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

Abstract

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.

Figure 1

The EIF1AX expression in endometrial carcinoma, precursor lesions, and normal endometrium. EIF1AX protein was positively expressed in normal proliferative phase endometrium ((a) cyt,-; nuc,++++), normal secretory phase endometrium ((b) cyt,-; nuc,+++), simple endometrial hyperplasia ((c) cyt,-; nuc,++++), complex endometrial hyperplasia ((d) cyt,-; nuc,++++), endometrial atypical hyperplasia ((e) cyt,+++; nuc,-), low-grade endometrial endometrioid carcinoma ((f) cyt,+++; nuc,-), high-grade endometrial endometrioid carcinoma ((g) cyt,++++; nuc,-), and serous carcinoma ((h) cyt,++++; nuc,-). Notes: normal endometrium (a, b). Precursor lesions (c–e). Endometrial carcinoma (f–h). Scale bar:100 μm in (a–h). (i) The protein expression of EIF1AX in normal endometrium and endometrial carcinoma tissue. GAPDH was used as a lane loading control. Student's t-test: ^∗∗∗P < 0.001. Bars indicate SD. Note: cyt: cytoplasmic expression pattern; nuc: nuclear expression pattern.

Figure 2

EIF1AX knockdown or translocation into the nucleus inhibits proliferation, migration, and invasion of endometrial carcinoma cells in vitro. (a) CCK-8 assay was used to detect cell proliferation activity of HEC-1A (left) and RL95-2 (right) cells at 24 h, 48 h, 72 h, and 96 h. (b) The protein expression of EIF1AX, E-cadherin, vimentin, beta-catenin, and Snail following shRNA transfection. GAPDH was used as a lane loading control. (c, d) HEC-1A cells were transfected with shRNA, and the motility of the cells was evaluated 12 h after transfection using a wound-healing assay. Scale bar: 100 μm. (e–h) HEC-1A and RL95-1 cells described in (c, d) were used in a transwell migration and invasion assay. Scale bar: 100 μm. One-way ANOVA: n = 3, ns: P > 0.05, ^∗P < 0.05, and ^∗∗P < 0.01. Bars indicate SD. Note: Ctrl group (empty vector plasmid), KD group (EIF1AX-shRNA), KD + Esm group (EIF1AX-shRNA+pcDNA3.1-EIF1AXsm), and KD + NLSsm group (EIF1AX-shRNA+pcDNA3.1-EIF1AXsm-SV40NLS).

Figure 3

The NLS sequence of EIF1AX. (a) The cNLS Mapper Software was used to predict nuclear localization signals (NLSs) of transporters for EIF1AX by calculating NLS scores. (b) Diagram of NLS mutation site in EIF1AX. (c, d) The protein expression of EIF1AX in the nucleus and cytoplasm upon mutation of the NLS sequence in EIF1AX (score 3.1). Scale bar: 100 μm. Scale bar: 50 μm in magnification. Student's t-test: n = 3, ^∗P < 0.05, and ^∗∗P < 0.01. Bars indicate SD.

Figure 4

The relationship between IPO13 and EIF1AX. (a) The expression of IPO13 protein after IPO13 siRNA transfection. (b) Immunofluorescence was used to detect the location of EIF1AX following IPO13 knockdown. Scale bar: 100 μm. Student's t-test: n = 3, ^∗∗P < 0.01. Bars indicate SD. (c) The protein expression of EIF1AX in the nucleus and cytoplasm following IPO13 knockdown. GAPDH was used as the reference gene for the cytoplasm, and HDAC1 was used as the reference gene for the nucleus. (d) Coimmunoprecipitation of EIF1AX with IPO13 in HEC-1A cells.

Figure 5

The relationship between XPO1 and EIF1AX. (a, b) The expression of EIF1AX in the nucleus and cytoplasm following XPO1 knockdown. Scale bar: 100 μm. Scale bar: 50 μm in magnification. Student's t-test: n = 3, ^∗P < 0.05, and ^∗∗P < 0.01. Bars indicate SD. (c, d) The expression of EIF1AX in the nucleus and cytoplasm following LMB treatment for 1 h. Scale bar: 100 μm. Scale bar: 50 μm in magnification. GAPDH was used as the reference gene for the cytoplasm, and HDAC1 was used as the reference gene for the nucleus. One-way ANOVA: n = 3, ^∗P < 0.05, and ^∗∗P < 0.01. Bars indicate SD.(e) Coimmunoprecipitation of XPO1 with EIF1AX in HEC-1A cells. (f) Coimmunoprecipitation of EIF1AX and EIF1AX-NLSmut with XPO1 in HEC-1A cells.

Figure 6

Lung metastases 5 weeks after injection of HEC1A cells in the mice that had received tail vein injection. (a) Ctrl group. (b) KD group. (c) KD + Esm group. (d) KD + NLSsm group. Scale bar: 100 μm. (e) The area of lung metastases were determined by ImageJ. One-way ANOVA: n = 3, ns: P > 0.05, ^∗P < 0.05. Bars indicate SD. Note: Ctrl group (empty vector plasmid), KD group (EIF1AX-shRNA), KD + Esm group (EIF1AX-shRNA+pcDNA3.1-EIF1AXsm), and KD + NLSsm group (EIF1AX-shRNA+pcDNA3.1-EIF1AXsm-SV40NLS).