TRIP13 Activates the PI3K/AKT Signal Pathway to Promote Melanoma Progression by Interacting With FLNA

Background: Skin melanoma is a malignant tumor originated from skin melanocytes. It is highly malignant and easy to relapse and metastasis. Finding new diagnostic and therapeutic targets has become a hot issue. Accumulating evidence now indicates that thyroid hormone receptor interactor 13 (TRIP13) plays important roles in tumor development. However, its role in melanoma remains unclear. Methods: Bioinformatic analyses were employed to excavate crucial genes in melanoma using several public databases. The expression of TRIP13 was detected by immunohistochemistry. MV3 cell and A2058 cell were steadily transfected with TRIP13 knock-down or overexpression lentiviruses, then the function and potential mechanism of TRIP13 were studied in vitro and in vivo. Co-immunoprecipitation (Co-IP) and mass spectrum were employed to screen out the interacting molecule of TRIP13. Results: Our results showed that TRIP13 was generally upregulated in melanoma tissues and was related to the poor prognosis of melanoma patients. The overexpression of TRIP13 promotes the invasion, migration and EMT transformation of melanoma cells in vitro, and promotes lung metastasis in vivo. Mechanismly, TRIP13 interacts with FLNA, and activates the PI3K/AKT pathway, and then induces melanoma migration, invasion and EMT transformation. Conclusion: Elevated TRIP13 drives tumor progression via the FLNA/PI3K/AKT axis, and TRIP13 is a innovative prognostic molecule and potential target of targeted therapy in melanoma.


Introduction
Melanoma is one of the fastest growing cutaneous malignant tumor, with an annual growth rate of 3%-5% [1] . There are about 200000 new cases worldwide every year, resulting in about 55000 deaths [2] .
Somatic mutations occur repeatedly in all types of melanoma, for example, genes which regulate proliferation: BRAF, NRAS [3] ; in uence growth and metabolism PTEN and KIT [4] ; resistance to apoptosis: TP53 [5] ; participate in cell cycle control: CDKN2A [6] . Therefore, targeted therapy for melanoma mutant genes came into being. For example, vemurafenib and dabrafenib were approved to treat metastatic and unresectable melanoma with BRAF mutation in 2011 and 2013 [7,8] . However, many patients will develop drug resistance in a short time, which affects the e cacy of targeted drugs. Therefore, nding the pathogenesis and key molecules of melanoma will help researchers nd new targeted therapies for melanoma.
ATPases Associated with Diverse Cellular Activity (AAA ATPase) is widely distributed in a variety of tissues and organs and affects a variety of life activities, such as DNA replication, protein degradation, membrane fusion, signal pathway transduction, regulation of gene expression and so on [9] . Studies have also found that it plays an important role in the occurrence and development of tumors [10] . RUVBL2, AAA ATPase, is necessary for the carcinogenic function of c-myb in AML by inhibiting myeloid differentiation [11] . ATAD3A (ATPase family AAA domain containing 3A) is affected by the up-regulation of miR-210-5P, which induces mitochondrial autophagy and sorafenib resistance in hepatoma cells [12] .
However, there are few studies on AAA ATPase in melanoma. In 2021, Jilei Ma et al. utilised melanoma database: GSE3189 and GSE7553 to screen out the differentially expressed genes (DEGs) and established a protein-protein interaction network (PPI), and AAA ATPase family proteins-TRIP13 with a relatively high degree of connectivity were selected as one of the hub-genes that remarkably correlated to a poor survival rate [13] . However, the speci c role of TRIP13 in the progression of melanoma has not been reported.
In this study, we explored the expression and clinical signi cance of TRIP13 in melanoma patients. Then we constructed TRIP13-Overexpression and TRIP13-knockdown cell line and further explore the biological function and underlying mechanism of TRIP13 both in in vivo and in vitro.

Materials And Methods
Expression level of DEGs and survival analysis GSE3189, GSE7553 and GSE15605 were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo). GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to calculate the adjusted p_values and logFC values among different groups. GEPIA(http://gepia.cancer-pku.cn/index.html), using the data of TCGA (cancer genome map) and GTEx (genotype tissue expression) can quickly provide useful information such as differential gene expression analysis between cancer and adjacent tissues and patient survival analysis [14] . The expression of TRIP13 in melanoma and the overall survival (OS) of patients was acquired from GEPIA.

Patients and follow-up
A total of 123 para n-embedded melanoma and matched peritumor tissues (TMA) was used for immunohistochemistry (IHC) analysis. All patients were received a complete excision then veri ed by a pathological examination. The patients did not receive radiotherapy and chemotherapy before operation. The clinical stage of patients was evaluated by the TNM staging system of the eighth edition of AJCC classi cation system. Ethical approval was obtained from the Ethics Committee of the Zhongshan Hospital Biomedical Research Department, and all patients signed informed consent.
For immuno uorescence staining, cells were xed with 4% formaldehyde for 15min, in ltrated with 0.3% Triton X-100 (Beyotime, Shanghai, China) for 30 min at room temperature, and blocked with 5% BSA. Cells were incubated with the primary antibodies overnight at 4°C, washed by PBS for three times and then incubated by uorescent secondary antibody. Then, cells were washed with PBS, stained with DAPI, and observed by Confocal uorescence microscope (Olympus, Japan).

Invasion and wound healing
For CCK-8 assay, cells were inoculated into 96-well plates (1000 cells/well). Then, cells were incubated with 10 µL Cell Counting Kit-8 (Yeasen, Shanghai, China) for 2 h at 37°C after the 1st, 2nd, 3rd and 4th days. The absorbance values were measured at 450 nm. For transwell invasion test, Matrigel (BD Biosciences, USA) was evenly spread into transwell chamber, and 10 4 cells were spread into the transwell chamber with 200µL serum-free DEME; DMEM medium containing 20% FBS was added to the lower chamber. After 48h, take out the chamber, wash it with PBS and x it in 4% paraformaldehyde solution for 30min; dyed with crystal violet for 15min and take photos under the microscope. For wound healing assays, When the cells are full, use 200 µL pipette tip scratch the bottom of the plate; Add serum-free DMEM medium and culture in 5% CO 2 cell incubator at 37°C; The cells were photographed again at 48h, the migration rate was calculated by image J.
In vivo assay A2058-TRIP13 and A2058-Vector were suspended in PBS and a total of 10 6 cells were injected subcutaneously into the left ank of mice. After 3 weeks, mice were sacri ced, and the tumors were collected and weighed. For the metastasis model, 10 6 cells were collected and were injected into the tail vein of nude mice. There were 6 mice in each group. The mice were killed 21 days after injection. The lungs were taken, xed with 10% formaldehyde, sliced and stained with hematoxylin and eosin, photographed, and lung metastases were counted under the microscope.

Co-immunoprecipitation (CO-IP) and MS
Melanoma cells were collected by IP lysis solution and centrifuged. The primary antibody and Protein A/G beads were added and mixed with the cell lysate at 4°C for 4 h. Then,the precipitate was added into 40µL loading buffer for suspension. Regular sample loading, electrophoresis and luminescence. As for LC-MS/MS analysis, take an appropriate amount of peptide from each sample and use the nano liter ow rate easy NLC 1200 chromatographic system (Thermo Scienti c) for chromatographic separation. Maxquant 1.6.1.0 mass spectrometry database retrieval software was used for retrieval.

Statistical analysis
The results are expressed as mean ± standard deviation (SD). Student's t tests was utilized to recognize the differences in two groups, one-way ANOVAs was utilized to recognize the differences among three or more groups. Kaplan-Meierand log-rank were utilized to analyze the overall survival and disease-free survival. A p < 0.05 was considered statistically signi cant.

Results
TRIP13 is highly expressed in melanoma and is associated with poor prognosis Three databases: GSE3189, GSE7553, GSE15605 were used to analyze differential genes between primary melanoma and normal skins. Among them, GSE3189 contains 7 normal skin and 45 primary melanoma; GSE7553 contained 4 normal skin and 14 primary melanoma; GSE15605 contained 16 normal samples and 46 primary melanoma. The expression pro les were analyzed by Limma package in R, and P_value < 0.05 and log 2 FC > 1.3 were used as thresholds. GSE3189 contained 7180 DEGs, 2841 upregulated genes and 4339 downregulated genes; GSE7553 contained 2857 DEGs, 1502 upregulated genes and 1355 downregulated genes; GSE15605 contained 8173 DEGs, 4428 upregulated genes and 3745 downregulated genes (Fig. 1A). We choose the upregulate genes in three databases( P_value < 0.05 Page 7/16 and log 2 FC > 1.3), and 65 DEGs overlapped in three databases (Fig. 1B). Then, overall survival analysis was performed based on 65 overlapped DEGs and the results showed that 24 DEGs are associated with poor prognosis of melanoma. Among them, TRIP13 has been reported to be involved in glioblastoma and chronic lymphocytic leukemia and overexpression TRIP13 signi ed poorer prognosis (Fig. 1D). [17,18] ; However, the expression level of TRIP13 in melanoma and its role in the progression of melanoma has not been studied.Therefore, we selected TRIP13 for further study. The level of TRIP13 mRNA in melanoma was tested with GEPIA. According to the database, the expression of TIRP13 raised in melanoma compared with normal skin (Fig. 1C). Then, we examined the expression of TRIP13 in our own cohorts of melanoma patients and revealed that TRIP13 was mainly in the cytoplasm (Fig. 1E). Through quanti cation analysis, We found that the content of TRIP13 in melanoma was higher than that in adjacent tissues (Fig. 1F). Importantly, patients in the TRIP13 High group (n=68) had poorer overall survival rates and disease-free survival than those in the TRIP13 Low group (n=55) (p=0.0042,p=0.0049, Fig. 1G-H).
In addition, high levels of TRIP13 were associated with lymph nodes metastasis (p = 0.002) and distant metastasis (p = 0.024) ( Table 1). Importantly, univariate and multivariate analyses showed that the TRIP13 levels were independent indicators of prognosis in patients with melanoma ( Table 2). Note: A chi-square test was used for comparing groups between low and high TRIP13 expression.

TRIP13 promotes the invasion and migration of melanoma cells in vitro and in vivo
Then we constructed lentivirus-mediated stable expression of sh-TRIP13 in MV3 cells and cDNA vectormediated stable expression of TRIP13 in A2058 cells. The transfection e ciency was detected by Western blot (Fig. 2A). Knockdown of TRIP13 decreased the proliferation, migration and invasion of MV3 cells, and overexpression of TRIP13 increased the proliferation, migration and invasion of A2058 cells (Fig. 2B-D). Next, we investigated the effect of TRIP13 on tumorigenesis in vivo. However, there was no signi cant difference in tumor size between nude mice injected with A2058-Vector and nude mice injected with A2058-TRIP13 (Fig. 2E). In the pulmonary metastasis model, TRIP13-A2058 formed more lung metastases than Vector (Fig. 2F). Collectively, these results show that TRIP13 upregulation promotes melanoma progression both in vitro and in vivo.
Overexpression TPIP13 lead to EMT by activating the PI3K/AKT pathway In order to explore the mechanism of TRIP13 induced EMT, we sequenced the cell lines overexpressing and knocking down TRIP13 (Fig. 4A). There were 399 upregulated DEGs and 415 downregulated DEGs in MV3-shNC cells compared with MV3-TRIP13 cells, while there were 1481 upregulated DEGs and 1640 downregulated DEGs in A2058-TRIP13 cells compared with A2058-Vector cells. Among them, 452 DEGs overlapped which KEGG analysis demonstrated were involved in pathways in cancer, focal adhesion, ECM-receptor interaction, regulation of actin cytoskeleton and PI3K/AKT signalling pathway (Fig. 4B).
According to the results of KEGG, we discovered that overexpression of TRIP13 enhanced the p-AKT, and that knock down of TRIP13 reduced p-AKT (Fig. 4C). Next, we found that TRIP13-induced migration, invasion were inhibited by LY-294002(AKT inhibitor, 50 µM, 24 h), an PI3K/AKT pathway inhibitor ( Fig. 4D-E). Further, we found that TRIP13-induced upregulation of p-AKT and EMT markers were also inhibited by LY-294002 (Fig. 4F). Altogether, we verdict that TRIP13 accelerates EMT and tumour progression via PI3K/AKT signaling.

TRIP13 binds to FLNA and activates the PI3K/AKT pathway
To nd the downstream protein of TRIP13, MS was conducted to nd latent TRIP13-combining molecule in A2058-Vector cells and A2058-TRIP13 cells. Finally, 82 and 64 molecule were recognized in A2058-Vector cells and A2058-TRIP13 cells, and twenty proteins only existed in A2058-TRIP13 cells (Fig. 5A). Among TRIP13 bound molecules, FLNA has been reported to be involved in the regulation of PI3K/AKT and EMT [21,22] . The interaction between TRIP13 and FLNA was con rmed by co-immunoprecipitation and immuno uorescence ( Fig. 5B-C). Next, when FLNA was inhibited, the migration, invasion, EMT transformation and the activation of PI3K/AKT by upregulated TRIP13 also restrained ( Fig. 5E-F). In sum, TRIP13 activates the PI3K/AKT pathway and induces EMT by interacting with FLNA.

High expression of TRIP13 and FLNA indicated poor prognosis
We then validated the relationship between TRIP13 and FLNA in our patient tissues, immunohistochemistry showed that TRIP13 was positively correlated with FLNA (r = 0.5949, p < 0.0001, Fig. 6A-B). Then, we examined the relationship between TRIP13 / FLNA and prognosis. it was found that patients with higher expression of TRIP13 / FLNA simultaneously had shorter overall survival and disease-free survival (Fig. 6C-D). This indicates that the high expression of TRIP13 and FLNA indicates bleak prognosis in patients with melanoma.Taken together, TRIP13 activates PI3K/AKT pathway, upregulates N-cadherin and down-regulates E-cadherin by combining with FLNA, and then affects melanoma cell migration, invasion and EMT (Fig. 6E).

Discussion
In this study, we explored the potential role and underlying mechanism of TRIP13 in melanoma. we demonstrates that TRIP13 is signi cantly upregulated in melanoma tissues, and is strongly correlated with poor prognosis in melanoma patients. Overexpression of TRIP13 is associated with a malignant phenotype of melanoma. Moreover, Our ndings indicate that, TRIP13 combined with FLNA and activates the PI3K/AKT pathway and induces cell EMT.
A large number of literature reports that TRIP13 plays a key role in regulating mitotic processes, including spindle assembly checkpoint and DNA repair and result in chromosome instability (CIN) [23,24] . Based on its own important physiological functions, abnormal expression of TRIP13 has been observed in various human cancers and related to several aspects of malignant phenotype, including excessive cell proliferation, drug resistance and tumor progression [25] . Liming Dong et al. found that TRIP13 regulates YWHAZ and promotes the proliferation, migration, invasion and EMT transformation of prostate cancer cells [19] . TRIP13 promotes lung adenocarcinoma cell proliferation, clonogenicity, and migration and inhibiting apoptosis and G2/M phase shift as well in vitro [26] . In our experiment, we found that upregulation of TRIP13 promoted the invasion, migration, proliferation and EMT of A2058 cells via PI3K/AKT pathway. High levels of TRIP13 correlated with lymph nodes metastasis and distant metastasis. Importantly, univariate and multivariate analyses indicated that the TRIP13 level were independent indicators for melanoma patient prognoses.
AAA ATPase can facilitate the assembly or degradation of protein complexes [27] . TRIP13 has been reported to interact with functional proteins to activate related pathways and induce malignant transformation. In HCC, TRIP13 is regulated by miR-192-5p, interacted with ACTN4 and positively regulated its expression, thus activating the AKT/mTOR pathway to drive tumor progression [28] . In CRC, TRIP13 interacted with a tyrosine kinase-FGFR4 and then activated the EGFR-AKT pathway and EMT [29] . In our experiment, we found that TRIP13 has a direct interaction with FLNA through CO-IP and mass spectrometry, and veri ed this interaction with Immuno uorescence. FLNA, the most abundant and widely distributed member of the lamin family (Filamin A, Filamin B and Filamin C), was rst identi ed as a non-muscle actin lament crosslinking protein in 1975 [30] . FLNA crosslinks actin laments and helps to stabilize the cytoskeleton network and supports cell integrity [31] . Several studies showed that FLNA associates with multiple functional non-cytoskeletal proteins and is involved in several unrelated pathways that regulate cell migration and adhesion and promote tumor progression [32] . Mengmeng Cheng et al. demonstrated that c-Met-AKT-FLNA-smad formed a positive feedback loop to enhance EMT, which contributes to 5-FU resistance of CRC cells [33] . Consistently, our experiments showed that FLNA knockdown partly abolished TRIP13-induced EMT and activation of the PI3K/AKT pathway. Taking together, these results indicate that TRIP13 triggers PI3K/AKT signaling to promote EMT by interacting with FLNA.
In conclusion, our ndings con rmed the important role of TRIP13 in tumour progression and EMT via FLNA/PI3K/AKT pathway. We provides new insights into the mechanisms underlying melanoma progression. Our study offers novel evidence that TRIP13 may serve as a prognostic indicator and potential therapeutic target for melanoma patients.  Cells, 2020,9(12). TRIP13 promotes the invasion and migration of melanoma cells in vitro and in vivo A. The e ciency of transfection were detected by western blot. B. CCK-8 was used to detect the proliferation of overexpressed / knockdown TRIP13 melanoma. C. The migration ability of overexpressed / knockdown TRIP13 melanoma was detected by Wound-healing assays. D. The Invasive abilities of overexpressed / knockdown TRIP13 melanoma was detected by transwell assays. E. Tumors in nude mice bearing A2058-shNC and A2058-TRIP13 cells and tumor weight were examined after 3 weeks. F. 10 6 A2058-shNC and A2058-TRIP13 cells were injected into the tail vein of nude mice(n=6). After 21 days, the lungs were obtained and Lung metastases were counted.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Migration and invasion assays were performed in A2058-shNC, A2058-TRIP13 cells after incubation with LY294002. F. Western blotting was used to detect the levels of TRIP13, p-AKT, AKT, E-cadherin and Ncadherin in A2058-shNC and A2058-TRIP13cells after incubation with LY294002. ***p < 0.001, ****p < 0.0001 Figure 5 TRIP13 binds to FLNA and activates the PI3K/AKT pathway.A. Co-IP and MS detected the latent TRIP13combining molecule in A2058-Vector cells and A2058-TRIP13 cells. B. Co-IP was applied to con rm the interaction of TRIP13 and FLNA. C. Immuno uorescence was applied to con rm the colocalization of TRIP13 and FLNA. D. Western blot analysis of TRIP13 or FLNA expression in A2058 cells transfected with shTRIP13 or siFLNA. E. Cell migration and invasion were detected in related cells. F. Western blotting was used to detect the levels of TRIP13, FLNA, p-AKT, AKT, N-cadherin in related cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001