Systematic expression analysis of m6A RNA methyltransferases in clear cell renal cell carcinoma

Abstract Objectives To investigate the regulation of the N‐6‐methyladenosine (m6A) methyltransferases METTL3, METTL14, WTAP, KIAA1429, and METTL4, referred to as “m6A writers,” in clear cell renal cell carcinoma (ccRCC), and other RCC subtypes in respect of the potential prognostic value. Patients and methods Tissue samples were collected within the framework of the Biobank at the Center for Integrated Oncology Bonn. The expression of the methyltransferases was systematically determined in clear cell renal carcinoma (ccRCC) on the RNA (real‐time PCR) and protein level (immunohistochemistry). Additionally, protein expression of the m6A writers was further investigated in papillary RCC, chromophobe RCC, sarcomatoid RCC, oncocytoma, and normal renal tissue (immunohistochemistry). Results The expression of all m6A‐methyltransferases was significantly downregulated in ccRCC compared to benign renal tissue. Low m6A‐methyltransferase levels were correlated with higher histological grade, advanced pT‐stage, pN‐stage, and metastatic disease. Reduced m6A‐methyltransferase expression was associated with shorter overall survival. Conclusion In conclusion, m6A‐methyltransferases are dysregulated in ccRCC and might act as tumor suppressor genes, which could be of particular importance for future diagnostic and therapeutic options.

(chRCC), sarcomatoid renal cell carcinoma (sRCC), and renal oncocytoma. Only a few patients present with the typical symptomatic triad of a renal malignant tumor (painless hematuria, a palpable abdominal mass, and flank pain). Since early stages of RCC frequently are asymptomatic, the majority of patients is diagnosed coincidentally with suspicious renal neoplasia through abdominal or thoracic imaging of an unrelated issue 3,4 ; however, up to 25% of all patients with RCC are diagnosed with metastatic disease. 5,6 Gold-standard treatment with curative intent for patients with localized RCC is partial nephron-sparing surgery or radical nephrectomy, while treatment of patients with metastatic disease or relapse is far more complicated due to low response rates to treatment. 7  Diverse post-transcriptional RNA modifications have been observed. N-6-methyladenosine (m 6 A), discovered in the 1970s, 8,9 is the most widespread and abundant internal modification of messenger RNA (mRNA) in eukaryotic RNA. 10 It is involved in numerous biological pathways, such as gene expression, cell growth, cell cycle, cellular differentiation and pluripotency, stem cell self-renewal, DNA damage response, and circadian rhythm-most of which play crucial roles in cancer progression and metastasis. 9,[11][12][13][14][15] The m 6 A modification is dynamically added by methyltransferases (so-called m 6 A writers) or removed by demethylases (erasers), and mediated by RNA binding enzymes (readers). Dysregulation of these m 6 A methylases, demethylases, and RNA binding enzymes contributes to a variety of human diseases, such as obesity, diabetes, infertility, growth retardation, neurological disorders, and cancer. 14, [16][17][18] The role of m 6 A methylases in RCC is largely unknown, and our study was designed to investigate systematically the expression of human m 6 A methyltransferases (METTL3, methyltransferase-like protein 3; METTL4, methyltransferase-like protein 4; METTL14, methyltransferase-like protein 14; WTAP, Wilms-tumor-1 associated protein; KIAA1429, vir like m 6 A methyltransferase associated).

| Patients
The collection of fresh-frozen renal tissue samples was performed within the framework of the Biobank at the Center for Integrated Oncology (CIO) Bonn as described earlier. 19 All benign and malign tissue samples were obtained after radical or partial nephrectomy at the Department of Urology at the University Hospital Bonn. The benign samples were obtained from the adjacent non-cancerous part of the kidneys. All patients provided written informed consent for the collection of biomaterials; the study was approved by the ethics committee of the University Bonn (approval number 127/17).
The renal tissue was stored at −80°C and used for mRNA expression studies. Immunohistochemistry was conducted with archival formalin-fixed and paraffin-embedded tissues. All tissue samples were reviewed by a uropathologist and classified according to the WHO classification of 2009. See Supporting Information Table S1 for detailed clinicopathological parameters.

| Immunohistochemistry
The expression of the methyltransferases was further investigated in ccRCC, papillary RCC (pRCC), chromophobe RCC (chRCC), sarcomatoid RCC (sRCC), oncocytoma, and benign renal parenchyma using a tissue microarray. Three tissue cores were arrayed to obtain a representative image of each tumor; see Supporting Information Table S3 for detailed clinicopathological data. The tissue microarray with formalin-fixed, paraffin-embedded archival tissues was cut 5 µm thick sections, placed down in a water bath at 45°C for ideal expansion, applied on slides, and dried at 65°C for 60 minutes.
Afterward, the slides were loaded in the Benchmark Ultra system  Signal detection was performed with the HRP Multimer technology of the UltraView DAB IHC Detection Kit (Ventana) and finally, the slides were counterstained using Mayer's hemalum and bluing reagent (Ventana). The staining intensity was evaluated using QuPath software. 21 A representative immunohistochemistry is shown in Supporting Information Figure S1.

| mRNA expression of m 6 A methyltransferases in ccRCC
The expression of the m 6 A-methyltransferases was investigated in ccRCC (n = 166) and normal renal tissues (n = 102). All m 6  Kaplan-Meier estimates indicated that decreased m 6 A methyltransferase expression was predictive of poor outcome in ccRCC patients (see Figure 2): progression-free survival (PFS; METTL14 log-rank P = .0042; KIAA1429 log-rank P = .008; WTAP log-rank P = .042), cancer-specific survival (CSS; METTL3 log-rank P = .044; METTL4 log-rank P = .031; METTL14 log-rank P = .041; KIAA1429 log-rank P < .001; WTAP log-rank P = .002), and overall survival (OS; METTL3 log-rank P = .001; METTL4 log-rank P = .013; METTL14 logrank P = .019; KIAA1429 log-rank P = .002; WTAP log-rank P = .027) were shortened in patients with low m 6 A-methyltransferase expression levels; see Supporting Information Tables S4-6  However, statistical significance was not observed in the multivariate Cox regression analysis. Parameters that did not show statistical significance in the univariate model (PFS, METTL3, and METTL4, both P > .05) were not included in the multivariate model; see Figure 3 and Supporting Information Table S7-9. We reviewed our findings with analysis of the bias-corrected and accelerated (BCa) bootstrap interval for progression-free survival, cancer-specific survival, and overall survival; for detailed information see Supporting Information Table S10-12.

| Protein expression of m 6 A methyltransferases
Immunohistochemical staining was performed to determine the m 6 Amethyltransferases' protein expression using a tissue microarray con-  Table S13. Cox regression analyses also demonstrated that low METTL3 expression levels were predictive for shortened progression-free survival (METTL3, P = .008; HR (95% CI) 5.278 (1.541-18.076)) in the univariate but not multivariate model, see   Table S14. We reviewed our findings with the analysis of the bias-corrected and accelerated (BCa) bootstrap interval for progression-free survival; for detailed information see Supporting Information Table S15.

| D ISCUSS I ON
The m 6 A modification influences almost every step of RNA metabolism: generally speaking, m 6 A modification of RNA fastens the RNA's

F I G U R E 2 Kaplan-Meier estimates indicate a poor outcome in patients with ccRCC and low m 6 A methyltransferases mRNA levels
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way from maturation to decomposition. It is involved in mRNA processing, splicing, exporting mRNA from nucleus to cytoplasm, mRNA translation, and decay. [13][14][15][23][24][25] Interestingly, the m 6 A modification can either play an oncogenic or tumor-suppressing role in human malignancies depending on the cellular context. 26,27 The m 6 A methylation is mediated by a multiprotein methylases complex composed of the methyltransferases METTL3, METTL4, METTL14, KIAA1429, and WTAP, either acting as catalyzing subunits or as auxiliary cofactors. 9,11,14,15,28 The methyltransferases METTL3 and METTL14 build a stable heterodimer core complex, the main part of the multiprotein methyltransferase complex, which is predominantly localized in nuclear speckles, regions enriched with pre-mRNA in processing to become mature mRNA. 25,29,30 METTL3, an S-adenosyl-L-methionine (SAM)-binding subunit of the multiprotein methyltransferase complex, has been detected in the nucleus and cytoplasm, 14 but was localized predominantly in nuclear speckles. 25,30 It has a major catalytic methyltransferase activity, by changing its localization from nucleus to cytoplasm, METTL3 can gain a function as a reader, thus also being able to promote translation of target mRNAs. 24,30 METTL3 may acts as oncogene or as tumor suppressor, depending on the cellular context. 27 In adenocarcinoma of the lung, 25 acute myeloid leukemia, 31   A close homolog of METTL3 and component of the multiprotein methyltransferase complex is METTL14. 14 METTL14 itself shows no catalytic activity, but stabilizes METTL3, maintaining complex integrity, and facilitates RNA binding. [36][37][38] The stable METTL3-METTL14 heterodimer core complex has an increased methyltransferase activity compared to each protein individually. 14,39 Our study demonstrates the downregulation of METTL14 in ccRCC, and low METTL14 expression go along with a shortened survival. Downregulation of METTL14 was also reported in glioma, 40 whereas high expression levels in acute myeloid leukemia cells enhanced leukemogenesis. 41 The Wilms tumor-1 associated protein (WTAP) is involved in the transcriptional and posttranscriptional regulation of cellular genes.
It is ubiquitously localized in nuclear speckles and cytoplasm, and partially colocalized with splicing factors. 29,42,43 WTAP acts as an adaptor molecule in the multiprotein methyltransferase complex, interacting with the METTL3-METTL14 heterodimer by facilitating its transport into nuclear speckles, 11,14,15,24 yet itself lacks methyltransferase activity. 29,39 Recent studies postulated that it might not only be mediating methylation as a writer, but also act as a reader binding mRNA. 44 Nevertheless, WTAP is required for efficient methylation of mRNA with m 6 A: its knockdown decreased the m 6 A levels in cellular mRNAs even more effectively than knockdown of either METTL3 or METTL14. 39,44 Even though WTAP only plays an auxiliary part, dysregulation also plays an important role for tumor progression in various malignancies 45 : an oncogenic role was described in lung 46 and pancreatic cancer. 47 In contrast, downregulation of WTAP was associated with poor survival in cancer of the bladder, eye, and soft tissue. 46 Our study demonstrates downregulation of WTAP in ccRCC, and a shortened survival related to low expression levels of WTAP.
The RNA-binding protein KIAA1429 became of interest for research due to its biochemically shown interaction with WTAP in Drosophila concerning sex-specific splicing. 48 It is an interacting part of the multiprotein methyltransferase complex. 49 KIAA1429 is localized in nuclear speckles similar to WTAP. 44 Knockdown of KIAA1429 causes decreased m6A levels, even more distinctively decreased than depletion observed by the knockdown of METTL3 or METTL4 individually, thereby underlining KIAA1429's pivotal role in the multiprotein methyltransferase complex. 44 Several studies propose that KIAA1429 promotes tumorigenesis and progression, for example, in breast cancer 50 and hepatocellular carcinoma. 49,51 We demonstrate that KIAA1429 is downregulated in ccRCC, and low RNAs. 53 Knockdown of METTL4 did not change cellular m 6 A levels 38,52 ; however, it seems to be involved in carcinogenesis. METTL4 is downregulated in colon adenocarcinoma 54 and upregulated in melanoma. 28 Further, METTL4 knockdown inhibited melanoma growth. 28 In ccRCC, we observed a decrease of METTL4, and low METTL4 mRNA expression was indicative of shortened survival.
In conclusion, the m 6

ACK N OWLED G M ENTS
The tissue samples were collected within the framework of the Biobank of the Center for Integrated Oncology Cologne Bonn (CIO) at the University Hospital Bonn.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.