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Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively.

Shojima K, Sato A, Hanaki H, Tsujimoto I, Nakamura M, Hattori K, Sato Y, Dohi K, Hirata M, Yamamoto H, Kikuchi A - Sci Rep (2015)

Bottom Line: Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity.For this action, Wnt5a-induced receptor endocytosis with clathrin is required.Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan.

ABSTRACT
Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively.

No MeSH data available.


Related in: MedlinePlus

Receptor-mediated endocytosis is not involved in Wnt5a-induced cell proliferation.(a) HeLaS3 cells treated with 25 μg/ml control Ab or mAb5A16 were subjected to the migration and invasion assays. (b) HeLaS3 cells were stimulated with 100 ng/ml Wnt5a for 60 min in the presence or absence of mAb5A16; cell lysates were subjected to the Rac assay. (c) HeLaS3 cells treated with control Ab or mAb5A16 were subjected to the proliferation assay. (d and e) HeLaS3 cells expressing (d) or not expressing (e) FLAG-Fz2 were incubated with 500 ng/ml Wnt5a* (labelled Wnt5a) at 4°C. FLAG-Fz2 is shown in green and Wnt5a* is in red. In the merged images, co-localization of FLAG-Fz2 and Wnt5a* appears yellow. (f) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; cells were then washed three times with an acid solution (100 mM NaCl and 100 mM glycine/HCl [pH 2.8]) to remove Wnt5a* bound to the receptors at the cell surface. (g and h) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; then the cells were incubated for 30 min at 37°C. The cells were stained with anti-FLAG (g and h) and anti-EEA1 (h) antibodies. Co-localization of FLAG-Fz2, EEA1, and Wnt5a* appears white. (i and j) HeLaS3 cells expressing FLAG-Fz2 were treated with Wnt5a* for 30 min in the presence of mAb5A16. FLAG-Fz2 is shown in green and Wnt5a* is in red. Co-localization of FLAG-Fz2 and Wnt5a* appears yellow. The representative confocal images (i) and quantification of internalized Wnt5a* (j) are shown. Results are shown as the mean ± SE of three independent experiments. Scale bars, 10 μm. *, P < 0.05. Cropped blots are used. Full scan images of immunoblots are presented in Supplementary Figure S9. All the gels were run under the same experimental condition as detailed in the Methods section.
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f4: Receptor-mediated endocytosis is not involved in Wnt5a-induced cell proliferation.(a) HeLaS3 cells treated with 25 μg/ml control Ab or mAb5A16 were subjected to the migration and invasion assays. (b) HeLaS3 cells were stimulated with 100 ng/ml Wnt5a for 60 min in the presence or absence of mAb5A16; cell lysates were subjected to the Rac assay. (c) HeLaS3 cells treated with control Ab or mAb5A16 were subjected to the proliferation assay. (d and e) HeLaS3 cells expressing (d) or not expressing (e) FLAG-Fz2 were incubated with 500 ng/ml Wnt5a* (labelled Wnt5a) at 4°C. FLAG-Fz2 is shown in green and Wnt5a* is in red. In the merged images, co-localization of FLAG-Fz2 and Wnt5a* appears yellow. (f) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; cells were then washed three times with an acid solution (100 mM NaCl and 100 mM glycine/HCl [pH 2.8]) to remove Wnt5a* bound to the receptors at the cell surface. (g and h) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; then the cells were incubated for 30 min at 37°C. The cells were stained with anti-FLAG (g and h) and anti-EEA1 (h) antibodies. Co-localization of FLAG-Fz2, EEA1, and Wnt5a* appears white. (i and j) HeLaS3 cells expressing FLAG-Fz2 were treated with Wnt5a* for 30 min in the presence of mAb5A16. FLAG-Fz2 is shown in green and Wnt5a* is in red. Co-localization of FLAG-Fz2 and Wnt5a* appears yellow. The representative confocal images (i) and quantification of internalized Wnt5a* (j) are shown. Results are shown as the mean ± SE of three independent experiments. Scale bars, 10 μm. *, P < 0.05. Cropped blots are used. Full scan images of immunoblots are presented in Supplementary Figure S9. All the gels were run under the same experimental condition as detailed in the Methods section.

Mentions: In HeLaS3 cells, mAb5A16 inhibited migration, invasion, and Wnt5a-dependent activation of Rac1 (Figures 4a and b), but not cell proliferation (Figure 4c). It did not affect proliferation of A549 cells, either (data not shown). The effect of mAb5A16 on Wnt5a-induced receptor internalization in cancer cells which proliferate in a Wnt5a-dependent manner was examined. Purified Wnt5a was labelled with AlexaFluor 546 carboxylic acid succinimidyl ester to directly detect the internalization of Wnt5a. AlexaFluor 546 was conjugated to Wnt5a at a molar ratio of 3.5:1 (Supplementary Figure S5A). Although the specific activity of labelled Wnt5a (Wnt5a*) that induces the phosphorylation of Dvl2 in NIH3T3 cells was partially reduced (Supplementary Figure S5A), we used this labelling condition in the following assays because the labelling of Wnt5a with AlexaFluor 546 at a higher molar ratio (5.5-6:1) resulted in the complete loss of Wnt5a activity (Supplementary Figure S5B).


Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively.

Shojima K, Sato A, Hanaki H, Tsujimoto I, Nakamura M, Hattori K, Sato Y, Dohi K, Hirata M, Yamamoto H, Kikuchi A - Sci Rep (2015)

Receptor-mediated endocytosis is not involved in Wnt5a-induced cell proliferation.(a) HeLaS3 cells treated with 25 μg/ml control Ab or mAb5A16 were subjected to the migration and invasion assays. (b) HeLaS3 cells were stimulated with 100 ng/ml Wnt5a for 60 min in the presence or absence of mAb5A16; cell lysates were subjected to the Rac assay. (c) HeLaS3 cells treated with control Ab or mAb5A16 were subjected to the proliferation assay. (d and e) HeLaS3 cells expressing (d) or not expressing (e) FLAG-Fz2 were incubated with 500 ng/ml Wnt5a* (labelled Wnt5a) at 4°C. FLAG-Fz2 is shown in green and Wnt5a* is in red. In the merged images, co-localization of FLAG-Fz2 and Wnt5a* appears yellow. (f) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; cells were then washed three times with an acid solution (100 mM NaCl and 100 mM glycine/HCl [pH 2.8]) to remove Wnt5a* bound to the receptors at the cell surface. (g and h) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; then the cells were incubated for 30 min at 37°C. The cells were stained with anti-FLAG (g and h) and anti-EEA1 (h) antibodies. Co-localization of FLAG-Fz2, EEA1, and Wnt5a* appears white. (i and j) HeLaS3 cells expressing FLAG-Fz2 were treated with Wnt5a* for 30 min in the presence of mAb5A16. FLAG-Fz2 is shown in green and Wnt5a* is in red. Co-localization of FLAG-Fz2 and Wnt5a* appears yellow. The representative confocal images (i) and quantification of internalized Wnt5a* (j) are shown. Results are shown as the mean ± SE of three independent experiments. Scale bars, 10 μm. *, P < 0.05. Cropped blots are used. Full scan images of immunoblots are presented in Supplementary Figure S9. All the gels were run under the same experimental condition as detailed in the Methods section.
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f4: Receptor-mediated endocytosis is not involved in Wnt5a-induced cell proliferation.(a) HeLaS3 cells treated with 25 μg/ml control Ab or mAb5A16 were subjected to the migration and invasion assays. (b) HeLaS3 cells were stimulated with 100 ng/ml Wnt5a for 60 min in the presence or absence of mAb5A16; cell lysates were subjected to the Rac assay. (c) HeLaS3 cells treated with control Ab or mAb5A16 were subjected to the proliferation assay. (d and e) HeLaS3 cells expressing (d) or not expressing (e) FLAG-Fz2 were incubated with 500 ng/ml Wnt5a* (labelled Wnt5a) at 4°C. FLAG-Fz2 is shown in green and Wnt5a* is in red. In the merged images, co-localization of FLAG-Fz2 and Wnt5a* appears yellow. (f) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; cells were then washed three times with an acid solution (100 mM NaCl and 100 mM glycine/HCl [pH 2.8]) to remove Wnt5a* bound to the receptors at the cell surface. (g and h) HeLaS3 cells expressing FLAG-Fz2 were incubated with Wnt5a* at 4°C; then the cells were incubated for 30 min at 37°C. The cells were stained with anti-FLAG (g and h) and anti-EEA1 (h) antibodies. Co-localization of FLAG-Fz2, EEA1, and Wnt5a* appears white. (i and j) HeLaS3 cells expressing FLAG-Fz2 were treated with Wnt5a* for 30 min in the presence of mAb5A16. FLAG-Fz2 is shown in green and Wnt5a* is in red. Co-localization of FLAG-Fz2 and Wnt5a* appears yellow. The representative confocal images (i) and quantification of internalized Wnt5a* (j) are shown. Results are shown as the mean ± SE of three independent experiments. Scale bars, 10 μm. *, P < 0.05. Cropped blots are used. Full scan images of immunoblots are presented in Supplementary Figure S9. All the gels were run under the same experimental condition as detailed in the Methods section.
Mentions: In HeLaS3 cells, mAb5A16 inhibited migration, invasion, and Wnt5a-dependent activation of Rac1 (Figures 4a and b), but not cell proliferation (Figure 4c). It did not affect proliferation of A549 cells, either (data not shown). The effect of mAb5A16 on Wnt5a-induced receptor internalization in cancer cells which proliferate in a Wnt5a-dependent manner was examined. Purified Wnt5a was labelled with AlexaFluor 546 carboxylic acid succinimidyl ester to directly detect the internalization of Wnt5a. AlexaFluor 546 was conjugated to Wnt5a at a molar ratio of 3.5:1 (Supplementary Figure S5A). Although the specific activity of labelled Wnt5a (Wnt5a*) that induces the phosphorylation of Dvl2 in NIH3T3 cells was partially reduced (Supplementary Figure S5A), we used this labelling condition in the following assays because the labelling of Wnt5a with AlexaFluor 546 at a higher molar ratio (5.5-6:1) resulted in the complete loss of Wnt5a activity (Supplementary Figure S5B).

Bottom Line: Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity.For this action, Wnt5a-induced receptor endocytosis with clathrin is required.Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan.

ABSTRACT
Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively.

No MeSH data available.


Related in: MedlinePlus