XPO1-Mediated EIF1AX Cytoplasmic Relocation Promotes Tumor Migration and Invasion in Endometrial Carcinoma

Dysregulation of eukaryotic translation initiation factor 1A, X-linked (EIF1AX), has been implicated in the pathogenesis of some cancers. However, the role of EIF1AX in endometrial carcinoma (EC) remains unknown. We investigated the EIF1AX expression in EC patients and assessed its tumorigenesis-associated function and nucleocytoplasmic transport mechanism in vitro and in vivo. The results indicated that the cytoplasmic EIF1AX expression showed a gradual increase when going from endometrium normal tissue, simple endometrial hyperplasia, complex endometrial hyperplasia, and endometrial atypical hyperplasia to EC, while vice versa for the nuclear EIF1AX expression. In addition, the cytoplasmic EIF1AX expression was positively correlated with histologic type, high International Federation of Gynecology and Obstetrics (FIGO) grade, advanced FIGO stage, deeper infiltration, high Ki67 index, and shorter recurrence-free survival in EC patients. In vitro, short hairpin RNA-mediated EIF1AX depletion or SV40NLS-mediated EIF1AX import into the nucleus in multiple human EC cells potently suppressed cell migration and invasion, epithelial-mesenchymal transition, and lung metastasis. Moreover, exportin 1 induced the transport of EIF1AX from the nucleus to the cytoplasm that could be inhibited by leptomycin B treatment or the mutation in the EIF1AX location sequence. These results demonstrate that cytoplasmic EIF1AX may play a key role in the incidence and promotion of EC, and thus, targeting EIF1AX or its nucleocytoplasmic transport process may offer an effective new therapeutic approach to EC.


Introduction
Endometrial carcinoma (EC) is the second most diagnosed gynecologic malignancy and the sixth most diagnosed cancer in women worldwide [1][2][3]. In China, which is undergoing rapid socioeconomic transitions, the incidence rates of EC have been increasing, and its onset shows a trend toward occurrence in younger women over the past decades [1,2,4]. Its risk factors include early menarche, late menopause, nulliparity, obesity, physical inactivity, tamoxifen, polycystic ovary syndrome, positive family history, and genetic alterations [5,6]. Although treatment strategies have greatly improved, outcomes in EC patients with advanced or recurrent disease are far from satisfactory [5,7]. Presently, it is more important to clarify the molecular mechanisms underlying the growth, metastasis, and recurrence of EC, which may foster research into potential targets for early diagnosis and gene therapy.
Eukaryotic translation initiation factors (eIFs), which are transported into and out of nuclei through the central channels of nuclear pore complexes (NPC) by nucleocytoplasmic transport receptors, regulate the gene expression. This can lead to the abnormal activation or inhibition of signaling pathways involved in tumor progression metastasis and drug resistance, suggesting eIFs as therapeutic target for various types of cancers [8][9][10]. Many initiation factors, especially eIF2, eIF3, and eIF4, have been implicated in the etiology of many human cancers [11][12][13][14][15]. However, little is understood regarding the exact role and mechanism of eukaryotic translation initiation factor 1A, X-linked (EIF1AX) in EC. EIF1AX, encoded on human chromosome X, includes a central domain with an oligonucleotide-/oligosaccharide-binding (OB) fold, a small helical subdomain, and two-charged basic and acidic unstructured N-terminal tails (NTTs) and C-terminal tails (CTTs), respectively [16][17][18]. It is essential for the initiation of protein synthesis, particularly for recruitment of the ternary complex and for assembling the 43S preinitiation complex (PIC) [19][20][21]. EIF1AX could also augment Ago-mediated Dicer-independent micro (mi) RNA biogenesis and RNA interference [22]. Additionally, it was reported to be one of the major marker genes of mammalian preimplantation zygote genome activation [23]. To our knowledge, EIF1AX is the only example of a PIC subunit that is recurrently mutated in cancer. EIF1AX mutations have been reported in uveal melanomas, thyroid tumors, and ovarian carcinomas and are frequently located in the NTT domain [24][25][26][27]. Recently, it was found that EIF1AX mutations primarily enhance translation of long 5′UTR mRNAs that mainly encode proteins related to cell proliferation, differentiation, angiogenesis, invasion, and metastasis [28]. The nucleocytoplasmic overexpression of EIF1AX was observed in breast cancer and positively associated with its aggressive behavior and worse prognosis. The EIF1AX overexpression might promote the G1/S phase transition through the transcriptional repression of p21 in a p53independent manner [29]. Further investigation revealed that eIF1A was exported by importin13 (IPO13) in HeLa cells, and an exportin 1 (XPO1)-dependent pathway might also be important for eIF1A localization [30].

EIF1AX Protein Sequence Analysis by cNLS Mapper.
Potential nuclear localization signal (NLS) was determinated using the open source software cNLS Mapper (http://nlsmapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). The NLS scores are calculated with four NLS profiles (for class 1/2, class 3, class 4, and bipartite NLSs), each of which represents a contribution of every amino acid residue at every position within an NLS class to the entire NLS activity [36]. Briefly, a GUS-GFP reporter protein fused to an NLS with a score of 8, 9, or 10 is exclusively localized to the nucleus, that with a score of 7 or 8 partially localized to the nucleus, that with a score of 3, 4, or 5 localized to both the nucleus and the cytoplasm, and that with a score of 1 or 2 localized to the cytoplasm.
2.10. Immunofluorescence Analysis. HEC-1A, ECC-1, and RL95-2 cells were treated as described above. Then, cells (1 × 10 5 ) were grown on glass coverslips attached to a 24well plate, subsequently washed three times with 0.1% PVA-PBA, and fixed in 4% paraformaldehyde at room temperature (RT) for 30 min. Cells were then treated with 0.5% Triton X-100 at RT for 30 min, blocked with 3% BSA at RT for 60 min, and then incubated overnight with diluted primary antibodies at 4°C. Cells were then washed with 0.1% 3 Oxidative Medicine and Cellular Longevity PVA-PBS, incubated with Alexa Fluor 488-labeled donkey anti-rabbit secondary antibodies (1 : 1,000 dilution) for 60 min, washed again, and treated with DAPI (1 : 5,000 dilution) for 30 min. Cells were immediately examined under a Leica TCS SP8 confocal microscope. The samples were imaged at lower magnification with high resolution using a ×20/0.80 numerical aperture (NA) objective lens and 2048 × 2048 image pixels with a resolution of 0.25 μm. Primary antibodies were used at the following dilutions: polyclonal rabbit anti-EIF1AX antibodies at 1 : 100 (Thermo Fisher Scientific, Cat#HPA002561).
Photoshop software was used to separate protein expressed in nucleus and cytoplasm according to DAPI images. ImageJ was used to analyze the gray values of immunostaining images. Briefly, images were normalized by the same parameter to subtract background staining. Then, gray values of the nucleus and cytoplasm of each cell were calculated separately.
2.12. Wound-Healing Scratch Assay. 5 × 10 6 target cells were transferred into 6-well plates and incubated at 37°C until 80-90% confluence. A 200 μL sterile plastic tip was used to create a wound line across the surface of the wells, and the suspended cells were removed with PBS. Cells were cultured in reduced serum MYCOY′S5A or DMEM/F12 medium in a humidified 5% CO 2 incubator at 37°C for 48 h, and then images were taken with a phase-contrast microscope. Each assay was replicated three times.

Transwell Migration and Invasion
Assay. The migration and invasion assays were performed using transwell chambers (Millipore, Billerica, MA, USA). For migration assays, the transfected cells were seeded into the upper chamber with serum-free medium (2:5 × 10 4 cells), and the bottom of the chamber contained the MYCOY′S5A or DMEM/F12 medium with 10% fetal bovine serum. For the invasion assay, the chamber was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), and the subsequent steps were similar to those used in the migration assay. After the cells migrated or invaded for 24 h, they were fixed and stained with crystal violet. Migrated and invaded EC cells were counted under an inverted light microscope. The number of migrated or invaded cells was quantified by counting the number of cells from 10 random fields at ×100 magnification.
2.15. Lung Metastases. 1 × 10 6 HEC-1A cells stably expressing plasmids were injected intravenously into the tail vein of nude mice. After 5 weeks (5 animals per groups), the mice were sacrificed. Lungs were collected, fixed in 10% neutral buffered formalin, and embedded in paraffin followed by serial sectioning. Five sections (100 μm apart) from each lung were stained with hematoxylin and eosin (H&E) and photographed. The area of lung metastases was determined by ImageJ.
2.16. Statistical Analysis. Statistical analyses were performed using the SPSS 22.0 software program and GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). The relationship between IHC expression and clinicopathologic parameters was analyzed using the χ 2 test. Survival analysis was performed using the Kaplan-Meier method and logrank test. Multivariate survival analysis was performed using the Cox proportional hazards model. Means between the groups or within the groups were compared with the oneway ANOVA. P values of <0.05 were considered to be significant.

EIF1AX Is Overexpressed in the Cytoplasm of Human EC and Has
No Mutations in the EIF1AX Coding Region. Using IHC, we detected the expression of EIF1AX in normal endometrium, precursor lesions, and EC tissues. As shown in Figure 1 and Table 2, compared with normal endometrial cells, the EC cells displayed stronger EIF1AX staining in the cytoplasm (P < 0:01), while weaker staining appeared in the nucleus (P < 0:01). Additionally, the cytoplasmic EIF1AX expression was higher in EEC than that in normal endometrium, SEH, CEH, or AEH (P < 0:01). The nuclear EIF1AX expression was lower in EEC than that in normal endometrium, SEH, CEH, or AEH (P < 0:01). Using western blotting, EIF1AX protein levels were found to be higher in EC than in normal tissue, which further confirmed the IHC results. However, Sanger DNA sequence analysis (30 EC patients) did not detect any point mutations, deletions, or insertions in the coding region of EIF1AX ( Figure S2).

Cytoplasmic EIF1AX Overexpression Is Correlated with
Adverse Clinical-Pathological Parameters and Poor Prognosis in Human EC. To determine the clinicopathological significance of the ectopic EIF1AX expression, a comprehensive data set of 315 EC cases was analyzed using a statistical tool. These analyses indicated that EIF1AX protein was mainly located in the cytoplasm of EC cells, with a cytoplasmic positive rate of 82.5%, while the nuclear positive rate was 12.9%. The cytoplasmic EIF1AX expression was positively correlated with 4 Oxidative Medicine and Cellular Longevity          Oxidative Medicine and Cellular Longevity histologic type (P < 0:01), high FIGO grade (P < 0:01), advanced FIGO stage (P < 0:01), deeper infiltration (P < 0:05 ), and a high Ki67 index (P < 0:01) as shown in Table 1. All cases were followed up periodically for no less than 3 years in total, and cases followed less than 1 year were recorded as lost to follow-up. A total of 143 patients with EC were enrolled in the survival analysis. At a median follow-up of 85 months, 128 patients (89.51%) were alive with no recurrence, four patients (2.80%) were alive but had relapsed, and 11 patients (7.69%) had died. The median survival time was 156 months, and 1-, 3-, and 5-year survival rates were 96.5%, 91.5%, and 86.9%, respectively. The higher the expression of EIF1AX, the worse the overall survival was in EC patients (P = 0:000, Figure S3). Univariate analysis using the Cox proportional hazards regression analysis for all parameters showed that high EIF1AX expression, histologic type, histological grade, FIGO stage, invasive depth, and Ki67 index were all prognostic variables in EC patients. However, multivariate analysis confirmed that only histologic type (P = 0:003, hazard ratio 1.755-17.022) and Ki67 index (P = 0:006, hazard ratio 1.611-17.117) were independent prognostic factors in EC (Table 3).

EIF1AX Knockdown or Relocation to the Nucleus Inhibits EC Cell Proliferation, Migration, and Invasion Capabilities.
Given that EIF1AX was mainly overexpressed in the cytoplasm in human EC tissues, we first investigated EIF1AX protein expression and localization in EC cell lines (HEC-1A, ECC-1, and RL95-2). Immunofluorescence staining revealed that the EIF1AX expression in the cytoplasm was stronger than that in the nucleus, especially for HEC-1A and RL95-2 cells; western blot results indicated that the EIF1AX protein expression in HEC-1A and RL95-2 cells was markedly higher than that in ECC-1 cells ( Figure S4A, B). Then, we selected HEC-1A and RL95-2 cells as model cell lines for further experiments. To clarify the role of EIF1AX in proliferation of EC cells, we successfully transfected the EIF1AX-NLS plasmid into HEC-1A cells resulting in relocation of the EIF1AX protein to the nucleus, as shown using immunofluorescence and western blotting ( Figure S4C, D).   11 Oxidative Medicine and Cellular Longevity group) with EIF1AX (Figure 2(a)). To further illustrate the role of EIF1AX on migration and invasion of EC cells, we detected the EMT marker gene expression and indicated that Snail and vimentin were decreased, while E-cadherin and beta-catenin were increased after EIF1AX knockdown, implying that EIF1AX may target Snail to promote the epithelial-mesenchymal transition process. Compared with the KD + Esm group, the rescue of EIF1AX in the nucleus did not change the expression of Snail, E-cadherin, vimentin, and beta-catenin more effectively (Figure 2(b)). In addition, we performed wound-healing assays and found that the wound-healing capability of EC cells was reduced after EIF1AX knockdown. Compared with the KD group, the rescue of EIF1AX in the cytoplasm improved its wound-healing capability but not in the KD + NLSsm group (Figures 2(c) and 2(d)). Furthermore, cell migration and invasion assays were performed in vitro, and the number of migrating and invading cells was counted. Consistent with the above results, significantly reduced migration and invasion (P < 0:05) in the KD and KD + NLSsm groups were observed, implying that downregulation of EIF1AX or relocating it to the nucleus slowed down migration and invasion of EC cells (Figures 2(e)-2(h)). Thus, the different location of EIF1AX between normal tissues and EC may demonstrate that the relocation of EIF1AX to the nucleus is an important factor in EC cell migration and invasion.

EIF1AX Is
Transported to the Cytoplasm by the XPO1-Mediated Nuclear Export Pathway in EC Cells. In present research, we found that the EIF1AX was located in nucleus in normal tissue. Therefore, the cNLS Mapper was used to predict the NLS sequences of EIF1AX, and the highest scores (score 3.1) of the sequence (RRRGKNENESEKRLVFKEDG-QEYAQVIKML) was selected (Figure 3(a)) [36]. Interestingly, the EIF1AX protein was located in the nucleus instead of cytoplasm after the mutant of EIF1AX NLS sequences in HEC-1A and RL95-2 cells (Figures 3(b)-3(d)). In general, small molecules, up to~20-40 kDa, can passively diffuse across the nuclear pore complexes (NPCs), while other molecules need to be actively transported [37]. Due to the small size (17 kDa), EIF1AX is thought to pas-sively diffuse through the NPCs without NLS sequence; its active export might therefore be required both to deplete EIF1AX from the nucleus and to maintain sufficient cytoplasmic levels [38]. We supposed that the possible overlapping between NLS and NES sequences of EIF1AX resulted in the protein translocation to the nucleus after the mutant of NLS/NES sequence. Michael et al. also found that the NES and NLS activities of M9 are either identical or overlapping as mutants which block M9 NLS activity and also abolish NES activity [39].
In order to test the hypothesis above, we further explored the export factors targeted EIF1AX. Previous research points out that IPO13 is a transporter of EIF1AX in HeLa cells [40]; we therefore devised a siRNA targeting IPO13 to suppress the transport process of the EIF1AX protein (Figure 4(a)). However, the location of EIF1AX was not changed after IPO13 knockdown (Figures 4(b) and 4(c)), and the results of the coimmunoprecipitation also showed that EIF1AX did not interact with IPO13 (Figure 4(d)).
Grunwald et al. predicted that eIF1A export from the nucleus is not unique to IPO13 and might involve other export factors, such as XPO1, and might occur in complex with other proteins [30]. Interestingly, the expression of EIF1AX was increased in the nucleus after XPO1 knockdown ( Figure S4E, Figures 5(a) and 5(b)). LMB, a XPO1cargo formation inhibitor [41], was also used to prove the relationship between EIF1AX and XPO1. EIF1AX was significantly increased (P < 0:05) in the nucleus with 40 nM LMB treatment in RL95-2 and HEC-1A cells (Figure 5(c)). Immunofluorescence also demonstrated that EIF1AX was inhibited from translocating to the cytoplasm in the 40 nM LMB groups ( Figure 5(d)). However, we also found that the EIF1AX protein expression was reduced in cytoplasm and nucleus after 80 nM leptomycin B treatment in HEC-1A cells but not in RL95-2 cells. Combined with the results of CCK-8, it may be the cytotoxicity of 80 nM leptomycin B to HEC-1A cells ( Figure S4F). It suggested that EIF1AX was transported to the cytoplasm by an XPO1-mediated nuclear export pathway in EC cells. In addition, results of the coimmunoprecipitation showed that XPO1 could interact with EIF1AX but not when the NLS (NES) of

EIF1AX Protein Knockdown or Translocation Causes
Attenuated Tumor Cell Extravasation In Vivo. The ability of tumor cells expressing wild type or mutant NLS sequence to extravasate into the lung was measured by injecting identical numbers of HEC-1A cells into the tail veins of nude mice. As expected, mice injected with cells expressing EIF1AX shRNA developed shrunken metastatic nodules evidenced both by gross and histological analysis. In addition, compared with the KD + Esm group, mice injected with cells expressing EIF1AX NLS sequence mutant also developed shrunken metastatic nodules ( Figure 6). The results also further indicated that EIF1AX translocated to cytoplasm may have an important role in the initiation and progression of EC.

Discussion
EIF1AX was identified as a cancer driver gene in thyroid cancer. EIF1AX mutations are present in 11% of poorly differentiated thyroid cancers and anaplastic thyroid cancers and are almost invariably associated with oncogenic RAS mutations [42]. Significant co-occurrence of mutations in NRAS and EIF1AX was also found in low-grade serous ovarian carcinomas. The coexpression of mutant NRAS and EIF1AX proteins promoted proliferation and clonogenicity survival in LGSC cells [24]. These results imply that EIF1AX and Ras may drive tumor progression synergistically. In this study, no mutations in the EIF1AX coding region were identified. Reasons for this finding may include the small sample number, the limited region assessed, and the sensitivity of the methods used. In agreement with the results in breast and ovarian cancer [24,29], to our knowledge, we demonstrated for the first time higher ectopic expression levels of EIF1AX protein in EC than in normal tissues and precancerous lesions. The cytoplasmic EIF1AX expression was positively linked to unfavorable clinicopathological characteristics and an adverse prognosis in EC.
The aberrant dysregulation of protein synthesis has been reported to be frequently associated with cancer [43,44]. EIF1AX plays a key role in scanning and AUG selection and differentially affects translation of distinct mRNAs [20]. EIF1AX also plays an important role in tumor pathogenesis. It has been known that the 5′UTR length is the main feature involved in the translational control by eIF1A in mammalian cells [28]. Cancer-associated eIF1A NTT mutants primarily enhance translation of long 5′UTR mRNAs regulating cell proliferation, differentiation, invasion, metastasis, and angiogenesis [24]. By using an embryonic fibroblast cell model, Urmila et al. showed that cell proliferation significantly declined with the majority of cells arrested in the G1 phase following siRNA-mediated downregulation of EIF1A expression levels [28]. In this study, EIF1AX was upregulated in the cytoplasm of EC cells, and EIF1AX knockdown or translocation into the nucleus markedly decreased the ability of EC cells to migrate and invade with E-cadherin overexpression, and Snail hypoexpression at protein levels in vitro, and EIF1AX knockdown also inhibited proliferation in vitro. Our findings were in partial agreement with previous results obtained in thyroid, ovarian, and breast cancer [24,26,27,29]. While the contribution of EIF1AX to tumorigenesis and cancer progression is not clear, EIF1AX has been found to have links to cancer-related signaling pathways including PI3K/AKT/mTOR and Ras/ ERK signaling pathways, as well as oncogene c-myc [26,[45][46][47][48]. With a deeper understanding of EIF1AX in cancer, EIF1AX may be a good molecular target for gene therapy in the future.
Proteins are known to exhibit diverse biological functions according to their subcellular location; thus, nucleocytoplasmic transport is an essential activity in eukaryotic cells. EIF1AX is localized to both nucleoli and cytoplasm, and its nuclear export process involves specific interactions of transporter IPO13 and EIF1AX localization sequences in HeLa cells [38]. Grunwald   14 Oxidative Medicine and Cellular Longevity 3.6-A°crystal structure of IPO13 in complex with RanGTP and with eIF1A and noted that at least a fraction of eIF1A might be exported via a XPO1-dependent pathway [30]. Less consistent with the above, our experiment in EC cells revealed that LMB treatment effectively inhibited XPO1mediated, but not IPO13-mediated, cytoplasmic export of EIF1AX. The discrepancy among these results may be related to the types of cells used and/or experimental conditions. XPO1 inhibitors are a unique class of drugs and are currently being evaluated in several phase I/II/III clinical studies [49][50][51]. Recent studies have pointed out that selinexor (an approved inhibitor of XPO1-mediated nuclear export) plus chemotherapy was a safe and tolerated treatment in advanced ovarian and endometrial cancer patients [52,53]. Our findings may partly provide a theoretical basis for the abovementioned clinical trial results. In addition, we identified the EIF1AX NLS sequence. Indeed, other factors including protein folding conformation, protein posttranscriptional modifications, and protein-protein interactions could influence the recognition and binding between transporters and substrates apart from the amino acid sequence [54,55].

Conclusion
In summary, the upregulated expression and nucleocytoplasmic translocation of EIF1AX protein occur in EC. The expression of cytoplasmic EIF1AX was positively correlated with aggressive clinicopathologic features and poor prognosis in EC patients, which might result from the ability of the ectopic EIF1AX expression to facilitate EC cell proliferation, migration, and invasion. The specific location signal sequence of EIF1AX was identified by XPO1 and then transported into the cytoplasm in EC cells. These results indicated that EIF1AX may have an important role in the initiation and progression of EC. Thus, EIF1AX may be employed as a potential target for gene therapy.

Data Availability
All the data is enclosed in the manuscript.

Ethical Approval
The Ethics Committee of Fujian Medical University has approved the animal study (Code of Ethics: LLSLBH-20210930-002). The Ethics Committee of the First Affiliated Hospital of Fujian Medical University has approved the human experiments (Code of Ethics: [2019]096).

Consent
The written informed consent was obtained from all of the participants.

Conflicts of Interest
None of the authors have any conflicts of interest to declare.

Authors' Contributions
S. W. and S.Z. designed the study. C. L. performed Co-IP, RIP, and RNA pull down. J. S., C. L., and X.L performed CCK-8 detection and western blot. Y. Y. and X. J. performed endometrial cancer cell administration. Z. L. and J. S. conducted RNA-seq. Y. L., H. L., and Y. Y. performed RT-qPCR. Y. H. and Y.C. prepared tissue samples. J. S. C.L. and Y. Y. wrote the manuscript. S. W. and S.Z. supervised the whole study. Yuhong Ye, Chengyu Lv, and Jiandong Sun contributed equally to this work.