Despite advancements in the care of RCC, there remains considerable room for improvement through identification of novel therapeutic targets and prognostic factors for assessing risk for patients. Ror2 has been shown to play a role in tumor progression in an increasing assortment of cancers including RCC through mediation of signaling cascades,
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resulting in increased cell migration, invasion, and tumor growth. Prior work in RCC showed the ECM remodeling protein MMP2 expression to be Ror2-dependent, in congruence with our findings here using gene expression microarrays (13).
We sought to further elucidate the mechanisms of Ror2-dependent regulation of MMP2. Although Ror2 has been primarily been identified as receptor for the non-canonical Wnt5a ligand, Ror2 expression in the context of RCC has been shown to result in a
heightened state of canonical β-catenin signaling which is further enhanced by the addition of Wnt3a (29). Furthermore, because MMP2 was shown to be induced downstream of the Wnt3a/β-catenin signaling cascade, we tested the effects on Wnt3a on MMP2 expression in RCC (31,32). Using both shRor2 knockdown and overexpression in 786-0 cells, we found that MMP2 expression mirrored the previous findings with the canonical Wnt target gene Axin2, suggesting that Ror2’s migratory/invasive phenotype is not limited to Wnt5a/Ror2 signaling. Although the consistent increased expression of MMPs has been noted with Ror2 expression in a multitude of cell contexts, here we have uniquely established the need for the Ror2 kinase domain in maintaining heightened MMP2 expression. These results suggest that Ror2 kinase activity is required to mediate regulation of downstream target genes such as MMP2.
MMP2 has a well-established role in mediating cell migration and invasion through degradation of the ECM and basement membrane, allowing for increased cell motility. In keeping with previous findings, suppression of Ror2 results in decreased migration in 786-0 cells. Here we also show that overexpression of Ror2 in HEK293T and 786-0 results in increased migration. However, as the Ror2-DM was unable to alter MMP2 expression, migration of RCC cells was likewise unaffected, strengthening the argument that Ror2 kinase activity is required for migration.
In addition to the Ror2-dependent expression of MMP2, the discovery of the Wnt antagonist/agonist SFRP2 regulation by Ror2 is an intriguing discovery. Although SFRP2 is
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typically thought of as a Wnt signaling antagonist, a recent study showed that SFRP2 expression in RCC cells enhances β-catenin-dependent signaling (18). As Ror2 expression likewise contributes to a poised state of β-catenin-dependent signaling in RCC, these
findings together raise the possibility that Ror2 effects on B-catenin-dependent signaling are partially mediated through SFRP2 (29). RCC is a highly vascularized cancer with anti- angiogenic therapies forming the backbone of RTK-targeted therapies currently used in the clinic. The development of resistance to RTK therapeutics, possibly through upregulation of other angiogenic factors, represents a continuing challenge to clinicians in the treatment of RCC. SFRP2’s additional function as an angiogenic factor presents a novel target for angiogenesis in RCC as previous studies have shown it to be expressed in the vasculature of RCC tumors (18,19). Our results which showed significant increases in tumor vascularity with overexpression in Ror2 xenografts in-vivo strengthens the proposal of SFRP2 as a potential target in RCC. Although overexpression of Ror2 only resulted in a trend towards increased tumor growth in xenografts, this trend was validated with the significant increase in tumor size observed with high Ror2 expression in primary RCC tumors highlighting Ror2’s role in promoting tumor growth and progression.
A key clinical characteristic of RCC is the 2:1 male:female ratio, but the biology driving this difference remains unresolved. Interestingly, we observe a shift between low- and high-expressing Ror2 tumors from the expected 2:1 male:female to ~3:1, respectively. Indeed, earlier work toward understanding this gender bias showed that men tend to present with larger, higher stage, higher grade RCC than women with a poorer overall survival (33). High Ror2 expression likewise exhibited a trend towards higher nuclear grade, clinical stage and tumor stage in RCC tumors. These results exhibit that Ror2 contribution to a more aggressive disease may also help drive differences seen between genders in RCC. Further studies are needed to more fully explore the interplay between Ror2 and gender in RCC.
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Our analysis of Ror2’s potential as a prognostic biomarker for RCC shows that Ror2 expression is capable of independently predicting overall survival. The inability of Ror2 to serve as a distinguishing factor for metastatic RCC is not unsurprising as Ror2 correlates with the aggressive ccB subtype of ccRCC. These findings highlight the role of Ror2 in promoting a more aggressive RCC and its ability to serve as a prognostic factor in RCC.
Materials and Methods
Cell Culture
786-0 and derivative cell lines, 786-0 RC3 and 786-0 WT8 (kindly provided by Dr. W. Kaelin, Boston, MA), 786-0 scramble, stable monoclonal knockdown 786-0 shRor2.1 and shRor2.2 cells, 786-0 pCDNA6 and hRor2 cells (13), 786-0 GFP, Ror2, and Ror2-DM (29) were grown in DMEM with 10% FBS, nonessential amino acids, L-glutamine, and
penicillin/streptomycin. HEK293T and derivative cell lines, 293T GFP, Ror2, Ror2-DM (29) were grown in DMEM with 10% FBS, nonessential amino acids, L-glutamine, and
penicillin/streptomycin. Expression of all GFP tagged constructs was visually confirmed 24- 48 hours after induction with doxycycline (500ng/mL).