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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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Related in: MedlinePlus

ClC-3 is Involved in β1 Integrin Recycling(A) Immunofluorescence of HeLa cells stained for ClC-3 (green) and β1 Integrin (red) indicates that ClC-3 colocalized with endogenous β1 integrin in ruffles (arrow or rectangle) in HeLa cells with or without EGF stimulation. The Pearson coefficient is 0.79±0.08 for without EGF stimulation and 0.71±0.04 for EGF stimulation (mean ± SEM, n=3 with 8 and 12 cells). (B) Indirect immunofluorescence indicates that ClC-3 and internalized β1-integrin colocalized together perfectly in the cytoplasm. The Pearson coefficient is 0.73±0.05 (mean ± SEM, n=3 with 10 cells). Surface β1 integrin was labeled with anti-β1 integrin antibody and the cells then incubated at 37°C for 2 h (pulse) to induce integrin-antibody complexes to be internalized. (C and D) Representative images(C) and quantitative analysis (D) show that down-regulation of ClC-3 expression did not affect membrane β1 integrin internalization. Before internalization assay HeLa cells were treated with shClC-3 or shNC for 48h. # P >0.05 VS shNC. Data are mean ± SEM. White arrows point transfected cells (green). (E-I) ClC-3 knockdown affects β1 integrin recycling in HeLa cells. Following β1 integrin internalization, recycling of anti-β1-integrin/β1-integrin complexes to the plasma membrane occurred by stimulation with serum. HeLa cells on plates (G-I) or coverslips (E and F) were mock-treated or treated with shClC-3 or ClC-3-RNAi. Immunofluorescence for detecting the fixed cells (E, arrows mark transfected cells) and mean fluorescence analysis of internal β1-integrin (F) suggests that internalized β1-integrin accumulates in the perinuclear region in ClC-3-knockdown cells transfected with shClC-3. β1-Integrin recycling to plasma membrane in live HeLa cells was visualized by immunofluorescence (H) and quantitatively measured by a flow cytometry recycling assay (I). ClC-3 knockdown (G) clearly impairs β1 integrin recycling to the plasma membrane. **P<0.01 VS shNC or mock. Data in (F and G) are mean ± SEM.
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Figure 5: ClC-3 is Involved in β1 Integrin Recycling(A) Immunofluorescence of HeLa cells stained for ClC-3 (green) and β1 Integrin (red) indicates that ClC-3 colocalized with endogenous β1 integrin in ruffles (arrow or rectangle) in HeLa cells with or without EGF stimulation. The Pearson coefficient is 0.79±0.08 for without EGF stimulation and 0.71±0.04 for EGF stimulation (mean ± SEM, n=3 with 8 and 12 cells). (B) Indirect immunofluorescence indicates that ClC-3 and internalized β1-integrin colocalized together perfectly in the cytoplasm. The Pearson coefficient is 0.73±0.05 (mean ± SEM, n=3 with 10 cells). Surface β1 integrin was labeled with anti-β1 integrin antibody and the cells then incubated at 37°C for 2 h (pulse) to induce integrin-antibody complexes to be internalized. (C and D) Representative images(C) and quantitative analysis (D) show that down-regulation of ClC-3 expression did not affect membrane β1 integrin internalization. Before internalization assay HeLa cells were treated with shClC-3 or shNC for 48h. # P >0.05 VS shNC. Data are mean ± SEM. White arrows point transfected cells (green). (E-I) ClC-3 knockdown affects β1 integrin recycling in HeLa cells. Following β1 integrin internalization, recycling of anti-β1-integrin/β1-integrin complexes to the plasma membrane occurred by stimulation with serum. HeLa cells on plates (G-I) or coverslips (E and F) were mock-treated or treated with shClC-3 or ClC-3-RNAi. Immunofluorescence for detecting the fixed cells (E, arrows mark transfected cells) and mean fluorescence analysis of internal β1-integrin (F) suggests that internalized β1-integrin accumulates in the perinuclear region in ClC-3-knockdown cells transfected with shClC-3. β1-Integrin recycling to plasma membrane in live HeLa cells was visualized by immunofluorescence (H) and quantitatively measured by a flow cytometry recycling assay (I). ClC-3 knockdown (G) clearly impairs β1 integrin recycling to the plasma membrane. **P<0.01 VS shNC or mock. Data in (F and G) are mean ± SEM.

Mentions: β1 Integrin has been revealed to be involved in membrane ruffling by internalizing, recycling and clustering in membrane ruffles [10]. We wondered whether ClC-3 has roles in β1 integrin trafficking during membrane ruffle formation. Using double labeling with immunofluorescence detection, we first found that ClC-3 colocalized with endogenous β1 integrin in ruffles in HeLa cells with or without EGF stimulation (Figure 5A). To follow β1 integrin trafficking, we next used 12G10 anti-β1 integrin antibody to label surface β1 integrin [35] and then incubated the cells at 37°C for 2 h (pulse) to induce integrin-antibody complexes to be internalized. Indirect immunofluorescence showed that ClC-3 and internalized β1-integrin colocalized closely in the cytoplasm (Figure 5B). These results imply that ClC-3 may mediate membrane ruffling by regulating β1 integrin internalizing and recycling. To confirm this, the effects of down-regulation of ClC-3 expression by transfecting with ClC-3 shRNA or SiRNA on β1 Integrin internalizing and recycling were examined. The results showed that internalized β1 integrin in ClC-3 shRNA-transfected cells mainly accumulated in the perinuclear region, and there was no difference with mock-treated cells (Figure 5C, D). This hints that ClC-3 may not be involved in the internalization of β1 integrin. Following 1 h incubation with medium containing 20% fetal bovine serum (FBS) to stimulate internalized β1 integrin recycling, most of the internalized β1 integrin in mock-treated cells were recycled to the membrane. However, cells treated with ClC-3 shRNA or SiRNA displayed sustained perinuclear β1 integrin aggregation and little immunostaining of recycled membrane β1 integrin (Figure 5E-H).


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 is Involved in β1 Integrin Recycling(A) Immunofluorescence of HeLa cells stained for ClC-3 (green) and β1 Integrin (red) indicates that ClC-3 colocalized with endogenous β1 integrin in ruffles (arrow or rectangle) in HeLa cells with or without EGF stimulation. The Pearson coefficient is 0.79±0.08 for without EGF stimulation and 0.71±0.04 for EGF stimulation (mean ± SEM, n=3 with 8 and 12 cells). (B) Indirect immunofluorescence indicates that ClC-3 and internalized β1-integrin colocalized together perfectly in the cytoplasm. The Pearson coefficient is 0.73±0.05 (mean ± SEM, n=3 with 10 cells). Surface β1 integrin was labeled with anti-β1 integrin antibody and the cells then incubated at 37°C for 2 h (pulse) to induce integrin-antibody complexes to be internalized. (C and D) Representative images(C) and quantitative analysis (D) show that down-regulation of ClC-3 expression did not affect membrane β1 integrin internalization. Before internalization assay HeLa cells were treated with shClC-3 or shNC for 48h. # P >0.05 VS shNC. Data are mean ± SEM. White arrows point transfected cells (green). (E-I) ClC-3 knockdown affects β1 integrin recycling in HeLa cells. Following β1 integrin internalization, recycling of anti-β1-integrin/β1-integrin complexes to the plasma membrane occurred by stimulation with serum. HeLa cells on plates (G-I) or coverslips (E and F) were mock-treated or treated with shClC-3 or ClC-3-RNAi. Immunofluorescence for detecting the fixed cells (E, arrows mark transfected cells) and mean fluorescence analysis of internal β1-integrin (F) suggests that internalized β1-integrin accumulates in the perinuclear region in ClC-3-knockdown cells transfected with shClC-3. β1-Integrin recycling to plasma membrane in live HeLa cells was visualized by immunofluorescence (H) and quantitatively measured by a flow cytometry recycling assay (I). ClC-3 knockdown (G) clearly impairs β1 integrin recycling to the plasma membrane. **P<0.01 VS shNC or mock. Data in (F and G) are mean ± SEM.
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Figure 5: ClC-3 is Involved in β1 Integrin Recycling(A) Immunofluorescence of HeLa cells stained for ClC-3 (green) and β1 Integrin (red) indicates that ClC-3 colocalized with endogenous β1 integrin in ruffles (arrow or rectangle) in HeLa cells with or without EGF stimulation. The Pearson coefficient is 0.79±0.08 for without EGF stimulation and 0.71±0.04 for EGF stimulation (mean ± SEM, n=3 with 8 and 12 cells). (B) Indirect immunofluorescence indicates that ClC-3 and internalized β1-integrin colocalized together perfectly in the cytoplasm. The Pearson coefficient is 0.73±0.05 (mean ± SEM, n=3 with 10 cells). Surface β1 integrin was labeled with anti-β1 integrin antibody and the cells then incubated at 37°C for 2 h (pulse) to induce integrin-antibody complexes to be internalized. (C and D) Representative images(C) and quantitative analysis (D) show that down-regulation of ClC-3 expression did not affect membrane β1 integrin internalization. Before internalization assay HeLa cells were treated with shClC-3 or shNC for 48h. # P >0.05 VS shNC. Data are mean ± SEM. White arrows point transfected cells (green). (E-I) ClC-3 knockdown affects β1 integrin recycling in HeLa cells. Following β1 integrin internalization, recycling of anti-β1-integrin/β1-integrin complexes to the plasma membrane occurred by stimulation with serum. HeLa cells on plates (G-I) or coverslips (E and F) were mock-treated or treated with shClC-3 or ClC-3-RNAi. Immunofluorescence for detecting the fixed cells (E, arrows mark transfected cells) and mean fluorescence analysis of internal β1-integrin (F) suggests that internalized β1-integrin accumulates in the perinuclear region in ClC-3-knockdown cells transfected with shClC-3. β1-Integrin recycling to plasma membrane in live HeLa cells was visualized by immunofluorescence (H) and quantitatively measured by a flow cytometry recycling assay (I). ClC-3 knockdown (G) clearly impairs β1 integrin recycling to the plasma membrane. **P<0.01 VS shNC or mock. Data in (F and G) are mean ± SEM.
Mentions: β1 Integrin has been revealed to be involved in membrane ruffling by internalizing, recycling and clustering in membrane ruffles [10]. We wondered whether ClC-3 has roles in β1 integrin trafficking during membrane ruffle formation. Using double labeling with immunofluorescence detection, we first found that ClC-3 colocalized with endogenous β1 integrin in ruffles in HeLa cells with or without EGF stimulation (Figure 5A). To follow β1 integrin trafficking, we next used 12G10 anti-β1 integrin antibody to label surface β1 integrin [35] and then incubated the cells at 37°C for 2 h (pulse) to induce integrin-antibody complexes to be internalized. Indirect immunofluorescence showed that ClC-3 and internalized β1-integrin colocalized closely in the cytoplasm (Figure 5B). These results imply that ClC-3 may mediate membrane ruffling by regulating β1 integrin internalizing and recycling. To confirm this, the effects of down-regulation of ClC-3 expression by transfecting with ClC-3 shRNA or SiRNA on β1 Integrin internalizing and recycling were examined. The results showed that internalized β1 integrin in ClC-3 shRNA-transfected cells mainly accumulated in the perinuclear region, and there was no difference with mock-treated cells (Figure 5C, D). This hints that ClC-3 may not be involved in the internalization of β1 integrin. Following 1 h incubation with medium containing 20% fetal bovine serum (FBS) to stimulate internalized β1 integrin recycling, most of the internalized β1 integrin in mock-treated cells were recycled to the membrane. However, cells treated with ClC-3 shRNA or SiRNA displayed sustained perinuclear β1 integrin aggregation and little immunostaining of recycled membrane β1 integrin (Figure 5E-H).

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