Limits...
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.

Show MeSH

Related in: MedlinePlus

ROR2 knockdown inhibits the in vivo invasion of melanoma cells in a murine modelB16 melanoma cells express the Ror2 receptor in a pattern, and at levels similar to that of UACC1273EV cells (A, CTRL). Treatment of B16 cells with rWnt5A causes redistribution of Ror2 to perinuclear foci, as seen with human cells (A, CTRL+rWnt5A). Treating B16 cells with Ror2 siRNA results in a decrease in Ror2 expression (A, Ror2 siRNA). In vivo tail vein metastasis assays demonstrate that Ror2 knockdown can significantly inhibit the pulmonary metastasis of melanoma cells, an effect that cannot be recovered by the addition of rWnt5A (B). Outliers in each group are shown and underlined. Immunohistochemical analysis of these tumors indicates that there are very few micrometastases in the ROR2 siRNA treated mice (C).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2803338&req=5

Figure 5: ROR2 knockdown inhibits the in vivo invasion of melanoma cells in a murine modelB16 melanoma cells express the Ror2 receptor in a pattern, and at levels similar to that of UACC1273EV cells (A, CTRL). Treatment of B16 cells with rWnt5A causes redistribution of Ror2 to perinuclear foci, as seen with human cells (A, CTRL+rWnt5A). Treating B16 cells with Ror2 siRNA results in a decrease in Ror2 expression (A, Ror2 siRNA). In vivo tail vein metastasis assays demonstrate that Ror2 knockdown can significantly inhibit the pulmonary metastasis of melanoma cells, an effect that cannot be recovered by the addition of rWnt5A (B). Outliers in each group are shown and underlined. Immunohistochemical analysis of these tumors indicates that there are very few micrometastases in the ROR2 siRNA treated mice (C).

Mentions: In B16 cells, Ror2 is largely cytoplasmic, but treatment of these cells with rWnt5A dramatically increased the number of foci (Figure 5A, +rWnt5A). We used mouse Ror2 siRNA to knockdown Ror2 expression, and confirmed its effectiveness (Figure 5A, Ror2 siRNA). Previous in vivo studies have indicated the effective use of naked duplex siRNAs in vivo. Lewis et al have shown that siRNA duplexes can be injected i.v. into animals and detected within 24 hours of injection in organs such as liver, lung kidney and spleen (Lewis et al., 2002). Other experiments have shown that even single-dose i.v. injections of siRNA duplexes can be effective over a period of as long as ten days, in diseases such as autoimmune hepatitis (Song et al., 2003). Liang et al (Liang et al., 2005) have shown that it is possible to use naked siRNA duplexes in vivo to inhibit cancer metastasis, and that this is most effective when siRNA is systemically- re-administered every 48–72 hours. Following this protocol, we treated B16 cells with control or Ror2 siRNA, and Ror2 siRNA treated cells were then treated with either rWnt5A or vehicle controls. To determine if Ror2 knockdown could decrease pulmonary metastases, we attempted to maximize the amount of metastases, by injecting the mice with 1 × 106 cells, which were injected via the tail vein, into 10 mice per group. Twice a week, rWnt5A treated mice received booster injections of rWnt5A. In addition, mice injected with siRNA transfected cells received booster injections of siRNA (i.e., all mice received either control siRNA or Ror2 siRNA injections) according to the protocol of Liang et al (Liang et al., 2005). Eight out of nine mice injected with control siRNA developed pulmonary metastases. In contrast, only 3 out of 10 mice treated with Ror2 siRNA developed metastases (p<0.02), and rWnt5A treatment could not significantly increase pulmonary metastases in these mice (4/10 mice developed metastases). Representative lung metastases are shown in Figure 5B. Immunohistochemistry revealed that Ror2 siRNA treated mice had very low levels of microscopic metastases. These data imply that fewer Ror2 negative cells either reach or survive at the sites of extravasation, 12 and only a subset of those have the propensity to establish tumorigenic colonies (Figure 5C).


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 knockdown inhibits the in vivo invasion of melanoma cells in a murine modelB16 melanoma cells express the Ror2 receptor in a pattern, and at levels similar to that of UACC1273EV cells (A, CTRL). Treatment of B16 cells with rWnt5A causes redistribution of Ror2 to perinuclear foci, as seen with human cells (A, CTRL+rWnt5A). Treating B16 cells with Ror2 siRNA results in a decrease in Ror2 expression (A, Ror2 siRNA). In vivo tail vein metastasis assays demonstrate that Ror2 knockdown can significantly inhibit the pulmonary metastasis of melanoma cells, an effect that cannot be recovered by the addition of rWnt5A (B). Outliers in each group are shown and underlined. Immunohistochemical analysis of these tumors indicates that there are very few micrometastases in the ROR2 siRNA treated mice (C).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: ROR2 knockdown inhibits the in vivo invasion of melanoma cells in a murine modelB16 melanoma cells express the Ror2 receptor in a pattern, and at levels similar to that of UACC1273EV cells (A, CTRL). Treatment of B16 cells with rWnt5A causes redistribution of Ror2 to perinuclear foci, as seen with human cells (A, CTRL+rWnt5A). Treating B16 cells with Ror2 siRNA results in a decrease in Ror2 expression (A, Ror2 siRNA). In vivo tail vein metastasis assays demonstrate that Ror2 knockdown can significantly inhibit the pulmonary metastasis of melanoma cells, an effect that cannot be recovered by the addition of rWnt5A (B). Outliers in each group are shown and underlined. Immunohistochemical analysis of these tumors indicates that there are very few micrometastases in the ROR2 siRNA treated mice (C).
Mentions: In B16 cells, Ror2 is largely cytoplasmic, but treatment of these cells with rWnt5A dramatically increased the number of foci (Figure 5A, +rWnt5A). We used mouse Ror2 siRNA to knockdown Ror2 expression, and confirmed its effectiveness (Figure 5A, Ror2 siRNA). Previous in vivo studies have indicated the effective use of naked duplex siRNAs in vivo. Lewis et al have shown that siRNA duplexes can be injected i.v. into animals and detected within 24 hours of injection in organs such as liver, lung kidney and spleen (Lewis et al., 2002). Other experiments have shown that even single-dose i.v. injections of siRNA duplexes can be effective over a period of as long as ten days, in diseases such as autoimmune hepatitis (Song et al., 2003). Liang et al (Liang et al., 2005) have shown that it is possible to use naked siRNA duplexes in vivo to inhibit cancer metastasis, and that this is most effective when siRNA is systemically- re-administered every 48–72 hours. Following this protocol, we treated B16 cells with control or Ror2 siRNA, and Ror2 siRNA treated cells were then treated with either rWnt5A or vehicle controls. To determine if Ror2 knockdown could decrease pulmonary metastases, we attempted to maximize the amount of metastases, by injecting the mice with 1 × 106 cells, which were injected via the tail vein, into 10 mice per group. Twice a week, rWnt5A treated mice received booster injections of rWnt5A. In addition, mice injected with siRNA transfected cells received booster injections of siRNA (i.e., all mice received either control siRNA or Ror2 siRNA injections) according to the protocol of Liang et al (Liang et al., 2005). Eight out of nine mice injected with control siRNA developed pulmonary metastases. In contrast, only 3 out of 10 mice treated with Ror2 siRNA developed metastases (p<0.02), and rWnt5A treatment could not significantly increase pulmonary metastases in these mice (4/10 mice developed metastases). Representative lung metastases are shown in Figure 5B. Immunohistochemistry revealed that Ror2 siRNA treated mice had very low levels of microscopic metastases. These data imply that fewer Ror2 negative cells either reach or survive at the sites of extravasation, 12 and only a subset of those have the propensity to establish tumorigenic colonies (Figure 5C).

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