KIF4A promotes genomic stability and progression of endometrial cancer through regulation of TPX2 protein degradation

Kinesin family member 4A (KIF4A) belongs to the kinesin superfamily proteins, which are closely associated with mitophagy. Nonetheless, the role of KIF4A in endometrial cancer (EC) remains poorly characterized. The present study showed that KIF4A not only was upregulated but also predicted poor prognosis in patients with EC. KIF4A knockdown in EC cells resulted in attenuated proliferative capacity in vitro and in vivo. Transcriptome sequencing and gene function analysis revealed that KIF4A contributed to the maintenance of EC cells' genomic stability and that KIF4A knockdown induced the DNA damage response, cell cycle arrest, and apoptosis. Mechanistically, KIF4A interacted with TPX2 (a protein involved in DNA damage repair to cope with the replication pressure) to enhance its stability via inhibition of TPX2 ubiquitination and eventually ensured the genomic stability of EC cells during mitosis. Taken together, our results indicated that KIF4A functions as a tumor oncogene that facilitates EC progression via the maintenance of genomic stability. Therefore, targeting the KIF4A/TPX2 axis may provide new concepts and strategies for the treatment of patients with EC.

Kinesins are microtubule-based motor proteins that mediate diverse functions within the cell, including the transport of vesicles, organelles, chromosomes, and protein complexes and the movement of microtubules. 8,9 Individual kinesin family members have specific functions, with some kinesins being involved in axonal transport and others functioning exclusively in mitosis. 10 To date, a total of 45 kinesin superfamily proteins (KIFs) with varying functions have been discovered in humans, 11 and at least 12 of them are required in various aspects of mitosis, including bipolar spindle assembly, chromosomal alignment and segregation, and cytokinesis. 12,13 Multiple kinesins also promote the proliferation and progression of pancreatic cancer, 14 hepatocellular carcinoma, [15][16][17] glioblastoma, 18,19 melanoma, 20,21 and lung adenocarcinoma. 22 KIFC1 and KIF2C are upregulated in EC and accelerate EC cell proliferation. 23,24 Nonetheless, the role of kinesins in EC has not yet been fully investigated.
DNA damage is a key factor in both the evolution and treatment of cancer, which gives rise to an abnormal nucleotide or nucleotide fragment, causing a break in one or both chains of the DNA strand. DNA damage increases the possibility of generated mutations. 25 Genomic instability, which represents one of the most important factors for cancer development, is a common feature of cancer cells and favors the accrual of driver mutations and expansion of tumor heterogeneity. 25,26 The DNA damage response (DDR) pathway contains a class of proteins that can detect DNA double-strand breaks (DSBs), single-strand breaks (SSB), base mismatch, and DNA adducts and can correct some alterations, which protect cancer cells against death due to endogenous and external DNA damage factors. 27 Consequently, DDR inhibition has become an ideal strategy for the development of new therapeutic agents in oncology, and drugs targeting the DDR pathway such as inhibitors of poly (ADP-ribose) polymerase (PARP), ATR Serine/Threonine Kinase (ATR), and ataxiatelangiectasia mutated kinase (ATM) have been developed. 28,29 Notably, as kinesins play an important role in cell division, they are crucial for the maintenance of the chromosomal structure and genomic stability, and kinesin-mediated DNA DSB repair has been reported. 30,31 However, the molecular mechanisms underlying DDR regulation by kinesins in EC have not been fully elucidated.
In the present study, we analyzed differentially expressed kinesins in The Cancer Genome Atlas-Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) and GSE17025 databases to evaluate the role of kinesins in EC. We observed that kinesin family member 4A  FBS, whereas HEC-1-B was maintained in MEM medium containing 10% FBS. Cells were cultured in a 5% CO 2 incubator at 37°C. Cycloheximide (CHX) (a protein synthesis inhibitor), MG132 (a proteasome inhibitor), and chloroquine (an autophagy inhibitor) were all purchased from SelleckChem, and these were diluted in dimethyl sulfoxide solvent to obtain a stock concentration. CHX was used at a concentration of 10 μmol/L，MG132 was used at a concentration of 20 μmol/L，chloroquin was used at a concentration of 50 μmol/L.

| Databases and clinical information
The mRNA levels of patients with UCEC and corresponding clinical information were obtained from the TCGA (https://portal.gdc.cancer. gov/) and GEO databases (https://www.ncbi.nlm.nih.gov/geo/). The R packages "edgeR" and "limma" were used for normalization and differential gene expression analysis with the R statistical software (version 4.1.0). The criteria for screening differentially expressed genes were as follows: FDR p < 0.05 and |log 2 Figure S1A,B). By detecting the mRNA level of kinesin in EC tissue samples, we found that KIF4A increased most significantly in EC (fold change = 3.64, p < 0.01) ( Figure 1A). Furthermore, in the TCGA and GSE17025 databases, the mRNA expression of KIF4A was significantly upregulated in EC tissues ( Figure 1B,C). We also selected 23 paired samples from the TCGA database and performed a paired ttest on the KIF4A expression level, which was consistent with the overall results ( Figure 1D). Interestingly, KIF4A was elevated in all cancer types, as compared with normal tissues ( Figure 1E). Regarding protein levels, western blot results showed that KIF4A was increased in human EC compared to adjacent normal tissues ( Figure 1F). In addition, immunohistochemical staining for KIF4A was performed in paraffin sections obtained from 20 normal and 35 tumor tissue samples, which suggested that KIF4A was highly expressed in EC tissues ( Figure 1G).    Figure S3A). Next, the effect of KIF4A on EC cell apoptosis was measured by flow cytometry apoptosis assay, which demonstrated that KIF4A knockdown significantly increased the apoptosis rate ( Figure 4C). Similarly, KIF4A

| KIF4A acts as an indicator of unfavorable outcomes in patients with EC
knockdown upregulated the expression of Bax, cleaved caspase 3, and decreased the expression of Bcl-2 ( Figure 4D). In addition, apoptosis was measured at different time points after KIF4A knockdown, and the results indicated that apoptotic cells were significantly increased with time compared to control (Supporting Information: Figure S3B). These results suggest that KIF4A knockdown induces G2/M phase arrest and promotes apoptosis of EC cells.  Figure 5A,B). To further analyze the differentially expressed genes, pathway enrichment analysis was performed, and the results revealed that mitotic G2-G2/M phases, cell cycle checkpoints, apoptosis, nucleotide excision repair, and DNA DSB repair pathways were simultaneously activated ( Figure 5C).

| KIF4A knockdown activates DDR signaling
Subsequently, Gene Ontology enrichment analysis indicated that these genes were predominantly enriched in cell cycle, DNA repair, apoptotic signaling pathway, G2/M transition of mitotic cell cycle, DSB repair, and ubiquitin (Ub)-dependent protein catabolic process ( Figure 5D). Furthermore, GSEA indicated that KIF4A was also highly associated with homology-directed repair, chromosomal maintenance, DNA DSB response, G2/M DNA damage checkpoint, and Hub-specific processing protease pathway in the TCGA-UCEC database (Supporting Information: Figure S2D-I  Figure 6A). We then set out to confirm whether an interaction exists between KIF4A and these 10 proteins. We used western blot analysis to examine the levels of these proteins in Ishikawa and KLE cells with KIF4A knockdown. Notably, CDK1 and TPX2 expressions were significantly downregulated in EC cells after KIF4A knockdown, as compared with those in control cells ( Figure 6B, Supporting Information: Figure S4A). Moreover, the qRT-PCR results indicated that KIF4A knockdown repressed CDK1 mRNA levels but had no effect on TPX2 mRNA levels ( Figure 6C, Supporting Information: Figure S4B). Furthermore, EC cells were treated with indicated G2/M inhibitors (Geldanamycin, 100 nM) for 24 or 48 h, and the results did not show a significant difference between treated and control groups (Supporting Information: Figure S4C). The inconsistency between the protein and mRNA levels of TPX2 suggests that KIF4A may regulate TPX2 levels by regulating TPX2 protein stability.
To further verify whether KIF4A interacts with TPX2, coimmunoprecipitation was performed, and the results revealed that endogenous KIF4A could bind to TPX2 in EC cells ( Figure 6D,E).

Subsequently, KLE and Ishikawa cells with stable KIF4A knockdown
were treated with CHX to inhibit protein synthesis, and TPX2 protein turnover was analyzed over time. Compared with that in the control cells, the TPX2 half-life was considerably decreased in EC cells with stable KIF4A knockdown, which were treated with CHX ( Figure 6F).
The Ub-proteasome and autophagy-lysosome pathways are the two main routes of protein degradation in eukaryotes. 37 To determine which pathway played a major role in this process, a proteasome inhibitor (MG132) and a lysosome inhibitor (chloroquine) were administered to KIF4A knockdown cells. Strikingly, only MG132 completely restored the TPX2 protein level in KIF4A knockdown EC cells compared to that in the untreated control ( Figure 6G).
Our data suggest that KIF4A knockdown represses TPX2 protein stability mainly by promoting protein degradation mediated by Ub/proteasomal pathway. Next, we analyzed the effect of KIF4A knockdown on endogenous TPX2 ubiquitination through both immunoprecipitation and western blot analysis, which showed that the level of ubiquitinated TPX2 was significantly higher in EC cells with stable knockout of KIF4A than in control cells ( Figure 6H). In general, the above results indicate that KIF4A regulates TPX2 protein stability by affecting TPX2 ubiquitination levels.
To further explore the regulatory domain, molecular docking was performed using the ClusPro, which suggested possible interactions between KIF4A and TPX2. The main binding interactions were found to be salt bridges, hydrogen bonds and hydrophobic contacts (Supporting Information: Figure S5A). To probe for a direct, physical interaction between KIF4A and TPX2, we constructed GST-KIF4A and purified GST and GST-KIF4A proteins from bacteria. GST pull-down experiments were carried out from supernatants containing TPX2. Compared to GST alone, GST-KIF4A can pull down a remarkable amount of TPX2 (Supporting Information: Figure S5B). To identify the domain of KIF4A responsible for TPX2 binding, full-length, and a variety of truncated forms of KIF4A were overexpressed in EC cells. We found that TPX2 was precipitated only when the C-terminal domain of KIF4A was used (Supporting Information: Figure S5C,D).
In addition, compared to the control and Flag-1000-1232deletion groups, the full length of KIF4A resulted in enhanced protein stability and decreased polyubiquitination levels of TPX2 (Supporting Information: Figure S6A,B). Taken together, these findings suggested that the C-terminus of KIF4A is required for the interaction with TPX2 and regulation of TPX2 stability. 3.7 | TPX2 is required for KIF4A-mediated tumor progression TPX2 is a multifunctional protein that includes one or more potential microtubule-binding sites that play a vital role in spindle assembly. 38 Other recent studies also reported that TPX2/Aurora A could protect DNA forks during replication stress. 39 To further verify whether

| DISCUSSION
EC is more likely to be diagnosed at an early stage, with good OS. 40 However, advanced-stage EC has a poor prognosis; the continued annual increase in morbidity and mortality of EC underscores a dire need for more efficient approaches for this malignancy. 41 Various kinesin genes have been reported to be upregulated in several cancers and promote tumor proliferation and progression; however, the role of kinesins in EC has not been fully elucidated.
KIF4A is a kinesin-4 family member, which is highly expressed in various tumors and promotes cancer progression. 9 Our study showed that KIF4A regulates TPX2 protein stability via the Ub-proteasome pathway. The ubiquitination process is a posttranslational modification considered crucial for the maintenance of protein homeostasis. 57 Normally, ubiquitination is a considerably dynamic process and is counterbalanced by deubiquitinating enzymes. 58 Remarkably, several E3 Ub ligases and deubiquitinating enzymes, such as FBXW7, 59