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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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AQP3 silencing reduces FGF-2-induced cell migration.MDA-MB-231 and Bcap-37 cells were stably transfected with lentiviral shRNA against AQP3, followed by treatment with or without FGF-2 (10 ng/ml) for 24 h. A scramble sequence was used as the negative control (NC). Cell migration was examined using wound scratch assay, photographed at 24 h, and represented as average gap (B and D). The results represent means ± SEM for triplicate experiments. For the cell migration experiment, at least 50 cell migration distances were counted for each experiment. #P<0.05 versus MOCK. *P<0.05 versus FGF-2 alone.
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pone-0056735-g004: AQP3 silencing reduces FGF-2-induced cell migration.MDA-MB-231 and Bcap-37 cells were stably transfected with lentiviral shRNA against AQP3, followed by treatment with or without FGF-2 (10 ng/ml) for 24 h. A scramble sequence was used as the negative control (NC). Cell migration was examined using wound scratch assay, photographed at 24 h, and represented as average gap (B and D). The results represent means ± SEM for triplicate experiments. For the cell migration experiment, at least 50 cell migration distances were counted for each experiment. #P<0.05 versus MOCK. *P<0.05 versus FGF-2 alone.

Mentions: To investigate whether AQP3 is involved in FGF-2 induced cell migration, RNAi experiment was performed. As expected, silencing AQP3 gene with a lentiviral shRNA (shRNA1) led to a prominent decrease of AQP3 basal expression, and inhibited FGF-2-induced AQP3 expression by 67% in MDA-MB-231 and 72% in Bcap-37 cells, respectively (Figs. 3E to 3H). The scramble control shRNA had no effect. Silence of the AQP3 gene also reduced FGF-2-induced cell migration (Figs. 4A and 4C). The shRNA treatment increased the average gap from 0.35-fold (FGF-2alone) to 0.56-fold (FGF-2 plus shRNA) in MDA-MB-231, and from 0.27-fold to 0.58-fold in Bcap-37 cells, respectively (Figs. 4B and 4D). Treatment with the scramble control shRNA had no effect. To further identify the role of AQP3 in FGF-2-induced cell migration, CuSO4, a water transport inhibitor of AQP3, was included. CuSO4 treatment at 10–500 µM decreased migration of MDA-MB-231 and Bcap-37 cells induced by 10 ng/ml FGF-2 (Figs. 5A and 5C). The inhibitory effect of CuSO4 was apparent at a concentration of 10 µM and was most prominent at 500 µM in both breast cancer cell lines (Figs. 5B and 5D).


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)

AQP3 silencing reduces FGF-2-induced cell migration.MDA-MB-231 and Bcap-37 cells were stably transfected with lentiviral shRNA against AQP3, followed by treatment with or without FGF-2 (10 ng/ml) for 24 h. A scramble sequence was used as the negative control (NC). Cell migration was examined using wound scratch assay, photographed at 24 h, and represented as average gap (B and D). The results represent means ± SEM for triplicate experiments. For the cell migration experiment, at least 50 cell migration distances were counted for each experiment. #P<0.05 versus MOCK. *P<0.05 versus FGF-2 alone.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056735-g004: AQP3 silencing reduces FGF-2-induced cell migration.MDA-MB-231 and Bcap-37 cells were stably transfected with lentiviral shRNA against AQP3, followed by treatment with or without FGF-2 (10 ng/ml) for 24 h. A scramble sequence was used as the negative control (NC). Cell migration was examined using wound scratch assay, photographed at 24 h, and represented as average gap (B and D). The results represent means ± SEM for triplicate experiments. For the cell migration experiment, at least 50 cell migration distances were counted for each experiment. #P<0.05 versus MOCK. *P<0.05 versus FGF-2 alone.
Mentions: To investigate whether AQP3 is involved in FGF-2 induced cell migration, RNAi experiment was performed. As expected, silencing AQP3 gene with a lentiviral shRNA (shRNA1) led to a prominent decrease of AQP3 basal expression, and inhibited FGF-2-induced AQP3 expression by 67% in MDA-MB-231 and 72% in Bcap-37 cells, respectively (Figs. 3E to 3H). The scramble control shRNA had no effect. Silence of the AQP3 gene also reduced FGF-2-induced cell migration (Figs. 4A and 4C). The shRNA treatment increased the average gap from 0.35-fold (FGF-2alone) to 0.56-fold (FGF-2 plus shRNA) in MDA-MB-231, and from 0.27-fold to 0.58-fold in Bcap-37 cells, respectively (Figs. 4B and 4D). Treatment with the scramble control shRNA had no effect. To further identify the role of AQP3 in FGF-2-induced cell migration, CuSO4, a water transport inhibitor of AQP3, was included. CuSO4 treatment at 10–500 µM decreased migration of MDA-MB-231 and Bcap-37 cells induced by 10 ng/ml FGF-2 (Figs. 5A and 5C). The inhibitory effect of CuSO4 was apparent at a concentration of 10 µM and was most prominent at 500 µM in both breast cancer cell lines (Figs. 5B and 5D).

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