Ror2, a Developmentally Regulated Kinase, Is Associated With Tumor Growth, Apoptosis, Migration, and Invasion in Renal Cell Carcinoma

Abstract

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiation and chemotherapy. Current therapies for RCC patients are inefficient due to the lack of diagnostic and therapeutic markers. The expression of novel tumor-associated kinases has the potential to dramatically shape tumor cell behavior. Identifying tumor-associated kinases can lend insight into patterns of tumor growth and characteristics. In the present study, we investigated the receptor tyrosine kinase-like orphan receptor 2 (Ror2), a new tumor-associated kinase, in RCC primary tumors and cell lines. Knockdown of Ror2 expression in RCC cells with specific shRNA significantly reduced cell proliferation and induced apoptosis. Using in vitro migration and Matrigel invasion assays, we found that cell migration and invasive ability were also significantly inhibited. In RCC, Ror2 expression correlated with expression of genes involved at the cell cycle and migration, including PCNA, CDK1, TWIST, and MMP-2. Furthermore, in vivo xenograft studies in nude mice revealed that administration of a Ror2 shRNA plasmid significantly inhibited tumor growth. These findings suggest a novel pathway of tumor-promoting activity by Ror2 within renal carcinomas, with significant implications for unraveling the tumorigenesis of RCC.

Figure 1

Expression of Ror2 in RCC tissues and cell lines. (A) The expression level of Ror2 detected by RT-PCR in 21 paired RCC tissues and adjacent normal kidney tissues. (B, C) Expression of Ror2 in four RCC cell lines and a normal renal cell line was detected by RT-PCR and Western blot. ***p < 0.001 compared with adjacent normal kidney tissues or HK-2 cell lines.

Figure 2

Ror2 downregulation inhibits cell proliferation. Expression of Ror2 in 786-0 (A, B) and CaKi-1 cells (C, D) was detected by RT-PCR and Western blot. The 786-0 cells were infected with shRNA-Ror2, and the CaKi-1 cells were infected with Ror2-expressing vector; 0, 24, 48, and 72 h later, cells were collected. (E, F) Cell proliferation was detected by CCK-8 assay. *p < 0.05, ***p < 0.001 compared with shRNA-NC.

Figure 3

Ror2 downregulation induces cell cycle arrest and apoptosis. The 786-0 cells were infected with shRNA-Ror2, and the CaKi-1 cells were infected with Ror2-expressing vector; 48 h later, cells were collected. (A) The 786-0 cell cycle profile was analyzed using flow cytometry and PI staining. The 786-0 (B, D) and CaKi-1 cell (C, E) apoptosis was analyzed using flow cytometry and annexin V-FITC/PI staining. ***p < 0.001 compared with shRNA-NC.

Figure 4

Ror2 downregulation inhibits cell migration and invasion. The 786-0 cells were infected with shRNA-Ror2, and the CaKi-1 cells were infected with Ror2-expressing vector; 48 h later, cells were collected. Cell migration (A–C) and invasion (D–F) were measured in 786-0 and CaKi-1 cells by Transwell assay coated without or with Matrigel.

Figure 5

Ror2 regulates proteins correlated with cell cycle and migration. The 786-0 cells were infected with shRNA-Ror2, and the CaKi-1 cells were infected with Ror2-expressing vector; 48 h later, cells were collected. (A, B) Expression of PCNA, CDK1, TWIST, and MMP-2 was evaluated in 786-0 (A, B) and CaKi-1 cells (C, D) by RT-PCR and Western blot. *p < 0.05, **p < 0.01, ***p < 0.001 compared with shRNA-NC.

Figure 6

Interacting proteins for Ror2 in vitro. (A, B) Coimmunoprecipitation showed that Ror2 interacts with WNT5A and WNT1 in the 786-0 cell lines.

Figure 7

Ror2 downregulation in 786-0 cells reduced tumor growth in vivo. The 786-0 cells infected with shRNA-NC or shRNA-Ror2 were subcutaneously injected in nude mice. (A) Expression of CTNNB1 was determined by immunohistochemistry staining. (B) Growth curve of tumor volumes. (C) Tumor weights. ***p < 0.001 compared with shRNA-NC.