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The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma.

O'Connell MP, Fiori JL, Xu M, Carter AD, Frank BP, Camilli TC, French AD, Dissanayake SK, Indig FE, Bernier M, Taub DD, Hewitt SM, Weeraratna AT - Oncogene (2009)

Bottom Line: We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2.WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels.ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.

ABSTRACT
Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we show, for the first time, the importance of the orphan tyrosine kinase receptor, ROR2, in melanoma progression. Using melanoma tissue microarrays, we show that ROR2 is expressed predominantly in metastatic melanoma. As ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our earlier data implicating Wnt5A as a mediator of melanoma metastasis. We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2. WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels. ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A. Using in vitro and in vivo metastasis assays, we show that ROR2 is necessary for the Wnt5A-mediated metastasis of melanoma cells. These data imply that ROR2 may represent a novel target for melanoma therapy.

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ROR2 is internalized via clathrin in a PKC-dependent mannerImmunofluorescent analysis indicates that ROR2 (green) co-localizes with clathrin (red, A, arrows). Wnt5A treatment causes an immediate internalization of ROR2 and co-localization of ROR2 and clathrin (B). Mono-dansyl cadaverine (MDC) inhibits the internalization of ROR2 via clathrin and causes its redistribution from foci to the periphery of the cell (C, arrows). MDC inhibition also decreases the invasion of melanoma cells through Matrigel (D). Clathrin siRNA decreases clathrin expression as demonstrated by Western blot analysis (E) and decreased clathrin inhibits the motility of melanoma cells as shown in a wound-healing assay (F). In Wnt5A-high cells, PKC inhibition results in the movement of ROR2 (green) from foci to a diffuse cytoplasmic distribution in cells, upon PKC inhibition (G). Foci reappear at 6h after treatment. Co-staining with clathrin (red) indicates that PKC inhibition may affect ROR2 internalization, as in the presence of PKC inhibitor, ROR2 does not co-localize with clathrin (H). Re-emergence of ROR2-positive foci at 6h of PKC inhibitor treatment corresponds to increased co-localization with the golgi (I, arrows).
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Figure 3: ROR2 is internalized via clathrin in a PKC-dependent mannerImmunofluorescent analysis indicates that ROR2 (green) co-localizes with clathrin (red, A, arrows). Wnt5A treatment causes an immediate internalization of ROR2 and co-localization of ROR2 and clathrin (B). Mono-dansyl cadaverine (MDC) inhibits the internalization of ROR2 via clathrin and causes its redistribution from foci to the periphery of the cell (C, arrows). MDC inhibition also decreases the invasion of melanoma cells through Matrigel (D). Clathrin siRNA decreases clathrin expression as demonstrated by Western blot analysis (E) and decreased clathrin inhibits the motility of melanoma cells as shown in a wound-healing assay (F). In Wnt5A-high cells, PKC inhibition results in the movement of ROR2 (green) from foci to a diffuse cytoplasmic distribution in cells, upon PKC inhibition (G). Foci reappear at 6h after treatment. Co-staining with clathrin (red) indicates that PKC inhibition may affect ROR2 internalization, as in the presence of PKC inhibitor, ROR2 does not co-localize with clathrin (H). Re-emergence of ROR2-positive foci at 6h of PKC inhibitor treatment corresponds to increased co-localization with the golgi (I, arrows).

Mentions: The perinuclear localization of Wnt5A/ROR2 was consistent with reports that Wnt signaling can occur from within endosomal compartments (Blitzer & Nusse, 2006). Neurotrophic tyrosine kinase receptors, to which ROR2 bears significant homology, have also been shown to signal from within endosomes upon ligand binding (Howe et al., 2001). This requires internalization via clathrin, and ligand-bound Trk receptors are often found in clathrin-coated endosomes in neuronal cells. Immunofluorescent analyses demonstrated that ROR2 (green) co-localized with clathrin (red) in Wnt5A high cell lines (Figure 3A), but not caveolin (Supplementary Figure 2A). In Wnt5A high cells, ROR2 also co-stains with both EEA1, a marker of the early endosome (Supplementary Figure 2B), supporting the theory that ROR2 is being internalized into endosomes. To determine if Wnt5A causes clathrin-mediated internalization of ROR2, cells were treated with rWnt5A. Indeed, after only 5 minutes of treatment with Wnt5A, ROR2 is internalized into foci, and colocalizes with clathrin (Figure 3B). To determine whether clathrin was required for ROR2 internalization, we inhibited clathrin using mono-dansyl cadaverine (MDC). Upon treatment of cells with 50µM of MDC for 1 hour, ROR2 internalization was inhibited in the majority of cells (Figure 3C, arrows). To demonstrate that clathrin-mediated receptor internalization is important for melanoma cell invasion, we treated cells with MDC (50µM for 1 hour), and subjected them to an invasion assay. Cells treated with MDC were unable to invade through Matrigel at the same rate as the untreated cells (Figure 3D). For an even more specific approach, we transfected cells with Clathrin 1 siRNA, which knocked down a large portion of the clathrin in the cell (Figure 3E), after which a wound-healing assay was performed. Cells treated with Clathrin 1 siRNA were also unable to close a wound as fast those transfected with a control siRNA (Figure 3F), indicating the importance of Clathrin in melanoma motility.


The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma.

O'Connell MP, Fiori JL, Xu M, Carter AD, Frank BP, Camilli TC, French AD, Dissanayake SK, Indig FE, Bernier M, Taub DD, Hewitt SM, Weeraratna AT - Oncogene (2009)

ROR2 is internalized via clathrin in a PKC-dependent mannerImmunofluorescent analysis indicates that ROR2 (green) co-localizes with clathrin (red, A, arrows). Wnt5A treatment causes an immediate internalization of ROR2 and co-localization of ROR2 and clathrin (B). Mono-dansyl cadaverine (MDC) inhibits the internalization of ROR2 via clathrin and causes its redistribution from foci to the periphery of the cell (C, arrows). MDC inhibition also decreases the invasion of melanoma cells through Matrigel (D). Clathrin siRNA decreases clathrin expression as demonstrated by Western blot analysis (E) and decreased clathrin inhibits the motility of melanoma cells as shown in a wound-healing assay (F). In Wnt5A-high cells, PKC inhibition results in the movement of ROR2 (green) from foci to a diffuse cytoplasmic distribution in cells, upon PKC inhibition (G). Foci reappear at 6h after treatment. Co-staining with clathrin (red) indicates that PKC inhibition may affect ROR2 internalization, as in the presence of PKC inhibitor, ROR2 does not co-localize with clathrin (H). Re-emergence of ROR2-positive foci at 6h of PKC inhibitor treatment corresponds to increased co-localization with the golgi (I, arrows).
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Related In: Results  -  Collection

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Figure 3: ROR2 is internalized via clathrin in a PKC-dependent mannerImmunofluorescent analysis indicates that ROR2 (green) co-localizes with clathrin (red, A, arrows). Wnt5A treatment causes an immediate internalization of ROR2 and co-localization of ROR2 and clathrin (B). Mono-dansyl cadaverine (MDC) inhibits the internalization of ROR2 via clathrin and causes its redistribution from foci to the periphery of the cell (C, arrows). MDC inhibition also decreases the invasion of melanoma cells through Matrigel (D). Clathrin siRNA decreases clathrin expression as demonstrated by Western blot analysis (E) and decreased clathrin inhibits the motility of melanoma cells as shown in a wound-healing assay (F). In Wnt5A-high cells, PKC inhibition results in the movement of ROR2 (green) from foci to a diffuse cytoplasmic distribution in cells, upon PKC inhibition (G). Foci reappear at 6h after treatment. Co-staining with clathrin (red) indicates that PKC inhibition may affect ROR2 internalization, as in the presence of PKC inhibitor, ROR2 does not co-localize with clathrin (H). Re-emergence of ROR2-positive foci at 6h of PKC inhibitor treatment corresponds to increased co-localization with the golgi (I, arrows).
Mentions: The perinuclear localization of Wnt5A/ROR2 was consistent with reports that Wnt signaling can occur from within endosomal compartments (Blitzer & Nusse, 2006). Neurotrophic tyrosine kinase receptors, to which ROR2 bears significant homology, have also been shown to signal from within endosomes upon ligand binding (Howe et al., 2001). This requires internalization via clathrin, and ligand-bound Trk receptors are often found in clathrin-coated endosomes in neuronal cells. Immunofluorescent analyses demonstrated that ROR2 (green) co-localized with clathrin (red) in Wnt5A high cell lines (Figure 3A), but not caveolin (Supplementary Figure 2A). In Wnt5A high cells, ROR2 also co-stains with both EEA1, a marker of the early endosome (Supplementary Figure 2B), supporting the theory that ROR2 is being internalized into endosomes. To determine if Wnt5A causes clathrin-mediated internalization of ROR2, cells were treated with rWnt5A. Indeed, after only 5 minutes of treatment with Wnt5A, ROR2 is internalized into foci, and colocalizes with clathrin (Figure 3B). To determine whether clathrin was required for ROR2 internalization, we inhibited clathrin using mono-dansyl cadaverine (MDC). Upon treatment of cells with 50µM of MDC for 1 hour, ROR2 internalization was inhibited in the majority of cells (Figure 3C, arrows). To demonstrate that clathrin-mediated receptor internalization is important for melanoma cell invasion, we treated cells with MDC (50µM for 1 hour), and subjected them to an invasion assay. Cells treated with MDC were unable to invade through Matrigel at the same rate as the untreated cells (Figure 3D). For an even more specific approach, we transfected cells with Clathrin 1 siRNA, which knocked down a large portion of the clathrin in the cell (Figure 3E), after which a wound-healing assay was performed. Cells treated with Clathrin 1 siRNA were also unable to close a wound as fast those transfected with a control siRNA (Figure 3F), indicating the importance of Clathrin in melanoma motility.

Bottom Line: We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2.WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels.ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.

ABSTRACT
Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we show, for the first time, the importance of the orphan tyrosine kinase receptor, ROR2, in melanoma progression. Using melanoma tissue microarrays, we show that ROR2 is expressed predominantly in metastatic melanoma. As ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our earlier data implicating Wnt5A as a mediator of melanoma metastasis. We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2. WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels. ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A. Using in vitro and in vivo metastasis assays, we show that ROR2 is necessary for the Wnt5A-mediated metastasis of melanoma cells. These data imply that ROR2 may represent a novel target for melanoma therapy.

Show MeSH
Related in: MedlinePlus