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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 Regulates β1 Integrin Recycling through Binding to K18(A) Besides membrane ruffles, ClC-3 and β1 integrin also colocalize in cord-like structures in the cytoplasm of HeLa cells stimulated with EGF. The Pearson coefficient is 0.87±0.04 (mean ± SEM, n=3 with 15 cells). (B) ClC-3 and β1 integrin do not bind to each other. Left: Immunoblots of co-IP with anti-ClC-3 antibody (B21) from extract of HeLa cells treated with EGF, probed with antibodies against β1 integrin or ClC-3 (Abcam). Right: co-IP with anti-β1 integrin antibody from the same extract probed with anti-ClC-3. (C) Immunofluorescence staining for ClC-3, β1 Integrin and pan-cytokeratin (P-CK) indicates that ClC-3 (Pearson coefficient: 0.87±0.05 for ruffles and 0.89±0.02 for cytoplasm, n=3 with 16 cells) or β1 integrin (Pearson coefficient: 0.89±0.02 for ruffles and 0.91±0.03 for cytoplasm, n=3 with 14 cells) colocalizes with P-CK in membrane ruffles and cytoplasm in HeLa cells stimulated with EGF. Bar: 20μM. (D and E) ClC-3 or β1 Integrin (Pearson coefficient: 0.78±0.04 for ruffles and 0.92±0.06 for cytoplasm, n=3 with 13 cells) colocalize with K18 in membrane ruffles and cytoplasm in cells with EGF stimulation. (D) Representative Immunofluorescence images. (E) A Pearson's coefficient was calculated to estimate the degree of co-localization of the different keratins with ClC-3. The results represent >20 cells from n = 2 independent experiments. (F) Co-IP experiment with anti-β1 integrin (right) or ClC-3 (left) antibody from the extract of ruffling HeLa cells (EGF treatment) probed with K18 showed that β1 Integrin or ClC-3 is binding to K18 respectively. (G) Co-IP with anti-K18 antibody from the extract of ruffling HeLa cells (EGF treatment) probed with anti-ClC-3 or β1 Integrin.
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Figure 6: ClC-3 Regulates β1 Integrin Recycling through Binding to K18(A) Besides membrane ruffles, ClC-3 and β1 integrin also colocalize in cord-like structures in the cytoplasm of HeLa cells stimulated with EGF. The Pearson coefficient is 0.87±0.04 (mean ± SEM, n=3 with 15 cells). (B) ClC-3 and β1 integrin do not bind to each other. Left: Immunoblots of co-IP with anti-ClC-3 antibody (B21) from extract of HeLa cells treated with EGF, probed with antibodies against β1 integrin or ClC-3 (Abcam). Right: co-IP with anti-β1 integrin antibody from the same extract probed with anti-ClC-3. (C) Immunofluorescence staining for ClC-3, β1 Integrin and pan-cytokeratin (P-CK) indicates that ClC-3 (Pearson coefficient: 0.87±0.05 for ruffles and 0.89±0.02 for cytoplasm, n=3 with 16 cells) or β1 integrin (Pearson coefficient: 0.89±0.02 for ruffles and 0.91±0.03 for cytoplasm, n=3 with 14 cells) colocalizes with P-CK in membrane ruffles and cytoplasm in HeLa cells stimulated with EGF. Bar: 20μM. (D and E) ClC-3 or β1 Integrin (Pearson coefficient: 0.78±0.04 for ruffles and 0.92±0.06 for cytoplasm, n=3 with 13 cells) colocalize with K18 in membrane ruffles and cytoplasm in cells with EGF stimulation. (D) Representative Immunofluorescence images. (E) A Pearson's coefficient was calculated to estimate the degree of co-localization of the different keratins with ClC-3. The results represent >20 cells from n = 2 independent experiments. (F) Co-IP experiment with anti-β1 integrin (right) or ClC-3 (left) antibody from the extract of ruffling HeLa cells (EGF treatment) probed with K18 showed that β1 Integrin or ClC-3 is binding to K18 respectively. (G) Co-IP with anti-K18 antibody from the extract of ruffling HeLa cells (EGF treatment) probed with anti-ClC-3 or β1 Integrin.

Mentions: We next sought to understand how ClC-3 regulates β1 integrin recycling. We first tested whether ClC-3 and β1 Integrin colocalized and combined in the cytoplasm. With EGF stimulation, ClC-3 and β1 integrin were found to colocalize in cord-like structures in the cytoplasm (Figure 6A). However, ClC-3 and β1 integrin were not bound to each other (Figure 6B). This suggests that ClC-3 does not directly regulate β1 integrin recycling. What is then the mediator between ClC-3 and β1 integrin? The cytoskeleton, including microtubules, microfilaments and intermediate filaments, is known to play key roles in intracellular protein traffic. We then speculated that one or more of cytoskeleton components may be involved in the regulation of β1 integrin recycling by ClC-3. Immunofluorescence detection showed that except for keratin, ClC-3 did not colocalize with microtubules, microfilaments or vimentin in the cytoplasm during membrane ruffling (Figure 6C and S4A-C, F). Keratin 18 (K18) was identified as the most possible keratin molecule involved in the regulation of β1 integrin recycling by immunofluorescence colocalization observation in HeLa cells and different types of cancer tissues (Figure 6D, E, Figure S4D-F). Co-IP analysis confirmed and showed that K18 and ClC-3 were associated with β1 integrin, respectively (Figure 6F, G). Together, these data suggest that ClC-3 may mediate β1 integrin recycling by regulating K18 organization.


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 Regulates β1 Integrin Recycling through Binding to K18(A) Besides membrane ruffles, ClC-3 and β1 integrin also colocalize in cord-like structures in the cytoplasm of HeLa cells stimulated with EGF. The Pearson coefficient is 0.87±0.04 (mean ± SEM, n=3 with 15 cells). (B) ClC-3 and β1 integrin do not bind to each other. Left: Immunoblots of co-IP with anti-ClC-3 antibody (B21) from extract of HeLa cells treated with EGF, probed with antibodies against β1 integrin or ClC-3 (Abcam). Right: co-IP with anti-β1 integrin antibody from the same extract probed with anti-ClC-3. (C) Immunofluorescence staining for ClC-3, β1 Integrin and pan-cytokeratin (P-CK) indicates that ClC-3 (Pearson coefficient: 0.87±0.05 for ruffles and 0.89±0.02 for cytoplasm, n=3 with 16 cells) or β1 integrin (Pearson coefficient: 0.89±0.02 for ruffles and 0.91±0.03 for cytoplasm, n=3 with 14 cells) colocalizes with P-CK in membrane ruffles and cytoplasm in HeLa cells stimulated with EGF. Bar: 20μM. (D and E) ClC-3 or β1 Integrin (Pearson coefficient: 0.78±0.04 for ruffles and 0.92±0.06 for cytoplasm, n=3 with 13 cells) colocalize with K18 in membrane ruffles and cytoplasm in cells with EGF stimulation. (D) Representative Immunofluorescence images. (E) A Pearson's coefficient was calculated to estimate the degree of co-localization of the different keratins with ClC-3. The results represent >20 cells from n = 2 independent experiments. (F) Co-IP experiment with anti-β1 integrin (right) or ClC-3 (left) antibody from the extract of ruffling HeLa cells (EGF treatment) probed with K18 showed that β1 Integrin or ClC-3 is binding to K18 respectively. (G) Co-IP with anti-K18 antibody from the extract of ruffling HeLa cells (EGF treatment) probed with anti-ClC-3 or β1 Integrin.
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Figure 6: ClC-3 Regulates β1 Integrin Recycling through Binding to K18(A) Besides membrane ruffles, ClC-3 and β1 integrin also colocalize in cord-like structures in the cytoplasm of HeLa cells stimulated with EGF. The Pearson coefficient is 0.87±0.04 (mean ± SEM, n=3 with 15 cells). (B) ClC-3 and β1 integrin do not bind to each other. Left: Immunoblots of co-IP with anti-ClC-3 antibody (B21) from extract of HeLa cells treated with EGF, probed with antibodies against β1 integrin or ClC-3 (Abcam). Right: co-IP with anti-β1 integrin antibody from the same extract probed with anti-ClC-3. (C) Immunofluorescence staining for ClC-3, β1 Integrin and pan-cytokeratin (P-CK) indicates that ClC-3 (Pearson coefficient: 0.87±0.05 for ruffles and 0.89±0.02 for cytoplasm, n=3 with 16 cells) or β1 integrin (Pearson coefficient: 0.89±0.02 for ruffles and 0.91±0.03 for cytoplasm, n=3 with 14 cells) colocalizes with P-CK in membrane ruffles and cytoplasm in HeLa cells stimulated with EGF. Bar: 20μM. (D and E) ClC-3 or β1 Integrin (Pearson coefficient: 0.78±0.04 for ruffles and 0.92±0.06 for cytoplasm, n=3 with 13 cells) colocalize with K18 in membrane ruffles and cytoplasm in cells with EGF stimulation. (D) Representative Immunofluorescence images. (E) A Pearson's coefficient was calculated to estimate the degree of co-localization of the different keratins with ClC-3. The results represent >20 cells from n = 2 independent experiments. (F) Co-IP experiment with anti-β1 integrin (right) or ClC-3 (left) antibody from the extract of ruffling HeLa cells (EGF treatment) probed with K18 showed that β1 Integrin or ClC-3 is binding to K18 respectively. (G) Co-IP with anti-K18 antibody from the extract of ruffling HeLa cells (EGF treatment) probed with anti-ClC-3 or β1 Integrin.
Mentions: We next sought to understand how ClC-3 regulates β1 integrin recycling. We first tested whether ClC-3 and β1 Integrin colocalized and combined in the cytoplasm. With EGF stimulation, ClC-3 and β1 integrin were found to colocalize in cord-like structures in the cytoplasm (Figure 6A). However, ClC-3 and β1 integrin were not bound to each other (Figure 6B). This suggests that ClC-3 does not directly regulate β1 integrin recycling. What is then the mediator between ClC-3 and β1 integrin? The cytoskeleton, including microtubules, microfilaments and intermediate filaments, is known to play key roles in intracellular protein traffic. We then speculated that one or more of cytoskeleton components may be involved in the regulation of β1 integrin recycling by ClC-3. Immunofluorescence detection showed that except for keratin, ClC-3 did not colocalize with microtubules, microfilaments or vimentin in the cytoplasm during membrane ruffling (Figure 6C and S4A-C, F). Keratin 18 (K18) was identified as the most possible keratin molecule involved in the regulation of β1 integrin recycling by immunofluorescence colocalization observation in HeLa cells and different types of cancer tissues (Figure 6D, E, Figure S4D-F). Co-IP analysis confirmed and showed that K18 and ClC-3 were associated with β1 integrin, respectively (Figure 6F, G). Together, these data suggest that ClC-3 may mediate β1 integrin recycling by regulating K18 organization.

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