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Ubiquitin-like protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1
Journal Pre-proof Ubiquitin-like protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1 Xuan Yi, Xueqiang Deng, Yanzhi Zhao, Binbin Deng, Jianyong Deng, Huimin Fan, Yunyan Du, Liang Hao PII:
S0014-4827(19)30692-5
DOI:
https://doi.org/10.1016/j.yexcr.2019.111804
Reference:
YEXCR 111804
To appear in:
Experimental Cell Research
Received Date: 26 October 2019 Revised Date:
19 December 2019
Accepted Date: 22 December 2019
Please cite this article as: X. Yi, X. Deng, Y. Zhao, B. Deng, J. Deng, H. Fan, Y. Du, L. Hao, Ubiquitinlike protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2019.111804. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Ubiquitin-like protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1 Xuan Yi1†, Xueqiang Deng1†, Yanzhi Zhao2, Binbin Deng1, Jianyong Deng1, Huimin Fan3, Yunyan Du4,5*, Liang Hao1* 1
Department of Orthopedics, Second Affiliated Hospital of Nanchang University,
Nanchang, China 2
The First Clinical Medical College of Nanchang University, Nanchang, China
3
Department of Ophthalmology, Second Affiliated Hospital of Nanchang University,
Nanchang, China 4
Department of Medical, Jiangxi Provincial People's Hospital, Nanchang, China
5
Department of Otorhinolaryngology, Jiangxi Provincial People's Hospital,
Nanchang, China †
Xuan Yi and Xueqiang Deng contributed equally to this work.
*
Corresponding author: Liang Hao, Department of Orthopedics, Second Affiliated
Hospital of Nanchang University, Nanchang, China. 1 Minde Road, Nanchang, Jiangxi Province, 330000, China. Tel: +86 13607008562; Fax: +86 86415785. E-mail address: [email protected]. Yunyan Du, Department of Medical, Jiangxi Provincial People's Hospital, Nanchang, China. No. 92 Aiguo Road, Nanchang, Jiangxi Province, 330000, China. E-mail: [email protected]. Abstract Osteosarcoma is a common malignancy of the bone tissue. The rapid growth exhibited by this cancer is a primary challenge in its treatment. In many types of cancers, FAT10, a ubiquitin-like protein, is involved in several biological activities, especially cell proliferation. Herein, we demonstrate that FAT10 plays a vital role in tumorigenesis and is overexpressed in tumor tissues compared to its expression in adjacent normal tissues. Functional assays revealed that knockdown of FAT10 expression significantly repressed the proliferation of osteosarcoma in vitro and in
vivo. Furthermore, our results indicate that FAT10 exhibits oncogenic functions by regulating the level of YAP1, a key protein of the Hippo/YAP signaling pathway, and a significant positive correlation exists between the levels of FAT10 and YAP1. Further analysis showed that FAT10-induced growth of osteosarcoma cells is dependent on YAP1. Mechanistically, FAT10 stabilizes YAP1 expression by regulating its ubiquitination and degradation. Taken together, our results link the two drivers of cell growth in osteosarcoma and reveal a novel pathway for FAT10 regulation. We provide new evidence for the biological and clinical significance of FAT10 as a potential biomarker for osteosarcoma. Key word: FAT10; YAP1; osteosarcoma; proliferation; ubiquitination Introduction Osteosarcoma (OS) is a primary malignant cancer of the bone tissue, with an incidence among primary bone tumors that is second only to that of plasma cell myeloma[1,2]. The cure rate of OS using surgery alone is only 15–20%[3]. However, since the adoption of effective adjuvant and neoadjuvant chemotherapeutic treatments in the 1970s[4], especially of neoadjuvant chemotherapy combined with limb-salvage surgery, the 5-year survival rate has reached 60% and the quality of life of OS patients has been greatly improved[5]. However, approximately 30–40% of the patients in the clinics have an OS that is resistant to chemotherapy[6,7]. Not only are these tumors prone to metastasis, the prognosis is not improved even if the tumor is completely removed. Before the major breakthroughs in immunotherapy happened and various biological treatments were devised, the ability of chemotherapy to cure most of the OS cases depended on enhancement of the effect of chemotherapy; further improvement of the cure rate of OS remains a top priority. The active discovery and development of new drugs and effort to increase the strength of drugs are important aspects for improving the effect of chemotherapy. More important is the identification of molecular targets of OS to provide a better theoretical basis for targeted therapies.
The human HLA-F adjacent transcript 10 (FAT10) protein plays an important role in the development of malignant tumors[8]. FAT10 was originally called diubiquitin for being a member of the ubiquitin-like protein family that has two fused ubiquitin-like regions[9]. In recent years, FAT10, which is a ubiquitin-like protein, has received increasing attention from researchers, with Merbl et al describing its structure and function
[10]
. Studies have shown that FAT10 is the only ubiquitin-like
protein that does not rely on ubiquitin to directly degrade the targeted substrate by the proteasome[11]. Under the action of the E1 and E2 enzymes, FAT10 is linked to the Lys residue of the substrate protein and is then recognized by the receptor subunit of the proteasome before being degraded[12]. A number of studies have suggested that the FAT10 proteasome system (FPS) is a novel protein degradation system that is different from the ubiquitin-proteasome system (UPS). It has been previously reported that the FAT10 gene is upregulated in many tumor tissues, such as in liver cancer[13,14], gastrointestinal cancer[15], and bladder cancer[16], with FAT10 overexpression in liver cancer tissues being closely associated with the independent poor prognosis of patients. Lower FAT10 expression reduces the activation of NF-κB by tumor necrosis factor, TNF-α, thereby inhibiting tumor cell proliferation[17]. However, the precise role and underlying signaling cascade of FAT10 in OS progression remains unclear. The highly conserved Hippo/YAP signaling pathway plays an important role in cell growth and apoptosis[18], and the upregulation of Hippo/YAP signaling in various tumor cells indicates that it is closely associated with tumorigenesis[19,20]. YAP1 is an important effector downstream of the Hippo signaling pathway that is overexpressed in hepatocellular carcinoma
[21]
, lung cancer[22], and osteosarcoma[23], suggesting that
it is closely associated with patient prognosis. In MCF-10A cells, YAP overexpression can induce the epithelial-to-mesenchymal transition (EMT) of cells, inhibits apoptosis, and promotes the growth factor-independent growth, and nonanchored growth[24]. YAP can interact with a number of transcription factors, such as with the WW domain of RUNX2[25] and p73[26], and it can upregulate the transcription of downstream genes that promote cell growth through its TEAD binding domain (TBD) domain family
transcription factor, TEAD[27]. Nevertheless, the mechanism and role of YAP1 in regulating the growth of OS remain unclear. The goal of this study was to elucidate the mechanism through which FAT10 contributes to the prognosis of OS patients. We also investigated the mechanism underlying this effect with regard to FAT10 and YAP1 in OS, showing that FAT10 stabilizes YAP1 expression by modifying its ubiquitination and degradation. Taken together, the results of this study may provide new potential prognostic and therapeutic targets for OS.
Materials and methods 1. Human tissue specimens This study was performed with the informed consent of the patients and their families. The study program was approved by the Medical Ethics Committee of Nanchang University, and we collected tissues from 49 OS patients and the corresponding adjacent tissues from the Second Affiliated Hospital and the First Affiliated Hospital of Nanchang University. The samples were stored at -80 °C for subsequent experiments. 2. Cell line and cell culture The OS cell lines (U2-OS, 143B, MG-63, and Saos2) and a normal bone cell line (hfoBI-19) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM medium containing 10% FBS and were incubated at 37 °C under an atmosphere containing 5% CO2. 3. qRT-PCR and western blot analyses The qRT-PCR and western blot analyses were performed as described previously[28]. The sequences of the primers used in this study are shown in Table 1. 4. Immunohistochemistry (IHC) assay Osteosarcoma and non-tumor tissues were fixed, paraffin embedded, sectioned, and incubated in appropriately diluted primary antibodies against FAT10 (Abcam; 1:200) and YAP1 (Abcam; 1:200) overnight at 4 °C. Subsequently, the tissues were labeled
using an EnVision HRP kit (DAKO) for 30 min at room temperature with DAB base (DAKO) and then incubated and counterstained with Mayer’s hematoxylin (DAKO). 5. Co-immunoprecipitation (Co-IP) Appropriate cells were collected and lysed on ice by incubation with 1.0 mL of pre-cooled IP buffer for 15 min. Thereafter, the cell lysate was centrifuged at 12,000 rpm for 15 min. The supernatant was then placed in a new microcentrifuge tube and incubated with the primary antibody (1:400) for 2–4 h. Next, protein A/G PLUS-Agarose (Santa Cruz) was added, after which the mixture was incubated at 4 °C for 24 h on a rocking platform and then centrifuged at 3000 rpm for 4 min to collect the immunoprecipitate. The pellet was washed three times with 600 µL of RIPA buffer, repeating the centrifugation step each time. Finally, the pellet was resuspended in 40 µL of 3% SDS solution. After the sample was boiled for 10 min, 20 µL of it was analyzed by SDS-PAGE and autoradiography. 6. shRNA plasmids and constructs The carrier encoding FAT10 and YAP1 was obtained from GenePharma (Shanghai, China). The shRNA plasmid and vector were then transfected into OS cells using Lipofectamine 3000 (Invitrogen), following the manufacturer’s instructions. 7. Cell growth assays EdU assays OS cells were incubated with 5-acetylene-20-deoxyuridine (EdU, RiboBio, Guangzhou, China) for 2 h, as described in the manufacturer’s instructions. The OS cells were then treated with 200 µL of 1X Apollo reaction mixture for 25 min. Subsequently, Hoechst 33342 (5 µg/mL) was added to each well and the cells were stained for 30 min, and then observed under a fluorescence microscope and imaged. CCK-8 assays Cell viability was assessed using the CCK-8 method. Logarithmically growing OS cells (1×104) were plated in 96-well plates, after which CCK-8 was added to each well and the cells were incubated for 2 h. The optical density was measured using an iMark microplate reader (Bio-Rad) at 450 nm. The average of the absorbance values for five wells was calculated for each group.
8. Flow cytometry Osteosarcoma cells were harvested by treating with 0.25% trypsin. The apoptosis rate of OS cells was determined using an Annexin V-PE and PI apoptosis detection kit (BD Biosciences, USA). 9. Tumorigenicity assay OS cells (5×106) were resuspended in 100 µL of the medium and subcutaneously injected into the dorsal side of nude mice (male BALB/c-nu/nu, 5-weeks old). Tumor size was measured every 5 days. After 35 days, the mice were imaged after the administration of anesthesia, and the tumors were individually weighed and collected. 10. Statistical analysis All data were analyzed using SPSS 19.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 7. The results of three independent experiments were averaged and are presented with the standard deviation. Student’s t-test was used to compare the two groups of data, and one-way ANOVA was used to compare the two groups, with a value of P < 0.05 considered to be significant. Results 1. FAT10 expression is upregulated in OS tissues and cells To elucidate the function and importance of FAT10 in OS, we first assessed its expression levels in OS and non-tumor tissues using quantitative real-time PCR, western blot, and immunohistochemical analyses. The qRT-PCR results showed that the FAT10 mRNA levels in OS were significantly higher than in the non-tumor tissues (Fig 1A). In addition, the FAT10 protein levels were also higher in OS compared to those in the adjacent tissues (Fig 1B). We also confirmed that FAT10 expression was elevated in OS tissue using immunohistochemistry (Fig 1C). Furthermore, we confirmed that the levels of FAT10 in OS cell lines (U2-OS, MG-63, Saos-2, and 143B) were significantly higher than those observed in normal bone cells (hfoBI-19) by quantitative real-time PCR and western blot analyses (Fig 1D-E). These results showed that FAT10 is upregulated in OS tissues and cells. 2. FAT10 positively promotes OS growth in vitro and in vivo Because high levels of FAT10 protein expression are significantly associated with
increased tumor size in liver and colorectal cancers, we speculated that FAT10 might influence the OS growth. The relationship between FAT10 protein expression and OS cell growth was assessed using shRNA to silence the FAT10 expression in the OS cell lines, U2-OS and Saos-2. Results of quantitative real-time PCR and western blot analyses showed that FAT10 expression was markedly decreased (Fig 2A-B). The results of colony formation, EdU, and CCK8 assays revealed that FAT10 knockdown markedly decreased the OS cell growth (Fig 2C-E). In addition, we overexpressed FAT10 in the OS cell lines, MG-63 and 143B, and confirmed the overexpression by qRT-PCR and western blot analyses (Fig 3A-B). The results of colony formation, EdU, and CCK8 assays results revealed that the upregulation of FAT10 markedly increased the growth of OS cells (Fig 3C-E). Finally, shFAT10 and P-FAT10 OS cells were injected into mice in a tumor xenograft experimental model. After 5 weeks of growth, the results of tumorigenicity assay showed that tumors from the U2OS-shFAT10 group had lower weights and volumes
than
those
of
the
shNC
group
(Fig
2F-H).
The
results
of
immunohistochemical analysis confirmed that the level of FAT10 was significantly decreased in the shFAT10 group (Supplementary Fig 2), whereas that observed in the mice injected with P-FAT10 cells was notably increased compared to that observed in the respective controls (Fig 3F-H). Taken together, these results demonstrate that FAT10 can promote the OS cell growth in vitro and in vivo. 3. FAT10 induces cell cycle progression and inhibits cell apoptosis To further determine the effects of FAT10 on the growth of OS cells, flow cytometry was performed, the results of which indicated that downregulation of FAT10 expression significantly arrested the OS cells in the G1 phase (Fig 4A). Similarly, the results of western blot analysis showed that decreased FAT10 expression led to decreased levels of cyclin D1 and PCNA (Fig 4B). Interestingly, the cell cycle was arrested at the G1 phase in the OS cells overexpressing FAT10 (Fig 4C). Similarly, the results of western blot analysis showed that upregulation of FAT10 led to an increase in cyclin D1 and PCNA levels (Fig 4D). Furthermore, we assessed the effect of FAT10 on the apoptosis of OS cells, with the results showing a significant
increase in the apoptosis ratio in shFAT10 cells (Fig 4E). Similarly, the results of western blot analysis showed that FAT10 knockdown led to decreased BCL-2 levels and increased BAX levels (Fig 4F). In contrast, the apoptosis rate was decreased in the P-FAT10 OS cells (Fig 4G), and immunoblotting showed that upregulated FAT10 levels in OS cells could increase BCL-2 expression and decrease the BAX protein levels (Fig 4H). These results show that FAT10 can arrest the cell cycle at the G1-phase and inhibit apoptosis. 4. FAT10 positive regulates the expression levels of YAP1 protein In a previous study, it was shown that YAP1 expression is elevated in OS and can positively regulate tumor growth, but the specific mechanism associated with this activity has not been elucidated. Thus, we analyzed the expression of YAP1 by western blot analysis of shFAT10 and shNC cells. The results showed that the YAP1 expression was inhibited in the shFAT10 OS cells. (Fig 5A-B), whereas it was upregulated in the P-FAT10 OS cells (Fig 5C-D). Furthermore, we analyzed the YAP1 expression in OS tissues and observed that YAP1 mRNA and protein levels were higher in OS tissues compared to that in non-tumor tissues (Fig 5E, F and H). In addition, scatter plots revealed that FAT10 and YAP1 mRNA and protein levels were positively correlated in OS tissues (Fig 5G and I). Taken together, these findings suggest that FAT10 can promote the expression of YAP1 in OS cells. 5. YAP1 is crucial for the FAT10-mediated regulation of OS cell proliferation As mentioned above, YAP1 has been shown to function downstream of FAT10. Therefore, we studied the effect of YAP1 on the promotion of OS cell growth. First, we overexpressed YAP1 in shFAT10 OS cells, and confirmed its overexpression using western blot analysis (Fig 6A-B). The results of CCK8 and EdU assays indicated that the decrease in OS cell growth was caused by the downregulation of FAT10 expression and was partially attributable to the introduction of P-YAP1 (Fig 6C-D). Next, we downregulated YAP1 expression in FAT10-overexpressing OS cells. The results of western blot analysis confirmed the silencing of YAP1 (Fig 6E-F), and CCK8 and EdU assays revealed that the increase in cell growth induced by FAT10 upregulation in 143B cells was inhibited by the introduction of shYAP1 (Fig 6G-H).
Taken together, these findings suggest that YAP1, as a downstream target of FAT10, mediates FAT10-induced OS cell proliferation. 6. FAT10 stabilizes YAP1 expression by modifying its ubiquitin-mediated degradation To decipher the mechanism through which FAT10 regulates the expression of YAP1, we first performed Co-IP assays to verify whether FAT10 and YAP1 could bind to each other. The results of Co-IP showed that FAT10 can directly bind to YAP1 (Fig 7A). Interestingly, it was reported previously that YAP1 undergoes degradation via the UPS[29], and the results of our previous studies confirmed that FAT10 is able to stabilize the ubiquitination-mediated degradation of substrate proteins[30]. Based on these observations, we hypothesized that FAT10 may promote the expression of YAP1 by inhibiting its ubiquitination and degradation. To test this hypothesis, we incubated the OS cells with the proteasome inhibitor, MG132, for a specific period of time, which resulted in significant accumulation of endogenous YAP1 protein in the OS cell lines, U2-OS and Saos-2 (Fig 7B). These results indicate that YAP1 is also degraded by the UPS in OS cells. In addition, to further elucidate whether FAT10 is associated with the regulation of YAP1 protein degradation, we transfected shROCK2 and P-FAT10 vectors into U2-OS and 143B cells and tested the effect of different expression levels of FAT10 on YAP1 expression, in the presence or absence of MG132. The results indicated that the upregulation or knockdown of FAT10 levels did not affect the YAP1 expression in OS cells after the addition of MG132 (Fig 7 C-D). In addition, analysis of the kinetics of YAP1 degradation indicated that the half-life of ectopically expressed YAP1 in the U2-OS and Saos-2 cells overexpressing FAT10 was significantly increased (Fig 7E-F). These results suggest that FAT10 is involved in the degradation of YAP1. We further evaluated the role of FAT10 in the degradation of YAP1. The results showed that the heterotopic dose-dependent effect of upregulation of FAT10 resulted in a significant increase in the level of endogenous YAP1 protein in U2-OS and Saos-2 cells (Fig. 7G-H). In addition, a single dose of HA-YAP Flag-FAT10 was
co-transfected into U2-OS and Saos-2 cells. A dose-dependent effect of upregulated FAT10 levels on the YAP1 expression was observed (Fig. 7I), indicating that FAT10 can stabilize the expression of YAP1. Finally, to elucidate the mechanism through which FAT10 stabilizes YAP1 expression, we treated the U2-OS/shFAT10 and 143B/pcDNA3.1(+)-FAT10 cells with MG132. The results of immunoprecipitation indicated that inhibition of FAT10 levels increased the levels of YAP1 ubiquitination, whereas the overexpression of FAT10 significantly reduced the YAP1 ubiquitination (Fig 7G). These results indicated that FAT10 is able to stabilize the ubiquitination and degradation of YAP1 to promote its expression. Discussion An important feature of OS is its rapid growth, and studies have shown that the proliferation of OS is closely associated with the abnormal expression of the related proto-oncogenes[31]. Despite the rapid development in surgical and oncological chemotherapeutic drugs, the prognosis and 5-year survival rate of OS patients have remained stagnant. Thus, we need to study and explore the growth mechanisms of OS to provide a more theoretical basis for targeted therapy of OS. The results of this study demonstrate that FAT10 plays a key role in OS cell proliferation. FAT10 belongs to Ubls and comprises an HLA-F site at the telomere end of MHC class I[32]. There has been increasing evidence that FAT10 is upregulated in a variety of malignancies, and abnormal FAT10 expression is closely associated with tumor growth and metastasis. Yuan and colleagues showed that FAT10 is closely associated with the prognosis of liver cancer[30], and silencing FAT10 expression could inhibit the growth and metastasis of this cancer. In contrast, the growth and metastasis of cells expressing FAT10 is significantly enhanced. Similarly, Dong et al. reported that FAT10 expression is closely associated with bladder cancer[16]. In addition, we found that FAT10 is highly expressed in many tumors, and there is a clinical subgroup with high FAT10 level in some tumors. We also found that FAT10 was related to the survival rate of many tumor patients in TCGA database. Although Ma et al. reported that silencing FAT10 could inhibit the invasion and migration of OS[33], the specific
mechanism associated with this activity has not been studied in detail, and FAT10 has not been reported to be involved in the growth of OS. Our results in the present study showed that FAT10 expression was significantly increased in OS specimens, and OS cell growth could be inhibited or enhanced by silencing and overexpressing FAT10 expression, respectively, in vivo and in vitro. Therefore, these findings suggest that FAT10 may function as an oncogene in OS. The Hippo/YAP signaling pathway plays a crucial role in tumor progression, and it was confirmed in a previous study that this pathway is abnormally activated in many cancers and is closely related to poor tumor prognosis[34]. As a downstream factor of the Hippo/YAP signaling pathway, YAP1 also plays an important role in regulating the growth and metastasis of tumors. Yang et al. studied the effect of silencing of YAP1 on the inhibition of OS growth in vitro and in vivo[35]. In addition, many studies have confirmed that YAP1 acts as a target for lncRNAs and circRNAs to regulate the growth of OS[36]. However, in our study, the data indicate that YAP1 is expressed in OS tissues and cells and is positively correlated with FAT10 expression. Knockdown of FAT10 expression could significantly reduce the mRNA and protein expression levels of YAP1, and FAT10 overexpression resulted in a significant increase in the expression of YAP1 mRNA and protein. Additionally, we have shown that the ability of FAT10 to regulate the growth of OS is dependent on the expression of YAP1. Previous studies have shown that FAT10 can affect the ubiquitination-mediated degradation of substrate proteins and that YAP1 can be degraded in response to ubiquitination. In this study, we confirmed the direct binding of FAT10 and YAP1 by immunoprecipitation, after which FAT10 was shown to significantly promote the level of YAP1 ubiquitination, as FAT10 overexpression suppressed YAP1 ubiquitination. Finally, FAT10 was shown to decrease the half-life of YAP1. In summary, the results of our study confirm that FAT10 is expressed at high levels in OS tissues and cells. In addition, our study also shows that FAT10 can promote the growth of OS in vitro and in vivo. More importantly, FAT10-induced OS cell growth was observed to be dependent on YAP1. Our findings also demonstrate that FAT10 inhibits the ubiquitination and degradation of YAP1. Based on these findings, FAT10
can serve as a candidate biomarker for the diagnosis and treatment of OS in the future. Acknowledgements We thank Elsevier’s English Language Editing service for editing this manuscript. Funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 81760487) and the Project of Jiangxi Provincial Department of Science and Technology (No. 2019ACBL20035). Consent for publication All authors have read and approved the final version of the manuscript. The authors and participants agreed to its publication. Competing interests The authors declare that they have no competing interests.
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Figure legends Figure 1 FAT10 expression is upregulated in osteosarcoma (OS) tissues and cells A-B. qRT-PCR and western blot analyses of FAT10 mRNA and protein expression in OS tissues (n = 30) and in their corresponding adjacent tissues (n = 30) (**p<0.01). C. Representative images and quantification of FAT10 staining in 49 paired OS and non-tumor tissues (**p<0.01). D-E. Relative FAT10 mRNA and protein levels in OS cells (U2-OS, Saos-2, MG-63, and 143B) compared to that observed in hfoBI-19 cells (**p<0.01). Figure 2 Knockdown of FAT10 expression inhibited the growth of osteosarcoma (OS) in vitro and in vivo A. Western blot analysis of FAT10 protein levels after shRNA transfection of the OS cell lines, U2-OS and Saos-2. B. Relative FAT10 mRNA levels in the OS cell lines, U2-OS and Saos-2, as detected by qRT-PCR (**p<0.01). C-D. Results of CCK8 and EdU assays showing that knockdown of FAT10 levels significantly decreases the growth of OS cells. E. Representative images for the clone formation assay of OS cells transfected with shFAT10. shFAT10/U2-OS and shNC/U2-OS cells were subcutaneously injected into nude mice, and at the end of the experiment, the tumors were imaged (F) and weighed (H, n = 5, *p<0.05, **p<0.01). G. The volumes of tumors were measured on the indicated days. Figure 3 Up-regulated expression of FAT10 promotes osteosarcoma (OS) growth in vitro and in vivo A-B. Western blot and qRT-PCR analyses of FAT10 mRNA and protein levels in OS cells after FAT10 overexpression (*p<0.05, **p<0.01). C-D. CCK8 and EdU assays
showing the increased proliferation of OS cells treated with MG-63 and 143B (*p<0.05, **p<0.01). E. Representative images of OS cells after upregulation of FAT10 expression in the clone formation assay. The vector and P-FAT10/U2-OS cells were subcutaneously injected into nude mice, and tumor volumes were measured on the indicated days (G). At the experimental endpoint, tumors were imaged (F) and weighed (H, n = 5, *p<0.05, **p<0.01). Figure 4 FAT10 induces cell cycle progression and inhibits cell apoptosis A. The percentage of osteosarcoma (OS) cells in different cell cycle phases was determined by FACS analysis, with the results showing that the number of U2-OS and Saos-2 OS cells in the G1 phase was increased after transfection with shFAT10 (*p<0.05). B. Western blot analysis for the detection of cyclin D1 and PANC protein levels in shFAT10 cells compared to that observed in shNC cells. C. The number of cells in the G1 phase was decreased in P-FAT10 cells compared to the vector cells (*p<0.05). D. The expression of cyclin D1 and PANC proteins was upregulated in the P-FAT10 cells. E-G. The apoptosis rate of OS cells was detected by flow cytometry and was significantly increased in the shFAT10 cells (E, **p<0.01) but decreased in the P-FAT10 cells (G, *p<0.05). F-H. Western blot analysis of the relative expression levels of the apoptosis-associated proteins, caspase3, BCL-2, and BAX in shFAT10 (F) or P-FAT10 cells. Figure 5 FAT10 positively regulates YAP1 protein expression A-B. The relative level of YAP1 expression in osteosarcoma (OS) cells was detected by qRT-PCR and western blot analyses after transfection with shFAT10 or shNC plasmids (**p<0.01). C-D. The mRNA and protein levels were increased in OS cells transfected with the P-FAT10 plasmid (**p<0.01). E-H. Observation (E) and quantification (H, **p<0.01) of FAT10 protein levels in the OS tissues (n = 30) compared to that observed in non-tumors (n = 30) by western blot analysis. F. Determination of FAT10 mRNA levels in OS tissues (n = 30) compared to that in non-tumor tissues (n = 30) by qRT-PCR (**p<0.01). G and I. Scatter plots of FAT10 and YAP1 mRNA and protein expression levels in OS. Figure 6 YAP1 is crucial for the FAT10-mediated regulation of osteosarcoma (OS)
cell proliferation A-B. Western blot analysis of FAT10 and YAP1 protein expression in U2-OS and Saos-2 OS cells, showing that YAP1 overexpression can mitigate the loss of YAP1 levels in shFAT10 cells. C-D. Results of CCK8 and EdU assays showing that the upregulation of YAP1 expression could restore the proliferation of OS cells transfected with the shFAT10 plasmid (*p<0.05, **p<0.01). E-F The relative levels of FAT10 and YAP1 were detected by western blot analysis, showing that the knockdown of YAP1 expression could decrease the YAP1 levels in P-FAT10 cells. G-H. Results of CCK8 and EdU assays showing the proliferation of OS cells stably transfected with P-FAT10 in the presence or absence of shYAP1 (*p<0.05, **p<0.01). Figure 7 FAT10 stabilizes YAP1 expression by modifying its ubiquitin-mediated degradation A. Co-immunoprecipitaion of endogenous FAT10 and YAP1 in U2-OS and Saos-2 osteosarcoma (OS) cells. B. Western blot analysis of YAP1 expression in cells treated with MG132 (15 µM) for the indicated times. C and D. After transfection with the shFAT10 or P-FAT10 plasmids, no significant change in YAP1 expression was observed upon addition of MG132. E and F. U2-OS and Saos-2 cells were transfected with expression plasmids encoding HA-YAP1 with or without the Flag-FAT10 plasmid. The cells were then exposed to CHX (20 µM) for the indicated times, and the degradation of YAP1 was detected using an anti-HA antibody. G-H. U2-OS and Saos-2 cells were transfected with increasing amounts of Flag-FAT10 plasmid, and endogenous YAP1 expression levels were detected using an anti-YAP1 antibody. U2-OS and Saos-2 cells were either untreated or transfected with a single dose of the expression plasmid encoding HA-YAP1 alone or in combination with the Flag-FAT10 plasmid. The level of YAP1 expression was detected using an anti-HA antibody. H and I. The level of ubiquitinated YAP1 in shFAT10 or P-FAT10 OS cells treated with MG132 (15 µM) was assessed by western blot analysis with a Ub antibody. 8. Figure 8 Proposed model for the mechanism through which FAT10 promotes the growth of osteosarcoma (OS) cells by regulating the expression of YAP1.
Primers for real-time PCR Gene
Primer sequences
FAT10
Forward:5'-CCGTTCCGAGGAATGGGATTT-3′ Reverse:5'-GCCATAAGATGAGAGGCTTCTCC-3′
YAP1
Forward:5'-GCAACTCCAACCAGCAGCAACA-3′ Reverse:5'-CGCAGCCTCTCCTTCTCCATCTG-3′
GAPDH
Forward:5'-AGCCTCAAGATCATCAGCAATG-3′ Reverse:5'-CCATCACGCCACAGTTTCC-3′
Highlights • • • •
FAT10 silencing inhibited cancer cell proliferation in vivo and in vitro YAP1 represents a novel target for the prevention and treatment of OS The expression of FAT10 and YAP1 are up-regulate in OS FAT10 stabilizes YAP1 expression by modifying its ubiquitination and degradation.
Authors’ contributions section Liang Hao and Yunyan Du conceived and designed the study. Xuan Yi, Xueqiang Deng, and Binbin Deng performed the experiments and collected data; Jianyong Deng and Huimin Fan performed the data analysis; All authors discussed and interpreted the data. Xueqiang Deng and Xuan Yi wrote the manuscript. All authors reviewed and edited the manuscript. All authors read and approved the final manuscript.
Conflict of interest statement The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted Sincerely, Liang Hao, M.D., Ph.D. Department of Orthopedics The Second Affiliated Hospital of Nanchang University No.1 Minde Road,Nanchang 330006,Jiangxi, China Phone: +86 791 86265564 E-mail: address: [email protected]
S0014-4827(19)30692-5
DOI:
https://doi.org/10.1016/j.yexcr.2019.111804
Reference:
YEXCR 111804
To appear in:
Experimental Cell Research
Received Date: 26 October 2019 Revised Date:
19 December 2019
Accepted Date: 22 December 2019
Please cite this article as: X. Yi, X. Deng, Y. Zhao, B. Deng, J. Deng, H. Fan, Y. Du, L. Hao, Ubiquitinlike protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2019.111804. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Ubiquitin-like protein FAT10 promotes osteosarcoma growth by modifying the ubiquitination and degradation of YAP1 Xuan Yi1†, Xueqiang Deng1†, Yanzhi Zhao2, Binbin Deng1, Jianyong Deng1, Huimin Fan3, Yunyan Du4,5*, Liang Hao1* 1
Department of Orthopedics, Second Affiliated Hospital of Nanchang University,
Nanchang, China 2
The First Clinical Medical College of Nanchang University, Nanchang, China
3
Department of Ophthalmology, Second Affiliated Hospital of Nanchang University,
Nanchang, China 4
Department of Medical, Jiangxi Provincial People's Hospital, Nanchang, China
5
Department of Otorhinolaryngology, Jiangxi Provincial People's Hospital,
Nanchang, China †
Xuan Yi and Xueqiang Deng contributed equally to this work.
*
Corresponding author: Liang Hao, Department of Orthopedics, Second Affiliated
Hospital of Nanchang University, Nanchang, China. 1 Minde Road, Nanchang, Jiangxi Province, 330000, China. Tel: +86 13607008562; Fax: +86 86415785. E-mail address: [email protected]. Yunyan Du, Department of Medical, Jiangxi Provincial People's Hospital, Nanchang, China. No. 92 Aiguo Road, Nanchang, Jiangxi Province, 330000, China. E-mail: [email protected]. Abstract Osteosarcoma is a common malignancy of the bone tissue. The rapid growth exhibited by this cancer is a primary challenge in its treatment. In many types of cancers, FAT10, a ubiquitin-like protein, is involved in several biological activities, especially cell proliferation. Herein, we demonstrate that FAT10 plays a vital role in tumorigenesis and is overexpressed in tumor tissues compared to its expression in adjacent normal tissues. Functional assays revealed that knockdown of FAT10 expression significantly repressed the proliferation of osteosarcoma in vitro and in
vivo. Furthermore, our results indicate that FAT10 exhibits oncogenic functions by regulating the level of YAP1, a key protein of the Hippo/YAP signaling pathway, and a significant positive correlation exists between the levels of FAT10 and YAP1. Further analysis showed that FAT10-induced growth of osteosarcoma cells is dependent on YAP1. Mechanistically, FAT10 stabilizes YAP1 expression by regulating its ubiquitination and degradation. Taken together, our results link the two drivers of cell growth in osteosarcoma and reveal a novel pathway for FAT10 regulation. We provide new evidence for the biological and clinical significance of FAT10 as a potential biomarker for osteosarcoma. Key word: FAT10; YAP1; osteosarcoma; proliferation; ubiquitination Introduction Osteosarcoma (OS) is a primary malignant cancer of the bone tissue, with an incidence among primary bone tumors that is second only to that of plasma cell myeloma[1,2]. The cure rate of OS using surgery alone is only 15–20%[3]. However, since the adoption of effective adjuvant and neoadjuvant chemotherapeutic treatments in the 1970s[4], especially of neoadjuvant chemotherapy combined with limb-salvage surgery, the 5-year survival rate has reached 60% and the quality of life of OS patients has been greatly improved[5]. However, approximately 30–40% of the patients in the clinics have an OS that is resistant to chemotherapy[6,7]. Not only are these tumors prone to metastasis, the prognosis is not improved even if the tumor is completely removed. Before the major breakthroughs in immunotherapy happened and various biological treatments were devised, the ability of chemotherapy to cure most of the OS cases depended on enhancement of the effect of chemotherapy; further improvement of the cure rate of OS remains a top priority. The active discovery and development of new drugs and effort to increase the strength of drugs are important aspects for improving the effect of chemotherapy. More important is the identification of molecular targets of OS to provide a better theoretical basis for targeted therapies.
The human HLA-F adjacent transcript 10 (FAT10) protein plays an important role in the development of malignant tumors[8]. FAT10 was originally called diubiquitin for being a member of the ubiquitin-like protein family that has two fused ubiquitin-like regions[9]. In recent years, FAT10, which is a ubiquitin-like protein, has received increasing attention from researchers, with Merbl et al describing its structure and function
[10]
. Studies have shown that FAT10 is the only ubiquitin-like
protein that does not rely on ubiquitin to directly degrade the targeted substrate by the proteasome[11]. Under the action of the E1 and E2 enzymes, FAT10 is linked to the Lys residue of the substrate protein and is then recognized by the receptor subunit of the proteasome before being degraded[12]. A number of studies have suggested that the FAT10 proteasome system (FPS) is a novel protein degradation system that is different from the ubiquitin-proteasome system (UPS). It has been previously reported that the FAT10 gene is upregulated in many tumor tissues, such as in liver cancer[13,14], gastrointestinal cancer[15], and bladder cancer[16], with FAT10 overexpression in liver cancer tissues being closely associated with the independent poor prognosis of patients. Lower FAT10 expression reduces the activation of NF-κB by tumor necrosis factor, TNF-α, thereby inhibiting tumor cell proliferation[17]. However, the precise role and underlying signaling cascade of FAT10 in OS progression remains unclear. The highly conserved Hippo/YAP signaling pathway plays an important role in cell growth and apoptosis[18], and the upregulation of Hippo/YAP signaling in various tumor cells indicates that it is closely associated with tumorigenesis[19,20]. YAP1 is an important effector downstream of the Hippo signaling pathway that is overexpressed in hepatocellular carcinoma
[21]
, lung cancer[22], and osteosarcoma[23], suggesting that
it is closely associated with patient prognosis. In MCF-10A cells, YAP overexpression can induce the epithelial-to-mesenchymal transition (EMT) of cells, inhibits apoptosis, and promotes the growth factor-independent growth, and nonanchored growth[24]. YAP can interact with a number of transcription factors, such as with the WW domain of RUNX2[25] and p73[26], and it can upregulate the transcription of downstream genes that promote cell growth through its TEAD binding domain (TBD) domain family
transcription factor, TEAD[27]. Nevertheless, the mechanism and role of YAP1 in regulating the growth of OS remain unclear. The goal of this study was to elucidate the mechanism through which FAT10 contributes to the prognosis of OS patients. We also investigated the mechanism underlying this effect with regard to FAT10 and YAP1 in OS, showing that FAT10 stabilizes YAP1 expression by modifying its ubiquitination and degradation. Taken together, the results of this study may provide new potential prognostic and therapeutic targets for OS.
Materials and methods 1. Human tissue specimens This study was performed with the informed consent of the patients and their families. The study program was approved by the Medical Ethics Committee of Nanchang University, and we collected tissues from 49 OS patients and the corresponding adjacent tissues from the Second Affiliated Hospital and the First Affiliated Hospital of Nanchang University. The samples were stored at -80 °C for subsequent experiments. 2. Cell line and cell culture The OS cell lines (U2-OS, 143B, MG-63, and Saos2) and a normal bone cell line (hfoBI-19) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM medium containing 10% FBS and were incubated at 37 °C under an atmosphere containing 5% CO2. 3. qRT-PCR and western blot analyses The qRT-PCR and western blot analyses were performed as described previously[28]. The sequences of the primers used in this study are shown in Table 1. 4. Immunohistochemistry (IHC) assay Osteosarcoma and non-tumor tissues were fixed, paraffin embedded, sectioned, and incubated in appropriately diluted primary antibodies against FAT10 (Abcam; 1:200) and YAP1 (Abcam; 1:200) overnight at 4 °C. Subsequently, the tissues were labeled
using an EnVision HRP kit (DAKO) for 30 min at room temperature with DAB base (DAKO) and then incubated and counterstained with Mayer’s hematoxylin (DAKO). 5. Co-immunoprecipitation (Co-IP) Appropriate cells were collected and lysed on ice by incubation with 1.0 mL of pre-cooled IP buffer for 15 min. Thereafter, the cell lysate was centrifuged at 12,000 rpm for 15 min. The supernatant was then placed in a new microcentrifuge tube and incubated with the primary antibody (1:400) for 2–4 h. Next, protein A/G PLUS-Agarose (Santa Cruz) was added, after which the mixture was incubated at 4 °C for 24 h on a rocking platform and then centrifuged at 3000 rpm for 4 min to collect the immunoprecipitate. The pellet was washed three times with 600 µL of RIPA buffer, repeating the centrifugation step each time. Finally, the pellet was resuspended in 40 µL of 3% SDS solution. After the sample was boiled for 10 min, 20 µL of it was analyzed by SDS-PAGE and autoradiography. 6. shRNA plasmids and constructs The carrier encoding FAT10 and YAP1 was obtained from GenePharma (Shanghai, China). The shRNA plasmid and vector were then transfected into OS cells using Lipofectamine 3000 (Invitrogen), following the manufacturer’s instructions. 7. Cell growth assays EdU assays OS cells were incubated with 5-acetylene-20-deoxyuridine (EdU, RiboBio, Guangzhou, China) for 2 h, as described in the manufacturer’s instructions. The OS cells were then treated with 200 µL of 1X Apollo reaction mixture for 25 min. Subsequently, Hoechst 33342 (5 µg/mL) was added to each well and the cells were stained for 30 min, and then observed under a fluorescence microscope and imaged. CCK-8 assays Cell viability was assessed using the CCK-8 method. Logarithmically growing OS cells (1×104) were plated in 96-well plates, after which CCK-8 was added to each well and the cells were incubated for 2 h. The optical density was measured using an iMark microplate reader (Bio-Rad) at 450 nm. The average of the absorbance values for five wells was calculated for each group.
8. Flow cytometry Osteosarcoma cells were harvested by treating with 0.25% trypsin. The apoptosis rate of OS cells was determined using an Annexin V-PE and PI apoptosis detection kit (BD Biosciences, USA). 9. Tumorigenicity assay OS cells (5×106) were resuspended in 100 µL of the medium and subcutaneously injected into the dorsal side of nude mice (male BALB/c-nu/nu, 5-weeks old). Tumor size was measured every 5 days. After 35 days, the mice were imaged after the administration of anesthesia, and the tumors were individually weighed and collected. 10. Statistical analysis All data were analyzed using SPSS 19.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 7. The results of three independent experiments were averaged and are presented with the standard deviation. Student’s t-test was used to compare the two groups of data, and one-way ANOVA was used to compare the two groups, with a value of P < 0.05 considered to be significant. Results 1. FAT10 expression is upregulated in OS tissues and cells To elucidate the function and importance of FAT10 in OS, we first assessed its expression levels in OS and non-tumor tissues using quantitative real-time PCR, western blot, and immunohistochemical analyses. The qRT-PCR results showed that the FAT10 mRNA levels in OS were significantly higher than in the non-tumor tissues (Fig 1A). In addition, the FAT10 protein levels were also higher in OS compared to those in the adjacent tissues (Fig 1B). We also confirmed that FAT10 expression was elevated in OS tissue using immunohistochemistry (Fig 1C). Furthermore, we confirmed that the levels of FAT10 in OS cell lines (U2-OS, MG-63, Saos-2, and 143B) were significantly higher than those observed in normal bone cells (hfoBI-19) by quantitative real-time PCR and western blot analyses (Fig 1D-E). These results showed that FAT10 is upregulated in OS tissues and cells. 2. FAT10 positively promotes OS growth in vitro and in vivo Because high levels of FAT10 protein expression are significantly associated with
increased tumor size in liver and colorectal cancers, we speculated that FAT10 might influence the OS growth. The relationship between FAT10 protein expression and OS cell growth was assessed using shRNA to silence the FAT10 expression in the OS cell lines, U2-OS and Saos-2. Results of quantitative real-time PCR and western blot analyses showed that FAT10 expression was markedly decreased (Fig 2A-B). The results of colony formation, EdU, and CCK8 assays revealed that FAT10 knockdown markedly decreased the OS cell growth (Fig 2C-E). In addition, we overexpressed FAT10 in the OS cell lines, MG-63 and 143B, and confirmed the overexpression by qRT-PCR and western blot analyses (Fig 3A-B). The results of colony formation, EdU, and CCK8 assays results revealed that the upregulation of FAT10 markedly increased the growth of OS cells (Fig 3C-E). Finally, shFAT10 and P-FAT10 OS cells were injected into mice in a tumor xenograft experimental model. After 5 weeks of growth, the results of tumorigenicity assay showed that tumors from the U2OS-shFAT10 group had lower weights and volumes
than
those
of
the
shNC
group
(Fig
2F-H).
The
results
of
immunohistochemical analysis confirmed that the level of FAT10 was significantly decreased in the shFAT10 group (Supplementary Fig 2), whereas that observed in the mice injected with P-FAT10 cells was notably increased compared to that observed in the respective controls (Fig 3F-H). Taken together, these results demonstrate that FAT10 can promote the OS cell growth in vitro and in vivo. 3. FAT10 induces cell cycle progression and inhibits cell apoptosis To further determine the effects of FAT10 on the growth of OS cells, flow cytometry was performed, the results of which indicated that downregulation of FAT10 expression significantly arrested the OS cells in the G1 phase (Fig 4A). Similarly, the results of western blot analysis showed that decreased FAT10 expression led to decreased levels of cyclin D1 and PCNA (Fig 4B). Interestingly, the cell cycle was arrested at the G1 phase in the OS cells overexpressing FAT10 (Fig 4C). Similarly, the results of western blot analysis showed that upregulation of FAT10 led to an increase in cyclin D1 and PCNA levels (Fig 4D). Furthermore, we assessed the effect of FAT10 on the apoptosis of OS cells, with the results showing a significant
increase in the apoptosis ratio in shFAT10 cells (Fig 4E). Similarly, the results of western blot analysis showed that FAT10 knockdown led to decreased BCL-2 levels and increased BAX levels (Fig 4F). In contrast, the apoptosis rate was decreased in the P-FAT10 OS cells (Fig 4G), and immunoblotting showed that upregulated FAT10 levels in OS cells could increase BCL-2 expression and decrease the BAX protein levels (Fig 4H). These results show that FAT10 can arrest the cell cycle at the G1-phase and inhibit apoptosis. 4. FAT10 positive regulates the expression levels of YAP1 protein In a previous study, it was shown that YAP1 expression is elevated in OS and can positively regulate tumor growth, but the specific mechanism associated with this activity has not been elucidated. Thus, we analyzed the expression of YAP1 by western blot analysis of shFAT10 and shNC cells. The results showed that the YAP1 expression was inhibited in the shFAT10 OS cells. (Fig 5A-B), whereas it was upregulated in the P-FAT10 OS cells (Fig 5C-D). Furthermore, we analyzed the YAP1 expression in OS tissues and observed that YAP1 mRNA and protein levels were higher in OS tissues compared to that in non-tumor tissues (Fig 5E, F and H). In addition, scatter plots revealed that FAT10 and YAP1 mRNA and protein levels were positively correlated in OS tissues (Fig 5G and I). Taken together, these findings suggest that FAT10 can promote the expression of YAP1 in OS cells. 5. YAP1 is crucial for the FAT10-mediated regulation of OS cell proliferation As mentioned above, YAP1 has been shown to function downstream of FAT10. Therefore, we studied the effect of YAP1 on the promotion of OS cell growth. First, we overexpressed YAP1 in shFAT10 OS cells, and confirmed its overexpression using western blot analysis (Fig 6A-B). The results of CCK8 and EdU assays indicated that the decrease in OS cell growth was caused by the downregulation of FAT10 expression and was partially attributable to the introduction of P-YAP1 (Fig 6C-D). Next, we downregulated YAP1 expression in FAT10-overexpressing OS cells. The results of western blot analysis confirmed the silencing of YAP1 (Fig 6E-F), and CCK8 and EdU assays revealed that the increase in cell growth induced by FAT10 upregulation in 143B cells was inhibited by the introduction of shYAP1 (Fig 6G-H).
Taken together, these findings suggest that YAP1, as a downstream target of FAT10, mediates FAT10-induced OS cell proliferation. 6. FAT10 stabilizes YAP1 expression by modifying its ubiquitin-mediated degradation To decipher the mechanism through which FAT10 regulates the expression of YAP1, we first performed Co-IP assays to verify whether FAT10 and YAP1 could bind to each other. The results of Co-IP showed that FAT10 can directly bind to YAP1 (Fig 7A). Interestingly, it was reported previously that YAP1 undergoes degradation via the UPS[29], and the results of our previous studies confirmed that FAT10 is able to stabilize the ubiquitination-mediated degradation of substrate proteins[30]. Based on these observations, we hypothesized that FAT10 may promote the expression of YAP1 by inhibiting its ubiquitination and degradation. To test this hypothesis, we incubated the OS cells with the proteasome inhibitor, MG132, for a specific period of time, which resulted in significant accumulation of endogenous YAP1 protein in the OS cell lines, U2-OS and Saos-2 (Fig 7B). These results indicate that YAP1 is also degraded by the UPS in OS cells. In addition, to further elucidate whether FAT10 is associated with the regulation of YAP1 protein degradation, we transfected shROCK2 and P-FAT10 vectors into U2-OS and 143B cells and tested the effect of different expression levels of FAT10 on YAP1 expression, in the presence or absence of MG132. The results indicated that the upregulation or knockdown of FAT10 levels did not affect the YAP1 expression in OS cells after the addition of MG132 (Fig 7 C-D). In addition, analysis of the kinetics of YAP1 degradation indicated that the half-life of ectopically expressed YAP1 in the U2-OS and Saos-2 cells overexpressing FAT10 was significantly increased (Fig 7E-F). These results suggest that FAT10 is involved in the degradation of YAP1. We further evaluated the role of FAT10 in the degradation of YAP1. The results showed that the heterotopic dose-dependent effect of upregulation of FAT10 resulted in a significant increase in the level of endogenous YAP1 protein in U2-OS and Saos-2 cells (Fig. 7G-H). In addition, a single dose of HA-YAP Flag-FAT10 was
co-transfected into U2-OS and Saos-2 cells. A dose-dependent effect of upregulated FAT10 levels on the YAP1 expression was observed (Fig. 7I), indicating that FAT10 can stabilize the expression of YAP1. Finally, to elucidate the mechanism through which FAT10 stabilizes YAP1 expression, we treated the U2-OS/shFAT10 and 143B/pcDNA3.1(+)-FAT10 cells with MG132. The results of immunoprecipitation indicated that inhibition of FAT10 levels increased the levels of YAP1 ubiquitination, whereas the overexpression of FAT10 significantly reduced the YAP1 ubiquitination (Fig 7G). These results indicated that FAT10 is able to stabilize the ubiquitination and degradation of YAP1 to promote its expression. Discussion An important feature of OS is its rapid growth, and studies have shown that the proliferation of OS is closely associated with the abnormal expression of the related proto-oncogenes[31]. Despite the rapid development in surgical and oncological chemotherapeutic drugs, the prognosis and 5-year survival rate of OS patients have remained stagnant. Thus, we need to study and explore the growth mechanisms of OS to provide a more theoretical basis for targeted therapy of OS. The results of this study demonstrate that FAT10 plays a key role in OS cell proliferation. FAT10 belongs to Ubls and comprises an HLA-F site at the telomere end of MHC class I[32]. There has been increasing evidence that FAT10 is upregulated in a variety of malignancies, and abnormal FAT10 expression is closely associated with tumor growth and metastasis. Yuan and colleagues showed that FAT10 is closely associated with the prognosis of liver cancer[30], and silencing FAT10 expression could inhibit the growth and metastasis of this cancer. In contrast, the growth and metastasis of cells expressing FAT10 is significantly enhanced. Similarly, Dong et al. reported that FAT10 expression is closely associated with bladder cancer[16]. In addition, we found that FAT10 is highly expressed in many tumors, and there is a clinical subgroup with high FAT10 level in some tumors. We also found that FAT10 was related to the survival rate of many tumor patients in TCGA database. Although Ma et al. reported that silencing FAT10 could inhibit the invasion and migration of OS[33], the specific
mechanism associated with this activity has not been studied in detail, and FAT10 has not been reported to be involved in the growth of OS. Our results in the present study showed that FAT10 expression was significantly increased in OS specimens, and OS cell growth could be inhibited or enhanced by silencing and overexpressing FAT10 expression, respectively, in vivo and in vitro. Therefore, these findings suggest that FAT10 may function as an oncogene in OS. The Hippo/YAP signaling pathway plays a crucial role in tumor progression, and it was confirmed in a previous study that this pathway is abnormally activated in many cancers and is closely related to poor tumor prognosis[34]. As a downstream factor of the Hippo/YAP signaling pathway, YAP1 also plays an important role in regulating the growth and metastasis of tumors. Yang et al. studied the effect of silencing of YAP1 on the inhibition of OS growth in vitro and in vivo[35]. In addition, many studies have confirmed that YAP1 acts as a target for lncRNAs and circRNAs to regulate the growth of OS[36]. However, in our study, the data indicate that YAP1 is expressed in OS tissues and cells and is positively correlated with FAT10 expression. Knockdown of FAT10 expression could significantly reduce the mRNA and protein expression levels of YAP1, and FAT10 overexpression resulted in a significant increase in the expression of YAP1 mRNA and protein. Additionally, we have shown that the ability of FAT10 to regulate the growth of OS is dependent on the expression of YAP1. Previous studies have shown that FAT10 can affect the ubiquitination-mediated degradation of substrate proteins and that YAP1 can be degraded in response to ubiquitination. In this study, we confirmed the direct binding of FAT10 and YAP1 by immunoprecipitation, after which FAT10 was shown to significantly promote the level of YAP1 ubiquitination, as FAT10 overexpression suppressed YAP1 ubiquitination. Finally, FAT10 was shown to decrease the half-life of YAP1. In summary, the results of our study confirm that FAT10 is expressed at high levels in OS tissues and cells. In addition, our study also shows that FAT10 can promote the growth of OS in vitro and in vivo. More importantly, FAT10-induced OS cell growth was observed to be dependent on YAP1. Our findings also demonstrate that FAT10 inhibits the ubiquitination and degradation of YAP1. Based on these findings, FAT10
can serve as a candidate biomarker for the diagnosis and treatment of OS in the future. Acknowledgements We thank Elsevier’s English Language Editing service for editing this manuscript. Funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 81760487) and the Project of Jiangxi Provincial Department of Science and Technology (No. 2019ACBL20035). Consent for publication All authors have read and approved the final version of the manuscript. The authors and participants agreed to its publication. Competing interests The authors declare that they have no competing interests.
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Figure legends Figure 1 FAT10 expression is upregulated in osteosarcoma (OS) tissues and cells A-B. qRT-PCR and western blot analyses of FAT10 mRNA and protein expression in OS tissues (n = 30) and in their corresponding adjacent tissues (n = 30) (**p<0.01). C. Representative images and quantification of FAT10 staining in 49 paired OS and non-tumor tissues (**p<0.01). D-E. Relative FAT10 mRNA and protein levels in OS cells (U2-OS, Saos-2, MG-63, and 143B) compared to that observed in hfoBI-19 cells (**p<0.01). Figure 2 Knockdown of FAT10 expression inhibited the growth of osteosarcoma (OS) in vitro and in vivo A. Western blot analysis of FAT10 protein levels after shRNA transfection of the OS cell lines, U2-OS and Saos-2. B. Relative FAT10 mRNA levels in the OS cell lines, U2-OS and Saos-2, as detected by qRT-PCR (**p<0.01). C-D. Results of CCK8 and EdU assays showing that knockdown of FAT10 levels significantly decreases the growth of OS cells. E. Representative images for the clone formation assay of OS cells transfected with shFAT10. shFAT10/U2-OS and shNC/U2-OS cells were subcutaneously injected into nude mice, and at the end of the experiment, the tumors were imaged (F) and weighed (H, n = 5, *p<0.05, **p<0.01). G. The volumes of tumors were measured on the indicated days. Figure 3 Up-regulated expression of FAT10 promotes osteosarcoma (OS) growth in vitro and in vivo A-B. Western blot and qRT-PCR analyses of FAT10 mRNA and protein levels in OS cells after FAT10 overexpression (*p<0.05, **p<0.01). C-D. CCK8 and EdU assays
showing the increased proliferation of OS cells treated with MG-63 and 143B (*p<0.05, **p<0.01). E. Representative images of OS cells after upregulation of FAT10 expression in the clone formation assay. The vector and P-FAT10/U2-OS cells were subcutaneously injected into nude mice, and tumor volumes were measured on the indicated days (G). At the experimental endpoint, tumors were imaged (F) and weighed (H, n = 5, *p<0.05, **p<0.01). Figure 4 FAT10 induces cell cycle progression and inhibits cell apoptosis A. The percentage of osteosarcoma (OS) cells in different cell cycle phases was determined by FACS analysis, with the results showing that the number of U2-OS and Saos-2 OS cells in the G1 phase was increased after transfection with shFAT10 (*p<0.05). B. Western blot analysis for the detection of cyclin D1 and PANC protein levels in shFAT10 cells compared to that observed in shNC cells. C. The number of cells in the G1 phase was decreased in P-FAT10 cells compared to the vector cells (*p<0.05). D. The expression of cyclin D1 and PANC proteins was upregulated in the P-FAT10 cells. E-G. The apoptosis rate of OS cells was detected by flow cytometry and was significantly increased in the shFAT10 cells (E, **p<0.01) but decreased in the P-FAT10 cells (G, *p<0.05). F-H. Western blot analysis of the relative expression levels of the apoptosis-associated proteins, caspase3, BCL-2, and BAX in shFAT10 (F) or P-FAT10 cells. Figure 5 FAT10 positively regulates YAP1 protein expression A-B. The relative level of YAP1 expression in osteosarcoma (OS) cells was detected by qRT-PCR and western blot analyses after transfection with shFAT10 or shNC plasmids (**p<0.01). C-D. The mRNA and protein levels were increased in OS cells transfected with the P-FAT10 plasmid (**p<0.01). E-H. Observation (E) and quantification (H, **p<0.01) of FAT10 protein levels in the OS tissues (n = 30) compared to that observed in non-tumors (n = 30) by western blot analysis. F. Determination of FAT10 mRNA levels in OS tissues (n = 30) compared to that in non-tumor tissues (n = 30) by qRT-PCR (**p<0.01). G and I. Scatter plots of FAT10 and YAP1 mRNA and protein expression levels in OS. Figure 6 YAP1 is crucial for the FAT10-mediated regulation of osteosarcoma (OS)
cell proliferation A-B. Western blot analysis of FAT10 and YAP1 protein expression in U2-OS and Saos-2 OS cells, showing that YAP1 overexpression can mitigate the loss of YAP1 levels in shFAT10 cells. C-D. Results of CCK8 and EdU assays showing that the upregulation of YAP1 expression could restore the proliferation of OS cells transfected with the shFAT10 plasmid (*p<0.05, **p<0.01). E-F The relative levels of FAT10 and YAP1 were detected by western blot analysis, showing that the knockdown of YAP1 expression could decrease the YAP1 levels in P-FAT10 cells. G-H. Results of CCK8 and EdU assays showing the proliferation of OS cells stably transfected with P-FAT10 in the presence or absence of shYAP1 (*p<0.05, **p<0.01). Figure 7 FAT10 stabilizes YAP1 expression by modifying its ubiquitin-mediated degradation A. Co-immunoprecipitaion of endogenous FAT10 and YAP1 in U2-OS and Saos-2 osteosarcoma (OS) cells. B. Western blot analysis of YAP1 expression in cells treated with MG132 (15 µM) for the indicated times. C and D. After transfection with the shFAT10 or P-FAT10 plasmids, no significant change in YAP1 expression was observed upon addition of MG132. E and F. U2-OS and Saos-2 cells were transfected with expression plasmids encoding HA-YAP1 with or without the Flag-FAT10 plasmid. The cells were then exposed to CHX (20 µM) for the indicated times, and the degradation of YAP1 was detected using an anti-HA antibody. G-H. U2-OS and Saos-2 cells were transfected with increasing amounts of Flag-FAT10 plasmid, and endogenous YAP1 expression levels were detected using an anti-YAP1 antibody. U2-OS and Saos-2 cells were either untreated or transfected with a single dose of the expression plasmid encoding HA-YAP1 alone or in combination with the Flag-FAT10 plasmid. The level of YAP1 expression was detected using an anti-HA antibody. H and I. The level of ubiquitinated YAP1 in shFAT10 or P-FAT10 OS cells treated with MG132 (15 µM) was assessed by western blot analysis with a Ub antibody. 8. Figure 8 Proposed model for the mechanism through which FAT10 promotes the growth of osteosarcoma (OS) cells by regulating the expression of YAP1.
Primers for real-time PCR Gene
Primer sequences
FAT10
Forward:5'-CCGTTCCGAGGAATGGGATTT-3′ Reverse:5'-GCCATAAGATGAGAGGCTTCTCC-3′
YAP1
Forward:5'-GCAACTCCAACCAGCAGCAACA-3′ Reverse:5'-CGCAGCCTCTCCTTCTCCATCTG-3′
GAPDH
Forward:5'-AGCCTCAAGATCATCAGCAATG-3′ Reverse:5'-CCATCACGCCACAGTTTCC-3′
Highlights • • • •
FAT10 silencing inhibited cancer cell proliferation in vivo and in vitro YAP1 represents a novel target for the prevention and treatment of OS The expression of FAT10 and YAP1 are up-regulate in OS FAT10 stabilizes YAP1 expression by modifying its ubiquitination and degradation.
Authors’ contributions section Liang Hao and Yunyan Du conceived and designed the study. Xuan Yi, Xueqiang Deng, and Binbin Deng performed the experiments and collected data; Jianyong Deng and Huimin Fan performed the data analysis; All authors discussed and interpreted the data. Xueqiang Deng and Xuan Yi wrote the manuscript. All authors reviewed and edited the manuscript. All authors read and approved the final manuscript.
Conflict of interest statement The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted Sincerely, Liang Hao, M.D., Ph.D. Department of Orthopedics The Second Affiliated Hospital of Nanchang University No.1 Minde Road,Nanchang 330006,Jiangxi, China Phone: +86 791 86265564 E-mail: address: [email protected]