YWHAE Influences the Malignant Behaviour of Ovarian Cancer by Regulating the PI3K/AKT and MAPK Pathways Via HE4


 Background: Malignant tumours of the female reproductive system threaten the lives and health of women worldwide, with ovarian cancer having the highest mortality rate among all malignant tumours. Based on previous work, this study analysed the expression and role of YWHAE in ovarian epithelial tumours. Methods: The interaction between YWHAE and HE4 was evaluated by immunoprecipitation, western blot analysis, and cellular immunofluorescence. Immunohistochemistry was used to address the relationship between YWHAE expression and clinicopathological parameters and patient prognosis. Changes in cell invasion, epithelial–mesenchymal transition, migration, proliferation, cell cycle, and apoptosis before and after differential expression of YWHAE were also explored in ovarian cancer cell lines and in vivo experiments. Results: YWHAE was found to directly interact with HE4, and its expression was positively correlated with HE4 expression. Moreover, YWHAE higher levels were associated with advanced ovarian cancer cases and with poorer patient outcome. YWHAE was found to enhance the invasion, migration, proliferation, and inhibition of apoptosis of ovarian cancer cells. These biological effects were found to be meditated by the activity of the PI3K/AKT and MAPK signalling pathways.Conclusions: Altogether, this study demonstrates that YWHAE is significantly increased in ovarian cancer tissues, representing a risk factor for the prognosis of ovarian cancer that is positively correlated with HE4 expression. Furthermore, YWHAE and its downstream signals may represent new therapeutic targets to tackle ovarian cancer.


Introduction
Ovarian cancer has the highest mortality rate among the most common malignant tumours of the female reproductive system. Due to the lack of obvious or speci c symptoms, as well as of ideal screening methods, the early detection and diagnosis rates of ovarian cancer are extremely low [1]. When the tumour progresses to an advanced stage, the tumour-free and overall survival times of patients are rapidly shortened [2]. Therefore, it is urgent to nd tumour markers with high sensitivity and speci city to guide early clinical screening, early diagnosis, and monitoring of ovarian cancer.
Human Epididymis Protein 4 (HE4) is an ovarian cancer marker that was identi ed by genomics and proteomics screenings [3]. Its high sensitivity and speci city has attracted the attention of researchers, who have proved that HE4 has more advantages than the common tumour marker CA125 for the early diagnosis of ovarian cancer and for monitoring disease progression [4]. In 2003, HE4 was designated as a serum marker for ovarian cancer and it was approved by the U.S. Food and Drug Administration in 2009 for monitoring the recurrence and progression of epithelial ovarian cancer [5]. Currently, several ongoing clinical studies are addressing the diagnostic potential of HE4, but only few are exploring its underlying molecular mechanisms.
Our previous work has proven that HE4 is highly expressed in ovarian cancer tissues, and that HE4 and Annexin A2 interaction can promote the invasion and metastasis of ovarian cancer. This mechanism is accomplished by activating adhesion signalling pathways such as MAPK and FOCAL [6][7]. Proteins perform their biological functions mainly by interacting with other proteins to form complexes or by working together with chaperone molecules. The tight coordination of proteins with different functions in time and space constitutes the basic process of life. Therefore, given the signi cant role of HE4 in ovarian cancer, it has become clear that recognition and improved knowledge on HE4-interacting proteins is an important step for an overall better understanding of this disease. A previous study using the twohybrid screening method and HE4 as bait, has identi ed the YWHAE protein as a partner of HE4.
X-ray diffraction analysis showed that YWHAE protein monomers form homodimers or heterodimers [15], which are bound by some highly conserved hydrophobic amino acids. Different YWHA subtypes can bind to the same target, granting these proteins the ideal conditions to regulate a large number of physiological processes, such as cell proliferation, apoptosis, protein transport, metabolic regulation, signal transduction, among others [16][17]. The particular structure of YWHAE may be the basis for its role as a "bridge protein", as well as for its contribution towards disease incidence [18].
Based on previous preliminary ndings, this study explored the relationship between YWHAE and HE4 and analysed the expression of YWHAE in ovarian epithelial tumors and its mechanism of action.
Enhanced knowledge on the underlying activity of YWHAE and HE4 may provide a basis to further explore the development and progression of ovarian cancer and develop new diagnostic strategies.

Primary samples
Ovarian tissue samples were derived from para n specimens surgically collected between 2008 and 2012 in the Department of Obstetrics and Gynecology, Shengjing Hospital, China Medical University. A total of 16 samples were derived from normal ovarian tissue removed due to uterine broids or cervical cancer (normal group), 18 samples were from benign cases, 24 samples were classi ed as borderline, and 105 samples were from malignant ovarian cancers. The pathological types among the malignant samples included 71 serous tumours, 7 mucinous tumours, 19 endometrioid tumours, and 8 clear cell carcinomas. The malignant group was also classi ed according to pathology assessment, with 51 cases identi ed as highly differentiated and 54 as poorly differentiated. The surgical pathological staging was performed in accordance with the International Union of Obstetrics and Gynecology (FIGO) standards: 44 cases in stage I-II and 61 in stage III-IV, of which a comprehensive exploratory operation was performed in the early stage and cytoreductive surgery was performed in the late stage. In the malignant group, 92 patients underwent lymph node dissection, with lymph node metastasis being con rmed in 28 cases.
Protein samples for western blotting analysis were derived from tissue specimens collected in 2018-2019 in the Department of Obstetrics and Gynecology, Shengjing Hospital A liated to China Medical University. A total of 33 specimens were surgically collected from 15 cases in the malignant group, 6 cases in the borderline group, 6 cases in the benign group, and 6 cases in the normal group. All cases were newly diagnosed and radiotherapy and chemotherapy naïve.

Immunochemistry
The histopathological specimens were xed with 10% formalin solution, embedded in para n, and then serially sectioned into 5 μm slices. The para n sections were depara nised with xylene and re-hydrated with gradient alcohol solutions, and the antigens were hot-recovered. Then, H 2 O 2 , goat serum blocking solution, and an anti-YWHAE antibody (1:100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-HE4 (1:1500, ab200828, Abcam, Cambridge, UK ) antibody were added dropwise sequentially, and the solutions were left to incubate overnight at 4 °C. On the next day, the slices were incubated in horseradish peroxidase (HRP)-labelled goat anti-rabbit/mouse secondary antibodies and stained using 3,3diaminobenzidine (Ultrasensitive TM SP Mouse/Rabbit IHC Kit, Maixin, Fuzhou, China). The cell nucleus was stained blue using haematoxylin. The sections were then dehydrated, cleared by xylene, and mounted.
The results were evaluated by two pathologists who did not know in advance the clinical information of the patients, and they independently observed and scored each sample. If discordant scoring results were obtained, a third pathologist would assess the sample for the nal decision. The samples were classi ed as positive when presenting with brownish-yellow or brown colour in the cell cytoplasm and/or membrane. If the proportion of positive cells was less than 5%, it was scored as 0 points, 5-25% was scored as 1 point, 26-50% was 2 points, 51-75% was 3 points, and more than 75% was counted as 4 points. According to the colour intensity, they were further scored with 3 points for brown, 2 points for brownish-yellow, 1 point for light yellow, and 0 points for no staining. To reach a nal score, the two classi cations were multiplied: 0-2 points were recorded as negative expression (−), 3-4 points as weak positive expression (+); 5-8 points as moderate positive expression (++), and 9-12 points as strong positive expression (+++).

Cellular immuno uorescence
The cells were seeded on a microscope slide and washed with PBS after they adhered to the glass. Goat serum blocking solution was added, followed by the anti-YWHAE (1:100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-HE4 (1:200, DF8160, A nity, OH, USA) primary antibody mix. A secondary antibody mixture containing tetramethylrhodamine-labelled goat anti-rabbit IgG (SA00007-2, Proteintech, Wuhan, China) and uorescein isothiocyanate-labelled goat anti-mouse IgG (SA00003-1, Proteintech, Wuhan, China) was dropped onto the slide, which was then incubated for 2 h in the dark. 4′,6diamidino-2-phenylindole (4083S; Cell Signaling Technology, Beverly, MA, USA) was used to stain the cell nucleus, and an anti-quenching agent was added dropwise onto the slide immediately before assessing the slices on a confocal microscope.

Establishment of stable over-expressing YWHAE cell lines and transient YWHAE-knockdown cell lines
The ovarian cancer cell lines CAOV3 and ES2 (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI 1640 medium (Biological Industries, Beit-Haemek, Israel) containing 10% foetal bovine serum (Biological Industries, Beit-Haemek, Israel). When the cells reached 80-90% con uence, the medium was discarded, the cells were washed with phosphatebuffered saline (PBS), trypsinised, and split to continue the culture. Lentivirus-mediated YWHAE over-expression vector was used to transfect OVCAR3 and A2780 cell lines, which had relatively low expression of YWHAE. To calculate the multiplicity of infection (MOI) of YWHAEexpressing lentivirus, 500 µL of complete medium were added to a 24-well plate, in addition to lentivirus supernatant and a corresponding volume of polybrene, to promote transfection. Puromycin at 50% lethal concentration (LC50) was used to select the e ciently transfected cells.

Western blotting
Total protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Brie y, 5-10 µL of protein sample was placed on each well of the gel and subjected to 80-120 V of constant electrical current for 40-100 min. The proteins were transferred onto a polyvinylidene uoride membrane (Millipore, Burlington, MA, USA), which was then blocked with 5% milk/bovine serum albumin solution for 2 h at 37 °C. The membrane was incubated overnight at 4 °C with primary antibodies antibody. An IgG antibody (5145S, Cell Signaling Technology, Beverly, MA) of the same species of the primary antibody was used as negative control. After mixing, the samples were left overnight at 4°C with slow rotation. Afterwards, beads (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA) were added to each tube and incubated for 6 h. The bound proteins were collected by centrifugation, (2×) Loading Buffer was added, and the samples were heated to denature the proteins.

Invasion test
A transwell insert was placed in the culture plate. Matrigel (1:7.5 dilution, 356234, BD Biosciences, New York, USA) and serum-free 4 × 10 4 cell suspension were added to the upper layer of the chamber, whereas the lower chamber contained complete medium (with serum). After culturing for 48-72 h, the cells in the lower chamber were collected, xed, and stained. Residual cells lefts on plate were observed under a microscope. The experiment was repeated thrice.

Scratch test
Cells were seeded in a 6-well culture plate and maintained at 37 °C in a 5% CO 2 incubator. After con rming under the microscope that the cells were at 90% con uence, the cell layer was scratched with a 100 µl pipette tip. The cells were cultured in serum-free medium for 24 h, then washed with PBS and photographed to monitor the healing of the scratches. The experiment was repeated thrice.

MTT assay
A total of 2,000 cells/well were seeded in a 96-well culture plate and maintained at 37 °C in a 5% CO 2 incubator. After the cells were adhered, 20 µL of sterile MTT (M8180, Solarbio, Beijing, China) working solution was added to each well, mixed well, and the cells were incubated at 37 °C for 4h. Following this, the medium was aspirated and 150 µL of DMSO (D8370, Solarbio, Beijing, China) was added to each well. The absorbance of each well was measured in a microplate reader after shaking for 5 min. The experiment was repeated thrice.

Cell cycle analysis
Cells in log phase were collected and washed, and pre-cooled ethanol was slowly added to x the cells for later use. Before the analysis, a totle of 500 µl PI/ RNase A (KGA512, KeyGen Biotech, Nanjing, China) staining solution were added to the cell suspension and the cells were left in the dark for 20 min, according to the manufacturer's instructions. The cells were analysed by ow cytometry. The experiment was repeated thrice.

Apoptosis assessment
The Annexin V/PI double staining method was used to evaluate the impact of YWHAE overexpression on ovarian cancer cells. The cells were trypsinized without EDTA, and the suspension was centrifuged to discard the supernatant. The cells were re-suspended in 500 µL of Binding Buffer added slowly and incubated with 5 µL of Annexin V-APC (550474, BD Biosciences, New York, USA) / FITC (KGA107, KeyGen Biotech, Nanjing, China) and PI in the dark at 37 ℃ for 15 minutes according to the manufacturer's instructions. The staining was evaluated by ow cytometry.

In vivo xenograft model of ovarian cancer
Twenty female nude mice (Huafukang Biosciences, Beijing, China) were randomly divided into two groups and injected with either OVCAR3-YWHAE-MOCK or OVCAR3-YWHAE-H ovarian cancer cell lines.
Approximately 100 µL of cell suspension containing 1×10 7 cells was injected subcutaneously into the armpit of the right forelimb of each nude mouse. The tumour progression and overall health status of the mice were observed every three days, and the diameter of the tumour and the weight of the mice were measured. The tumour volume was calculated as V = (a × b 2 )/2, where a represents the largest diameter and b the shortest diameter. On day 7 post-injection the newly formed tumours were detectable, while on day 21 the largest tumour was nearly 1 cm in diameter, and the mice had started to shown poor health symptoms.The tumor samples were then xed in 4% paraformaldehyde and embedded in para n. Continuous 5 μm-thick sections were cut and analyzed using hematoxylin and eosin (HE) or immunohistochemical staining. The animal study was approved by the Institutional Animal Research Committee of China Medical University.

Bioinformation analysis
The YWHAE co-expression gene set was downloaded from cBioPortal (www.cbioportal.org), and the top 300 genes were annotated according to the gene ontology (GO) (BP, biological process; CC, cellular component; and MF, molecular function) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis using the tools available in DAVID (www.david.ncifcrf.gov). The interaction network among the top 100 genes was constructed using STRING (www.string-db.org) and Cytoscape (www.cytoscape.org).
The Oncomine database (http://www.oncomine.org), which is currently the world's largest oncogene chip database integrated with a data mining platform, was used to analyze the mRNA expression level of YWHAE in the different cancer cell types.
The protein-protein interaction (PPI) network was constructed using the STRING (http://string.embl.de/) online tool. The visualization plot was generated using Cytoscape software with a con dence score of ≥0.1 de ned as the cutoff. The core modules of the PPI network were screened using Molecular Complex Detection (MCODE) with the following parameters: degree threshold = 2, node threshold = 0.2, kcore = 2, and maximum depth = 100.

Statistical analysis
Statistical differences between two groups were evaluated by the Student's t test, and one-way analysis of variance was used for the comparison of more than two groups. The data were counted using the χ 2 and Fisher's exact probability tests, and measurements of the data were performed using the single factor analysis of variance. P<0.05 was considered statistically signi cant.

YWHAE and HE4 are interacting proteins in ovarian cancer
The levels of YWHAE and HE4 were evaluated in the CAOV3 and ES2 ovarian cancer cell lines by cellular immunohistochemistry, revealing that both proteins were expressed in the cytoplasm and cell membrane (Fig. 1a). Co-localizing in the cytoplasm and cell membrane of CAOV3 and ES2 (Fig. 1b). Coimmunoprecipitation of both YWHAE and HE4 in these cell lines further demonstrated that they are interacting proteins (Fig. 1c), siRNA-mediated knock-down of YWHAE led to reduced levels of HE4 in both CAOV3 and ES2 transiently transfected cells, whereas HE4 knock-down had no signi cant effect on YWHAE levels. These results suggest that YWHAE is upstream of HE4 (Fig. 1d) and it can regulate HE4 expression.
David analyzed the GO (BP/CC/MF) and KEGG pathways of the top 300 gene set, and enriched 36 BP, such as RNA scattering, Wnt signaling pathway, cell adhesion, 23 CC, 13 MF (all P<0.05), and 2 KEGG signaling pathways (all P<0.05), such as spliceosome, RNA transport, etc., and drew the bubble diagram of the top 5 go (BP/cc/MF) and KEGG pathways. The results showed that YWHAE might interact with PAFAH1B1, EIF5A, PITPNA, TIMM22, PFN1, WFDC2(HE4) (Fig. 1e). that YWHAE-positive expression was signi cantly higher in the malignant group (P<0.05); however, the junction group YWHAE-positive rate was also higher than that of the benign and normal groups (P<0.05). The expression rate of YWHAE in the benign group was higher than in the normal group, but the difference was not statistically signi cant (P>0.05) ( Table 1, Fig. 2a,b)

Relationship between YWHAE expression and clinicopathological parameters of ovarian cancer
In order to compare clinicopathological parameters with the expression of YWHAE in ovarian tissue, we reviewed the clinical information of the 105 patients with primary ovarian epithelial malignant tumours. Analysis of the pathological data showed that YWHAE-strong positive expression rate was signi cantly higher in FIGO III-IV stage ovarian epithelial malignancies than in the early stage group (80.33% versus 56.82%, P<0.01). No statistical differences were observed in their other clinicopathological parameters ( Table 2, Fig. 2c).

Relationship between YWHAE expression and survival prognosis of patients with ovarian cancer
Follow-up of the patients further showed that only four deaths occurred in the YWHAE low expression group (n=31), while 21 deaths were recorded in the YWHAE high expression group (n=74). Kaplan-Meier survival analysis showed that the survival rate among patients with high YWHAE levels was signi cantly lower compared with that in the YWHAE low expression group. This result followed the same trend as observed when comparing patients with early or late FIGO staging (P<0.05) (Fig. 2d). Univariate and multivariate Cox regression analysis of YWHAE expression with age, pathological type, degree of differentiation, FIGO stage, and lymph node metastasis demonstrated that YWHAE expression and FIGO staging are risk factors for the prognosis of epithelial ovarian malignancies (Fig. 2e).
YWHAE and HE4 expressions are related in ovarian cancer tissues Next, the co-expression of YWHAE and HE4 was evaluated in 80 cases of ovarian cancer. A total of 0, 3, 9, and 68 cases were YWHAE−/HE4−, YWHAE−/HE4+, YWHAE+/HE4−, and YWHAE+/HE4+, respectively. Spearman correlation analysis con rmed that YWHAE and HE4 expressions are positively correlated in ovarian cancer (correlation coe cient Rs=0.277, P=0.013) ( Table 3). Linear regression analysis showed that the expression of YWHAE and HE4 can in uence each other (P<0.05), and that the late FIGO stage is an important factor affecting the expression of HE4 (Table 4). Multivariate linear regression analysis also showed that HE4 expression score was an independent in uencing factor of YWHAE expression, as well as YWHAE expression was an independent in uencing factor of HE4 expression (Fig. 2e).
YWHAE and HE4 expressions are related in ovarian cancer tissues Study of YWHAE expression in Oncomine database showed that YWHAE was highly expressed in the many cancer group compared to the normal tissue group, for ovarian cancer, YWHAE was signi cantly highly expressed in 185 ovarian carcinoma tissues compared with 10 ovarian surface epithelium tissues (Fig. 2g,h).

YWHAE promotes ovarian cancer cell invasion, migration, and epithelial-mesenchymal transition potential
Analysis of the levels of YWHAE in several ovarian cancer cell lines revealed that it was higher in CAOV3 and ES2 than in OVCAR3 and A2780. Based on these results, CAOV3 and ES2 cells with relatively high expression of YWHAE were used to establish a cell line model with low YWHAE expression, whereas OVCAR3 and A2780 ovarian cancer cells with relatively low YWHAE were used to establish a stable overexpressing cell line model of YWHAE (Fig. 3a-f).
The effect on invasion and migration of ovarian cancer cells upon transient knock-down or stable overexpression of YWHAE was evaluated next by transwell and scratch experiments. Overall, the data revealed that both OVCAR3 and A2780 cells overexpressing YWHAE had signi cantly enhanced invasion and migration capacity than mock-transduced and untransduced cells. In contrast, CAOV3 and ES2 cells lacking YWHAE showed weaker invasion and migration abilities compared with control cells (all P<0.05) (Fig. 3g-n). To further explore the impact of YWHAE on the behaviour of ovarian cancer cells, the levels of cell epithelial and mesenchymal markers were evaluated in these cells by western blot. YWHAEoverexpression was found to be associated with higher levels of the N-cadherin, Vimentin, MMP2, and MMP9 (cell mesenchymal markers), whereas the epithelial marker E-cadherin was found to be reduced compared with that of cells of the control groups. The opposite trend was seen when YWHAE was knocked-down (both P<0.05), with higher levels of E-cadherin and reduced levels of N-cadherin, Vimentin, MMP2, and MMP9 (Fig. 4a-d). Altogether, these results demonstrate that YWHAE promotes invasion, migration, and epithelial-mesenchymal transition of epithelial ovarian cancer cells.

YWHAEpromotes ovarian cancer via inducing cell proliferation, cell cycle progression, and apoptosis inhibition
Additional assessment of the OVCAR3 and A2780 YWHAE-over-expressing cells further showed that more of these cells were in G2/M phase, suggesting that they were actively proliferating compared with mocktransduced and untransduced cells (all P<0.05). Furthermore, these cells had higher expression of Ki67, Cyclin D1, and Bcl-2 and reduced levels of Bax (all P<0.05). CAOV3 and ES2 cells lacking YWHAE showed the opposite results, with signi cantly lower proportion of cells in the G2/M phase, reduced levels of proliferation markers, and increased levels of the Bax apoptosis marker. Furthermore, ow cytometry results revealed that compared with the control group, the overall apoptosis of OVCAR3 and A2780 cells was signi cantly reduced after YWHAE overexpression (P<0.05) , while apoptosis was signi cantly increased in OVCAR3 and A2780 cells compared to the control group when YWHAE expression was inhibited ( Fig. 4e-h, Fig. 5). Taken together, these results demonstrate that the expression of YWHAE can enhance the proliferation of ovarian cancer cells, while it also promotes cell cycle progression and inhibits cell apoptosis.

Effect of YWHAE on the in vivo tumorigenesis of ovarian cancer cells
In order to explore the effect of YWHAE on the tumorigenic ability of ovarian cancer cells, OVCAR3 stably overexpressing YWHAE or a mock control were injected into athymic nude mice. Assessment of the tumours formed 21 days after the cells were injected revealed that cells overexpressing YWHAE were signi cantly bigger and weighed about 2.83 times more than the tumours seen in the control group. Moreover, the growth rate of the tumours produced by YWHAE-overexpressing cells was also signi cantly higher compared with those in the control group (Fig. 6a-c). Tumour biopsies collected and evaluated by immunohistochemistry revealed that the signals of activated AKT and ERK (p-AKT and p-ERK, respectively) were stronger in OVCAR3 cells over-expressing YWHAE, further suggesting that YWHAE can promote the proliferation of tumour cells in vivo (Fig. 6d).

YWHAE-induced cellular effects are mediated by the PI3K/AKT and MAPK signalling pathways
We used the STRING database to predict the relevant molecules of YWHAE by PPI, and the results showed that YWHAE had direct or indirect interaction with PI3K, MAPK, ACTR1A, cAMP-dependent protein kinase and other molecules (Fig. 7a).
To have a more detailed perspective on the underlying mechanisms triggered by YWHAE, the levels of several critical signalling molecules were evaluated by western blotting. The results showed that the ratio of p-PI3K/PI3K, p-AKT/AKT, mTOR/p-m-TOR, p-ERK/ERK, and p-MEK/MEK increased signi cantly in the presence of high levels of YWHAE, but they were reduced upon YWHAE-knock-down (all P<0.05). The above results prove that YWHAE can activate PI3K/AKT and MAPK signal pathways (Fig. 7b-e).
To further explore the role of these two signalling pathways on YWHAE-induced cellular effects, PI3K inhibitor (GDC-0941) or MEK inhibitor (PD98059) was used. The invasion and migration experiments were repeated in the presence of these chemical inhibitors, revealing that blockage of both PI3K/AKT and MAPK signals signi cantly weakened the pro-invasion and pro-migration effect promoted by YWHAE over-expression (P<0.05). Moreover, the results of the MTT experiment showed that the proliferation ability of OVCAR3 cells overexpressing YWHAE-H was signi cantly reduced in the presence of GDC-0941 or PD98059 (P<0.05) (Fig. 8, Fig. 9).
The above described results demonstrate that YWHAE can impact on the invasion, migration, and proliferation potentials of ovarian cancer cells, as well as other malignant biological behaviours, through the PI3K/AKT and the MAPK signalling pathways.

Discussion
Ovarian cancer is the tumour of the female reproductive system with the highest mortality rate. Although a variety of targeted drugs for ovarian cancer have been used in the clinical setting, its high mortality rate still represents a serious threat to the lives and health of women worldwide.
YWHAE protein is widely expressed in eukaryote cells, and has been detected in wheat [19], giant trematodes in goat blood cells [20], liver ukes [21], and mosquitoes [22]. Moreover, in the physiological state of the human body, YWHAE was described as an important element in retinal photoreceptor rod cells [23]. Some researchers have found that YWHAE is also involved in the differentiation of adiposederived mesenchymal stem cells into osteoblasts, enhancing the body's osteogenic ability [24]. In contrast, some studies have detected a peak of YWHAE expression 168 h after partial liver resection, preventing cell cycle and negatively regulating liver regeneration [25]. Therefore, these discordant ndings suggest that YWHAE may have a two-way regulatory effect on the cell cycle.
Since YWHAE was originally identi ed in the brain, its pathological effects were initially investigated in the eld of neurological diseases, such as Parkinson's [26] and Alzheimer's disease [27], brain excitotoxic injury [28], and myocardial ischemia reperfusion [29], among others. These studies agreed that the mechanism by which YWHAE could impact on nerve cells could be related to mitochondrial dysfunction and regulation of apoptosis.
In recent years, studies have suggested that abnormal expression of YWHAE may also play an important role in the occurrence and development of tumours. Liang et al. found that YWHAE is highly expressed in kidney cancer tissues, and in vitro experiments demonstrated that YWHAE can promote the abnormal proliferation of tumour cells [30]. In gastric cancer cell lines, the expression of YWHAE is signi cantly increased and it can inhibit cell proliferation, invasion, and migration by reducing the expression of MYC and CDC25B, whereas MYC induces cell proliferation, invasion, and migration by enhancing CDC25B and reducing YWHAE expression [31][32]. In breast cancer, YWHAE expression was also related to tumour size, lymph node metastasis, and patient survival prognosis, as well as to breast cancer cell resistance to chemotherapy. Indeed, YWHAE overexpression signi cantly increases breast cancer cell proliferation, migration, and invasion, whereas reduced YWHAE expression prevents Snail and Twist expression in breast cancer cells [33]. Although high expression of YWHAE has been described in colorectal, liver, kidney, breast, gastric, and oesophageal cancers, its speci c mechanism of action remains unclear. Among the malignant tumours of the female reproductive system, YWHAE is more commonly reported upon genetic testing of the uterine sarcoma cells. Endometrial stromal sarcoma carrying the YWHAE-NUTM2 (or YWHAE-FAM22) fusion gene has obvious malignant biological effects, such as enhanced invasion and drug resistance, and the prognosis of patients harbouring such genetic abnormality is worse [34][35]. Sylvain et al. [36] performed gene chip detection on matched tumour samples from six patients with advanced high-grade epithelial ovarian cancer before and after chemotherapy. The results showed that 54 genes that recurred after chemotherapy showed a down-regulation trend, whereas 121 genes, including YWHAE, showed an up-regulation trend. This change in the expression pro le suggests that YWHAE may be related to ovarian cancer invasion, proliferation, and drug resistance. Sun et al. used the Gene Expression Omnibus database to analyse the relationship between ovarian cancer and diabetes, nding that 10 key genes, including YWHAE, are important links in the regulation of the redox reaction process and carboxylic acid metabolism in the body [37]. Based on these results, they believe that ovarian cancer is related to sugar metabolism, and that certain key metabolism-related genes and proteins could be used as potential targets for the treatment of ovarian cancer.
Altogether, the results described in the present study demonstrate that YWHAE and HE4 are interacting proteins. And YWHAE is signi cantly associated with advanced stage cancers and poorer patient outcomes, thereby speculating that high YWHAE expression may represent a risk factor for the prognosis of ovarian cancer. Overall, YWHAE showed a similar cancer-promoting effect as seen with HE4, partaking in the occurrence and development of ovarian cancer.
Through induced differential expression of YWHAE and in vivo experiments, it was also demonstrated that YWHAE contributes to ovarian cancer cell invasion, epithelial-mesenchymal transition, and migration, as well as to enhancing their proliferative and anti-apoptotic responses.
Previous studies have con rmed that HE4 mainly plays an important role in the spreading and adhesion of ovarian cancer cells. Moreover, low levels of HE4 prevented the activation of ERK and EGFR in ovarian cancer cells. Therefore, it is believed that HE4 may in uence the biological behaviour of cancer cells in the ovaries through the EGFR and MAPK signalling pathways, but the speci c underlying mechanism is still unclear. Studies have reported that HE4 can affect the cell cycle (G0/G1 phase), migration, and invasion capabilities by regulating the ERK/MAPK signals and the expression of MMP-9, MMP-2, and cathepsin B [38]. In accordance with its role as an interaction protein of HE4, YWHAE was also shown to affect the malignant biological behaviour of ovarian cancer cells through the above-mentioned signalling pathways.
In breast cancer, YWHAEτ acts together with 1,3-DCQA (eicosanylquinic acid) to prevent the proliferation and metastasis of cancer cells through the Jak/PI3K/AKT and Raf/ERK pathways and by inducing the Bad/Bax/caspase 9 apoptosis pathway [39]. Overexpression of YWHAEζ can regulate the expression of Snail protein by activating PI3K/AKT signals, thereby signi cantly promoting the proliferation, migration, and invasion of glioma cells, representing a potential prognostic marker therapeutic target for glioma [40]. In colorectal cancer, YWHAEσ acts as a tumour suppressor gene. However, COPS5 and LASP1 through PI3K/AKT-dependent signals stimulate YWHAEσ ubiquitination and degradation, making it lose its tumour suppressor activity, thereby promoting the progression of colorectal cancer [41].
Relevant studies have shown that YWHAE can inhibit cell apoptosis in colorectal cancer , and this process can be reversed by non-steroidal anti-in ammatory drugs. The inhibition of apoptosis may be related to the ability of YWHAE to interfere with mitochondrial pro-apoptotic mechanisms and activate the transcription factors FKHRL1 and Bad [43]. Moreover, ATPR (4-amino-2-tri uoromethylphenylretinoic acid) can induce G0/G1 phase arrest in gastric cancer SGC-7901 cells by down-regulating YWHAE [44]. We found that PI3K/AKT pathway node proteins (PI3K, AKT, and mTOR) and MAPK pathway node proteins (MEK and ERK) were signi cantly activated in ovarian cancer cells over-expressing YWHAE.
Importantly, inhibition of these pathways with speci c inhibitors prevented the pro-invasion, promigration, and pro-proliferation effects induced by YWHAE. Therefore, these results suggest that YWHAE promotes the malignant biological behaviour of epithelial ovarian cancer through activation of the PI3K/AKT and MAPK pathways.

Conclusions
This study described for the rst time that YWHAE and HE4 were interacting proteins and underlying a colocalization relationship. It was proved in tissues that the expression of YWHAE was signi cantly increased in ovarian cancer tissues, which was a risk factor for the prognosis of ovarian cancer.
Moreover, it was discovered for the rst time that YWHAE could promote the invasion, migration, proliferation of epithelial ovarian cancer through PI3K/AKT and MAPK pathways. In the future, YWHAE may be used as a prognostic factor and trigger new research ideas for further understanding the underlying pathogenesis and improving the diagnosis and treatment of ovarian cancer.      The impact of YWHAE on tumor formation and proliferation ability in vivo. a Subcutaneous xenograft of nude mice model was performed using YWHAE stable overexpression OVCAR3 cells; b,c Volume and quality changes of tumors;d Hematoxylin-Eosin staining, immunohistochemical staining of p-AKT and p-ERK in YWHAE-overexpressed group and control group.   PI3K and MEK inhibitors reduce migration ability in ovarian cancer cells.