A Zic2/Runx2/NOLC1 signaling axis mediates tumor growth and metastasis in clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies with rapid growth and high metastasis, but lacks effective therapeutic targets. Here, using public sequencing data analyses, quantitative real-time PCR assay, western blotting, and IHC staining, we characterized that runt-related transcription factor 2 (Runx2) was significantly upregulated in ccRCC tissues than that in normal renal tissues, which was associated with the worse survival of ccRCC patients. Overexpression of Runx2 promoted malignant proliferation and migration of ccRCC cells, and inversely, interfering Runx2 with siRNA attenuates its oncogenic ability. RNA sequencing and functional studies revealed that Runx2 enhanced ccRCC cell growth and metastasis via downregulation of tumor suppressor nucleolar and coiled-body phosphoprotein 1 (NOLC1). Moreover, increased Zic family member 2 (Zic2) was responsible for the upregulation of Runx2 and its oncogenic functions in ccRCC. Kaplan-Meier survival analyses indicated that ccRCC patients with high Zic2/Runx2 and low NOLC1 had the worst outcome. Therefore, our study demonstrates that Zic2/Runx2/NOLC1 signaling axis promotes ccRCC progression, providing a set of potential targets and prognostic indicators for patients with ccRCC.

Fig. 1. High expression of Runx2 is related to the worse outcome of ccRCC patients.

A TCGA and Oncomine (Yusenko’s cohort) database analyses showed the high level of Runx2 in ccRCC tissues than that in non-tumor renal tissues. B qRT-PCR was used to test the mRNA level of Runx2 in ccRCC and paired non-tumor renal tissues (n = 29). C Western blot analyses of the expression of Runx2 in ccRCC and corresponding non-tumor renal tissues at protein level (n = 6). β-Actin was also tested as a loading control. D High expression of Runx2 was associated with the poorer tumor grade and patient stage in ccRCC based on the TCGA dataset analysis. E Survival cures from TCGA cohort showed that ccRCC patients with high Runx2 had a short overall survival (P < 0.001). F Representative images of IHC staining demonstrated the different expression level of Runx2 in ccRCC tissues. Scale bar, 40 μm. G Score analysis of IHC staining showed the high expression of Runx2 in ccRCC tissues (n = 208) than that in non-tumor renal tissues (n = 301). H Kaplan–Meier survival analysis indicated that high expression of Runx2 was associated with poorer survival of patients with ccRCC (P = 0.031). In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 2. Exogenous overexpression of Runx2 enhances the proliferation and migration of ccRCC cells.

A The expression level of Runx2 in one human immortalized renal epithelial cell and five ccRCC cells was analyzed by qRT-PCR and western blot analyses, respectively. β-Actin was also tested as a loading control. B Plasmid-mediated transfection was used to overexpress the Runx2 in CAKI-1 and SKRC39 cells, and the expression level of Runx2 was confirmed by qRT-PCR and western blot analyses. C Foci formation assay showed that overexpression of Runx2 promoted ccRCC cells growth. D Migration assay suggested that the migration ability of ccRCC cells was increased after overexpression of Runx2, compared to control cells. In all panels, *, P < 0.05; **, P < 0.01.

Fig. 3. Knockdown of Runx2 attenuates the growth and metastasis of ccRCC cells.

A Two siRNAs were used to downregulate the expression of Runx2 in ACHN and 786-O cells, and the knockdown of Runx2 was confirmed by qRT-PCR and western blot analyses, respectively. B Interference of Runx2 decreased the foci formation frequencies of ccRCC cells. C Transwell migration assay was performed to explore the effect of Runx2 on the migration ability of ccRCC cells. D ACHN and 786-O cells after knockdown of Runx2 were subcutaneously injected into nude mice, and the tumor volumes were measured every week (n = 4 mice per group). E IHC staining with antibodies against Runx2 and Ki67 in xenograft tumors derived from ACHN and 786-O cells with or without Runx2 silencing. Scale bar, 25 μm. F Nude mice were treated by tail intravenous injection of 786-O cells with or without Runx2 knockdown. Hematoxylin–eosin staining confirmed the lung metastasis, and the incidence of metastasis was indicated in right panel (n = 6 mice per group). Scale bar, 50 μm. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 4. NOLC1 mediates the regulation of Runx2 in ccRCC cell growth and metastasis.

A RNA-sequencing was performed on ACHN cells transfected with two siRNA targeting Runx2, and the genes with significant changes were indicated by heat map. B The mRNA expressions of Runx2 and NOLC1 in ACHN and 786-O cells after knockdown of Runx2 were analyzed by qRT-PCR. C Western blot analysis showed the downregulation of NOLC1 in SKRC39 and CAKI-1 cells after overexpression of Runx2. β-Actin was also tested as a loading control. D Luciferase report assay showed that high expression of Runx2 inhibited the transcription of NOLC1. E IHC staining analysis in ccRCC tissue array showed the negative correlation between the expressions of Runx2 and NOLC1. F qRT-PCR was used to analyze the expression level of NOLC1 in ccRCC and corresponding normal renal tissues at mRNA level. G Survival analyses of ccRCC patients with different expression level of NOLC1 based on TCGA datasheet. H–J MTT test (H), EdU incorporation (I), and foci formation (J) assays were performed to analyze the proliferation of 786-O and ACHN cells after knockdown of NOLC1 or/and Runx2, respectively. K The migration ability of ccRCC cells after interference of NOLC1 or/and Runx2 was analyzed by Transwell migration assay. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 5. Zic2 upregulates the expression of Runx2 promoting ccRCC cell growth and metastasis.

A IHC staining analysis in ccRCC tissue array showed the positive correlation of Zic2 and Runx2 expressions. B Western blot analysis showed the downregulation of Runx2 in 786-O and ACHN cells after knockdown of Zic2. β-Actin was also tested as a loading control. C qRT-PCR was used to analyze the expression level of Zic2 in ccRCC and paired normal renal tissues (n = 29). D Survival curves based on IHC staining indicated the poorer outcome of ccRCC patients with high expression of Zic2 than those with low level of Zic2 (P < 0.0001). E–G MTT test (E), EdU incorporation (F), and foci formation (G) assays were performed to analyze the proliferation of 786-O and ACHN cells after knockdown of Zic2 or/and Runx2 overexpression, respectively. H The migration ability of ccRCC cells after interference of Zic2 or/and overexpression of Runx2 was tested by Transwell migration assay. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.