YWHAH activates the HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect the proliferation of gastric cancer cells

Fos-related antigen 1 (Fra-1) is a nuclear transcription factor that regulates cell growth, differentiation, and apoptosis. It is involved in the proliferation, invasion, apoptosis and epithelial mesenchymal transformation of malignant tumor cells. Fra-1 is highly expressed in gastric cancer (GC), affects the cycle distribution and apoptosis of GC cells, and participates in GC occurrence and development. However, the detailed mechanism of Fra-1 in GC is unclear, such as the identification of Fra-1-interacting proteins and their role in GC pathogenesis. In this study, we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) as a Fra-1-interacting protein in GC cells using co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry. Experiments showed that YWHAH positively regulated Fra-1 mRNA and protein expression, and affected GC cell proliferation. Whole proteome analysis showed that Fra-1 affected the activity of the high mobility group AT-hook 1 (HMGA1)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway in GC cells. Western blotting and flow cytometry confirmed that YWHAH activated HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect GC cell proliferation. These results will help to discover new molecular targets for the early diagnosis, treatment, and prognosis prediction of GC.

Fra-1 is a member of the activator protein-1 (AP-1) transcription factor superfamily and Fos family of proteins, and its basic domain is highly homologous with c-Fos [17,18]. Fos family proteins encode leucine zipper proteins that can form dimers with JUN family proteins to form the transcription factor complex AP-1 [18]. Fra-1 is an important nuclear transcription factor that regulates the growth, differentiation, and apoptosis of normal cells. It is abnormally expressed in many cancer cells and tissues, and plays an important role in the tumorigenesis and progression or maintenance of many tumor types [6,7,[19][20][21]. Fra-1 levels are frequently elevated in a variety of human cancers because of carcinogenic signal transduction, and Fra-1 is closely related to metastasis and poor prognosis [20][21][22]. Overexpression of Fra-1 can lead to morphological changes of fibroblasts and is related to mesenchymal characteristics and E-cadherin downregulation in cancer cells [23][24][25]. In the cell cycle, Fra-1 is recruited to the promoter of CCNA2 (encoding cyclin A2). Fra-1 induces the expression of Jun B, which interacts with the CCNA2 promoter to enhance cell proliferation [26,27]. Previous studies suggest that Fra-1 plays an important role in the occurrence and development of malignant tumors such as GC.
In a previous study, we used quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and western blotting to show that Fra-1 is highly expressed in GC tissues. In vitro experiments confirmed that Fra-1 overexpression inhibited the apoptosis of GC cells, increased the proportion of S-phase cells, and was related to an imbalance of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway [6]. However, the detailed mechanism of Fra-1 in GC has not been fully defined. In this study, we aimed to identify molecules that interact with Fra-1 in GC cells using coimmunoprecipitation (Co-IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and clarified their mutual regulation relationship. Then, western blotting and flow cytometry were used to confirm that the newly identified Fra-1 interacting molecule, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH), could promote the proliferation of GC cells by positively regulating Fra-1 to activate the high mobility group AT-hook 1 (HMGA1)/PI3K/AKT/ mechanistic target of rapamycin (mTOR) signaling pathway. Our results provide a new molecular target to explore the pathogenesis of GC.

Materials and Methods
Cell lines and plasmids Gastric cancer cells SGC7901 and AGS were preserved and provided by the Cancer Research Institute, Basic School of Medicine, Central South University. The cells were cultured via adherent growth in an incubator set at 37°C with 5% CO 2 in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-Life Technologies at Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco-Life Technologies) and 1% Penicillin-Streptomycin Solution.

Immunoprecipitation
After transfection, the cells were cultured in a 100 mm dish for 48 h and then lysed using IP lysis buffer containing a mixture of protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology, P0013, Shanghai, China). Then, 1 µg of primary antibody was incubated with 30 µL of protein A+G beads on a rotator at room temperature for 2 h, the protein lysate was then added and incubated on the rotator at 4°C overnight. Next day, the beads and immune complexes were washed five times with IP lysis buffer by rotating at room temperature for five minutes at a speed (2000 rpm/min) of 20 s per round. The sample was added to the corresponding SDS loading buffer and heated at 95°C for 5 min. The immunoprecipitated samples were detected using western blotting.

Mass spectrometry analysis
The purified protein complex eluent was concentrated using IP beads, separated by SDS-PAGE, and stained with Coomassie brilliant blue (Beyotime Biotechnology). The corresponding protein band was excised from the gel, reduced, alkylated, and digested overnight using trypsin at 37°C (Thermo Fisher Scientific, Waltham, MA, USA). The digested peptides were dried and resuspended in MS compatible buffers, and the mixture was analyzed using an LTQ Orbitrap velos MS instrument (Thermo Fisher Scientific) in combination with a UltiMate RSLC Nano LC system (Dionex, Sunnyvale, CA, USA). Proteome Discoverer 1.4 software (Thermo Fisher Scientific) was used to identify proteins, import files, and search the UniProtKB/Swiss-Prot databases. The mass tolerances of precursors and fragments were set to 10 ppm and 0.8 Da, respectively. Peptide data with an error detection rate of <1% (p < 0.01) were discarded.
For siRNA transfection, the riboFECT CP Transfection Kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China) was used according to the manufacturer's instructions, and then the silencing effect was detected. The most successful siRNA sequence for each gene was used for subsequent experiments.
5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assay Cells (1 × 10 5 to 3 × 10 6 ) were inoculated in 6-well plates and cultured at 37°C in a 5% CO 2 incubator to the required cell density. Two hours before transfection, serum-free 1640 medium was used. The transfected cells were divided into groups according to the corresponding experiments. After 6 h, the mixed solution was sucked out and replaced with normal culture medium, and culture was continued for 48 h. Referring to the operating instructions of the EdU-647 cell proliferation test kit (Beyotime biotechnology, C0081S), the samples were processed before flow cytometry [28,29].
Finally, the samples were tested by flow cytometry at a 488 nm excitation wavelength and 520 nm emission wavelength. Cells were analyzed with Moflo XDP High-Performance Cell Sorter (Beckman Coulter). Data were acquired and analyzed with Summit v5.2 software.
Protein extraction and western blotting Cells were digested with trypsin and then lysed with Radioimmunoprecipitation assay (RIPA) buffer (CWBio, Beijing, China). The sample was centrifuged at 12000 × g at 4°C for 15 min to remove insoluble matter. The protein concentration of the retained supernatant was detected, and protein electrophoresis was carried out using a 10% SDS-PAGE gel at 50 V for 40 min, and then at 120 V for 60 min (PowerPac Universal, Bio-Rad, Hercules, CA, USA). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA) at 100 V for 90 min, and then incubated in phosphate-buffered saline (PBS)-Tween20 with 5% skimmed milk for 1-2 h. The membrane was then incubated with primary antibody at 4°C overnight.

Statistical analysis
GraphPad Prism 5 software (GraphPad Inc., La Jolla, CA, USA) was used perform the statistical analyses. T-tests were used to analyze whether the difference between any two groups was significant. One way analysis of variance (ANOVA) was used to analyze whether the data of more than two groups were significantly different. All experiments were set with three or more replicates, and p < 0.05 indicated a significant difference.

Identification of YWHAH as an interacting protein of Fra-1 in GC cells by Co-IP and LC-MS/MS technologies
Our previous research results showed that Fra-1 was upregulated in GC tissue and is involved in its occurrence and development. To systematically explore the role and possible mechanism of Fra-1 in GC, we first used Co-IP combined with LC-MS/MS to identify proteins that interact with Fra-1 in GC cells. After the Fra-1 interaction proteins were separated by SDS-PAGE, stained with Coomassie blue, and decolorized, we observed obvious difference bands at 25~40, 40~70, and 70~180 kDa on the gel (Fig. 1A). The bands were then analyzed by endogel enzymolysis and mass spectrometry. Among them, 25 specific proteins of the FLAG lane were identified at 25~40 kDa, among which 6 protein molecules belonged to the 14-3-3 protein family, including YWHAE, YWHAH, YWHAG, YWHAZ, and YWHAQ, suggesting that the 14-3-3 protein family might interact with Fra-1 (Suppl. Table 1). To verify the interaction between the 14-3-3 protein family and Fra-1, we performed IP experiments for YWHAE, YWHAH, YWHAG, YWHAZ, and YWHAQ with Fra-1, respectively. The results showed that Fra-1 interacted only with YWHAH in SGC7901 GC cells (Fig. 1B). To further explore the interaction domain between YWHAH and Fra-1, we constructed vectors expressing Fra-1 3-segment domains D1 (1-107 aa), D2 (1-127 aa) and D3 (107-271 aa) with HA tags (Fig. 1C), and conducted IP experiments to verify the domain of interaction. The results showed that YWHAH interacted with full length Fra-1 and all three segment domains; however, the interaction between YWHAH and D2 (1-127 aa) was stronger (Fig. 1D), suggesting that the Bag domain of Fra-1 plays an important role in the interaction between the two proteins. In conclusion, we identified YWHAH as a interacting protein of Fra-1 in SGC7901 GC cells.
YWHAH positively regulates the mRNA and protein expression of Fra-1 To clarify the mutual regulation relationship between YWHAH and Fra-1, we first designed three siRNA sequences for the YWHAH gene, and transiently transfected the three siRNAs into SGC7901 GC cells. Western blotting showed that si-YWHAH-01 had the best effect in reducing the protein level of YWHAH (Suppl. Fig. 1); therefore, si-YWHAH-01 was used in subsequent experiments. Then, we overexpressed and silenced YWHAH, separately, in SGC7901 GC cells. Western blotting and qRT-PCR assays showed that the mRNA and protein levels of Fra-1 were upregulated after YWHAH overexpression (Figs. 2A and 2B) but downregulated after YWHAH silencing (Figs. 2B and 2C), suggesting that YWHAH further affected the translation level after regulating the transcription of Fra-1.
To further verify our experimental results, we repeated the above experiments in AGS cells, and the results were consistent with those in SGC7901 cells (Figs. 2D-2F). The above results showed that YWHAH interacted with and positively regulated Fra-1, and affected its protein level by regulating Fra-1 transcription.
YWHAH affects the proliferation of GC cells by positively regulating Fra-1 Based on the discovery that YWHAH interacts with and positively regulates the mRNA and protein levels of Fra-1, we assessed whether YWHAH could affect the proliferation of GC cells by regulating Fra-1. Cell proliferation assays combined with flow cytometry showed that the cell proliferation increased after the stable overexpression of YWHAH in SGC7901 GC cells. However, when Fra-1 was silenced in SGC7901 cells stably overexpressing YWHAH, cell proliferation was inhibited (Fig. 3A), suggesting that YWHAH might affect GC cell proliferation by regulating Fra-1. When we silenced with YWHAH in SGC7901 cells, their proliferation ability was inhibited. However, in cell silenced for YWHAH, Fra-1 overexpression restored cell proliferation (Fig. 3B). The above results showed that YWHAH affected the proliferation of GC cells by regulating Fra-1. To further verify our experimental results, we Whole proteome analysis suggests that Fra-1 affects the activity of the PI3K/AKT/mTOR signal pathway in GC cells To explore the possible mechanism by which YWHAH regulates Fra-1 to affect the GC cell proliferation, the whole proteome was analyzed after vector (blank control) and Fra-1 plasmids were transfected into SGC7901 GC cells, separately. The results showed that 206 protein molecules were upregulated (Suppl. Table 2) and 146 proteins were downregulated after overexpression of Fra-1 in GC cells SGC7901 (Fig. 4A, Suppl. Table 3). Based on the analysis of these differentially abundant proteins, we performed functional classification, functional enrichment, and cluster analysis for all differentially abundant proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of upregulated proteins showed that the PI3K/AKT signaling pathway, the mitogen activated protein kinase (MAPK) signal transduction pathway, and the human papillomavirus infection signal transduction pathway were three main signal transduction pathways affected by Fra-1 (Fig. 4B). It has been reported that Fra-1 regulates the expression of HMGA1, and HMGA1 is a regulator of the PI3K/AKT/mTOR signaling pathway. Combined with our previous research results, we clarified whether YWHAH could promote the proliferation of GC cells by positively regulating Fra-1 to activate the HMGA1/PI3K/AKT/mTOR signaling pathway.
YWHAH activates the HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect the proliferation of gastric cancer cells To confirm whether YWHAH can affect the proliferation of GC cells by positively regulating Fra-1 to activate the HMGA1/PI3K/AKT/mTOR signaling pathway, we first transfected NC+siNC, OE-YWHAH+siNC, and OE-YWHAH+siFra-1 plasmids into SGC7901 GC cells, and detected the mRNA levels of PI3K/AKT/mTOR signal pathway related molecules (PI3K, AKT, PDK1, MTOR, etc.) using qRT-PCR. The results showed that the mRNA expression levels of PI3K, AKT, PDK1, and MTOR were upregulated after overexpression of YWHAH. However, after Fra-1 silencing in a background of YWHAH overexpression, upregulation of the mRNA expression levels of PI3K, AKT, PDK1, and MTOR molecules was inhibited (Fig. 5A). In addition, western blotting analysis also showed that the protein levels of PI3K and PDK1 were upregulated, as were the levels of phosphorylated (p)-AKT and p-mTOR after YWHAH overexpression. However, after Fra-1 silencing in a background of YWHAH overexpression, the upregulation of PI3K and PDK1 protein levels was inhibited, as were the upregulation of p-AKT and p-mTOR levels (Fig. 5B). The results suggest that YWHAH can activate the PI3K/AKT/mTOR signaling pathway through Fra-1.
At the same time, we silenced the expression of YWHAH, in which siNC+NC, siYWHAH+NC, and siYWHAH+OE-Fra-1 plasmids were transfected into SGC7901 GC cells, and the related molecules were detected using qRT-PCR. The results showed that the mRNA expression levels of PI3K, AKT, PDK1, and MTOR were downregulated after YWHAH silencing. However, overexpression of Fra-1 in a background of YWHAH silencing restored the mRNA expression levels of PI3K, AKT, PDK1, and MTOR (Fig. 5C). In addition, the protein levels of PI3K/AKT/mTOR signaling pathway-related molecules were detected by western blotting. The results showed that after YWHAH silencing, the protein levels of PI3K and PDK1 were downregulated, and the levels of p-AKT and p-mTOR were downregulated. Overexpression of Fra-1 in a background of YWHAH silencing restored the protein levels of PI3K and PDK1, as FIGURE 3. YWHAH affects the proliferation of GC cells by positively regulating Fra-1. (A) YWHAH+siNC, NC+siNC, and YWHAH+siFra-1 plasmids were transfected into SGC7901 GC cells, separately. Cell proliferation was detected using an EdU-647 cell proliferation assay combined with flow cytometry. (B) siYWHAH+ NC, siNC + NC, and siYWHAH+Fra-1 plasmids were transfected into SGC7901 GC cells, separately. Cell proliferation was detected using an EdU-647 cell proliferation assay combined with flow cytometry. (C) YWHAH+siNC, NC+siNC, and YWHAH+siFra-1 plasmids were transfected into AGS GC cells, separately. Cell proliferation was detected using an EdU-647 cell proliferation assay combined with flow cytometry. (D) siYWHAH+ NC, siNC + NC, and siYWHAH+Fra-1 plasmids were transfected into AGS GC cells, separately. Cell proliferation was detected using an EdU-647 cell proliferation assay combined with flow cytometry. ***p < 0.001. Three independent experiments were conducted.
well as the levels of p-AKT and p-mTOR (Fig. 5D). Our results suggested that YWHAH can promote the proliferation of gastric cancer cells by activating the HMGA1/PI3K/AKT/ mTOR signaling pathway through Fra-1.
To further confirm that YWHAH promotes gastric cancer cells proliferation by activating the PI3K/AKT/ mTOR signaling pathway, we used rapamycin, an inhibitor of the PI3K/AKT/mTOR signaling pathway to treat gastric cancer cells. Firstly, we divided gastric cancer cells SGC7901 into three groups: OE-YWHAH+siNC+DMSO, OE-YWHAH +siFra-1+DMSO, OE-YWHAH+siFra-1+Rapamycin by transfecting the different plasmids and whether treating cells with rapamycin. Then, we tested the gastric cancer cells proliferation using an EDU proliferation detection kit and flow cytometry. Our results showed that compared to the OE-YWHAH+siNC+DMSO group, OE-YWHAH+siFra-1 +DMSO group had a decrease in the proportion of proliferating cells, OE-YWHAH+siFra-1+Rapamycin group had a more significant reduction in the proportion of proliferating cells (Fig. 6A). To further confirm the effect of silencing YWHAH, we divided gastric cancer cells SGC7901 into three groups: siYWHAH+NC+DMSO, siYWHAH +OE-Fra-1+DMSO, siYWHAH+OE-Fra-1+Rapamycin by transfecting the different plasmids and whether treating cells with rapamycin. Then we tested the gastric cancer cells proliferation using an EDU proliferation detection kit and flow cytometry. Our results showed that compared to the siYWHAH+NC+DMSO group, the siYWHAH+OE-Fra-1 +DMSO group had an increase in the proportion of proliferating cells. But, the siYWHAH+OE-Fra-1 +Rapamycin group had a decrease in the proportion of proliferating cells (Fig. 6B). In AGS cells, we obtained results with the same trend (Figs. 6C and 6D). Above of all, our results suggested that YWHAH could promote the proliferation of gastric cancer cells by activating the PI3K/ AKT/mTOR signaling pathway.

Discussion
The pathogenesis of GC is complex. Although some progress has been made in its early diagnosis, surgical resection, and adjuvant chemotherapy, GC is still difficult to identify at the early stage of onset. The 5-year survival rate of patients with GC is not high. GC is one of the most aggressive cancers of the human digestive system [30][31][32][33]. Previous studies have shown that the pathogenesis of GC is closely related to Helicobacter pylori infection, proto oncogene activation, and tumor suppressor gene inactivation [6][7][8][12][13][14]34]; however, the detailed mechanism is unclear. More experimental evidence is needed to clarify its pathogenesis and potential molecular targets, to provide an experimental basis for early GC diagnosis, the identification of potential molecular therapeutic targets, and prognosis prediction.
Fra-1 is a member of the FOS family and is an important nuclear transcription factor that regulates the growth, differentiation, and apoptosis of normal cells [6,7,[19][20][21]. Fra-1 is highly expressed in a variety of malignant tumors and plays an important role in cell transformation, proliferation, invasion, and metastasis [18][19][20][21]35]. Our previous study found that Fra-1 was highly expressed in GC tissues compared with that in adjacent non-cancerous tissues. In vitro experiments confirmed that Fra-1 overexpression inhibited GC cell apoptosis and increased the proportion of S-phase cells [6,36]. To further clarify the role and possible mechanism of Fra-1 in GC cell proliferation, we identified and confirmed YWHAH as a Fra-1-interacting protein in GC cells. Protein interaction is widely involved in cell-cell interactions, metabolic and developmental controls and other biological processes [37,38]. Protein interaction also plays a key role in predicting the function of target proteins [39,40]. We further studied the function of YWHAH, which provided a new perspective on the role and possible mechanism of Fra-1 in GC.
Based on the discovery that YWHAH interacts with Fra-1, further qRT-PCR and western blotting experiments showed that YWHAH positively regulates the transcription of Fra-1 and then affects the protein level of Fra-1. In addition, YWHAH overexpression in SGC7901 GC cells enhanced their proliferation. Compared with the vector group, cell proliferation was inhibited in the group transfected with YWHAH and siFra-1. The results of silencing YWHAH were opposite to those of YWHAH overexpression. These results suggested that YWHAH promotes GC cell proliferation by activating Fra-1. YWHAH is a 14-3-3 eta protein and a member of the 14-3-3 protein family [41,42]. A remarkable feature of 14-3-3 proteins is that they can bind to a variety of signal proteins with diverse functions, including kinases, phosphatases, and transmembrane receptors [43,44]. 14-3-3 proteins play an important role in biological processes, such as mitotic signal transduction, apoptotic cell death, and cell cycle control [45,46]. Wu et al. found that the miR-660-5p/YWHAH axis could activate the PI3K/AKT pathway to promote epithelial mesenchyme transition and the cell cycle process in hepatoma cells [47]. In thyroid cancer, the long noncoding RNA MAPKAPK5-AS1 promoted the proliferation and migration of thyroid cancer cell lines by targeting miR-519e-5p/YWHAH [48]. Our results provide new ideas and an experimental basis to clarify the role and possible mechanism of YWHAH in malignant tumors.
To further explore the specific mechanism by which YWHAH affects GC cell proliferation via Fra-1, we overexpressed Fra-1 in SGC7901 GC cells for whole proteome analysis. KEGG enrichment analysis of all differentially abundant proteins showed that the PI3K/AKT signaling pathway was abnormally activated. Then, we confirmed that YWHAH promoted GC cell proliferation by positively regulating Fra-1 to activate the HMGA1/PI3K/ AKT/mTOR signaling pathway using western blotting, flow cytometry, and other technologies (Fig. 7). The PI3K/AKT signaling pathway plays an important role in basic intracellular functions, such as cell growth, apoptosis, translation, and cell metabolism [49][50][51]. It is important not only in carcinogenesis, but also in identifying potential new therapeutic targets [52][53][54]. The PI3K/AKT pathway is stimulated by receptor tyrosine kinases and by cytokine receptor activation. Tyrosine residues are subsequently phosphorylated and provide anchor sites for PI3K translocation to the membrane, thus participating in the transduction of various extracellular matrix molecules and cytokines [55,56]. Other studies have shown that changes in the activity of the PI3K/AKT/mTOR signaling pathway affect the proliferation of tumor cells [57][58][59]. Our results also confirmed that activation of the PI3K/AKT/mTOR signaling pathway promoted GC cell proliferation, which was consistent with the above related research results.
The experimental results provide new molecular targets to explore the pathogenesis of GC and provide an experimental basis to clarify the pathogenesis of GC.
Funding Statement: This work was supported by the Hunan Provincial Natural Science Foundation (2021JJ30915).
Author Contributions: JH, FZ and XJ performed the experiments. LL, MG and WL analyzed the data. GL and YZ designed the study and wrote the manuscript. All authors read and approved the final manuscript.
Availability of Data and Materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics Approval: Not applicable.

Conflicts of Interest:
The authors declare that they have no conflicts of interest to report regarding the present study.