PAK5‐stabilized Smuc confers renal cell carcinoma metastasis

Dear Editor, Abundant evidence has demonstrated that PAK5 confers an oncogenic potential in carcinogenesis and tumor progression. Our previous discoveries have confirmed that PAK5 promotes the growth of breast cancer andmetastasis of cervical cancer respectively through phosphorylating NF-κB-p65 and SATB1.1,2 And microRNA-106-5p targets PAK5 to inhibit cell migration and invasion in RCC.3 However, the mechanism by which RCC acquires malignant phenotypes conferred by PAK5 remains unclear. Snail and Slug are extremely unstable proteins, which can be explained by posttranslational modification.4 We speculated that whether Smuc possesses the potential characteristics similar to the other two members, and there is a previously unknown direct linkage between serine/threonine PAK5 kinase and Smuc. Western blot was performed to confirm the overexpression of PAK5 and Smuc (Figure S1A,B). Notably, reciprocal immunoprecipitation and co-localization of immunofluorescence staining indicated the physical interaction between PAK5 and Smuc (Figure 1A; Figure S1C,D). We calculated and predicted four putative PAK5 phosphorylation sites (Ser63, Ser98, Ser125, Ser278) of Smuc according to the consensus PAKs phosphorylation residues,5 whereas only the motif surrounding Ser278 is evolutionarily conserved across other species (Figure 1B). We performed the in vitro phosphorylation assay using recombinant PAK5 proteins and various synthetic peptides containing unphosphorylated or phosphorylated residues of Smuc. PAK5 promoted the phosphorylation of the peptide containing the potential PAK5-phosphorylation site (Ser278) of Smuc (Figure 1C). However, phosphorylation level of Smuc was effectively inhibited by the addition of homolog peptides harboring the random disruption of Smuc sequences or single-site mutation of Ser278 replaced by alanine(Figure 1C). Snail and Slug are short half-life proteins targeted by the ubiquitin-proteasome system.6 It has been reported that PAK1-mediated phosphorylation of Snail regulates its subcellular localization and functions.7 Addition of

wileyonlinelibrary.com/journal/ctm2 1 of 6 https://doi.org/10.1002/ctm2.559 F I G U R E 1 PAK5 phosphorylates and stabilizes Smuc. (A) Exogenous interaction between PAK5 and Smuc was determined by immunoprecipitation with the anti-Smuc antibody in RCC cells. Immunoglobulin (Ig) G serves as negative control. (B) The motif surrounding Ser278 of Smuc is evolutionarily conserved across other species. (C) In vitro phosphorylation assay was performed in RCC cells using recombinant PAK5 proteins and various synthetic peptides (peptide #1: random disruption of the potential PAK5-phosphorylation site/Ser278 of Smuc; peptide #2: potential PAK5-phosphorylation site/Ser287 of Smuc; peptide #3: single-site mutation of Ser278 to alanine of Smuc; peptide #4 [positive control]: identified PAK5-phosphorylation site/Ser39 of E47; peptide #5: single-site mutation of Ser39 to alanine of E47). E47 that has been reported to be phosphorylated at Ser39 by PAK5 in colon cancer was adopted as a positive control. (D) After transfecting PAK5 in a dose-dependent manner, the expression of Smuc and PAK5 in protein levels was examined by western blot. (E) PAK5 interference decreased the protein expression of Smuc in RCC cells, which was blocked by the proteasome inhibitor MG132. (F) After the treatment with cycloheximide (CHX, a protein synthesis inhibitor) in RCC cells, lysates were collected at the indicated time and endogenous Smuc expression was tested by immunoblot. (G) After the indicated transfection, cells were treated with MG132. The ubiquitinated Smuc was detected. *p < 0.05; **p < 0.01; ***p < 0.001 frequently observed in RCC tissues using IHC, but sparse or negative staining was found in adjacent normal tissues ( Figure 2I). In addition, 50.6% and 41.6% of RCC tissues presented increased PAK5 and Smuc expression (Figure 2J), respectively. Increased PAK5 was associated with histological grade (p = 0.029), gender (p = 0.027), and TNM stage (p = 0.009) (Table S1). Inconsistently, increased Smuc was correlated with TNM stage (p = 0.025) and perirenal involvement (p = 0.029) (Table S1). Simultaneously, PAK5 positively correlated with Smuc (Table S2, R = 0.411), which indicated that coordination between Smuc and PAK5 might elicit a positive action on RCC progression. Both PAK5 and Smuc predicted a shorter overall survival for RCC patients (Table 2K). And they may function as novel prognostic markers in RCC (Table S3; Figure 2L).
In conclusion, we identify Smuc as a novel downstream partner of PAK5 and demonstrate that PAK5-mediated Smuc phosphorylation impairs its ubiquitination degradation ( Figure 3K). Mechanism dissections support the crucial potential for the PAK5-Smuc axis in promoting the EMT and metastasis of RCC. Our study provides evidence of a de novo PAK5-Smuc pathway in RCC progression and novel therapeutic targets for tumor metastasis.

C O N F L I C T O F I N T E R E S T
The authors declare that they have no competing interests.

A U T H O R C O N T R I B U T I O N S
Fu-Chun Huo, Zhi-Man Zhu and Qiu-Ying Du performed and analyzed experiments. Fu-Chun Huo and Zhi-Man Zhu wrote the paper. Qiu-Ying Du analyzed and interpreted the data. Dong-Sheng Pei obtained funding and designed the research.

AVA I L A B I L I T Y O F D ATA A N D M AT E R I A L S
All data in our study are available upon request.

E T H I C S A P P R O VA L A N D C O N S E N T T O PA R T I C I PAT E
The use of human subjects was approved by ethics committee of the Affiliated Hospital of Xuzhou Medical University, and written informed consent was obtained from the participants. All animal experiments were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University and in accordance with institutional guidelines.