Aberrant MEK5 Signalling Promotes Renal Cancer Development via mTOR Activation


 Purpose: Renal cell carcinoma is one of the most incident malignancies globally. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma. However, comprehensive clinical treatment has some limitations. Therefore, exploring the pathogenesis and identifying novel therapeutic targets are required urgently. MEK5 has been reported to play an essential role in the development of various cancers. However, no study has evaluated its role and specific mechanisms in ccRCC. Methods: Using the ONCOMINE database, MEK5 expression in ccRCC and normal tissues was compared. ccRCC and adjacent normal tissues were collected from fourteen ccRCC patients, and ccRCC expression was assessed by qPCR and immunohistochemistry. MEK5 overexpression and knockdown plasmids were constructed and transfected into ccRCC cells. CCK8, wound-healing assay, and clone formation assay were performed to examine the cell proliferation, migration, and clone formation ability of ccRCC cells. Furthermore, a western blot was performed to verify the regulation and influence of MEK5 on the mTOR signaling pathway. The MEK5 small molecule inhibitor BIX was used to treat cells, followed by CCK8, wound-healing assay, clone formation, and flow cytometry assay to examine the cell proliferation, migration, clone formation ability, apoptosis, and cell cycle. Finally, a murine subcutaneous tumor model was constructed, and the effect and safety of BIX were evaluated in-vivo.Results: The ONCOMINE database indicated that the MEK5 expression in ccRCC was significantly higher than the normal tissues, further confirmed in clinical specimens. The knockdown of MEK5 markedly inhibited the ability of ccRCC cell proliferation, colony formation, and migration. In contrast, MEK5 overexpression promoted cell proliferation, colony formation, and migration. Western blotting showed that overexpression of MEK5 can further activate the mTOR signaling pathway. The MEK5 inhibitor, BIX treatment of ccRCC, significantly inhibited cell proliferation, arrested the cell cycle in the G0/G1 phase, induced apoptosis, and effectively inhibited cell migration and clone formation. BIX also showed an excellent anti-tumor effect and favorable safety profile in murine models.Conclusions: MEK5 regulated the mTOR signal pathway and regulated the cell proliferation, cycle, migration, clone formation of ccRCC. Targeted inhibition of MEK5 had a good anti-tumor effect and favorable safety profile, providing new directions for ccRCC therapy.


Introduction
The incidence of renal cell carcinoma is steadily increasing annually. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma and accounts for most renal cancer deaths [1]. Localized renal cell carcinoma can be treated by partial or radical nephrectomy [2], ablation [3], and active monitoring [4]. However, approximately 30% of patients have metastases or relapses after partial or radical nephrectomy [5,6].
Conventional cancer therapies, including radiation and chemotherapy, are ineffective in treating metastatic ccRCC [7]. Therefore, comprehensive treatment is crucial. High-dose interleukin 2 (IL2) is one of the rst cytokines, which, along with interferon α (IFN-α), was used for the treatment of RCC, but only 15-25% responded to this treatment [8]. The treatment options for ccRCC currently include targeted therapy and checkpoint inhibitor (CPI) immunotherapy. The targeted therapies include vascular endothelial growth factor receptor, tyrosine kinase inhibitors (VEGFR, TKIs), and the mammalian target of the rapamycin (mTOR) pathway signaling inhibitors. Further, a majority of patients initially respond to therapy, most eventually relapse or progress [9]. Therefore, the dynamic development of new therapeutic drugs or the search for novel therapeutic targets is essential for the clinical treatment of ccRCC.
MEK5/ERK5 was found to be associated with cancer due to its abnormal expression in human tumors, including colon cancer [12], breast cancer [13], prostate cancer [14], hepatocellular carcinoma [15]. The overexpression of MEK5/ERK5 in various tumors makes it an ideal target for targeted therapy. The role of MEK5 in ccRCC and the underlying mechanism is still unclear. This study systematically described the function and role of MEK5 and its downstream signaling pathways in ccRCC for the rst time. Thus, con rming that MEK5 is speci cally and highly expressed in tumor tissues, regulating tumor cell proliferation, cycle, invasion, and migration, and targeting MEK5 has a good anti-tumor effect in-vivo and in-vitro.

Western blotting
The whole-cell lysates were prepared by direct lysis with 1× SDS sample buffer and separated using 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose (Bio-Rad, Hercules, CA, USA). After blocking the membrane with 5% non-fat milk prepared in +0.1% Tris-buffered saline, the membranes were incubated with primary antibodies overnight at 4°C. This was followed by an hour incubation with horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling Technology, Beverly, MA, USA). The protein bands were detected using a chemiluminescence phototope-HRP kit (Cell Signaling Technology).

Detection of apoptosis
The eBioscience TM Annexin V-APC apoptosis detection kit (Thermo Fisher Scienti c, Waltham, MA, USA) was used to detect apoptosis. Brie y, ccRCC cells were treated with BIX solution for the indicated times, ccRCC cells (1 × 10 6 ) were harvested, washed with PBS, and resuspended in the binding buffer. Annexin V-APC (5 µL) and PI (5 µL) were sequentially added to the cell suspension and incubated for 15 min in the dark at room temperature. Cells were analyzed using a ow cytometer (Fortessa, San Francisco, CA, USA) and DIVA™ v8.0 software. Approximately 10,000 cells were analyzed from each sample.

Cell cycle assay
Cell cycle distribution was analyzed by estimating the DNA content using ow cytometry. Harvested cells were xed with 75% ethanol at −20°C for 24 h. The samples were then incubated with 50 µg/mL RNase A for 30 min at 37°C, followed by 100 µg/mL propidium iodide (PI) at 37°C for 30 min. The DNA content was analyzed by a ow cytometer (Fortessa), and cell cycle distribution was estimated using FlowJo software.
Wound-healing assay Cells were grown to con uence in 100 mm 2 dishes, and arti cial wounds were created using a sterile 200 µL pipette tip by scraping. The cells were allowed to close the wound and photographed at the indicated times.

Colony Formation Assay
The cells were subcultured until reaching the logarithmic growth phase, the stable transfected cells were digested with 0.25% trypsin and gently pipetted to make them single cells. The cells were counted and reseeded at a density of 1000-2000 cells per well into a 6-well plate and incubated at 37°C with 5% CO 2 in a humidi ed incubator for 10-15 days. When the cell formed visible clones, cells were terminated cultured, and washed twice with PBS, xed using 4% paraformaldehyde for 15 min, and then stained using Crystal Violet for 5 min. The cells were then washed with PBS and dried.

Immunohistochemical analysis
Isolated tumors was formalin-xed and para n-embedded and sectioned into slices. The tissue sections were subsequently stained with hematoxylin and eosin(HE) using standard techniques and used for the MEK5 and anti-Ki67 immunoassays. . A human ccRCC model was established by inoculating subcutaneously 1 × 106 A498 cells near the armpits of the forelimbs of experimental mice. The mice were randomly divided into the BIX and control groups when tumor masses were visible. BIX was intraperitoneally administrated at a dose of 30 mg/kg once a day for subsequent 10 days. The tumor growth was monitored by daily measurements by electronic cliper, in two dimensions(length(L) and width(W) ), and tumor volume(V) was estimated using with the formula: V=L/2 ×W2. After ten days treatments, the mice were sacri ced by cervical dislocation, and the tumor masses were removed, photographed and weighed.

Patient's Samples and Analysis
Under the supervision of the local ethics committee and pathologists, and without interfering with histological evaluation, fresh samples of four cases were obtained from ccRCC patients, diagnosed and treated at the Department of Urology, Renji Hospital. T test was used to evaluate the difference between MEK5 expression in ccRCC and adjacent normal tissues.

Statistical analysis
The statistically signi cant differences observed in drug-treated versus control cultures were determined using the Wilcoxon signed-rank test. The minimum level of signi cance was p < 0.05.

MEK5 is highly expressed in ccRCC tissues
The MEK5 mRNA expression in ccRCC patient samples was determined using the ONCOMINE database. MEK5 mRNA expression in normal and tumor tissues was compared in the Oncomine database. MEK5 mRNA expression was signi cantly upregulated in tumor tissues compared to the normal kidney tissues (Fig. 1A). ccRCC samples were collected to detect MEK5 at mRNA levels. The mRNA levels of MEK5 expression in ccRCC tissues were signi cantly higher than in adjacent tissues (p<0.05) (Fig. 1B). Immunohistochemical staining data were available from fourteen normal renal and ccRCC tissues. MEK5 exhibited a higher expression in the ccRCC tissues than normal tissues (p<0.05) (Fig. 1C). Combined with the database and clinical specimen data, we found that MEK5 was speci cally and highly expressed in ccRCC tumor tissues.
MEK5 promoted proliferation, migration, and colony formation MEK5 was speci cally and highly expressed in ccRCC tumor tissues. The biological function of MEK5 in ccRCC was validated further. Two MEK5 knockdown plasmids were generated, and the knockdown e ciency of MEK5 was measured at the protein and mRNA levels ( Fig. 2A). MEK5 knockdown suppressed the proliferation of ccRCC cells compared with the control (Fig. 2B). Conversely, overexpression MEK5 signi cantly promoted cell proliferation, as shown in Fig. 2D. The cell cycle effect of MEK5 was evaluated( Fig. 2E and 2F), suggesting that knockdown of MEK5 can induced cell cycle arrested in G0/G1 phase. Cell migration is one of the important processes in tumor development and metastasis. The migration effects of MEK5 on the ccRCC cell lines were evaluated ( Fig. 2G and 2H), suggesting that MEK5 can promote the migration of ccRCC cells. Additionally, the effect of MEK5 on colony formation of ccRCC cells was examined. Consistent with the results of the CCK-8 assay, colony formation analysis demonstrated that the MEK5 knockdown suppressed the colony formation of ccRCC cells, whereas MEK5 overexpression increased its colony formation capacity ( Fig. 2I and 2J).
MEK5 regulates the mTOR signaling pathway MEK5 plays an important oncogene effect in ccRCC, and its mechanism was explored further. It has been reported that MEK5 can regulate the mTOR signaling pathway, which plays a vital role in the development and progression of ccRCC. Thus, the effects of MEK5 on the mTOR signaling were examined. The knockdown of MEK5 inhibited the mTOR signaling pathway, while overexpression activated the mTOR signaling pathway (Fig. 2K and 2L).
Targeted inhibition of MEK5 has a good anti-tumor effect invitro MEK5 activates the mTOR signaling pathway and promotes proliferation, migration, colony formation, and tumor growth. The effects of MEK5 inhibitors on ccRCC cells were evaluated. The MEK5 inhibitor BIX e ciently induced cell apoptosis and inhibited cell proliferation of ccRCC ( Fig. 3A and 3B), consistent with the knockdown of MEK5. The G0/G1 arrest effect of BIX on ccRCC cells in the G0/G1 phase was dose-dependent (Fig. 3C). The front experiments con rmed that MEK5 knockdown suppressed colony formation and migration of ccRCC cells. The same phenomenon was observed in the experiment of BIX targeted inhibition of MEK5 ( Fig. 3D and 3E). BIX effectively inhibited the activation of the mTOR signaling pathway in ccRCC cells (Fig. 3F). The results of the experiments showed that the MEK5 inhibitor BIX has a good anti-tumor effect in-vitro.

Targeted inhibition of MEK5 has a good anti-tumor effect in murine models
We further validated the experiment in-vivo to evaluate the anti-tumor effect and safety of BIX. The murine's subcutaneous tumor models were constructed using A498 cell lines, and the tumor of murine in the DMSO group and the MEK5 inhibitor BIX group were observed dynamically. MEK5 inhibitor BIX effectively reduced tumor growth and load in murine ( Fig. 4A and 4B). Besides, there was no signi cant difference in body weight (Fig. 4C), excretion, and life status between the two murine groups, suggesting a favorable safety pro le of MEK5 inhibitor BIX in-vivo. The tumor tissues were processed for immunohistochemical analysis, and the results are shown in Fig. 4D. MEK5 inhibitor BIX effectively reduced tumor growth in murine by reducing Ki67 expression.

Discussion
MEK5/ERK5 pathway is involved in multiple processes, such as cell proliferation, differentiation, motility, and apoptosis. It has attracted attention because of its role in tumors [11,16,17]. High expression of MEK5 in metastasis prostate cancer may be correlated to bone metastasis, and the AP-1induced by MEK5/ERK5 makes prostate cancer more aggressive and harbors poorer prognosis [14]. In small cell lung cancer (SCLC), the abnormal expression of MEK5/ERK5 plays a pivotal role in driving tumors through modulating lipid metabolism pathways [18]. In this study, we con rmed for the rst time that the MEK5 was highly expressed in ccRCC and played a crucial role in cell proliferation, migration, and colony formation, and also demonstrated that MEK5 activated the mTOR signaling pathway in ccRCC.
Tumor treatment and immunotherapy can be improved because of the effect of mTOR inhibitors on speci c T cell subsets. In a phase I clinical trial of combined use of everolimus and low-dose cyclophosphamide in the treatment of metastatic RCC, it was observed that changes in various immune cells populations promoted anti-tumor immunity [29,30]. In a mouse model of renal cell carcinoma, everolimus combined with anti-PD-L1 was more effective in reducing tumor burden than PD-L1 treatment alone [31]. Our research con rmed that MEK5 is involved in the abnormal expression and activation of mTOR and the occurrence and development of ccRCC.
MEK5 inhibitors have a good application prospect in various tumors by targeting the biological activity of MEK5. MEK5/ERK5 inhibitors combined with PI3K/Akt inhibitors were more effective than either inhibitor alone in reducing the proliferation and survival of triple-negative breast cancer (TNBC) [32]. MEK5 inhibitor may cause FLT3-ITD-positive AML cellular damage such as apoptosis and serve as an alternate target for therapy [33]. ERK5 inhibitors signi cantly enhanced the anti-tumor effect of 5-FU, increased cell apoptosis, and inhibited the growth of tumors in colon cancer [34]. However, no study reported the role of MEK5 inhibitors in ccRCC. Our experiment applied MEK5 inhibitor BIX to ccRCC for the rst time and demonstrated that BIX exhibited signi cant anti-tumor effects in-vitro and in-vivo with excellent safety.
Our results suggest that MEK5 could be a target for developing a new anti-cancer systemic treatment for ccRCC.
In summary, we proved that MEK5 expression aberrantly increased in ccRCC, which activated the mTOR signal pathway and regulated cell proliferation, cycle, migration, clone formation of ccRCC. Targeted inhibition of MEK5 has the potential for widespread clinical application in patients with ccRCC.

Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
manuscript. Yonghui Chen and Qi Chen supervised the study. All authors read and approved the nal manuscript.
Data availability: The datasets generated during the current study are not publicly available due to ethical restrictions, but are available from the corresponding author on reasonable request.
Ethical approval: Human sample collection and the study protocol were approved by the Committee for the Ethical Review of Research, Renji Hospital, Shanghai Jiao Tong University School of Medicine.