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. 2022 Aug 1;29(3):159-174.
doi: 10.32604/or.2022.03458. eCollection 2021.

RNF43 is a novel tumor-suppressor and prognostic indicator in clear cell renal cell carcinoma

Dawei Zhu  1 , Lei Zhang  1 , Xiaokai Shi  1 , Shenglin Gao  1 , Chuang Yue  1 , Lifeng Zhang  1 , Y U Bai  1 , Qifeng Wang  2 , Atsushi Okada  3 , Takahiro Yasui  3 , Chao Wang  1   4 , Xingang Cui  4   5 , L I Zuo  1
Affiliations

RNF43 is a novel tumor-suppressor and prognostic indicator in clear cell renal cell carcinoma

Dawei Zhu et al. Oncol Res. .

Abstract

Identifying prognostic indicators of clear cell renal cell carcinoma (ccRCC) and elucidating the mechanisms underlying ccRCC progression are crucial for improving ccRCC patient prognosis. This study investigated the clinical significance and biological role of Ring finger protein 43 (RNF43) in ccRCC. Two independent cohorts of patients with ccRCC were employed to determine the prognostic significance of RNF43 by immunohistochemistry and statistical analyses. In vitro and in vivo experiments, RNA-seq, and other techniques were used to determine the biological role of RNF43 in ccRCC and related molecular mechanisms. RNF43 expression was commonly decreased in ccRCC specimens, and low expression of RNF43 indicated a higher TNM stage, SSIGN score, and WHO/ISUP grade and short survival in patients with ccRCC. Additionally, RNF43 overexpression suppressed the proliferation, migration, and targeted drug resistance of ccRCC cells, while the knockdown of RNF43 enhanced these characteristics of ccRCC. RNF43 knockdown activated YAP signaling by decreasing YAP phosphorylation by p-LATS1/2 and increasing the transcription and nuclear distribution of YAP. By contrast, RNF43 overexpression showed the opposite effects. Decreasing YAP abolished the effect of RNF43 knockdown in promoting the malignant features of ccRCC. Additionally, restoring RNF43 expression suppressed the resistance of the targeted drug pazopanib in in vivo orthotopic ccRCC. Furthermore, combining the expression of RNF43 and YAP with TNM stage or the SSIGN score exhibited greater accuracy than any of these indicators alone in assessing the postoperative prognosis of ccRCC patients. In summary, our study identified a novel tumor suppressor, RNF43, which is also a prognostic indicator and potential target for ccRCC.

Keywords: Clear cell renal cell carcinoma; RNF43, Prognosis; YAP, Tumor progression.

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Conflict of interest statement

The authors have no conflict of interest.

Figures

Figure 1
Figure 1. RNF43 is downregulated in ccRCC specimens, and low RNF43 expression predicts malignant features of ccRCC. (a) The expression of RNF43 was analyzed in normal renal tissues (n = 72) and ccRCC tissues (n = 533) from TCGA datasets. (b) The expression of RNF43 was compared between paired tumor and normal tissues (n = 72) in TCGA ccRCC datasets. (c) The expression of RNF43 in matched ccRCC and normal samples was determined by real-time PCR (n = 48). (d) Representative images of H&E staining and IHC staining for RNF43 in ccRCC tissues and adjacent tissues are shown (scale bar = 50 μm, 200× and 20 μm, 400×), and the expression level of RNF43 was calculated using the H-score. (e) Representative images of H&E and IHC staining for RNF43 in ccRCC specimens with different TNM stages are presented (scale bar = 50 μm, 200× and 20 μm, 400×). (f) Real-time PCR was performed to detect the expression of RNF43 in various ccRCC cell lines (786-O, 769-P, OS-RC-2, ACHN) and a normal cell line (HK-2). (g) Real-time PCR was conducted to determine the expression of RNF43 in 786-O-SR or 786-O-PR cells and control naïve 786-O cells. (h) Representative images of H&E and IHC staining for RNF43 in 786-O-derived pazopanib-resistant orthotopic tumors and naïve orthotopic tumors are shown (scale bar = 50 μm, 200× and 20 μm, 400×). ***p < 0.001.
Figure 2
Figure 2. Low expression of RNF43 is associated with an unfavorable prognosis in ccRCC patients. (a) Representative H&E and IHC staining for RNF43 in ccRCC tissues from the RNF43low and RNF43high groups (scale bar = 50 μm, 200× and 20 μm, 400×). (b) Time-dependent ROC analysis was conducted to evaluate the optimum cut-off value of RNF43 in cohort 1. (c–f) Kaplan-Meier curves for the OS and PFS of ccRCC patients were compared between the RNF43low and RNF43high groups in cohort 1 (n = 193; c, d) and cohort 2 (n = 127; e, f).
Figure 3
Figure 3. Overexpression of RNF43 inhibits the proliferation, migration, and pazopanib resistance of ccRCC. (a) Western blot assays were performed to detect the expression of RNF43 in 786-O or 769-P cells with or without RNF43 overexpression (RNF43OE). (b) CCK-8 proliferation assays were employed to determine the proliferation of 786-O cells without or with RNF43 overexpression(RNF43OE). (c) Representative images and statistical analysis of migration assays in 786-O cells without or with RNF43 overexpression(RNF43OE) are presented (scale bar = 200 µm). (d, e) 786-O cells without and with RNF43 overexpression were treated with pazopanib (3 μM) for 36 hours, cell proliferation was examined using CCK-8 assays (d), and cell apoptosis was detected using flow cytometry assays (e). **p < 0.01 (n = 3); NS, no significance.
Figure 4
Figure 4. Knock down the expression of RNF43 facilitates the proliferation, migration, and pazopanib resistance of ccRCC. (a) Western blot assays were performed to examine the expression of RNF43 in 786-O or 769-P cells with or without RNF43 knockdown. (b) CCK-8 proliferation assays were used to detect the proliferation of 786-O cells without or with RNF43 knockdown. (c) Representative images and statistical analysis of the migration assays in 786-O cells without or with RNF43 knockdown are shown (scale bar = 200 µm). (d, e) 786-O cells without and with RNF43 knockdown were treated with pazopanib (3μM) for 36 h, cell proliferation was examined by CCK-8 assays (d), and cell apoptosis was detected by flow cytometry assays (e). **p < 0.01 and ***p < 0.001 (n = 3).
Figure 5
Figure 5. RNF43 inhibits the malignant features of ccRCC by suppressing YAP signaling. (a, b) RNA sequencing was performed in 786-O cells with or without RNF43 knockdown. A heatmap depicting the significantly differentially expressed genes (a) and an analysis of differentially expressed pathways (b) are presented. (c, d) Real-time PCR was performed to examine the expression of YAP, AMOTL1, CTGF, AXL, CYR61, ANKRD1, and TEAD1 in 786-O cells without or with RNF43 knockdown (c) or with RNF43 overexpression (RNF43OE) (d). (e, f) Luciferase assays were performed to determine the transcriptional activity of YAP in 786-O cells without or with RNF43 knockdown (e) or with RNF43 overexpression (RNF43OE) (f). (g, h) Western blot analysis of p-LATS1/2, LATS1/2, p-YAP, and YAP in cytoplasmic (Cyt) and nuclear (Nuc) fractions of 786-O cells without or with RNF43 knockdown (g) or with RNF43 overexpression (RNF43OE) (h). β-Actin and histone H3 were used as internal controls for the cytoplasmic and nuclear fractions, respectively. ***p < 0.001 (n = 3).
Figure 6
Figure 6. RNF43 mediates the malignant features of ccRCC in a YAP-dependent manner. (a) Real-time PCR was conducted to detect the expression of YAP in RNF43-knockdown 786-O or 769-P cells without or with decreasing YAP by using siRNAs. (b) CCK-8 assays were conducted to detect the proliferation of RNF43-knockdown 786-O or 769-P cells without or with decreasing YAP. (c) Representative images and statistical analysis of the migration assays in RNF43-knockdown 786-O or 769-P cells in the absence or presence of decreasing YAP are shown (scale bar = 200 µm). (d) 786-O cells and RNF43-knockdown 786-O cells without or with decreasing YAP were treated with pazopanib (3 μM) for 36 h, and cell proliferation was evaluated at different times using CCK-8 assays. (e, f) 786-O cells and RNF43-knockdown 786-O cells without or with decreasing YAP were subcutaneously injected into nude mice (n = 6/group). The volumes of tumor xenografts from the different groups were compared at the indicated times (e), and tumor xenografts are shown (f). **p < 0.01 and ***p < 0.001 (n = 3).
Figure 7
Figure 7. Overexpression of RNF43 inhibits the Pazopanib resistance in orthotopic ccRCC mice (a, b) Stable luciferase-expressing 786-O-PR cells without or with RNF43 overexpression were injected under the renal capsule of mice, and tumor growth was monitored using an in vivo imaging system. After 3 weeks, the mice injected with 786-O-PR cells were treated with normal saline or pazopanib (80 mg/kg), and the mice injected with RNF43-overexpressing 786-O-PR cells were treated with pazopanib (80 mg/kg). Images of luciferase intensity, orthotopic xenografts, and H&E and IHC staining of RNF43 and YAP in tumor specimens from different groups are presented (scale bar = 20 µm) (a). Photon flux levels were examined in the different groups of mice, and the results are presented as the fold increase in tumor growth (b). **p < 0.01 (n = 3); NS, no significance.
Figure 8
Figure 8. Incorporation of RNF43 and YAP with current clinical prognostic parameters better predicts the prognosis of ccRCC patients. (a) A flow chart of the research design is shown. (b) Correlation analysis between RNF43 and YAP in ccRCC is shown. (c) Time-dependent ROC curve analysis was performed to examine the optimal cut-off value of YAP in cohort 1. (d–g) Kaplan-Meier analyses of OS and PFS were performed in ccRCC patients from cohort 1 (n = 193; d, e) and cohort 2 (n = 127; f, g) **p < 0.01; NS, no significance.
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