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Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu B, Jin X, Min L, Li Q, Deng L, Wu H, Lin G, Chen L, Zhang H, Li C, Wang L, Zhu J, Wang W, Chu F, Shen J, Li H, Mao J - Oncotarget (2015)

Bottom Line: High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients.We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis.ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

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ClC-3 Mediates Recycling of β1 Integrin by Inducing Keratin 18 Phosphorylation(A) Immunofluorescence images (Left) and evaluation of fluorescence intensity of K18 (right) in HeLa cells treated with shK18 (pGPU6/GFP-K18 shRNA) or shRNA negative control (shNC). **P<0.01; n=3 with >15 cells. Data are mean ± SEM. White arrows mark transfected cells. (B) K18 knockdown by transfection of shK18 inhibits membrane ruffling induced by EGF. (Left) Fluorescent CTXB labeling photographs of live cells. Insets in images show transfected cells by detection of GFP. Yellow arrows indicate membrane ruffles. (Right) Percentage of cells with membrane ruffling was calculated in control or transfected conditions (data are mean ± SEM, **P < 0.001 n=3 with >50 cells). (C) K18 down-regulation prevents K18 and ClC-3 trafficking between cytoplasm and membrane leads them to gather in perinuclear region. After HeLa cells transfected with shK18 or shNC were stimulated by EGF for 5 min immunofluorescence analysis for ClC-3 and β1 integrin carried out. Green arrows point transfected cells. (D) 3D reconstruction of subcellular localization of Ser-52 phosphorylated K18 (K18 pS52) in ruffling cells. Immunofluorescence of Ser-52 phosphorylated K18 in EGF-stimulated HeLa cells was detected with an anti- K18 pS52 antibody and an Alexa Fluor 488-conjugated secondary antibody. White arrows indicate membrane ruffles. See also Movie S2. (E) Co-IP experiment with anti-K18 pS52 antibody from extract of ruffling cells probed with anti-ClC-3 and β1 Integrin. (F and G) Co-IP with anti-ClC-3 (F) or anti-β1 Integrin (G) antibody from extract of ruffling cells probed with anti-K18 pS52. (H) Manipulation of ClC-3 expression by overexpression or knockdown of ClC-3 results in altered Ser-52 phosphorylation of K18. (I) Effects of blocked phosphorylation of K18 at Ser-52 on interaction between ClC-3 or β1 integrin and K18 in EGF-stimulated HeLa cells by mutation of Ser-52 to Ala. Cells were transfected with vectors expressing wild-type (WT) or Ser-33 and Ser-52 (Ala) mutant GFP-K18.
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Figure 7: ClC-3 Mediates Recycling of β1 Integrin by Inducing Keratin 18 Phosphorylation(A) Immunofluorescence images (Left) and evaluation of fluorescence intensity of K18 (right) in HeLa cells treated with shK18 (pGPU6/GFP-K18 shRNA) or shRNA negative control (shNC). **P<0.01; n=3 with >15 cells. Data are mean ± SEM. White arrows mark transfected cells. (B) K18 knockdown by transfection of shK18 inhibits membrane ruffling induced by EGF. (Left) Fluorescent CTXB labeling photographs of live cells. Insets in images show transfected cells by detection of GFP. Yellow arrows indicate membrane ruffles. (Right) Percentage of cells with membrane ruffling was calculated in control or transfected conditions (data are mean ± SEM, **P < 0.001 n=3 with >50 cells). (C) K18 down-regulation prevents K18 and ClC-3 trafficking between cytoplasm and membrane leads them to gather in perinuclear region. After HeLa cells transfected with shK18 or shNC were stimulated by EGF for 5 min immunofluorescence analysis for ClC-3 and β1 integrin carried out. Green arrows point transfected cells. (D) 3D reconstruction of subcellular localization of Ser-52 phosphorylated K18 (K18 pS52) in ruffling cells. Immunofluorescence of Ser-52 phosphorylated K18 in EGF-stimulated HeLa cells was detected with an anti- K18 pS52 antibody and an Alexa Fluor 488-conjugated secondary antibody. White arrows indicate membrane ruffles. See also Movie S2. (E) Co-IP experiment with anti-K18 pS52 antibody from extract of ruffling cells probed with anti-ClC-3 and β1 Integrin. (F and G) Co-IP with anti-ClC-3 (F) or anti-β1 Integrin (G) antibody from extract of ruffling cells probed with anti-K18 pS52. (H) Manipulation of ClC-3 expression by overexpression or knockdown of ClC-3 results in altered Ser-52 phosphorylation of K18. (I) Effects of blocked phosphorylation of K18 at Ser-52 on interaction between ClC-3 or β1 integrin and K18 in EGF-stimulated HeLa cells by mutation of Ser-52 to Ala. Cells were transfected with vectors expressing wild-type (WT) or Ser-33 and Ser-52 (Ala) mutant GFP-K18.

Mentions: To further confirm the roles of K18, we examined the effects of down-regulation of K18 expression on membrane ruffling and β1 integrin and ClC-3 trafficking. The results showed that down-regulation of K18 expression with transfection of K18 shRNA almost completely inhibited EGF-induced membrane ruffling and prevented β1 integrin and ClC-3 trafficking between the cytoplasm and membrane, leading them to gather in the perinuclear region (Figure 7A-C).


Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu B, Jin X, Min L, Li Q, Deng L, Wu H, Lin G, Chen L, Zhang H, Li C, Wang L, Zhu J, Wang W, Chu F, Shen J, Li H, Mao J - Oncotarget (2015)

ClC-3 Mediates Recycling of β1 Integrin by Inducing Keratin 18 Phosphorylation(A) Immunofluorescence images (Left) and evaluation of fluorescence intensity of K18 (right) in HeLa cells treated with shK18 (pGPU6/GFP-K18 shRNA) or shRNA negative control (shNC). **P<0.01; n=3 with >15 cells. Data are mean ± SEM. White arrows mark transfected cells. (B) K18 knockdown by transfection of shK18 inhibits membrane ruffling induced by EGF. (Left) Fluorescent CTXB labeling photographs of live cells. Insets in images show transfected cells by detection of GFP. Yellow arrows indicate membrane ruffles. (Right) Percentage of cells with membrane ruffling was calculated in control or transfected conditions (data are mean ± SEM, **P < 0.001 n=3 with >50 cells). (C) K18 down-regulation prevents K18 and ClC-3 trafficking between cytoplasm and membrane leads them to gather in perinuclear region. After HeLa cells transfected with shK18 or shNC were stimulated by EGF for 5 min immunofluorescence analysis for ClC-3 and β1 integrin carried out. Green arrows point transfected cells. (D) 3D reconstruction of subcellular localization of Ser-52 phosphorylated K18 (K18 pS52) in ruffling cells. Immunofluorescence of Ser-52 phosphorylated K18 in EGF-stimulated HeLa cells was detected with an anti- K18 pS52 antibody and an Alexa Fluor 488-conjugated secondary antibody. White arrows indicate membrane ruffles. See also Movie S2. (E) Co-IP experiment with anti-K18 pS52 antibody from extract of ruffling cells probed with anti-ClC-3 and β1 Integrin. (F and G) Co-IP with anti-ClC-3 (F) or anti-β1 Integrin (G) antibody from extract of ruffling cells probed with anti-K18 pS52. (H) Manipulation of ClC-3 expression by overexpression or knockdown of ClC-3 results in altered Ser-52 phosphorylation of K18. (I) Effects of blocked phosphorylation of K18 at Ser-52 on interaction between ClC-3 or β1 integrin and K18 in EGF-stimulated HeLa cells by mutation of Ser-52 to Ala. Cells were transfected with vectors expressing wild-type (WT) or Ser-33 and Ser-52 (Ala) mutant GFP-K18.
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Show All Figures
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Figure 7: ClC-3 Mediates Recycling of β1 Integrin by Inducing Keratin 18 Phosphorylation(A) Immunofluorescence images (Left) and evaluation of fluorescence intensity of K18 (right) in HeLa cells treated with shK18 (pGPU6/GFP-K18 shRNA) or shRNA negative control (shNC). **P<0.01; n=3 with >15 cells. Data are mean ± SEM. White arrows mark transfected cells. (B) K18 knockdown by transfection of shK18 inhibits membrane ruffling induced by EGF. (Left) Fluorescent CTXB labeling photographs of live cells. Insets in images show transfected cells by detection of GFP. Yellow arrows indicate membrane ruffles. (Right) Percentage of cells with membrane ruffling was calculated in control or transfected conditions (data are mean ± SEM, **P < 0.001 n=3 with >50 cells). (C) K18 down-regulation prevents K18 and ClC-3 trafficking between cytoplasm and membrane leads them to gather in perinuclear region. After HeLa cells transfected with shK18 or shNC were stimulated by EGF for 5 min immunofluorescence analysis for ClC-3 and β1 integrin carried out. Green arrows point transfected cells. (D) 3D reconstruction of subcellular localization of Ser-52 phosphorylated K18 (K18 pS52) in ruffling cells. Immunofluorescence of Ser-52 phosphorylated K18 in EGF-stimulated HeLa cells was detected with an anti- K18 pS52 antibody and an Alexa Fluor 488-conjugated secondary antibody. White arrows indicate membrane ruffles. See also Movie S2. (E) Co-IP experiment with anti-K18 pS52 antibody from extract of ruffling cells probed with anti-ClC-3 and β1 Integrin. (F and G) Co-IP with anti-ClC-3 (F) or anti-β1 Integrin (G) antibody from extract of ruffling cells probed with anti-K18 pS52. (H) Manipulation of ClC-3 expression by overexpression or knockdown of ClC-3 results in altered Ser-52 phosphorylation of K18. (I) Effects of blocked phosphorylation of K18 at Ser-52 on interaction between ClC-3 or β1 integrin and K18 in EGF-stimulated HeLa cells by mutation of Ser-52 to Ala. Cells were transfected with vectors expressing wild-type (WT) or Ser-33 and Ser-52 (Ala) mutant GFP-K18.
Mentions: To further confirm the roles of K18, we examined the effects of down-regulation of K18 expression on membrane ruffling and β1 integrin and ClC-3 trafficking. The results showed that down-regulation of K18 expression with transfection of K18 shRNA almost completely inhibited EGF-induced membrane ruffling and prevented β1 integrin and ClC-3 trafficking between the cytoplasm and membrane, leading them to gather in the perinuclear region (Figure 7A-C).

Bottom Line: High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients.We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis.ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Show MeSH
Related in: MedlinePlus