FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10

FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10 Siwei Hu, Yihong Chen, Yuting Wen, Linglan Tu, Wenhu Chen, Kangsheng Tu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6568148/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract objectives Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and its incidence and mortality rates remain high. Therefore, new diagnostic and therapeutic approaches are urgently required. FAM188B mRNA encodes an evolutionarily conserved protein that is highly expressed in various cancers. While FAM188B has been implicated in the progression of several tumors, its role in HCC progression remains unknown. Methods We analyzed FAM188B expression in HCC using TCGA and UALCAN databases. Functional studies included in vitro proliferation, migration, and invasion assays, as well as in vivo xenograft models. Co-immunoprecipitation (Co-IP), Western blotting, and immunofluorescence were used to investigate the FAM188B-USP10-YAP/TAZ interaction. Results FAM188B was found highly expressed in HCC cells and associated with poor prognosis. Both in vitro and in vivo, FAM188B promoted the proliferation, migration, and invasion of HCC. FAM188B directly interacts with and stabilizes USP10 and the downregulation of FAM188B by shRNA led to decreased USP10 and YAP/TAZ protein levels, suggesting that FAM188B may regulate the YAP/TAZ pathway through its interaction with USP10. Conclusion This study suggests that FAM188B is involved in the proliferation, migration and invasion of hepatocellular carcinoma (HCC) and the mechanism may involve the regulation of the USP10/YAP/TAZ signalling pathways in vitro and in vivo. Hepatocellular carcinoma FAM188B USP10 YAP/TAZ pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, after lung and colorectal cancer[ 1 ]. Surgical resection, liver transplantation and local ablation are effective treatment options for early-stage HCC. However, due to its late symptom onset, most patients are diagnosed with advanced-stage HCC and have a poor prognosis[ 2 ]. The multi-kinase inhibitor sorafenib remains a systemic therapy for advanced-stage HCC, although it only prolongs patient survival by a few months and has a low response rate[ 3 ]. Moreover, the treatment benefit of regorafenib, lenvatinib and cabozantinib is suboptimal[ 4 – 6 ], and it is clinically important to understand the pathogenesis of HCC and identify effective interventions. Family with sequence similarity 188 member B (FAM188B) is a novel gene encoding an evolutionarily conserved protein in mammals, and its mRNA is highly expressed in a variety of solid tumors. In colon cancer, FAM188B knockdown induces p53 activation and accumulation and increases apoptosis[ 7 , 8 ]. Furthermore, FAM188B suppression sensitises lung cancer cells to anoikis by downregulating epidermal growth factor receptor (EGFR) expression and inhibits tumor metastasis in vivo. It also co-precipitated with forkhead box M1 (FOXM1), and FOXM1 ubiquitination levels increased with FAM188B knockdown but decreased with FAM188B overexpression[ 9 , 10 ]. However, the role of FAM188B in HCC progression remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a member of the ubiquitin-specific protease family and is a highly conserved deubiquitinase in mammals[ 11 ]. Further, it has been implicated in the progression of various malignancies, including HCC, pancreatic cancer, breast cancer and colorectal cancer[ 12 – 15 ]. In addition, USP10 promotes HCC proliferation by deubiquitinating and stabilising of YAP/TAZ[ 16 ]. The Hippo pathway and its downstream effectors, transcriptgional coactivator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP), are overexpressed in human cancers[ 17 ]. In the liver, acute inactivation of Hippo signalling in vivo leads to the dedifferentiation of adult hepatocytes into progenitor cells with single-cell self-renewal and cellular plasticity[ 18 ]. Recent studies have shown that YAP/TAZ is associated with tumorigenesis in most solid tumors and that its activation induces proliferation, metastasis, and chemotherapy resistance in cancer stem cells[ 19 ]. Additionally, US Food and Drug Administration-approved drugs that indirectly block YAP/TAZ activation or its key downstream targets have significantly reduced drug resistance[ 20 ]. In this study, we investigated the role of FAM188B in promoting HCC progression and the potential underlying mechanisms. Our analysis suggests that FAM188B interacts with USP10 to regulate YAP/TAZ. These data indicate that FAM188B may be an important diagnostic and prognostic biomarker for controlling HCC progression. 2. Materials and Methods 2.1 Datasets from the public databases Datasets for liver cancer cells were obtained from TCGA (The Cancer Genome Atlas). The UALCAN (The University of Alabama at Birmingham Cancer data analysis Portal) and GEPIA (Gene Expression Profiling Interactive Analysis) websites were used for data visualization. The Kaplan-Meier method and the log-rank test were used to analyse the survival of patients with liver cancer. 2.2 Cell Culture and Transfection The normal human liver cell line L02 and the human liver tumor-derived cell lines MHCC97H and huh7 were obtained from the Whelab Biotechnology LTD (Shanghai, China) in 2021. Human liver tumor-derived cell lines SNU387 and Hep3B, human embryonic kidney HEK392T cells were obtained from the American Type Culture Collection (Manassas, VA, USA) in 2021. All cell lines were maintained in DMEM, MEM or RPMI-1640 media (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Sigma- Aldrich, St. Louis, MO, USA) at 37℃, in a 5% CO₂ humid atmosphere. The cell incubator was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The cells used for the experiments were passaged no more than10 times after thawing. Lentivirus-mediated FAM188B shRNA (sh-FAM188B) and negative control shRNA (sh-NC) were purchased from Genechem (Shanghai, China). The sequence of sh-FAM188B is shown in Table S1 . The pcDNA3.1-FAM188B-Flag and pcDNA3,1-USP10-Myc plasmids were obtained from GuanNan. Co, Ltd (Hangzhou, China). Plasmids were delivered into the cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and lentivirally encapsulated sh-FAM188B or sh-NC was transfected into cells using polyberene (Beyotime, Shanghai, China) according to the manufacturers’ instructions. Finally purinomycin (ST551-50 mg, Beyotime, Shanghai, China) was screened with 2 µg/ml for one week to obtain stable transfection cell lines. 2.3 Cell proliferation and Transwell assays Transfected HCC cells were added to a 96-well plate at a density of 5 × 10 3 /well, and 10 µl of CCK-8 solution (Beyotime, Shanghai, China) was added to each well at the same time every day for 4 days. After 1.5 hours of incubation, the absorbance was measured at 450 nm using a microplate reader (Thermo-Fisher Scientific, Waltham, MA, USA). FAM188B knockdown HCC cells were seeded at a density of 1 × 10 5 cells/well in the upper chamber of the Transwell insert. For the invasion experiment, each upper chamber was coated with Matrigel matrix (Corning, Corning, NY, USA). Each of the lower chambers contained 500 µl of medium supplemented with 10% FBS. After 24 hours of cultivation, migrating and invading cells were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.4% ammonium oxalate crystal violet and counted microscopically in five random fields. Transwell chambers were purchased from Corning (Corning, NY, USA), and 0.4% ammonium oxalate crystal violet was purchased from YuanyeBio (Shanghai, China). 2.4 Reagents The proteasome inhibitor MG-132 and protein synthesis inhibitor CHX (Cycloheximide) were purchased from Selleck Chem (Houston, TX, USA). RIPA lysis buffer, Enhanced BCA (Bicinchoninic acid) Protein Assay Kits, TBS with Tween-20, Phosphate Buffered Saline (PBS, premixed powder), Antifade Mounting Medium with DAPI and Western rapid transfer buffer were purchased from Beyotime (Shanghai, China). Protease and phosphatase inhibitors were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The FastPAGE precast gel and MOPS-SDS running buffer were purchased from Tsingke (Beijing, China). Polyvinylidene difluoride (PVDF) membranes and an ECL kit were purchased from Millipore (Bedford, MA, USA). The skim milk powder and neutral buffered formalin (10%) were purchased from Biosharp (Hefei, China). The Triton X-100 was purchased from Solarbio (Beijing, China). The Bovine Serum Albumin (BSA) lyophilized powder was obtained from Sigma-Aldrich (St. Louis, MO, USA). The following primary antibodies were used: anti-FAM188B (WB, 1:1000; IHC, IF, 1:200; ab122097) was purchased from Abcam (Cambridge, UK), anti-β-actin (WB, 1:1000; 13E5) was purchased from Cell Signaling Technology (Beverly, MA, USA), anti-TAZ (1:5000; 23306-1-AP) was purchased from Proteintech (Wuhan, China), anti-ubiquitin (1:2000; PTM-1106RM) was purchased from PTMBIO (Hangzhou, China), anti-USP10 (WB, 1:1000; IHC, IF, 1:200; ET1706-12), anti-YAP (1:1000; ET1608-30), anti-E-cadherin (1:5000; ET1607-75), anti-N-cadherin (1:2000; ET1607-37), anti-Vimentin (1:10000; ET1610-39), anti-Flag-tag (1:1000; 0912-1), anti-Myc-tag (1:1000; R1208-1) and anti-Ki67 (1:200; ET1609-34) were purchased from HuaBio (Hangzhou, China). 2.5 qRT-PCR Cells were harvested in TRIzol reagent (Ambion, Austin, TX, USA) for total RNA extraction, and 1 µg RNA was used for cDNA transcription as following the instructions of the Hifair III 1st Strand cDNA Synthesis SuperMix Kit(Yeasen, Shanghai, China). Amplification and quantification were carried out with a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green Master Mix (Yeasen, Shanghai, China). The relative expression of mRNAs was normalized to GAPDH using 2 −△△Ct method. The sequences of all primers are listed in the Supplementary Table 1. 2.6 Immunoblotting and co-immunoprecipitation For immunoblotting, proteins were collected using a lysis buffer containing protease and phosphatase inhibitors, and boiled at 95°C for 5 minutes after the addition of loading buffer. The protein concentration in each sample was determined using the BCA protein assay kit. Protein samples were separated on 4%~20% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline and Tween-20 (TBS-T) for 1hour at approximately 25°C, and then incubated with primary antibodies overnight (8h ~ 12h) at 4°C and washed three times for 10 min with TBS-T each time. HRP-conjugated antibodies (Cell Signaling Technology, Beverly, MA, USA) were incubated with the membranes for 1 hour at approximately 25°C and then washed three times with TBS-T for 15 minutes each time. The labelled proteins were visualized by enhanced chemiluminescence using the ChemiDoc Imaging System (Hercules, CA, USA). Finally, protein levels were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalised to β-actin. The pcDNA-FAM188B-Flag and pcDNA-USP10-Myc plasmids were transfected into HCC cells. After 48 hours, the cells were harvested with lysis adding cocktail and the Flag-tag or Myc-tag Protein IP Assay Kit (Beyotime, Shanghai, China) was used for co-immunoprecipitation according to the manufacturer's instructions. 2.7 Immunofluorescence MHCC97H and Huh7 cells (1×10 5 /100 µl) with sh-FAM188B or sh-NC were cultured in confocal dishes (Biosharp, Hefei, China) for 24 hours. The cells were washed with PBS solution and fixed with neutral buffered formalin (10%) for 15 minutes and 0.1% Triton (diluted with PBS) for 20 minutes at approximately 25°C. Cells were blocked with 10% BSA (diluted with PBS) for 1 hour at 25°C and incubated with primary antibodies at 4°C overnight (8h ~ 12h). The next day, cells were washed three times with PBS for 15 minutes each time and incubated with fluorescent antibodies for 1 hour at 25°C in the dark. The dishes were washed three times with PBS for 15 minutes each time. Finally, an appropriate amount of Antifade Mounting Medium containing DAPI was dripped onto the dishes and images were captured using the confocal fluorescence microscope (Nikon, Tokyo, Japan). 2.8 In vivo Experiments and Immunohistochemical Staining of Tumor Tissues Four-week-old BALB/c nude mice were obtained from the Laboratory Animal Center of Hangzhou Medical College. Huh7 cells (1×10 6 /100 µl) with sh-FAM188B or sh-NC were injected subcutaneously into the mice (n = 5 in each group). The tumor volume (V) was calculated as follows: V = π/6×L×W×H, where L, W and H represent the tumor length, width and height, respectively. For immunohistochemical staining, tissue samples were deparaffinized, rehydrated and washed in PBS solution three times for five times. The tissue sections were placed in boiling citrate buffer for 6 minutes for antigen retrieval, then removed after natural cooling to about 25℃, and washed with PBS solution three times for 5 minutes. The samples were blocked at 37℃ with 3% H 2 O 2 solution for 20 minutes, and then washed in PBS solution three times for five minutes. Slides were blocked in 5% BSA solution for 1 hour and incubated with primary antibodies at 4 ℃ overnight. After blocking, slides were washed with TBS-T and incubated with secondary antibodies (HuaBio, Hangzhou, China) for 1 hour. DAB horseradish peroxidase color development Kit (Beyotime, Shanghai, China) was used to visualize staning. IHC images were captured with a microscope (Leika, Weztlar, Germany). 2.9 Statistical Analysis Data from three independent experiments are presented as mean ± standard error (SEM). Comparisons between two or more groups were made using one-way analysis of variance or Student's t-test. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Inc., San Diego, CA, USA). p-values < 0.05 were considered statistically significant. 3. RESULTS 3.1 FAM188B expression is upregulated in HCC and predictive of poor patient survival Previous studies have shown that FAM188B expression is upregulated in human colorectal and lung cancers and promotes the malignant progression of cancer. However, its role in HCC has not yet been determined. To investigate the effect of FAM188B on HCC, we analysed its expression in various cancers. An analysis of The Cancer Genome Atlas (TCGA) data showed that FAM188B is highly expressed in liver cancer and closely related to the HCC tumor grade and cancer stage according to UALCAN (uab.edu) (Fig. 1 A). We also extracted 50 sets of RNA-seq data from TCGA-LIHC and analysed them statistically using the Wilcoxon signed-rank test. The results showed that FAM188B mRNA expression was up-regulated in tumour tissue compared to para-carcinoma tissue. (Fig. 1 B). In addition, the joint analysis based on TCGA and GTEx databases showed that FAM188B expression in HCC tissues was significantly higher than that in normal liver tissues (Fig. 1 B). We found that high FAM188B mRNA levels predicted shorter survival using Kaplan-Meier survival analysis of the TCGA data (Fig. 1 C). Furthermore, FAM188B protein levels were markedly elevated in HCC cell lines (MHCC97H and Huh7) compared to normal hepatocyte cell lines (L02). However, this elevation was not observed in the other two cell lines (Hep3B and SNU387) (Fig. 1 D). To further investigate the role of FAM188B in the malignant progression of HCC, we used immunofluorescence to verify its localisation in HCC cells. L02, MHCC97H and Huh7 cells were stained with an anti-FAM188B antibody and DAPI, which was followed by confocal microscopy analysis. FAM188B was found to be located in the nucleus (Fig. 1 E). These data suggested that FAM188B has a significant oncogenic effect in HCC. 3.2 FAM188B promotes HCC cells proliferation, migration and invasion To investigate the effect of FAM188B on malignant progression, we generated FAM188B knockout cell lines (MHCC97H and Huh7) (Fig. 2 A). Transwell results showed that FAM188B knockdown suppressed metastasis (Fig. 2 B) and CCK-8 results suggested inhibition of proliferation with decrease of FAM188B (Fig. 2 C). Since epithelial–mesenchymal transition (EMT) contributes to the enhanced migration and invasion of tumor cells, we also investigated whether FAM188B mediates this process. Immunoblotting results showed that FAM188B expression was negatively correlated with E-cadherin but positively correlated with N-cadherin and vimentin (Fig. 2 D). Next, FAM188B-overexpressing HCC cells (SNU387 and Hep3B) were generated (Fig. 3 A); in these two cell lines, transwell experiments showed that FAM188B promoted the tumor cell migration and invasion of tumor cells (Fig. 3 B). Experimental results from the CCK-8 assay further demonstrated that FAM188B overexpression could enhance HCC cell proliferation (Fig. 3 C). Taken together, these data indicate that FAM188B expression is important for the growth and metastasis of liver cancer cell lines. 3.3 FAM188B interacts with USP10 in HCC cells To confirm the molecular mechanism underlying the effects of FAM188B in HCC cells, immunoprecipitation (IP) was performed based on FAM188B-Flag expression in MHCC97H and Huh7 cells. We then searched for potential FAM188B-binding proteins using liquid chromatography/mass spectrometry (LC/MS)-MS (Fig. 4 A and B). Based on the results, we speculated that FAM188B was related to the level of ubiquitination in HCC cells and that it might bind to ubiquitination-related enzymes. MS was further used to screen four deubiquitinating enzymes that potentially interact with FAM188B in HCC cells, including USP7, USP9X, USP10, and USP47 (Fig. 4 B). Among the four deubiquitinating enzymes, USP10 attracted our attention because it is a novel regulator of YAP/TAZ signalling and promotes HCC proliferation by deubiquitinating and stabilizing YAP/TAZ. Moreover, analysis revealed that USP10 is highly expressed in HCC and positively correlated with FAM188B expression according to GEPIA (cancer-pku.cn) (Fig. 4 C). Thus, we selected this protein as a potential FAM188B interactor for further experiments. The protein level of USP10 was found to be elevated in hepatocellular carcinoma (HCC) cells (MHCC97H, Huh7, Hep3B and SNU387) in comparison to normal liver cells (L02) (Fig. 4 D). Immunofluorescence experiments were also performed to demonstrate the localisation of USP10 in the cytosol and nucleoplasm of HCC cells (Fig. 4 E). We also performed co-IP assays, and the results showed that USP10 can interact with FAM188B in HCC cells (Fig. 4 F). 3.4 FAM188B positively regulates USP10-YAP/TAZ signalling pathway To verify the regulatory relationship between FAM188B and USP10, we performed immunoblotting and immunofluorescence assays using two HCC cell lines transfected with FAM188B shRNA (sh-FAM188B) and found that endogenous FAM188B deficiency noticeably reduced the protein abundance and fluorescence intensity of USP10 (Fig. 5 A, B). Furthermore, we explored the effect of FAM188B knockdown on endogenous YAP/TAZ levels using immunoblotting and qRT-PCR. We found that FAM188B suppression reduced YAP/TAZ protein levels without affecting their mRNA levels in MHCC97H and Huh7 cells (Fig. 5 C, D). To further demonstrate the positive regulatory relationship between FAM188B and the USP10–YAP/TAZ signalling pathways. HEK293T cells were transfected with a plasmid expressing FAM188B with a Flag-tag and a plasmid encoding USP10 with a Myc-tag. Subsequently, immunoblotting and qRT-PCR experiments were conducted. Our results show that exogenous FAM188B can increase the abundance of USP10 and YAP/TAZ proteins. Consistently, exogenous USP10 increased FAM188B and YAP/TAZ protein levels (Fig. 6 A). Of note, the qRT-PCR results showed that the overexpression of FAM188B had no effect on USP10 and YAP mRNA levels, but significantly increased TAZ mRNA levels (Fig. 6 B). This suggests that other pathways regulate FAM188B and YAP/TAZ signalling at the transcriptional level. To further ascertain whether FAM188B affects the stability of the USP10 protein, MHCC97H and Huh7 cells were treated with cycloheximide (CHX) for the indicated times following FAM188B knockdown. The results showed that FAM188B knockdown promoted USP10 degradation (Fig. 6 C). To acquire a deeper understanding of the regulatory relationship between FAM188B and USP10–YAP/TAZ, we restored the protein levels of USP10 and YAP/TAZ using the proteasome inhibitor MG132 in FAM188B-knockdown cells (Fig. 6 D). Moreover, the ubiquitination levels of total cellular proteins were significantly higher in the sh-FAM188B-treated group than in the control group (Fig. 6 E). Taken together, these results demonstrate that FAM188B positively regulates YAP/TAZ protein levels through its interaction with USP10. 3.5 FAM188B knockdown inhibits HCC growth in vivo To assess whether FAM188B could be a novel target for controlling HCC growth, Huh7 cells with or without sh-FAM188B transfection were injected subcutaneously into BALB/c nude mice (n = 5) and tumor growth was monitored. Mice were euthanised by cervical dislocation under ether anaesthesia on day 30 after inoculation. Consistent with the results of the in vitro experiments, tumor burden was lower in the shFAM188B treatment group than in the control group (negative control shRNA; sh-NC) (Fig. 7 A). Isolated tumors were then subjected to total protein extraction and immunohistochemistry. Immunoblotting results showed FAM188B protein expression was decreased in the shFAM188B treatment group (Fig. 7 B). Immunohistochemical analysis further demonstrated that the protein levels of USP10, YAP and TAZ, as well as Ki-67, a proliferation marker, were downregulated following FAM188B knockdown in the tumor tissue (Fig. 7 C). These data demonstrate that FAM188B knockdown inhibits HCC growth in vivo. 4. Discussion The lack of targets for HCC intervention remains a major challenge in its treatment. Due to the critical and widespread functions of the YAP/TAZ signalling pathway in HCC cells, the exploration of upstream regulatory factors to develop potential therapeutic strategies has become more feasible. Previous studies have shown that YAP/TAZ is involved in tumor glucose metabolism, fatty acid metabolism, α-ketoglutarate metabolism and glutamine catabolism, all of which provide energy for tumor growth and synthetic materials necessary for tumor cell activity[ 21 ]. Genetic studies have demonstrated that the Hippo pathway is a critical regulator of liver size, regeneration, development, metabolism and homeostasis. Therefore, dysregulation of the Hippo pathway can lead to liver diseases, such as fatty liver and cancer. Drugs that target the Hippo pathway promote liver regeneration and prevent the development or progression of liver disease[ 22 ]. Furthermore, studies have shown that a pair of proteins synergistically promote cancer progression and co-targeting these proteins, YAP/TAZ, has achieved significant efficacy in anticancer therapy[ 23 ]. In the present study, we demonstrated that FAM188B interacts with USP10 and indirectly regulates YAP/TAZ expression, thereby promoting HCC progression. USP10 has multiple roles in tumor initiation and progression, and its oncogenic and anticancer effects depend primarily on its substrates in different malignant tumors[ 24 – 27 ]. A recent study showed that USP10 interacts with ANLN and positively regulates its protein levels, thereby promoting cytoplasmic division and proliferation in oesophageal squamous cell carcinoma cells[ 24 ]. Additionally, USP10 has shown efficacy in preclinical models of oncogenic FLT3, including cell lines, primary patient samples and mouse models of oncogenic-FLT3-driven leukaemia[ 25 ]. Previous studies have also demonstrated that USP10 regulates the localisation and stability of p53 by deubiquitinating it[ 27 ]. Through a comprehensive analysis of HCC patient samples, PDX models and TCGA data, studies have shown that USP10 exerts oncogenic effects in HCC through the deubiquitination and stabilisation of YAP/TAZ[ 26 ]. FAM188B, also known as MINDY-4, is distantly related to FAM63A (MINDY-1)[ 28 ]. Although FAM188B is annotated in gene databases as a putative ubiquitin carboxyl-terminal hydrolase, its functions are not fully undestood[ 29 ]. Notably, FAM188B has been identified as an interactor of USP7, a deubiquitinating enzyme that has been shown to stabilize p53 through deubiquitination. This interaction is significant as it implicates FAM188B in the regulation of cell survival and apoptosis, which are critical biological processes in cancer development. The functional implications of elevated FAM188B expression in tumors are further substantiated by the observation that FAM188B knockdown results in a significant increase in apoptotic cellular phenotypes across different cancer types, thereby reinforcing its role as a potential therapeutic target in cancer treatment. Our data showing that FAM188B has no effect on USP10 and YAP/TAZ mRNA and but affects protein levels, particularly in the context of protein synthesis inhibition (CHX) and proteasome suppression (MG132), suggest that FAM188B regulates USP10 and YAP/TAZ levels by affecting protein stability rather than transcription. Notably, a previous study demonstrated the role of FAM63A (MINDY-1) in stabilizing YAP, via deubiquitination in bladder cancer cells[ 30 ]. Further investigation is required to determine whether FAM188B plays a similar role in HCC. Finally, Huh7 cells transfected with sh-FAM188B or sh-NC were injected subcutaneously into nude mice and the results showed that low expression of FAM188B resulted in slow tumor growth of xenografted Huh7 in BALB/c nude mice. Immunohistochemical analyses also showed that FAM188B was an important proliferation gene. The study revealed that FAM188B is a putative regulator of YAP/TAZ in HCC cells, which could be used as a putative target for the treatment of HCC. However, the role of FAM188B in HCC is still not fully understood, and the regulatory mechanism of FAM188B needs to be further investigated in the future. In conclusion, we have shown that FAM188B is highly expressed in HCC and is associated with a poor prognosis. FAM188B promotes HCC progression both in vitro and in vivo by interacting with USP10. Importantly, the regulation of USP10 and YAP/TAZ by FAM188B does not occur at the transcriptional level, suggesting a potential involvement of carboxyl-terminal ubiquitin hydrolase activity. Further investigations are needed to elucidate how FAM188B directly or indirectly modulates oncogenic genes. Our findings suggest that targeting FAM188B is a potential strategy for controlling the progression of HCC. Abbreviations BCA, Bicinchoninic acid CHX, Cycloheximide EGFR, Epidermal growth factor receptor FAM188B, Family with sequence similarity 188 member B FAM63A, Family with sequence similarity 63 member A FLT3, FMS-like tyrosine kinase 3 FOXM1, Forkhead box M1 GEPIA, Gene Expression Profiling Interactive Analysis HCC, Hepatocellular carcinoma IHC, Immunohistochemistry MINDY-1, MINDY lysine 48 deubiquitinase-1 MINDY-4, MINDY lysine 48 deubiquitinase-4 NC, Negative control PDX, Patient derived tumor xenogeneic animal model TAZ, Transcriptional coactivator with PDZ-binding motif UALCAN, The University of Alabama at Birmingham Cancer data analysis Portal USP10, Ubiquitin-specific peptidase 10 YAP, Yes-associated protein Declarations Funding This work was supported by the Natural Science Foundation of Zhejiang Province (LR22H160008), Department of Education of Zhejiang Province (Y202044568, Y202146077), Precision Medicine Research in Oncology of Hangzhou Medical College (00004ACCXLJ202102), Key Research and Development Select Projects of Zhejiang Provincial Department of Science and Technology (2020C03008) and Fundamental Research Funds for the Central Universities (xzy012022095). Author contributions Qiuran Xu proposed the original design of this study. Siwei Hu, Yihong Chen performed most of the bench work. Linglan Tu, Wenhu Chen, Yuting Wen performed the animal experiments. Siwei Hu, Yuting Wen performed the IHC assay. Siwei Hu wrote the manuscript. Xiaoyan Li revised the manuscript. Kangsheng Tu, Xin Liu, Qiuran Xu, Dongsheng Huang and Xiaoyan Li provide research ideas and supervise the process of the study. The author(s) read and approved the final manuscript. Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose. Ethical statement All animal studies and experiments were approved by the Experimental Animal Ethics Committee of Hangzhou Medical College (2022-181) and performed in accordance with the Declaration of Helsinki. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Journal of Experimental & Clinical Cancer Research. 2021;40(1). Zhu H, Yan F, Yuan T, Qian M, Zhou T, Dai X, et al. USP10 Promotes Proliferation of Hepatocellular Carcinoma by Deubiquitinating and Stabilizing YAP/TAZ. Cancer Research. 2020;80(11):2204-16. Moya IM, Halder G. Hippo–YAP/TAZ signalling in organ regeneration and regenerative medicine. Nature Reviews Molecular Cell Biology. 2018;20(4):211-26. Yimlamai D, Christodoulou C, Galli Giorgio G, Yanger K, Pepe-Mooney B, Gurung B, et al. Hippo Pathway Activity Influences Liver Cell Fate. Cell. 2014;157(6):1324-38. Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ at the Roots of Cancer. Cancer Cell. 2016;29(6):783-803. Nguyen CDK, Yi C. YAP/TAZ Signaling and Resistance to Cancer Therapy. Trends in Cancer. 2019;5(5):283-96. Zhang X, Zhao H, Li Y, Xia D, Yang L, Ma Y, et al. The role of YAP/TAZ activity in cancer metabolic reprogramming. Molecular Cancer. 2018;17(1). Driskill JH, Pan D. The Hippo Pathway in Liver Homeostasis and Pathophysiology. Annual Review of Pathology: Mechanisms of Disease. 2021;16(1):299-322. Hayashi H, Higashi T, Yokoyama N, Kaida T, Sakamoto K, Fukushima Y, et al. An Imbalance in TAZ and YAP Expression in Hepatocellular Carcinoma Confers Cancer Stem Cell–like Behaviors Contributing to Disease Progression. Cancer Research. 2015;75(22):4985-97. Cao Y-F, Xie L, Tong B-B, Chu M-Y, Shi W-Q, Li X, et al. Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma. Cell Death & Differentiation. 2022;30(2):527-43. Weisberg EL, Schauer NJ, Yang J, Lamberto I, Doherty L, Bhatt S, et al. Inhibition of USP10 induces degradation of oncogenic FLT3. Nature Chemical Biology. 2017;13(12):1207-15. Deng M, Yang X, Qin B, Liu T, Zhang H, Guo W, et al. Deubiquitination and Activation of AMPK by USP10. Molecular Cell. 2016;61(4):614-24. Yuan J, Luo K, Zhang L, Cheville JC, Lou Z. USP10 Regulates p53 Localization and Stability by Deubiquitinating p53. Cell. 2010;140(3):384-96. Abdul Rehman Syed A, Kristariyanto Yosua A, Choi S-Y, Nkosi PJ, Weidlich S, Labib K, et al. MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes. Molecular Cell. 2016;63(1):146-55. Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, et al. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Current Protocols in Bioinformatics. 2016;54(1). Luo Y, Zhou J, Tang J, Zhou F, He Z, Liu T, et al. MINDY1 promotes bladder cancer progression by stabilizing YAP. Cancer Cell International. 2021;21(1). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6568148","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453734959,"identity":"3e44c0aa-bb6e-4c51-94b9-5125385c7848","order_by":0,"name":"Siwei Hu","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Siwei","middleName":"","lastName":"Hu","suffix":""},{"id":453734960,"identity":"93c4521e-db7f-4631-8ad9-e9b431bd28e4","order_by":1,"name":"Yihong Chen","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yihong","middleName":"","lastName":"Chen","suffix":""},{"id":453734961,"identity":"dc8de835-00bd-45bb-8764-3238f92ff391","order_by":2,"name":"Yuting Wen","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Wen","suffix":""},{"id":453734962,"identity":"e1bfca23-d134-4f4c-b22b-8cc8a1916076","order_by":3,"name":"Linglan Tu","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Linglan","middleName":"","lastName":"Tu","suffix":""},{"id":453734963,"identity":"0eb650e2-f46c-4e9f-adfb-3d9b60040c24","order_by":4,"name":"Wenhu Chen","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wenhu","middleName":"","lastName":"Chen","suffix":""},{"id":453734964,"identity":"20086e6d-eb7f-45d8-88a5-a231ee8666b2","order_by":5,"name":"Kangsheng Tu","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Kangsheng","middleName":"","lastName":"Tu","suffix":""},{"id":453734965,"identity":"4f84459b-4aef-4253-9235-53772ca18c31","order_by":6,"name":"Xin Liu","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Liu","suffix":""},{"id":453734966,"identity":"6ca8adb2-6f50-43d4-b168-f93622b2f3f5","order_by":7,"name":"Qiuran Xu","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Qiuran","middleName":"","lastName":"Xu","suffix":""},{"id":453734967,"identity":"cdcb8dc8-63f4-4c2a-8660-d0f78605a67e","order_by":8,"name":"Dongsheng Huang","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Dongsheng","middleName":"","lastName":"Huang","suffix":""},{"id":453734968,"identity":"0c69577f-4de3-4232-a8ab-538d9394ffe0","order_by":9,"name":"Xiaoyan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACPmYw9Y/H/nhj48MPxGhhg2g5IMdw5nCzsQRRWiDUAWOGG+ltAjxEaWHnMfxcwHAnsXHmwzYGCQY7Od0Ggg7jMZaewfAssVk6se1BAUOysdkBwloMpHkYmBPbpBPbDSQYDiRuI0KL8W+Qlh7Jg20SPERqMQPacthYQoKRaC1sZdY8DGlyBjyJwEA2IMIv/PyHN9/mYbDhMWA//vDhhwo7OYJaGBg4DBgY/8E4BgSVgwD7A6KUjYJRMApGwQgGAItuN7BxD4FVAAAAAElFTkSuQmCC","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":true,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-04-30 22:55:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6568148/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6568148/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82585101,"identity":"01e3aaf2-73d5-4706-a02e-1be66dacea69","added_by":"auto","created_at":"2025-05-13 07:02:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":772507,"visible":true,"origin":"","legend":"\u003cp\u003eFAM188B expression is upregulated and associated with poor prognosis in hepatocellular carcinoma (HCC). (A) Expression of FAM188B mRNA in tumor and non-tumor tissues and its association with cancer stage and tumor grade in UALCAN database. (B) mRNA expression of FAM188B in paired HCC tissues and adjacent non-tumor tissues in TCGA database (n = 50). (C) Kaplan–Meier survival analysis of overall survival in different groups based on FAM188B expression. (D) The expression of FAM188B protein was evaluated in normal hepatocytes (L02) and in hepatocellular carcinoma (HCC) cells (MHCC97H, Huh7, Hep3B and SNU387) by western blot. (E) Localization of FAM188B in L02 and HCC cells (scale bar, 20 μm). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns, not significant. The data are expressed as the mean ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/5052940fae355f235e62abdd.png"},{"id":82585099,"identity":"12411ac9-adc4-4bca-8ea3-5f133d7442b8","added_by":"auto","created_at":"2025-05-13 07:02:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2311986,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of FAM188B inhibits proliferation, migration and invasion of HCC cells in vitro. (A) Validation of FAM188B expression after knockdown in the indicated cell lines using western blotting (n = 3). (B) Migratory and invasive capacities of MHCC97H and Huh7 cells within 24 hours, as evaluated by performing Transwell assays (scale bar, 50 μm) (n = 3). (C) Proliferation of MHCC97H and Huh7 cells, as detected via CCK -8 assays (n = 10). (D) Protein levels of epithelial–mesenchymal transition markers in MHCC97H and Huh7 cells, detected through western blotting (n = 3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. The data are expressed as the mean±SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/2c9285636ca560b41eeae026.png"},{"id":82586520,"identity":"01691140-8f3e-4b2d-a3c4-b1ff0c0a5bfa","added_by":"auto","created_at":"2025-05-13 07:18:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3273154,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of FAM188B promotes cell proliferation, migration and invasion of HCC cells in vitro. (A) Validation of FAM188B expression after overexpression in the indicated cell lines using western blotting (n = 3). (B) Migratory and invasive capacities of MHCC97H and Huh7 cells within 24 hours, as evaluated using Transwell assays (scale bar, 50 μm) (n = 3). (C) Proliferation of MHCC97H and Huh7 cells, as detected by performing CCK - 8 assays (n = 10). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. The data are expressed as the mean ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/34308f210b3d118a0aa66820.png"},{"id":82585916,"identity":"b9d4a15a-4870-4009-9916-ed7c1fcda18d","added_by":"auto","created_at":"2025-05-13 07:10:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2132163,"visible":true,"origin":"","legend":"\u003cp\u003eFAM188B interacts with USP10. (A) IP and Coomassie brilliant blue staining were performed using MHCC97H and Huh7 cells expressing FAM188B-Flag. (B) Schematic illustration of IP and GO enrichment of identified proteins (using all proteins in the species database as the background). Fisher's exact test was used to analyze the significance and P value \u0026lt; 0.05 were considered significant. (C) Expression levels of USP10 mRNA in tumor (n = 374) and non-tumor (n = 50) tissues and its relationship with FAM188B, based on a public database. (D) The protein expression level of USP10 was evaluated in normal hepatocytes (L02) and hepatocellular carcinoma (HCC) cells (MHCC97H, Huh7, Hep3B and SNU387) by western blot. (E) Localization of USP10 in HCC cells (scale bar, 20 μm). (F) Co-IP assays to confirm the protein-protein interaction between FAM188B and USP10 in vitro. **p \u0026lt; 0.01, ***p \u0026lt; 0.001. The data are expressed as the mean ± SD of three independent experiments. IP, immunoprecipitation. Co-IP, co-immunoprecipitation.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/d848c50ea9ed718a17ace9d8.png"},{"id":82585097,"identity":"0631b29e-5eb3-4552-b892-f0e5df568a44","added_by":"auto","created_at":"2025-05-13 07:02:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1968751,"visible":true,"origin":"","legend":"\u003cp\u003eFAM188B knockdown decreases the protein expression levels of USP10 and YAP/TAZ HCC cells. (A) Protein levels of USP10 and FAM188B in MHCC97H and Huh7 cells transfected with negative control shRNA (sh-NC) or shRNA targeting FAM188B (sh-FAM188B), as detected via western blotting (n = 3). (B) Immunofluorescence assay showing decreased fluorescence intensity of USP10 in MHCC97H and Huh7 cells upon FAM188B knockdown (scale bar, 20 μm). (C) Protein levels of FAM188B, USP10, and YAP/TAZ in MHCC97H and Huh7 cells after transfection with sh -NC or shFAM188B, as detected via western blotting (n = 3). (D) mRNA levels of FAM188B, USP10, and YAP/TAZ in MHCC97H and Huh7 cells transfected with sh -NC or sh-FAM188B, as evaluated through qRT-PCR (n = 3). **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns, not significant. The data are expressed as the mean ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/8fa52b3a9614ee1457c6e7c1.png"},{"id":82586519,"identity":"7fea0147-fd96-4424-a28d-93cb30791875","added_by":"auto","created_at":"2025-05-13 07:18:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2035531,"visible":true,"origin":"","legend":"\u003cp\u003eFAM188B regulates and stabilizes USP10. (A) Protein levels of FAM188B, USP10, and YAP/TAZ in HEK293T cells transfected with FAM188B-Flag or USP10-Myc plasmids for 48 hours, as detected via western blotting (n = 3). (B) mRNA levels of FAM188B, USP10, and YAP/TAZ in HEK293T cells transfected with FAM188B-Flag or USP10-Myc plasmids for 48 hours, as evaluated through qRT-PCR (n = 3). (C) MHCC97H and Huh7 cells with stable FAM188B knockdown were treated with 100 μg/ml dimethyl sulfoxide (DMSO) and cycloheximide (CHX) for the indicated times (0h, 1h, 2h and 4h) and then lysed and subjected to immunoblotting. (D) MHCC97H and Huh7 cells transfected with sh-FAM188B were left untreated or treated with MG132 (10 μmol/l) for 12 h, and then cell lysates were subjected to immunoblotting, as indicated (n = 3). (E) MHCC97H and Huh7 cells with stable FAM188B knockdown were lysed and then subjected to immunoblotting using an anti-ubiquitin antibody. ****p \u0026lt; 0.0001, ns, not significant. The data are expressed as the mean ± SD of three independent experiments. Ub, ubiquitin.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/47f2e5ba4afb0ef60f5ddfe0.png"},{"id":82585917,"identity":"44cb4689-8ff1-4742-8a1c-67bb98aedb57","added_by":"auto","created_at":"2025-05-13 07:10:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3418846,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of FAM188B inhibits hepatocellular carcinoma tumor growth in a xenograft mouse model. (A) Representative image of xenograft tumors in nude mice, and statistical analyses of tumor volumes and weights in the different groups (n = 5). (B) Expression of FAM188B protein in the FAM188B-knockdown tumors compared to that in controls. (C) Representative xenograft tumor tissues (scale bar 100 μm). *p ﹤ 0.05, **p ﹤ 0.01, ***p ﹤ 0.001. The data are expressed as the mean ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/7f2b78484f90a3f60272e089.png"},{"id":85084503,"identity":"a176f093-7806-4046-9bb0-b16bd9a45c85","added_by":"auto","created_at":"2025-06-20 18:47:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16442701,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/92214633-ff43-4f5c-9831-52bf7b690513.pdf"},{"id":82585094,"identity":"15b52963-e986-423d-9777-8efafd439037","added_by":"auto","created_at":"2025-05-13 07:02:35","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16596,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6568148/v1/c43dd63e7a5e4297ce175a53.docx"}],"financialInterests":"","formattedTitle":"FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, after lung and colorectal cancer[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Surgical resection, liver transplantation and local ablation are effective treatment options for early-stage HCC. However, due to its late symptom onset, most patients are diagnosed with advanced-stage HCC and have a poor prognosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The multi-kinase inhibitor sorafenib remains a systemic therapy for advanced-stage HCC, although it only prolongs patient survival by a few months and has a low response rate[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, the treatment benefit of regorafenib, lenvatinib and cabozantinib is suboptimal[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and it is clinically important to understand the pathogenesis of HCC and identify effective interventions.\u003c/p\u003e \u003cp\u003eFamily with sequence similarity 188 member B (FAM188B) is a novel gene encoding an evolutionarily conserved protein in mammals, and its mRNA is highly expressed in a variety of solid tumors. In colon cancer, FAM188B knockdown induces p53 activation and accumulation and increases apoptosis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, FAM188B suppression sensitises lung cancer cells to anoikis by downregulating epidermal growth factor receptor (EGFR) expression and inhibits tumor metastasis in vivo. It also co-precipitated with forkhead box M1 (FOXM1), and FOXM1 ubiquitination levels increased with FAM188B knockdown but decreased with FAM188B overexpression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the role of FAM188B in HCC progression remains unclear.\u003c/p\u003e \u003cp\u003eUbiquitin-specific peptidase 10 (USP10) is a member of the ubiquitin-specific protease family and is a highly conserved deubiquitinase in mammals[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Further, it has been implicated in the progression of various malignancies, including HCC, pancreatic cancer, breast cancer and colorectal cancer[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, USP10 promotes HCC proliferation by deubiquitinating and stabilising of YAP/TAZ[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Hippo pathway and its downstream effectors, transcriptgional coactivator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP), are overexpressed in human cancers[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the liver, acute inactivation of Hippo signalling in vivo leads to the dedifferentiation of adult hepatocytes into progenitor cells with single-cell self-renewal and cellular plasticity[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent studies have shown that YAP/TAZ is associated with tumorigenesis in most solid tumors and that its activation induces proliferation, metastasis, and chemotherapy resistance in cancer stem cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, US Food and Drug Administration-approved drugs that indirectly block YAP/TAZ activation or its key downstream targets have significantly reduced drug resistance[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of FAM188B in promoting HCC progression and the potential underlying mechanisms. Our analysis suggests that FAM188B interacts with USP10 to regulate YAP/TAZ. These data indicate that FAM188B may be an important diagnostic and prognostic biomarker for controlling HCC progression.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Datasets from the public databases\u003c/h2\u003e \u003cp\u003eDatasets for liver cancer cells were obtained from TCGA (The Cancer Genome Atlas). The UALCAN (The University of Alabama at Birmingham Cancer data analysis Portal) and GEPIA (Gene Expression Profiling Interactive Analysis) websites were used for data visualization. The Kaplan-Meier method and the log-rank test were used to analyse the survival of patients with liver cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell Culture and Transfection\u003c/h2\u003e \u003cp\u003eThe normal human liver cell line L02 and the human liver tumor-derived cell lines MHCC97H and huh7 were obtained from the Whelab Biotechnology LTD (Shanghai, China) in 2021. Human liver tumor-derived cell lines SNU387 and Hep3B, human embryonic kidney HEK392T cells were obtained from the American Type Culture Collection (Manassas, VA, USA) in 2021. All cell lines were maintained in DMEM, MEM or RPMI-1640 media (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Sigma- Aldrich, St. Louis, MO, USA) at 37℃, in a 5% CO₂ humid atmosphere. The cell incubator was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The cells used for the experiments were passaged no more than10 times after thawing.\u003c/p\u003e \u003cp\u003eLentivirus-mediated FAM188B shRNA (sh-FAM188B) and negative control shRNA (sh-NC) were purchased from Genechem (Shanghai, China). The sequence of sh-FAM188B is shown in Table\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The pcDNA3.1-FAM188B-Flag and pcDNA3,1-USP10-Myc plasmids were obtained from GuanNan. Co, Ltd (Hangzhou, China). Plasmids were delivered into the cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and lentivirally encapsulated sh-FAM188B or sh-NC was transfected into cells using polyberene (Beyotime, Shanghai, China) according to the manufacturers\u0026rsquo; instructions. Finally purinomycin (ST551-50 mg, Beyotime, Shanghai, China) was screened with 2 \u0026micro;g/ml for one week to obtain stable transfection cell lines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell proliferation and Transwell assays\u003c/h2\u003e \u003cp\u003eTransfected HCC cells were added to a 96-well plate at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/well, and 10 \u0026micro;l of CCK-8 solution (Beyotime, Shanghai, China) was added to each well at the same time every day for 4 days. After 1.5 hours of incubation, the absorbance was measured at 450 nm using a microplate reader (Thermo-Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003eFAM188B knockdown HCC cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in the upper chamber of the Transwell insert. For the invasion experiment, each upper chamber was coated with Matrigel matrix (Corning, Corning, NY, USA). Each of the lower chambers contained 500 \u0026micro;l of medium supplemented with 10% FBS. After 24 hours of cultivation, migrating and invading cells were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.4% ammonium oxalate crystal violet and counted microscopically in five random fields. Transwell chambers were purchased from Corning (Corning, NY, USA), and 0.4% ammonium oxalate crystal violet was purchased from YuanyeBio (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Reagents\u003c/h2\u003e \u003cp\u003eThe proteasome inhibitor MG-132 and protein synthesis inhibitor CHX (Cycloheximide) were purchased from Selleck Chem (Houston, TX, USA). RIPA lysis buffer, Enhanced BCA (Bicinchoninic acid) Protein Assay Kits, TBS with Tween-20, Phosphate Buffered Saline (PBS, premixed powder), Antifade Mounting Medium with DAPI and Western rapid transfer buffer were purchased from Beyotime (Shanghai, China). Protease and phosphatase inhibitors were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The FastPAGE precast gel and MOPS-SDS running buffer were purchased from Tsingke (Beijing, China). Polyvinylidene difluoride (PVDF) membranes and an ECL kit were purchased from Millipore (Bedford, MA, USA). The skim milk powder and neutral buffered formalin (10%) were purchased from Biosharp (Hefei, China). The Triton X-100 was purchased from Solarbio (Beijing, China). The Bovine Serum Albumin (BSA) lyophilized powder was obtained from Sigma-Aldrich (St. Louis, MO, USA).\u003c/p\u003e \u003cp\u003eThe following primary antibodies were used: anti-FAM188B (WB, 1:1000; IHC, IF, 1:200; ab122097) was purchased from Abcam (Cambridge, UK), anti-β-actin (WB, 1:1000; 13E5) was purchased from Cell Signaling Technology (Beverly, MA, USA), anti-TAZ (1:5000; 23306-1-AP) was purchased from Proteintech (Wuhan, China), anti-ubiquitin (1:2000; PTM-1106RM) was purchased from PTMBIO (Hangzhou, China), anti-USP10 (WB, 1:1000; IHC, IF, 1:200; ET1706-12), anti-YAP (1:1000; ET1608-30), anti-E-cadherin (1:5000; ET1607-75), anti-N-cadherin (1:2000; ET1607-37), anti-Vimentin (1:10000; ET1610-39), anti-Flag-tag (1:1000; 0912-1), anti-Myc-tag (1:1000; R1208-1) and anti-Ki67 (1:200; ET1609-34) were purchased from HuaBio (Hangzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 qRT-PCR\u003c/h2\u003e \u003cp\u003eCells were harvested in TRIzol reagent (Ambion, Austin, TX, USA) for total RNA extraction, and 1 \u0026micro;g RNA was used for cDNA transcription as following the instructions of the Hifair III 1st Strand cDNA Synthesis SuperMix Kit(Yeasen, Shanghai, China). Amplification and quantification were carried out with a CFX96 Touch\u0026trade; real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green Master Mix (Yeasen, Shanghai, China). The relative expression of mRNAs was normalized to GAPDH using 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method. The sequences of all primers are listed in the Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunoblotting and co-immunoprecipitation\u003c/h2\u003e \u003cp\u003eFor immunoblotting, proteins were collected using a lysis buffer containing protease and phosphatase inhibitors, and boiled at 95\u0026deg;C for 5 minutes after the addition of loading buffer. The protein concentration in each sample was determined using the BCA protein assay kit. Protein samples were separated on 4%~20% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline and Tween-20 (TBS-T) for 1hour at approximately 25\u0026deg;C, and then incubated with primary antibodies overnight (8h\u0026thinsp;~\u0026thinsp;12h) at 4\u0026deg;C and washed three times for 10 min with TBS-T each time. HRP-conjugated antibodies (Cell Signaling Technology, Beverly, MA, USA) were incubated with the membranes for 1 hour at approximately 25\u0026deg;C and then washed three times with TBS-T for 15 minutes each time. The labelled proteins were visualized by enhanced chemiluminescence using the ChemiDoc Imaging System (Hercules, CA, USA). Finally, protein levels were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalised to β-actin.\u003c/p\u003e \u003cp\u003eThe pcDNA-FAM188B-Flag and pcDNA-USP10-Myc plasmids were transfected into HCC cells. After 48 hours, the cells were harvested with lysis adding cocktail and the Flag-tag or Myc-tag Protein IP Assay Kit (Beyotime, Shanghai, China) was used for co-immunoprecipitation according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunofluorescence\u003c/h2\u003e \u003cp\u003eMHCC97H and Huh7 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/100 \u0026micro;l) with sh-FAM188B or sh-NC were cultured in confocal dishes (Biosharp, Hefei, China) for 24 hours. The cells were washed with PBS solution and fixed with neutral buffered formalin (10%) for 15 minutes and 0.1% Triton (diluted with PBS) for 20 minutes at approximately 25\u0026deg;C. Cells were blocked with 10% BSA (diluted with PBS) for 1 hour at 25\u0026deg;C and incubated with primary antibodies at 4\u0026deg;C overnight (8h\u0026thinsp;~\u0026thinsp;12h). The next day, cells were washed three times with PBS for 15 minutes each time and incubated with fluorescent antibodies for 1 hour at 25\u0026deg;C in the dark. The dishes were washed three times with PBS for 15 minutes each time. Finally, an appropriate amount of Antifade Mounting Medium containing DAPI was dripped onto the dishes and images were captured using the confocal fluorescence microscope (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 In vivo Experiments and Immunohistochemical Staining of Tumor Tissues\u003c/h2\u003e \u003cp\u003eFour-week-old BALB/c nude mice were obtained from the Laboratory Animal Center of Hangzhou Medical College. Huh7 cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/100 \u0026micro;l) with sh-FAM188B or sh-NC were injected subcutaneously into the mice (n\u0026thinsp;=\u0026thinsp;5 in each group). The tumor volume (V) was calculated as follows: V\u0026thinsp;=\u0026thinsp;π/6\u0026times;L\u0026times;W\u0026times;H, where L, W and H represent the tumor length, width and height, respectively. For immunohistochemical staining, tissue samples were deparaffinized, rehydrated and washed in PBS solution three times for five times. The tissue sections were placed in boiling citrate buffer for 6 minutes for antigen retrieval, then removed after natural cooling to about 25℃, and washed with PBS solution three times for 5 minutes. The samples were blocked at 37℃ with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution for 20 minutes, and then washed in PBS solution three times for five minutes. Slides were blocked in 5% BSA solution for 1 hour and incubated with primary antibodies at 4 ℃ overnight. After blocking, slides were washed with TBS-T and incubated with secondary antibodies (HuaBio, Hangzhou, China) for 1 hour. DAB horseradish peroxidase color development Kit (Beyotime, Shanghai, China) was used to visualize staning. IHC images were captured with a microscope (Leika, Weztlar, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData from three independent experiments are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM). Comparisons between two or more groups were made using one-way analysis of variance or Student's t-test. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Inc., San Diego, CA, USA). p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 FAM188B expression is upregulated in HCC and predictive of poor patient survival\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that FAM188B expression is upregulated in human colorectal and lung cancers and promotes the malignant progression of cancer. However, its role in HCC has not yet been determined. To investigate the effect of FAM188B on HCC, we analysed its expression in various cancers. An analysis of The Cancer Genome Atlas (TCGA) data showed that \u003cem\u003eFAM188B\u003c/em\u003e is highly expressed in liver cancer and closely related to the HCC tumor grade and cancer stage according to UALCAN (uab.edu) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We also extracted 50 sets of RNA-seq data from TCGA-LIHC and analysed them statistically using the Wilcoxon signed-rank test. The results showed that FAM188B mRNA expression was up-regulated in tumour tissue compared to para-carcinoma tissue. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, the joint analysis based on TCGA and GTEx databases showed that FAM188B expression in HCC tissues was significantly higher than that in normal liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We found that high FAM188B mRNA levels predicted shorter survival using Kaplan-Meier survival analysis of the TCGA data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, FAM188B protein levels were markedly elevated in HCC cell lines (MHCC97H and Huh7) compared to normal hepatocyte cell lines (L02). However, this elevation was not observed in the other two cell lines (Hep3B and SNU387) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To further investigate the role of FAM188B in the malignant progression of HCC, we used immunofluorescence to verify its localisation in HCC cells. L02, MHCC97H and Huh7 cells were stained\u003c/p\u003e \u003cp\u003e with an anti-FAM188B antibody and DAPI, which was followed by confocal microscopy analysis. FAM188B was found to be located in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These data suggested that FAM188B has a significant oncogenic effect in HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 FAM188B promotes HCC cells proliferation, migration and invasion\u003c/h2\u003e \u003cp\u003eTo investigate the effect of FAM188B on malignant progression, we generated FAM188B knockout cell lines (MHCC97H and Huh7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Transwell results showed that FAM188B knockdown suppressed metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and CCK-8 results suggested inhibition of proliferation with decrease of FAM188B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Since epithelial\u0026ndash;mesenchymal transition (EMT) contributes to the enhanced migration and invasion of tumor cells, we also investigated whether FAM188B mediates this process. Immunoblotting results showed that FAM188B expression was negatively correlated with E-cadherin but positively correlated with N-cadherin and vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Next, FAM188B-overexpressing HCC cells (SNU387 and Hep3B) were generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA); in these two cell lines, transwell experiments showed that FAM188B promoted the tumor cell migration and invasion of tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Experimental results from the CCK-8 assay further demonstrated that FAM188B overexpression could enhance HCC cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Taken together, these data indicate that FAM188B expression is important for the growth and metastasis of liver cancer cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 FAM188B interacts with USP10 in HCC cells\u003c/h2\u003e \u003cp\u003eTo confirm the molecular mechanism underlying the effects of FAM188B in HCC cells, immunoprecipitation (IP) was performed based on FAM188B-Flag expression in MHCC97H and Huh7 cells. We then searched for potential FAM188B-binding proteins using liquid chromatography/mass spectrometry (LC/MS)-MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Based on the results, we speculated that FAM188B was related to the level of ubiquitination in HCC cells and that it might bind to ubiquitination-related enzymes. MS was further used to screen four deubiquitinating enzymes that potentially interact with FAM188B in HCC cells, including USP7, USP9X, USP10, and USP47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Among the four deubiquitinating enzymes, USP10 attracted our attention because it is a novel regulator of YAP/TAZ signalling and promotes HCC proliferation by deubiquitinating and stabilizing YAP/TAZ. Moreover, analysis revealed that USP10 is highly expressed in HCC and positively correlated with FAM188B expression according to GEPIA (cancer-pku.cn) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Thus, we selected this protein as a potential FAM188B interactor for further experiments. The protein level of USP10 was found to be elevated in hepatocellular carcinoma (HCC) cells (MHCC97H, Huh7, Hep3B and SNU387) in comparison to normal liver cells (L02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Immunofluorescence experiments were also performed to demonstrate the localisation of USP10 in the cytosol and nucleoplasm of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). We also performed co-IP assays, and the results showed that USP10 can interact with FAM188B in HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 FAM188B positively regulates USP10-YAP/TAZ signalling pathway\u003c/h2\u003e \u003cp\u003eTo verify the regulatory relationship between FAM188B and USP10, we performed immunoblotting and immunofluorescence assays using two HCC cell lines transfected with FAM188B shRNA (sh-FAM188B) and found that endogenous FAM188B deficiency noticeably reduced the protein abundance and fluorescence intensity of USP10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Furthermore, we explored the effect of FAM188B knockdown on endogenous YAP/TAZ levels using immunoblotting and qRT-PCR. We found that FAM188B suppression reduced YAP/TAZ protein levels without affecting their mRNA levels in MHCC97H and Huh7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). To further demonstrate the positive regulatory relationship between FAM188B and the USP10\u0026ndash;YAP/TAZ signalling pathways. HEK293T cells were transfected with a plasmid expressing FAM188B with a Flag-tag and a plasmid encoding USP10 with a Myc-tag. Subsequently, immunoblotting and qRT-PCR experiments were conducted. Our results show that exogenous FAM188B can increase the abundance of USP10 and YAP/TAZ proteins. Consistently, exogenous USP10 increased FAM188B and YAP/TAZ protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Of note, the qRT-PCR results showed that the overexpression of FAM188B had no effect on \u003cem\u003eUSP10\u003c/em\u003e and \u003cem\u003eYAP\u003c/em\u003e mRNA levels, but significantly increased \u003cem\u003eTAZ\u003c/em\u003e mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This suggests that other pathways regulate FAM188B and YAP/TAZ signalling at the transcriptional level. To further ascertain whether FAM188B affects the stability of the USP10 protein, MHCC97H and Huh7 cells were treated with cycloheximide (CHX) for the indicated times following FAM188B knockdown. The results showed that FAM188B knockdown promoted USP10 degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To acquire a deeper understanding of the regulatory relationship between FAM188B and USP10\u0026ndash;YAP/TAZ, we restored the protein levels of USP10 and YAP/TAZ using the proteasome inhibitor MG132 in FAM188B-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Moreover, the ubiquitination levels of total cellular proteins were significantly higher in the sh-FAM188B-treated group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Taken together, these results demonstrate that FAM188B positively regulates YAP/TAZ protein levels through its interaction with USP10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 FAM188B knockdown inhibits HCC growth in vivo\u003c/h2\u003e \u003cp\u003eTo assess whether FAM188B could be a novel target for controlling HCC growth, Huh7 cells with or without sh-FAM188B transfection were injected subcutaneously into BALB/c nude mice (n\u0026thinsp;=\u0026thinsp;5) and tumor growth was monitored. Mice were euthanised by cervical dislocation under ether anaesthesia on day 30 after inoculation. Consistent with the results of the in vitro experiments, tumor burden was lower in the shFAM188B treatment group than in the control group (negative control shRNA; sh-NC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Isolated tumors were then subjected to total protein extraction and immunohistochemistry. Immunoblotting results showed FAM188B protein expression was decreased in the shFAM188B treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Immunohistochemical analysis further demonstrated that the protein levels of USP10, YAP and TAZ, as well as Ki-67, a proliferation marker, were downregulated following FAM188B knockdown in the tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These data demonstrate that FAM188B knockdown inhibits HCC growth in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe lack of targets for HCC intervention remains a major challenge in its treatment. Due to the critical and widespread functions of the YAP/TAZ signalling pathway in HCC cells, the exploration of upstream regulatory factors to develop potential therapeutic strategies has become more feasible. Previous studies have shown that YAP/TAZ is involved in tumor glucose metabolism, fatty acid metabolism, α-ketoglutarate metabolism and glutamine catabolism, all of which provide energy for tumor growth and synthetic materials necessary for tumor cell activity[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Genetic studies have demonstrated that the Hippo pathway is a critical regulator of liver size, regeneration, development, metabolism and homeostasis. Therefore, dysregulation of the Hippo pathway can lead to liver diseases, such as fatty liver and cancer. Drugs that target the Hippo pathway promote liver regeneration and prevent the development or progression of liver disease[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, studies have shown that a pair of proteins synergistically promote cancer progression and co-targeting these proteins, YAP/TAZ, has achieved significant efficacy in anticancer therapy[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the present study, we demonstrated that FAM188B interacts with USP10 and indirectly regulates YAP/TAZ expression, thereby promoting HCC progression.\u003c/p\u003e \u003cp\u003eUSP10 has multiple roles in tumor initiation and progression, and its oncogenic and anticancer effects depend primarily on its substrates in different malignant tumors[\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A recent study showed that USP10 interacts with ANLN and positively regulates its protein levels, thereby promoting cytoplasmic division and proliferation in oesophageal squamous cell carcinoma cells[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, USP10 has shown efficacy in preclinical models of oncogenic FLT3, including cell lines, primary patient samples and mouse models of oncogenic-FLT3-driven leukaemia[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous studies have also demonstrated that USP10 regulates the localisation and stability of p53 by deubiquitinating it[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Through a comprehensive analysis of HCC patient samples, PDX models and TCGA data, studies have shown that USP10 exerts oncogenic effects in HCC through the deubiquitination and stabilisation of YAP/TAZ[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFAM188B, also known as MINDY-4, is distantly related to FAM63A (MINDY-1)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although FAM188B is annotated in gene databases as a putative ubiquitin carboxyl-terminal hydrolase, its functions are not fully undestood[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Notably, FAM188B has been identified as an interactor of USP7, a deubiquitinating enzyme that has been shown to stabilize p53 through deubiquitination. This interaction is significant as it implicates FAM188B in the regulation of cell survival and apoptosis, which are critical biological processes in cancer development. The functional implications of elevated FAM188B expression in tumors are further substantiated by the observation that FAM188B knockdown results in a significant increase in apoptotic cellular phenotypes across different cancer types, thereby reinforcing its role as a potential therapeutic target in cancer treatment. Our data showing that FAM188B has no effect on USP10 and YAP/TAZ mRNA and but affects protein levels, particularly in the context of protein synthesis inhibition (CHX) and proteasome suppression (MG132), suggest that FAM188B regulates USP10 and YAP/TAZ levels by affecting protein stability rather than transcription. Notably, a previous study demonstrated the role of FAM63A (MINDY-1) in stabilizing YAP, via deubiquitination in bladder cancer cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Further investigation is required to determine whether FAM188B plays a similar role in HCC.\u003c/p\u003e \u003cp\u003eFinally, Huh7 cells transfected with sh-FAM188B or sh-NC were injected subcutaneously into nude mice and the results showed that low expression of FAM188B resulted in slow tumor growth of xenografted Huh7 in BALB/c nude mice. Immunohistochemical analyses also showed that FAM188B was an important proliferation gene. The study revealed that FAM188B is a putative regulator of YAP/TAZ in HCC cells, which could be used as a putative target for the treatment of HCC. However, the role of FAM188B in HCC is still not fully understood, and the regulatory mechanism of FAM188B needs to be further investigated in the future.\u003c/p\u003e \u003cp\u003eIn conclusion, we have shown that FAM188B is highly expressed in HCC and is associated with a poor prognosis. FAM188B promotes HCC progression both in vitro and in vivo by interacting with USP10. Importantly, the regulation of USP10 and YAP/TAZ by FAM188B does not occur at the transcriptional level, suggesting a potential involvement of carboxyl-terminal ubiquitin hydrolase activity. Further investigations are needed to elucidate how FAM188B directly or indirectly modulates oncogenic genes. Our findings suggest that targeting FAM188B is a potential strategy for controlling the progression of HCC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBCA, Bicinchoninic acid\u003c/p\u003e\n\u003cp\u003eCHX, Cycloheximide\u003c/p\u003e\n\u003cp\u003eEGFR, Epidermal growth factor receptor\u003c/p\u003e\n\u003cp\u003eFAM188B, Family with sequence similarity 188 member B\u003c/p\u003e\n\u003cp\u003eFAM63A, Family with sequence similarity 63 member A\u003c/p\u003e\n\u003cp\u003eFLT3, FMS-like tyrosine kinase 3\u003c/p\u003e\n\u003cp\u003eFOXM1, Forkhead box M1\u003c/p\u003e\n\u003cp\u003eGEPIA, Gene Expression Profiling Interactive Analysis\u003c/p\u003e\n\u003cp\u003eHCC, Hepatocellular carcinoma\u003c/p\u003e\n\u003cp\u003eIHC, Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eMINDY-1, MINDY lysine 48 deubiquitinase-1\u003c/p\u003e\n\u003cp\u003eMINDY-4, MINDY lysine 48 deubiquitinase-4\u003c/p\u003e\n\u003cp\u003eNC, Negative control\u003c/p\u003e\n\u003cp\u003ePDX, Patient derived tumor xenogeneic animal model\u003c/p\u003e\n\u003cp\u003eTAZ, Transcriptional coactivator with PDZ-binding motif\u003c/p\u003e\n\u003cp\u003eUALCAN, The University of Alabama at Birmingham Cancer data analysis Portal\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUSP10, Ubiquitin-specific peptidase 10\u003c/p\u003e\n\u003cp\u003eYAP, Yes-associated protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Zhejiang Province (LR22H160008), Department of Education of Zhejiang Province (Y202044568, Y202146077), Precision Medicine Research in Oncology of Hangzhou Medical College (00004ACCXLJ202102), Key Research and Development Select Projects of Zhejiang Provincial Department of Science and Technology (2020C03008) and Fundamental Research Funds for the Central Universities (xzy012022095).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQiuran Xu proposed the original design of this study. Siwei \u0026nbsp;Hu,\u0026nbsp;Yihong Chen performed most of the bench work. Linglan Tu, Wenhu Chen, Yuting Wen performed the animal experiments. Siwei Hu, Yuting Wen performed the IHC assay. Siwei Hu wrote the manuscript. Xiaoyan Li revised the manuscript. Kangsheng Tu, Xin Liu, Qiuran Xu, Dongsheng Huang and Xiaoyan Li provide research ideas and supervise the process of the study. The author(s) read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies and experiments were approved by the Experimental Animal Ethics Committee of Hangzhou Medical College (2022-181) and performed in accordance with the Declaration of Helsinki.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Cancer Cell International. 2021;21(1).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, FAM188B, USP10, YAP/TAZ pathway","lastPublishedDoi":"10.21203/rs.3.rs-6568148/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6568148/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eobjectives\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and its incidence and mortality rates remain high. Therefore, new diagnostic and therapeutic approaches are urgently required. FAM188B mRNA encodes an evolutionarily conserved protein that is highly expressed in various cancers. While FAM188B has been implicated in the progression of several tumors, its role in HCC progression remains unknown.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe analyzed FAM188B expression in HCC using TCGA and UALCAN databases. Functional studies included in vitro proliferation, migration, and invasion assays, as well as in vivo xenograft models. Co-immunoprecipitation (Co-IP), Western blotting, and immunofluorescence were used to investigate the FAM188B-USP10-YAP/TAZ interaction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFAM188B was found highly expressed in HCC cells and associated with poor prognosis. Both in vitro and in vivo, FAM188B promoted the proliferation, migration, and invasion of HCC. FAM188B directly interacts with and stabilizes USP10 and the downregulation of FAM188B by shRNA led to decreased USP10 and YAP/TAZ protein levels, suggesting that FAM188B may regulate the YAP/TAZ pathway through its interaction with USP10.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study suggests that FAM188B is involved in the proliferation, migration and invasion of hepatocellular carcinoma (HCC) and the mechanism may involve the regulation of the USP10/YAP/TAZ signalling pathways in vitro and in vivo.\u003c/p\u003e","manuscriptTitle":"FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 07:02:30","doi":"10.21203/rs.3.rs-6568148/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad9ef6cd-fad6-4780-82c5-c16200b6988d","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-20T18:38:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 07:02:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6568148","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6568148","identity":"rs-6568148","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}