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

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.

Figure 1. ROR2 expression in human melanoma

Staining of a melanoma tissue microarray demonstrates that the majority of primary melanomas do not express ROR2 (A), whereas subcutaneous or visceral metastases of melanoma stained very strongly for ROR2 in the cytoplasm and nucleus (B, arrows demonstrate nuclear staining). Lymph node metastases for the most part had very light staining of ROR2, and in these specimens ROR2 appeared to be largely at the membrane of the cell (C, arrows). Overall staining results are represented as percentage of samples positive per group (D). The increase in ROR2 expression in metastatic melanoma as compared to primary melanoma is significant to p<0.0001.

Figure 2. ROR2 expression and localization in human melanoma cell lines

ROR2 mRNA is highly expressed in Wnt5A high lines (UACC903 and M93-047), but not Wnt5A low UACC1273EV and G361 cells (A). ROR2 protein levels are also higher in Wnt5A high lines than Wnt5A-low G361 cells, as shown by Western analysis (B). Treating ROR2-low UACC1273EV cells with rWnt5A increases ROR2 expression (C). Immunofluorescent analysis demonstrates that in Wnt5A-low lines, ROR2 is expressed in a diffuse cytoplasmic pattern in most cells. In Wnt5A-high lines, ROR2 is expressed in both a diffuse pattern as well as in perinuclear/ nuclear foci in the vast majority of the cells (D). Treatment of Wnt5A-low cells with rWnt5A increases the focal localization of ROR2 (E). Wnt5A and ROR2 expression are both expressed in the perinuclear foci, as shown by immunolocalization analysis (F).

Figure 3. ROR2 is internalized via clathrin in a PKC-dependent manner

Immunofluorescent 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).

Figure 4. Wnt5A expression modulates ROR2 expression, and requires ROR2 for signaling

WNT5A knockdown results in a decrease in ROR2 mRNA (A, solid lines) in both UACC903 and M93-047 cells as analyzed by real time PCR (A) and in ROR2 protein as analyzed by immunofluorescence (B). Wnt5A protein is maximally decreased at 72hours, coincident with the greatest levels of ROR2 knockdown (A, inset). ROR2 knockdown results in a decrease in ROR2 mRNA (C) and protein (D), but not WNT5A 28 mRNA (C, dashed lines). However ROR2 knockdown does inhibit Wnt5A signaling as demonstrated by a decrease in PO₄-PKC (E) and an increase in MART-1 expression by Western analysis (F).

Figure 5. ROR2 knockdown inhibits the in vivo invasion of melanoma cells in a murine model

B16 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).

Figure 6. ROR2 decreases motility and invasiveness in metastatic in human melanoma cell lines in vitro and in vivo

Using 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.