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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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Related in: MedlinePlus

ROR2 decreases motility and invasiveness in metastatic in human melanoma cell lines in vitro and in vivoUsing a Matrigel invasion assay, ROR2 knockdown inhibits the invasion of UACC903 and M93-047 cells by over 50% (A). Two different siRNAs were used to demonstrate the requirement for ROR2, see also Supplementary Figure 3. G361 cells are similarly affected in a matrigel invasion assay, where Wnt5A treatment can significantly (**=p<0.01, ***=p<0.001) increase their migration through Matrigel, but not in the presence of ROR2 siRNA (B). When M93-047 29 cells are injected via the tail vein into nude mice, ROR2 knockdown results in a decreased ability of M93-047 cells to form pulmonary metastases (C). Two lungs with evidence of macroscopic metastases are shown and a histology section from a third mouse (C, arrow) is shown in order to demonstrate the hemorrhaging seen in these tumors.
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Figure 6: ROR2 decreases motility and invasiveness in metastatic in human melanoma cell lines in vitro and in vivoUsing a Matrigel invasion assay, ROR2 knockdown inhibits the invasion of UACC903 and M93-047 cells by over 50% (A). Two different siRNAs were used to demonstrate the requirement for ROR2, see also Supplementary Figure 3. G361 cells are similarly affected in a matrigel invasion assay, where Wnt5A treatment can significantly (**=p<0.01, ***=p<0.001) increase their migration through Matrigel, but not in the presence of ROR2 siRNA (B). When M93-047 29 cells are injected via the tail vein into nude mice, ROR2 knockdown results in a decreased ability of M93-047 cells to form pulmonary metastases (C). Two lungs with evidence of macroscopic metastases are shown and a histology section from a third mouse (C, arrow) is shown in order to demonstrate the hemorrhaging seen in these tumors.

Mentions: In order to determine if ROR2 is important in Wnt5A-mediated invasion in human melanoma cells as well, a Matrigel invasion assay was performed after 48 hours of ROR2 siRNA treatment in Wnt5A-high cell lines (UACC903 and M93-047). ROR2 siRNA inhibits migration of these highly metastatic melanoma cells through Matrigel by over 50% (Figure 6A). Further, treatment of G361 melanoma cells with rWnt5A in the presence of control siRNA increased their invasion through Matrigel, but in the presence of ROR2 siRNA, rWnt5A was unable to increase migration (Figure 6B). ROR2 siRNA appears to have little effect by itself, but this is likely due to the fact that G361 cells are so non-metastatic, there is already minimal invasion through Matrigel (Supplementary Figure 3A). We also performed wound-healing assays that supported these observations (Supplementary Figure 3B–D).


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 decreases motility and invasiveness in metastatic in human melanoma cell lines in vitro and in vivoUsing a Matrigel invasion assay, ROR2 knockdown inhibits the invasion of UACC903 and M93-047 cells by over 50% (A). Two different siRNAs were used to demonstrate the requirement for ROR2, see also Supplementary Figure 3. G361 cells are similarly affected in a matrigel invasion assay, where Wnt5A treatment can significantly (**=p<0.01, ***=p<0.001) increase their migration through Matrigel, but not in the presence of ROR2 siRNA (B). When M93-047 29 cells are injected via the tail vein into nude mice, ROR2 knockdown results in a decreased ability of M93-047 cells to form pulmonary metastases (C). Two lungs with evidence of macroscopic metastases are shown and a histology section from a third mouse (C, arrow) is shown in order to demonstrate the hemorrhaging seen in these tumors.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803338&req=5

Figure 6: ROR2 decreases motility and invasiveness in metastatic in human melanoma cell lines in vitro and in vivoUsing a Matrigel invasion assay, ROR2 knockdown inhibits the invasion of UACC903 and M93-047 cells by over 50% (A). Two different siRNAs were used to demonstrate the requirement for ROR2, see also Supplementary Figure 3. G361 cells are similarly affected in a matrigel invasion assay, where Wnt5A treatment can significantly (**=p<0.01, ***=p<0.001) increase their migration through Matrigel, but not in the presence of ROR2 siRNA (B). When M93-047 29 cells are injected via the tail vein into nude mice, ROR2 knockdown results in a decreased ability of M93-047 cells to form pulmonary metastases (C). Two lungs with evidence of macroscopic metastases are shown and a histology section from a third mouse (C, arrow) is shown in order to demonstrate the hemorrhaging seen in these tumors.
Mentions: In order to determine if ROR2 is important in Wnt5A-mediated invasion in human melanoma cells as well, a Matrigel invasion assay was performed after 48 hours of ROR2 siRNA treatment in Wnt5A-high cell lines (UACC903 and M93-047). ROR2 siRNA inhibits migration of these highly metastatic melanoma cells through Matrigel by over 50% (Figure 6A). Further, treatment of G361 melanoma cells with rWnt5A in the presence of control siRNA increased their invasion through Matrigel, but in the presence of ROR2 siRNA, rWnt5A was unable to increase migration (Figure 6B). ROR2 siRNA appears to have little effect by itself, but this is likely due to the fact that G361 cells are so non-metastatic, there is already minimal invasion through Matrigel (Supplementary Figure 3A). We also performed wound-healing assays that supported these observations (Supplementary Figure 3B–D).

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