Phosphorylated MAPK11 promotes the progression of clear cell renal cell carcinoma by maintaining RUNX2 protein abundance

Abstract Previous studies have demonstrated that mitogen‐activated protein kinase 11 (MAPK11) functions as an important point of integration in signalling transduction pathways and controlling endocellular processes, including viability of cells, differentiation, proliferation and apoptosis, through the sequence phosphorylation of the substrate protein Ser/Thr kinase protein cascade. Though MAPK 11 plays an important role in various tumours, especially in the invasive and metastatic processes, its expression and molecular mechanism in clear cell renal cell carcinoma (ccRCC) remain unclear. Runt‐associated transcription factor 2 (RUNX2), a main transcription factor for osteoblast differentiation and chondrocyte maturation, has high expression in a number of tumours. In this study, the mRNA and protein levels of targeted genes in ccRCC tissues and adjacent tissues are analysed using the Cancer Genome Atlas (TCGA) database and western blotting. The ccRCC cell proliferation was measured with colony formation and EdU assay, and cell migration was examined through transwell assay. The interactive behaviour between proteins was detected with immunoprecipitation. Half‐life period of RUNX2 protein was measured with cycloheximide chase assay. The results of the study indicated overexpression of MAPK11 and RUNX2 in ccRCC tissues and cell lines. MAPK11 and RUNX2 promoted the ccRCC cell proliferation and migration. Additionally, physical interaction took place between RUNX2 and P‐MAPK11, which functioned to sustain the stability of RUNX2 protein. The high expression of RUNX2 could neutralize the functional degradation in MAPK11. And the outcomes of the study suggest that the P‐MAPK11/RUNX2 axis may be used as a potential therapeutic target of ccRCC.


| INTRODUC TI ON
Renal cell carcinoma (RCC) ranks the sixth commonest carcinoma in the male population and 10th in the female population globally, which account for 5% and 3% respectively among all malignancies clinically. 1 While the diagnostic and managerial techniques for RCC have been improved in the past two decades, RCC remains one of the leading causes of death. An increasing number of cases of RCC are identified accidentally during routine imaging intended for other diseases. 2 Clear cell renal carcinoma (ccRCC) is the commonest subcategory of RCC. Surgical treatment of ccRCC can remove the diseased tissue, but relapse and metastasis are found in about 40% of postoperative ccRCC patients, and poor prognostic effects are particularly found in patients with advanced and metastatic disease, and merely 12% could reach a survival time of 5 years after diagnosis. 3,4 Given that conventional chemoradiotherapy does not work for ccRCC, 5  phosphorylation of MAPK proteins. 6 P38 MAPK contains four isoforms p38α, p38β, p38δ and p38γ. They can promote the cell growth and motility and are highly homologous. 7,8 The expressions of p38α and p38β, which share approximately 75% homology, are ubiquitous, while the expressions of p38δ and p38γ are differential in the tissues. 9 Overexpression of MAPK11 has been detected in a number of malignancies. Furthermore, MAPK11 plays a crucial role in the occurrence, invasion and migration of cancerous cells in breast carcinoma, endometrial carcinoma, hepatocellular carcinoma and other types of malignancies. [10][11][12] Nevertheless, the impact of p38β (MAPK11) on ccRCC remains unclear and requires further exploration.
RUNX2 is a key transcription factor in osteogenesis, performs an important function in osteoblast differentiation. 13,14 Studies have suggested that RUNX2 is associated with malignancy progression, functioning in the invasive and migrating behaviour of a number of carcinomas, including cancers in prostate, bladder and mantle cell lymphoma. [15][16][17] The mechanism of RUNX 2 in ccRCC remains unclear and requires further exploration.
Studies indicate p38β is also related to a few transcription factors. 18 MEFs (Myocite Enhancer Factors, for instance, play the regulatory role in various aspects of cells, such as apoptosis, proliferation, differentiation as well as migration and metabolism.
Phosphorylation of MEF2A and MEF2C by p38β occurs in the transcriptional activation domain of MEF2 and stimulates the transcriptional activity in vivo. 19 The absence of MAPK14 is apt to result in decline of the protein level in RUNX2. 20 The results of this study showed that P-MAPK11 and RUNX2 were overly expressed in ccRCC, and P-MAPK11 could have physical interaction with RUNX2. In addition, P-MAPK11 helps keep RUNX2 protein stable by blocking its pathway of ubiquitination degradation.

| Bioinformatics analyses
The Cancer Genome Atlas (TCGA) was used for the analysis of the mRNA expression levels of particular genes in ccRCC tissues.
Relevant information to TCGA is available on this website: http:// gepia.cance r-pku.cn.

| Tissue samples
The tissue samples were derived from 32 patients hospitalized at the First Hospital of China Medical University from November 2021 to February 2022. All tissues were histologically examined and diagnosed as ccRCC. The study won approval from the hospital and all patients signed a consent form.

| Western blotting
RIPA buffer combined with protease inhibitor cocktail were applied to lysed tissues and cells, and a BCA protein assay kit was utilized for protein concentration detection. SDS-PAGE (140 V, 10%) was employed for the separation of protein fractions. A Mini Transblot Cell device was selected to transfer resolved protein (350 mA) to PVDF membranes (0.2 μm). The membranes were blocked in a capsule with fat-free milk (5%) at 37°C for 60 minutes and cultured at 4°C overnight with antibodies. The antibodies included anti-RUNX2 (1:1000, 12,556) and antiβ-tubulin (1:1000, 2128S) from Cell Signalling Technology, anti-MAPK11 (1:1000, PHT5450M) and anti-P-MAPK11 (1:1000, T40076) from Abmart. The membranes were afterwards cultured with the anti-rabbit secondary antibody for 60 min at 37°C. Quantification of the immunoblots was performed using ImageJ software (version 1.51).

| Quantitative real-time PCR
The separation of aggregate RNA was via TRIzol reagent and

| Lentiviral transduction
Lentiviral-based plasmids for MAPK11 knockdown and those for the overexpression of RUNX2 were synthesized by and purchased from Shanghai-based GeneChem. MAPK11 knockdown stable cell lines were produced with the lentivirus vector containing short hairpin (sh) RNAs targeting MAPK11 (shRNA-MAPK11) and negative control vector (shRNA-Ctrl). Virosome that contains RUNX2 fragment in full length were deployed to produce LV-RUNX2. The negative control vectors (LV-Ctrl) were produced as well. Lentivirus infection technology was used to make stable cell lines and the infected cells were selected after treatment of the growth medium with 10 μg/mL puromycin (for no less than four passages).

| Co-immunoprecipitation assay
RIPA buffer combined with protease inhibitor cocktail were employed to lysed cells and centrifugation was performed with the cells (12,000 g) for 20 min at 4°C for immunoprecipitation.
Subsequently, the cell lysate was cultured with a mixture of antibodies composed of anti-P-MAPK11, anti-RUNX2 and anti-IgG overnight at 4°C in rotation. The antibodies were obtained from Abmart or Cell Signalling Technology, respectively. The pyrolysis solution underwent rotation for 4 h with magnetic beads in. After the beads were taken out with a magnetic rack, protein loading buffer (5×) was added and denatured at 100°C for 10 min. Western blotting was carried out subsequently.

| Colony formation assays
Trypsinization treatment was performed on cells growing to 80% confluence were trypsinized. The cells were removed to a newly prepared medium in single-cell suspension. Dilution-treated cells were seeded on six-well plates, each plate containing 500 cells.
They were then cultivated at 37°C with 5%CO2 for 10 days, and subsequently treated with crystal violet staining solution for 10 min. ImageJ software were drawn upon for the quantification of Colony areas.

| EdU Assay
Cells were placed in six-well plates with EdU (BeyoClick™, EDU-488) for labelling. Culture medium was taken away followed by EdU staining after being labelled, the cells were treated for half an hour with click reaction cocktail in dark environment at normal room temperature in conformity with the manufacturer's guidelines. A fluorescent microscope (Olympus Corporation) was used for imaging.

| P-MAPK11 Regulates the RUNX2 Protein Expression
Previous studies have been shown that MAPK14 depletion could result in a decline in RUNX2 protein level. 20,24 Therefore, stable MAPK11 knockdown ccRCC cell lines were prepared for the purpose of determining whether P-MAPK11 could regulate the RUNX2 protein in ccRCC cells in our study. When MAPK11 was absent, the mRNA expression level of MAPK11 dropped considerably, while in the expression of RUNX2 mRNA, no change in statistical significance occurred ( Figure 3A). It was indicated that RUNX2 protein expression dropped significantly ( Figure 3B,C). The ccRCC cell lines overexpressing RUNX2 were used for determining the correlation between RUNX2 and the expression of MAPK11 and P-MAPK11. No significant change showed in the mRNA expression of MAPK11 as RUNX2 was overly expressed ( Figure 3D). And western blotting revealed that increased expression of RUNX2 did not cause change in the protein levels of MAPK11 and P-MAPK11 ( Figure 3E, F). These results showed that MAPK11 or P-MAPK11 could regulate the RUNX2 protein expression, while the regulation mechanism remained to be explored.

| P-MAPK11 Regulated the RUNX2 Protein Expression and Maintained the Stability of the RUNX2 Protein
To determine whether the decline of RUNX2 protein was a consequence of that of P-MAPK11, the study selected the inhibitor Losmapimod (GW856553X) of P-MAPK11, which could downgrade the phosphorylation level of MAPK11 while maintaining the protein level in MAPK11. 25 Figure 4F,G). Hence, the conclusion was that the half-life period of the RUNX2 protein was 4 h.
CHX treatment (100 μg/mL) was also performed to cells with stable expression of shRNA-Ctrl and shRNA-MAPK11 for a specific duration and then the protein of cells was collected for immunoblotting analysis. As MAPK11/P-MAPK11 decreased markedly, the half-life period of RUNX2 protein decreased significantly ( Figure 4H,I). The TA B L E 1 Correlation between P-MAPK11 and RUNX2 expression and the clinicopathological parameters of 32 ccRCC patients.  findings indicate that MAPK11/P-MAPK11 could stabilize RUNX protein presumably by blocking its pathway of ubiquitination degradation.

| RUNX2 restored the effect of MAPK11 and P-MAPK11 in part in ccRCC progression
In order to explore the role RUNX2 played in MAPK11/P-MAPK11mediated cellular proliferating, migrating and invasive behaviour of ccRCC, the MAPK11 knockdown cells were transfected with virosome that contains RUNX2 in full length (shRNA-MAPK11/ LV-RUNX2). As shown by migration assay (Figure 5A,B), the overexpression of RUNX2 led to incomplete reverse in the diminishment of cellular migrating capability resulting from the decrease of MAPK11 and P-MAPK11. Through EdU assay and colony formation assay, it was found that the deficiency of MAPK11 and P-MAPK11 led to the impairment of the proliferating ability of ccRCC cells, which was restored partially when RUNX2 was overly expressed ( Figure 5C-F). The above data showed that ccRCC progression could be promoted to some extent through the stabilization of RUNX2 protein. It has been reported that a variety of proteins and substrates could be phosphorylated by P38 MAPK including many transcription factors. 28 And MAPK11 is thought that it relates to the proliferating, invasive and metastatic behaviour of a variety of cancerous cells. [29][30][31] Aside from that, this study examined the mechanism of P-MAPK11 in ccRCC cells in 786-O and ACHN. When shRNA-MAPK11 diminished the abundance of P-MAPK11 protein, the cellular proliferating, migrating and clone-forming capabilities showed significant decline.
In view of the fact that P-MAPK11 is the main functional form of MAPK11, the overexpression of the protein could be found in both ccRCC tissues and cell lines. P-MAPK11 was presumably conducive to the development of ccRCC.
RUNX2 could push osteoblast phenotype to change from immaturity to maturity and hence promote skeletal development 32,33 Additionally, RUNX2 plays the crucial role in the migrating, invasive and metastatic process of a variety of cancers. [34][35][36][37] Recently, the correlation between MAPKs and RUNX2 has been recorded in different tumours. 38,39 And study has shown that RUNX2 could be phosphorylated and activated by P38 MAPK. 40  In conclusion, P-MAPK11 could intermodulate with RUNX2 to facilitate the progress of ccRCC. Our research outcomes reveal that RUNX2 is a substrate of P-MAPK11, and P-MAPK11 could play a preventative role through binding. As a consequence, it is in need of exploring the regulatory mechanism of P-MAPK11 in the ubiquitination pathway degradation of RUNX2.

ACK N OWLED G EM ENTS
Not applicable.

FU N D I N G I N FO R M ATI O N
No funding from any individual or organization is involved in this study.

CO N FLI C T O F I NTER E S T S TATEM ENT
We declare that our work involves no conflict of interest of any nature with any individual or professional organization.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets involved in this study are obtained from relevant authors at the reasonable request.