Introduction

In 2018, 400,000 new diagnoses and over 170,000 deaths due to renal cancer were reported [1]. Renal cell carcinoma (RCC) comprises about 90% of all renal-originated tumors [2], with 35% of RCC patients develop metastases [3]. About 25–30% of patients with local metastases who receive radical nephrectomy develop metastases within 5 years [4]. Thus, new therapeutic approaches should be developed.

The N6-methyladenosine modification was first discovered in 1974 [5]. Since then, the mechanisms of RNA modification have remained elusive, until the first RNA demethylase, the fat mass, and obesity-associated protein (FTO), revealed that m6A is reversible in 2011 [6]. Using the methylated RNA immunoprecipitation-sequencing method, m6A modifications were found to be enriched in the 3’-untranslated regions (3’UTRs) and 5’-untranslated regions (5’UTRs) [7], which regulate RNA alternative splicing, mRNA degradation, and translation [8].

The m6A modification is determined by the methyltransferase complex (MTC), which contains methyltransferases called “writers” [9]. The Wilms tumor 1-associating protein (WTAP) stabilizes MTC localization and substrate recruitment [10,Full size image

We further determined whether IGF2BP-mediated S1PR3 regulation is m6A-dependent. As reported previously, IGF2BP proteins preferentially bind to the “UGGAC” consensus sequence and the bind sites are highly enriched near stop codons and in the 3’ UTRs [14]. Thus, we exploited SRAMP prediction tool to predict the potential bind sites of S1PR3. Interestingly, one of the putative binding sites contained “UGGAC” consensus sequence and located near stop codons (Fig. 6A). Methylation RNA immunoprecipitation (MeRIP) assay was performed with negative control IgG and m6A antibodies, and RT-PCR was performed using the primers specific for the predicted S1PR3-binding site. As expected, we observed a decrease in m6A methylation level of S1PR3 in WTAP KO cells which confirmed that m6A modification of S1PR3 is catalyzed by the predominant catalytic subunit WTAP (Fig. 6B). Especially, S1PR3 was highly expressed in 21 (87.5%) clinical samples (Fig. 6C). We next inserted the 44-nt wild-type or mutant sequence containing the binding site into a dual-luciferase reporter (Supplementary Fig. 3E). The dual-luciferase reporter assay showed decreased luciferase activity following individual IGF2BPs knockout in wild-type reporter, and such decrease was almost completely abrogated by mutation in the m6A consensus sites (Fig. 6D). Consistently, WTAP knockout, similar to individual IGF2BPs knockout, inhibited firefly luciferase activity. In addition, IGF2BP knockdown-mediated decrease of luciferase activity was completely blocked by WTAP knockout (Fig. 6D). Taken together, these results revealed that WTAP-mediated m6A modification maintained the enhancement of S1PR3 stability by IGF2BP proteins.

Fig. 6: WTAP-mediated m6A modification of S1PR3 maintains the IGF2BPs-induced regulation of RCC proliferation and metastasis.
figure 6

A The predicted sites in S1PR3 mRNA in the SRAMP database. B Enrichment of m6A modification on S1PR3 as detected by MeRIP-PCR assay. C S1PR3 were elevated in 21 (87.5%) RCC tissues relative to adjacent nonmalignant tissues. D Relative luciferase activities of S1PR3-WT of S1PR3-MUT in WTAP KO 786-O cells with IGF2BPs knockdown. E The protein levels of S1PR3 and PI3K/AKT pathway following S1PR3 knockdown. F, G Knockdown of S1PR3 markedly suppressed migration and proliferation viabilities of RCC cells. Scale bar 250 μm. H Knockout of WTAP and IGF2BPs regulate PI3K/AKT pathway. I Schematic diagram of IGF2BP-mediated regulation of m6A-modified S1PR3 mRNA. Values in B and D are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

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S1PR3 is responsible for the IGF2BPs-induced regulation of RCC proliferation and metastasis

To determine whether IGF2BPs-induced regulation of cell proliferation and migration in RCC relies on S1PR3, the role of S1PR3 on the proliferation and metastasis of RCC cells by knocking down its expression by siRNAs. The efficiency of siRNAs silencing is shown in Fig. 6E. We found that S1PR3 knockdown markedly inhibited proliferation and migration abilities of 786-O and CAKI-1 cells in vitro (Fig. 6F, G). S1PR3 has been reported to regulate PI3K/AKT pathway and our results consistently indicated that downregulation of S1PR3 regulated the PI3K/AKT signaling pathway (Fig. 6E). Notably, knockdown or knockout of IGF2BPs or WTAP regulated the PI3K/AKT signaling pathway (Fig. 6H and Supplementary Fig. 2C). Similar results were observed with EGR2 knockdown (Supplementary Fig. 3D). In contrast, knockdown of EGR2 had no effect on WTAP expression (Supplementary Fig. 3D). To test the hypothesis that IGF2BPs-induced regulation of RCC proliferation and migration is relevant for S1PR3 downregulation, we performed rescue experiments by overexpressing S1PR3 in WTAP KO and IGF2BPs KO cells. Results showed that forced expression of S1PR3 partly abrogated the inhibitory effect WTAP KO and IGF2BP KO on colony-formation rates (Supplementary Fig. 2B). Similar results were obtained in transwell experiments in which S1PR3 was used to rescue WTAP KO and IGF2BPs KO (Supplementary Fig. 2A). The protein expression levels of S1PR3 in the above rescue experiments are shown in Supplementary Fig. 1C. In summary, WTAP and IGF2BP proteins promote tumorigenesis and metastasis by enhancing the stability of S1PR3 and regulating S1PR3-PI3K/AKT pathway (Fig. 6I).