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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-mediated Membrane Ruffling Is Related to Cancer Cell Migration(A) Representative Western blot probed for total ClC-3 to detect expression differences between high- (MHCC97H and HO8910PM) and low- (MHCC97Land HO8910) metastatic potential cancer cell lines.**P<0.01; n=3. Data are mean ± SEM. (B-D) Comparison of membrane ruffling capability between high- and low-metastatic potential liver (B) and ovarian (C) cancer cell lines and quantification of multiple visual fields (n = 10, D). Bright-field photographs of live cells were taken at 20 min after subculturing of cells. Pink arrows indicate membrane ruffles. (E -G) Observation of cell migration ability for high- and low-metastatic potential liver (E) and ovarian (F) cancer cell lines. Confluent monolayers were scratched and then cultured in the medium along with EGF (10 ng/ml) for different time. Data shown in (G) are mean ± SEM. (H-J) Altering ClC-3 expression affects cell migration in an in vitro wound assay. Representative photographs of scratch wound-healing motility assays obtained from HeLa cells (HeLa/ClC-3) stably transfected with PCDNA3.1-ClC-3 vectors (H) or HeLa/ClC-3 cells transiently treated with shRNA against ClC-3 (shClC-3, I). Average migratory width of three independent experiments is shown (J, data are mean ± SEM, ** P < 0.01). (K) Positive correlation between ClC-3 expression and membrane ruffling. Percentage of cells with ruffling is plotted against the level of ClC-3 protein expression. Fitting the data with the equation, f = y0 + ax, results in a linear correlation coefficient (r) of 0.98 (P < 0.01; y0 = 4.5, a = 395.3). (L) A positive correlation between ClC-3 expression and cell migration is obtained by plotting the migration rate against the ClC-3 expression level under the same treatments and by fitting the data with the equation f = y0 + ax. Fitting yields a linear correlation coefficient of r = 0.81 (P < 0.05; y0 = 14.5, a = 110.2).
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Figure 4: ClC-3-mediated Membrane Ruffling Is Related to Cancer Cell Migration(A) Representative Western blot probed for total ClC-3 to detect expression differences between high- (MHCC97H and HO8910PM) and low- (MHCC97Land HO8910) metastatic potential cancer cell lines.**P<0.01; n=3. Data are mean ± SEM. (B-D) Comparison of membrane ruffling capability between high- and low-metastatic potential liver (B) and ovarian (C) cancer cell lines and quantification of multiple visual fields (n = 10, D). Bright-field photographs of live cells were taken at 20 min after subculturing of cells. Pink arrows indicate membrane ruffles. (E -G) Observation of cell migration ability for high- and low-metastatic potential liver (E) and ovarian (F) cancer cell lines. Confluent monolayers were scratched and then cultured in the medium along with EGF (10 ng/ml) for different time. Data shown in (G) are mean ± SEM. (H-J) Altering ClC-3 expression affects cell migration in an in vitro wound assay. Representative photographs of scratch wound-healing motility assays obtained from HeLa cells (HeLa/ClC-3) stably transfected with PCDNA3.1-ClC-3 vectors (H) or HeLa/ClC-3 cells transiently treated with shRNA against ClC-3 (shClC-3, I). Average migratory width of three independent experiments is shown (J, data are mean ± SEM, ** P < 0.01). (K) Positive correlation between ClC-3 expression and membrane ruffling. Percentage of cells with ruffling is plotted against the level of ClC-3 protein expression. Fitting the data with the equation, f = y0 + ax, results in a linear correlation coefficient (r) of 0.98 (P < 0.01; y0 = 4.5, a = 395.3). (L) A positive correlation between ClC-3 expression and cell migration is obtained by plotting the migration rate against the ClC-3 expression level under the same treatments and by fitting the data with the equation f = y0 + ax. Fitting yields a linear correlation coefficient of r = 0.81 (P < 0.05; y0 = 14.5, a = 110.2).

Mentions: As shown above, ClC-3 expression promotes membrane ruffle formation. Membrane ruffling has been shown to be an indicator of tumor cell motility and metastatic potential [2]. Accordingly, cells with high metastatic potential should have much more membrane ruffles and higher migration potential. To validate this, differences in ClC-3 expression, membrane ruffling ability and cell migration potential between high- and low-metastatic potential cell lines were assessed. Both high-metastatic MHCC97H and HO-8910PM cells possessed higher ClC-3 expression, more membrane ruffles and greater migration distance than corresponding low-metastatic MHCC97L and HO-8910 (Figure 4A-G). The effects of up-regulation or down-regulation of ClC-3 expression on cell migration were also evaluated. HeLa cells with stably overexpressing ClC-3 had faster movement. However, silencing ClC-3 expression stopped the HeLa/ClC-3 cells' locomotion (Figure 4H-J). Analysis of the relationship to each other of ClC-3 expression and membrane ruffling and cell migration showed that the former were positively correlated with the latter two (Figure 4K, L). The positive relationship provides additional evidence for the involvement of ClC-3-mediated membrane ruffling in cancer cell migration. Together, these data indicate that ClC-3-regulated membrane ruffling is closely related to cancer cell migration.


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-mediated Membrane Ruffling Is Related to Cancer Cell Migration(A) Representative Western blot probed for total ClC-3 to detect expression differences between high- (MHCC97H and HO8910PM) and low- (MHCC97Land HO8910) metastatic potential cancer cell lines.**P<0.01; n=3. Data are mean ± SEM. (B-D) Comparison of membrane ruffling capability between high- and low-metastatic potential liver (B) and ovarian (C) cancer cell lines and quantification of multiple visual fields (n = 10, D). Bright-field photographs of live cells were taken at 20 min after subculturing of cells. Pink arrows indicate membrane ruffles. (E -G) Observation of cell migration ability for high- and low-metastatic potential liver (E) and ovarian (F) cancer cell lines. Confluent monolayers were scratched and then cultured in the medium along with EGF (10 ng/ml) for different time. Data shown in (G) are mean ± SEM. (H-J) Altering ClC-3 expression affects cell migration in an in vitro wound assay. Representative photographs of scratch wound-healing motility assays obtained from HeLa cells (HeLa/ClC-3) stably transfected with PCDNA3.1-ClC-3 vectors (H) or HeLa/ClC-3 cells transiently treated with shRNA against ClC-3 (shClC-3, I). Average migratory width of three independent experiments is shown (J, data are mean ± SEM, ** P < 0.01). (K) Positive correlation between ClC-3 expression and membrane ruffling. Percentage of cells with ruffling is plotted against the level of ClC-3 protein expression. Fitting the data with the equation, f = y0 + ax, results in a linear correlation coefficient (r) of 0.98 (P < 0.01; y0 = 4.5, a = 395.3). (L) A positive correlation between ClC-3 expression and cell migration is obtained by plotting the migration rate against the ClC-3 expression level under the same treatments and by fitting the data with the equation f = y0 + ax. Fitting yields a linear correlation coefficient of r = 0.81 (P < 0.05; y0 = 14.5, a = 110.2).
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Figure 4: ClC-3-mediated Membrane Ruffling Is Related to Cancer Cell Migration(A) Representative Western blot probed for total ClC-3 to detect expression differences between high- (MHCC97H and HO8910PM) and low- (MHCC97Land HO8910) metastatic potential cancer cell lines.**P<0.01; n=3. Data are mean ± SEM. (B-D) Comparison of membrane ruffling capability between high- and low-metastatic potential liver (B) and ovarian (C) cancer cell lines and quantification of multiple visual fields (n = 10, D). Bright-field photographs of live cells were taken at 20 min after subculturing of cells. Pink arrows indicate membrane ruffles. (E -G) Observation of cell migration ability for high- and low-metastatic potential liver (E) and ovarian (F) cancer cell lines. Confluent monolayers were scratched and then cultured in the medium along with EGF (10 ng/ml) for different time. Data shown in (G) are mean ± SEM. (H-J) Altering ClC-3 expression affects cell migration in an in vitro wound assay. Representative photographs of scratch wound-healing motility assays obtained from HeLa cells (HeLa/ClC-3) stably transfected with PCDNA3.1-ClC-3 vectors (H) or HeLa/ClC-3 cells transiently treated with shRNA against ClC-3 (shClC-3, I). Average migratory width of three independent experiments is shown (J, data are mean ± SEM, ** P < 0.01). (K) Positive correlation between ClC-3 expression and membrane ruffling. Percentage of cells with ruffling is plotted against the level of ClC-3 protein expression. Fitting the data with the equation, f = y0 + ax, results in a linear correlation coefficient (r) of 0.98 (P < 0.01; y0 = 4.5, a = 395.3). (L) A positive correlation between ClC-3 expression and cell migration is obtained by plotting the migration rate against the ClC-3 expression level under the same treatments and by fitting the data with the equation f = y0 + ax. Fitting yields a linear correlation coefficient of r = 0.81 (P < 0.05; y0 = 14.5, a = 110.2).
Mentions: As shown above, ClC-3 expression promotes membrane ruffle formation. Membrane ruffling has been shown to be an indicator of tumor cell motility and metastatic potential [2]. Accordingly, cells with high metastatic potential should have much more membrane ruffles and higher migration potential. To validate this, differences in ClC-3 expression, membrane ruffling ability and cell migration potential between high- and low-metastatic potential cell lines were assessed. Both high-metastatic MHCC97H and HO-8910PM cells possessed higher ClC-3 expression, more membrane ruffles and greater migration distance than corresponding low-metastatic MHCC97L and HO-8910 (Figure 4A-G). The effects of up-regulation or down-regulation of ClC-3 expression on cell migration were also evaluated. HeLa cells with stably overexpressing ClC-3 had faster movement. However, silencing ClC-3 expression stopped the HeLa/ClC-3 cells' locomotion (Figure 4H-J). Analysis of the relationship to each other of ClC-3 expression and membrane ruffling and cell migration showed that the former were positively correlated with the latter two (Figure 4K, L). The positive relationship provides additional evidence for the involvement of ClC-3-mediated membrane ruffling in cancer cell migration. Together, these data indicate that ClC-3-regulated membrane ruffling is closely related to cancer cell migration.

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