Receptor tyrosine kinase-like orphan receptor 2 (Ror2) expression creates a poised state of Wnt signaling in renal cancer

Abstract

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.

Keywords: Cancer; RCC; Receptor Tyrosine Kinase; Renal Cell Carcinoma; Ror2; Signaling; Wnt Signaling; Wnt3a; β-Catenin.

FIGURE 1.

Expression of Ror2 in primary human RCC tumors correlates with expression of Wnt signaling component genes. A, heat map of 721 probes exhibiting significant (false discovery rate < 0.043%) correlation and anticorrelation with Ror2 expression, as determined by significance of analysis of microarrays in a panel of 95 primary human RCC tumors. Shown are magnified heat maps for the following significantly enriched gene ontologies identified using DAVID from genes positively correlating with Ror2 expression, skeletal development (B), and the Wnt signaling pathway (C).

FIGURE 2.

Expression of Ror2 in RCC cells results in changes of canonical Wnt regulators and target genes. A, RNA expression was paneled across various Wnt component and target genes using the human Wnt signaling RT² Profiler PCR array with cDNA from 786-0 cells that endogenously express Ror2, 786-0 shRor2 where Ror2 is stably suppressed, and the Ror2 overexpression cell line 786-0 + TAP-hRor2. Transcript values for each sample were normalized to the geometric mean of five internal housekeeping genes. Changes in the level of expression correlating with Ror2 expression are displayed for the indicated 24 genes, with fold change calculated relative to 786-0. B, representative quantitative RT-PCR showing Ror2 expression levels for all 786-0 cell lines relative to 786-0. Quantitative RT-PCR shows a decrease in 786-0 shRNA Ror2 knockdown relative to 786-0 cells for FZD1 (C) and JUN (D). For all quantitative RT-PCR assays, transcript values were normalized to the β-actin RNA internal standard. Error bars represent S.E. across triplicates in triplicate experiments. p values were calculated using one-way ANOVA. *, p < 0.05; **, < 0.001.

FIGURE 3.

Suppression of Ror2 results in decreased β-catenin-mediated transcription in RCC cells. Quantitative RT-PCR in 786-0 cells for Ror2 (A) shows a significant increase in control RC3 cells with Wnt3a (100 ng/ml) stimulation, whereas levels were reduced significantly in both shRNA Ror2 knockdown cell lines. B, likewise, a significant attenuation in Axin2 transcription in response to Wnt3a (100 ng/ml) is seen in both shRNA Ror2 knockdowns using quantitative RT-PCR. For all quantitative RT-PCR assays, transcript values were normalized to an 18 S RNA internal standard, with fold change calculated in reference to unstimulated 786-0 RC3. C, a significant reduction of β-catenin-mediated transcriptional response following Wnt3a (100 ng/ml) stimulation with the TOPFlash luciferase reporter in 786-0 Ror2-suppressed cells in comparison to control 786-0 pRS cells. Relative luciferase units (RLU) were calculated by normalizing TOPFlash luciferase to internal Renilla luciferase activity. Error bars represent S.E. across triplicates in duplicate experiments. p values were calculated using one-way ANOVA. *, p < 0.05; **, < 0.001.

FIGURE 4.

Overexpression of Ror2 enhances β-catenin-mediated transcription in renal and RCC cells. Quantitative RT-PCR for Ror2 in 786-0 (A) and HEK293Tcells (E) shows a strong induction of Ror2 following treatment with doxycycline (500 ng/ml) and concordant significant increase in basal expression of the canonical Wnt target gene Axin2 that is further heightened upon the addition of Wnt3a (100 ng/ml) relative to the control cells (B and F). Quantitative RT-PCR in 786-0 (C) and 293T (G) cells for Axin2 again shows an increase in basal levels with Ror2 expression and a significant increase in response to treatment with LiCl (10 mm). For all quantitative RT-PCR assays, transcript values were normalized to the β-actin RNA internal standard, with fold change calculated in reference to unstimulated GFP-expressing cells. D, the expression of Ror2 in 786-0 transfected with a TOPFlash luciferase reporter exhibited a significant increase in activity basally, with Ror2 expression leading to a significant increase in response to Wnt3a (100 ng/ml) stimulation. H, HEK293T cells transfected with a TOPFlash luciferase reporter displayed a significantly enhanced response to Wnt3a (100 ng/ml) stimulation. Error bars represent S.E. across triplicates in duplicate experiments. p values were calculated using one-way ANOVA. *, p < 0.05; **, p < 0.001.

FIGURE 5.

Ror2 expression results in an increased pool of stable β-catenin independent of exogenous Wnt stimulation. Immunoblot analysis for the indicated antibodies of whole cell lysates or concanavalin A-treated lysates (asterisk) following stimulation with either control or Wnt3a CM (dilution 1:2) for 1 h. 786-0 (A) and HEK293T (B) cells show increased levels of phosphorylated DVL2, DVL3, and stable β-catenin with Ror2 expression and an additional increase of stable β-catenin following treatment with Wnt3a CM. C, 786-0 shRor2 knockdown cell lines exhibited attenuated levels of stable β-catenin basally and in response to Wnt3a CM stimulation in comparison to 786-0 RC3 control cells. Changes in pDVL2, pDVL3, and stable β-catenin relative to unphosphorylated DVL or total β-catenin are shown with densitometric quantification below the corresponding bands. LC, loading control.

FIGURE 6.

Ror2-dependent stabilization of β-catenin is independent of LRP6. A, immunoblot analysis for the indicated antibodies of either whole cell lysates or concanavalin A bead-treated lysates (asterisk) from 293T cells 48 h post-transfection with siCon (nonspecific control) or siLRP6 and stimulated with either control or Wnt3a CM (dilution 1:2) for 1 h shows that increased levels of β-catenin are maintained with Ror2 expression. B, immunoblot analysis for the indicated antibodies of whole cell lysates or concanavalin A-treated lysates (asterisk) following stimulation with either Wnt3a CM (dilution 1:2) or DKK1 (400 ng/ml) for 1 h followed by Wnt3a CM stimulation showed increased levels of β-catenin concurrent with Ror2 expression. LC, loading control. Quantitative RT-PCR for Axin2 in HEK293T cells (C) transfected with siCon or siLRP6 and stimulated 48 h post transfection with Wnt3a (100 ng/ml) show that LRP6 is needed for mediating Wnt3a/β-catenin-dependent transcription. D, Axin2 expression is shown in 293T cells stimulated with either Wnt3a (100 ng/ml) or DKK1 (400 ng/ml) for 1 h followed by Wnt3a stimulation, with high DKK1 inhibiting the response to Wnt3a stimulation in both GFP- and Ror2-expressing cells. For all quantitative RT-PCR assays, transcript values were normalized to the β-Actin RNA internal standard, with fold change calculated in reference to unstimulated GFP-expressing cells. Error bars represent S.E. across triplicates in duplicate experiments. p values were calculated using one-way ANOVA. *, p < 0.05; *, p < 0.001.

FIGURE 7.

Ror2 activity is required for a poised Wnt signaling state. Immunoblot analysis of whole cell lysates or concanavalin A-treated lysates (asterisk) from 786-0 (A) or HEK293T cells (B) expressing GFP, Ror2, or the double mutant Ror2-DM 48 h post induction with doxycycline (500 ng/ml) show increased levels of stable β-catenin with wild-type Ror2 compared with Ror2-DM. Densitometry quantification of stabilized β-catenin relative to total β-catenin is provided below the corresponding bands. LC, loading control. Quantitative RT-PCR for Ror2 in 786-0 (C) or HEK293T (D) cells shows expression of Ror2 and Ror2-DM cells 24 h post-induction but a significant decrease of Axin2 expression in Ror2-DM expression in both cell lines. For all quantitative RT-PCR assays, transcript values were normalized to the β-actin RNA internal standard, with fold change calculated in reference to unstimulated GFP-expressing cells. Error bars represent S.E. across triplicates of a representative duplicated experiment. p values were calculated using one-way ANOVA. *, p < 0.05; **, p < 0.001.

FIGURE 8.

Ror2/Wnt3a signaling in RCC cells. Schematic overview of Ror2/Wnt3a signaling in RCC cells. A, in canonical Wnt signaling in the absence of either Wnt ligand or Ror2 expression, β-catenin is degraded by the proteasome. B, however, with the expression of Ror2, DVL becomes phosphorylated, leading to stabilization of β-catenin and its translocation into the nucleus, driving transcription of target genes (i.e. Axin2). C, in cells expressing Ror2 and stimulated with Wnt3a, a further enhancement of β-catenin stabilization and amplified transcription of target genes is observed.