harvested at (2,5, 15, 30, 60 min). Phospho-AKT (p-AKT) and phospho-ERK1/2 (p-ERK1/2) were analyzed by Western blot and normalized to total AKT(t-AKT) and total-ERK1/2 (t-ERK1/2), respectively. The results represented mean ± SEM for triplicate experiments.
Mentions: To further investigate the signal pathway of FGF-2-induced AQP3 expression and cell migration, additional experiments were conducted using kinase inhibitors. As expected, FGF-2 induced AKT and ERK1/2 phosphorylation (Figs. 8A and 8D). ERK1/2 phosphorylation reached a peak at 5 min after FGF-2 treatment; AKT phosphorylation peaked as early as 2 min; the phosphorylation of AKT and ERK1/2 remained sustained for 1 h (Figs. 8B, 8C, 8E and 8F). Western blot analyses showed that pretreatment of PI3K inhibitor LY294002 or MEK1/2 inhibitor PD98059 inhibited, but not fully blocked FGF-2-induced AQP3 expression (Figs. 9A and 9C). In MDA-MB-231 cells, AQP3 expression was decreased from 1.74-(FGF-2alone) to 0.87-fold by LY294002 and 0.51-fold by PD98059. In Bcap-37 cells, AQP3 expression was reduced from 2.89-(FGF-2 alone) to 1.11-fold by LY294002 and 0.71-fold by PD98059. Likewise, FGF-2-induced cell migration was also reduced (Figs. 10A and 10C). In MDA-MB-231 cells, the average gap was increased from 0.32-(FGF-2 alone) to 0.50-fold by LY294002 and0.58-fold by PD98059. In Bcap-37 cells, the gap was increased from 0.27-(FGF-2alone) to 0.45-fold by LY294002 and 0.55-fold by PD98059 (Figs. 10B and 10D).

Bottom Line: The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration.The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Breast Cancer Prevention and Treatment, Cancer Institute and Hospital, Tianjin, China.

ABSTRACT

Purpose: The aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers.

Methods: Potential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO4. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression.

Results: FGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO4, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.

Conclusions: AQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.

Show MeSH
Related in: MedlinePlus