TRIM65 Triggers Β-Catenin Signaling Via Ubiquitylation of Axin1 To

Home , AXIN1

RESEARCH ARTICLE TRIM65 triggers β-catenin signaling via ubiquitylation of Axin1 to promote hepatocellular carcinoma Yu-Feng Yang1,*, Mei-Fang Zhang2,*, Qiu-Hong Tian3,* and Chris Zhiyi Zhang2,‡

ABSTRACT cellular processes, including cell development, proliferation and Deregulation of ubiquitin ligases contributes to the malignant differentiation. Several TRIM members, such as TRIM24 and progression of human cancers. Tripartite motif-containing protein 65 TRIM29, participate in the progression of solid tumors by inducing (TRIM65) is an E3 ubiquitin ligase and has been implicated in human ubiquitylation of their downstream targets (Dükel et al., 2016; Groner diseases, but its role and clinical significance in hepatocellular et al., 2016). For example, TRIM65 is upregulated in lung cancer and carcinoma (HCC) remain unknown. Here, we showed that TRIM65 promotes tumor growth via ubiquitylation of p53 (also known as expression was increased in HCC tissues and associated with poor TP53) to inhibit its antitumor activity (Li et al., 2016; Wang et al., outcome in two independent cohorts containing 888 patients. In vitro 2016). Li et al. reported that TRIM65 was capable of modulating the and in vivo data demonstrated that overexpression of TRIM65 microRNA profile in lung and cervical cancer cells (Li et al., 2014). promoted cell growth and tumor metastasis, whereas knockdown of However, the role of TRIM65 in HCC remains unclear. β TRIM65 resulted in opposite phenotypes. Further studies revealed Aberrant activation of -catenin signaling contributes to the β that TRIM65 exerted oncogenic activities via ubiquitylation of Axin1 to initiation and progression of HCC. The activation of -catenin activate the β-catenin signaling pathway. TRIM65 directly bound to signaling is dependent on two protein families: Wnt and Akt β Axin1 and accelerated its degradation through ubiquitylation. (Monga, 2015). Canonically, -catenin is activated by Wnt/Frizzled β Furthermore, HMGA1 was identified as an upstream regulator of signaling. Cytoplasmic -catenin is phosphorylated by a complex β β TRIM65 in HCC cells. In clinical samples, TRIM65 expression was with glycogen synthase kinase-3 (GSK-3 ), Axin1/2 and positively correlated with the expression of HMGA1 and nuclear adenomatosis polyposis coli (APC), and is subsequently targeted β-catenin. Collectively, our data indicate that TRIM65 functions as an for proteosomal degradation. Once Wnt signaling is activated β oncogene in HCC. The newly identified HMGA1/TRIM65/β-catenin by Frizzled, -catenin is dissociated from the degradation complex, axis serves as a promising prognostic factor and therapeutic target. and accumulates in the nucleus to form a transcriptional complex with TCF/LEF proteins to induce the transcription of downstream KEY WORDS: TRIM65, Axin1, β-catenin, HMGA1, Hepatocellular genes implicated in carcinogenesis (Nusse and Clevers, 2017). carcinoma Previous studies have reported that Wnt/β-catenin signaling could be triggered by HMGA1 (Akaboshi et al., 2009; Xian et al., 2017), an INTRODUCTION architectural transcriptional factor. HMGA1 is a nonhistone nuclear The outcomes of hepatocellular carcinoma (HCC) remain extremely protein involved in genetic recombination, chromosomal modulation, poor. Although efforts have been made to develop new strategies for DNA repair and cell apoptosis (Sumter et al., 2016). Overexpression clinical intervention, the 5 year survival of patients with HCC has of HMGA1 in human cancers, such as medulloblastoma, colorectal barely improved (Coleman, 2014; Torre et al., 2015). The cancer and HCC, promotes malignant progression via transcriptional identification of potential biomarkers for disease diagnosis and regulation of several oncogenes or tumor suppressor genes prognostic prediction is a priority in precision medicine, especially (Andreozzi et al., 2016; Lau et al., 2012; Liang et al., 2013). for tumors lacking targeted therapy. As a result, identifying proteins In this study, we evaluated the expression of TRIM65 and its that promote the malignant progression of HCC has been attracting clinical significance in two independent cohorts of 888 patients with increasing attention. HCC. We investigated the effects of TRIM65 on HCC progression Deregulation of ubiquitin ligase is responsible for many human and the underlying mechanisms. Our data suggest that TRIM65, diseases. Tripartite motif-containing (TRIM) proteins, containing a regulated by HMGA1, exerts oncogenic activity in HCC through RING, a B box type 1 and B box type 2, and a coiled-coil region, ubiquitylation of Axin1 to activate β-catenin signaling. have been identified as E3 ubiquitin ligases (Reymond et al., 2001). TRIM proteins have been shown to have important roles in various RESULTS TRIM65 expression is increased in HCC and correlates with poor outcome 1Department of Pathology, Dongguan Third People’s Hospital, Dongguan, China. To determine the expression of TRIM65 in HCC, 48 pairs of fresh 2Department of Pathology, Sun Yat-sen University Cancer Center, State Key TRIM65 Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer tissue were collected. We observed that mRNA was Medicine, Guangzhou 510060, China. 3Department of Oncology, First Affiliated overexpressed in HCC tissues, compared with the corresponding Hospital of NanChang University, NanChang, Jiangxi 330006, China. adjacent nontumorous tissues (Fig. 1A). Consistently, the level of *These authors contributed equally to this work TRIM65 protein was much higher in HCC tissues than in nontumorous ‡Author for correspondence ([email protected]) tissues (Fig. 1B). To validate the observation of increased expression of TRIM65 in HCC, a cohort of 516 patients was recruited. Results from Y.-F.Y., 0000-0001-9901-3263; Q.-H.T., 0000-0003-3348-2889; C.Z.Z., 0000- 0002-1944-8281 tissue microarray-based immunohistochemistry (IHC) showed that TRIM65 mainly localized in the cytoplasm. In 56.6% (292/516) of the

Received 27 May 2017; Accepted 25 July 2017 cases, TRIM65 expression was upregulated in HCC (Fig. 1C). In Journal of Cell Science

3108 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

Fig. 1. TRIM65 expression is increased in HCC. (A) TRIM65 mRNA levels were significantly higher in HCC tissues than in the corresponding nontumor tissues in 48 samples, as shown by qRT-PCR. (B) Western blots showing TRIM65 expression in six of the 15 HCC samples, normalized to GADPH expression. T, tumor; N, nontumor. (C) IHC staining was performed on samples from a cohort of 516 HCC patients and another cohort of 87 HCC patients with embolus metastasis to assess the expression of TRIM65 in nontumorous (N), primary (T) and metastatic (M) tumors. Representative images are shown and the relevant IHC scores indicated.

another cohort of patients with venous metastasis, TRIM65 expression To reveal the clinical implications of TRIM65 in HCC, the was higher in the portal vein embolus than in the primary tumor in relationship between TRIM65 and pathological features of the 83.9% (73/87) of the cases (Fig. 1C). disease was evaluated. Results showed that the IHC score for

Fig. 2. High TRIM65 expression is correlated with poor outcome in HCC. (A) TRIM65 expression in HCC steadily increased from G1 to G4 in both the DSN cohort (left) and TCGA cohort (right). (B) Scatter diagram showing increased expression of TRIM65 in HCC patients with vascular invasion, compared with those without vascular invasion, in the two cohorts. (C,D) Kaplan–Meier analyses were conducted to determine the prognostic value of TRIM65 protein (DSN cohort) and mRNA (TCGA cohort) for overall (C) and disease-free (D) survival. Journal of Cell Science

3109 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

TRIM65 steadily increased from G1 to G4 in the DSN cohort (see Bel-7402 cells with HMGA1 overexpression, but decreased in Materials and Methods) (Fig. 2A). Patients with tumor vascular MHCC-97H cells with HMGA1 silencing (Fig. 3A). Luciferase invasion had increased TRIM65 expression (Fig. 2B). These reporter assays confirmed that HMGA1 was capable of modulating findings were further confirmed in the TCGA cohort (see the activity of the TRIM65 promoter. The related luciferase activity Materials and Methods) (Fig. 2A,B). According to the median of the TRIM65 promoter was enhanced by HMGA1 IHC score (DSN cohort) and mRNA expression value (TCGA overexpression, but attenuated by HMGA1 depletion, in Bel-7402 cohort) for TRIM65, patients were separated into two groups: high cells (Fig. 3B). In clinical samples, the expression of TRIM65 was or low TRIM65. High TRIM65 expression was significantly positively correlated with HMGA1 expression. According to the associated with tumor size (P=0.022), tumor differentiation TCGA data, TRIM65 mRNA coexpressed with HMGA1 mRNA in (P=0.004), clinical stage (P=0.001) and vascular invasion 423 cases (Fig. 3C). In 19 pairs of fresh HCC specimens, TRIM65 (P=0.024) in the DSN cohort (Table S1). Kaplan–Meier analyses mRNA expression was associated with HMGA1 protein expression indicated that HCC patients with high expression of TRIM65 were (Fig. 3D). As indicated by IHC staining, patients with high TRIM65 associated with shorter overall and disease-free survival in both the expression frequently expressed more HMGA1 than those with low DSN and TCGA cohorts (Fig. 2C,D). Multivariate analysis revealed TRIM65 expression (Fig. 3E). that TRIM65 was an independent factor for overall survival (hazard ratio, 1.354; 95% confidence interval, 1.099–1.668; P=0.004) TRIM65 exerts oncogenic activities in HCC (Table S2), but not for disease-free survival (data not shown). Because TRIM65 expression correlated with poor outcome, we next investigated whether TRIM65 was involved in the malignant TRIM65 is transcriptionally regulated by HMGA1 progress of HCC. According to the expression of TRIM65 mRNA Using bioinformatics programs, HMGA1 was predicted to be an (Fig. S1), TRIM65 was induced in QGY-7703 and Bel-7402 cells, upstream regulator of TRIM65. The in vitro experiments showed and reduced in MHCC-97H cells (Fig. 4A). As shown by MTT that the expression of TRIM65 was increased in QGY-7703 and assays, cell growth rates were increased in cells with TRIM65

Fig. 3. TRIM65 is transcriptionally upregulated by HMGA1. (A) According to the results from qRT-PCR and western blot analyses, TRIM65 expression was upregulated by HMGA1 overexpression, but downregulated by HMGA1 siRNAs. (B) Luciferase reporter assays were performed to show the effects of HMGA1 overexpression or knockdown on the activity of the TRIM65 promoter. (C) TRIM65 mRNA was associated with HMGA1 mRNA in the TCGA cohort of 423 patients with HCC. (D) TRIM65 mRNA was correlated with HMGA1 protein expression in 19 HCC cases. (E) The correlation between HMGA1 and TRIM65 protein expression in the DSN cohort, as shown by IHC. Representative images (top) and statistical analyses (bottom) are shown. *P<0.05, ***P<0.001. Journal of Cell Science

3110 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

Fig. 4. TRIM65 facilitates HCC cell proliferation and migration in vitro. (A) The overexpression or knockdown of TRIM65 protein in HCC was confirmed by western blot analysis. (B) Cells were transfected with TRIM65 overexpression vector or siRNAs on day (D) 1 and D3. The related cell growth rates assessed by MTT assay indicated that TRIM65 increased HCC cell proliferation. (C) Colony formation assays were used to confirm the effects of TRIM65 on cell growth. (D) Transwell assays were performed to show that TRIM65 was capable of enhancing cell migration. *P<0.05, **P<0.01. overexpression, but decreased in cells with TRIM65 depletion TRIM65 depletion group were lighter (Fig. 5B). For detection of (Fig. 4B). The effect of TRIM65 on cell proliferation was further tumor metastasis, a caudal vein injection model was used. On day confirmed by colony formation assays, which showed that more foci 42, lungs were sectioned and stained with Hematoxylin–Eosin were formed by cells with TRIM65 overexpression than those with (H&E). H&E staining of lung nodules was often observed in the TRIM65 depletion (Fig. 4C). Transwell assays were performed to TRIM65 overexpression groups, but rarely in the TRIM65-deficient assess the impact of TRIM65 on cell migration. Exogenous group. Statistically, the frequency of lung metastasis was TRIM65 remarkably enhanced cell movement, resulting in greater significantly higher in the TRIM65 overexpression groups than in numbers of migrating cells (Fig. 4D). These data indicated that the TRIM65-silenced groups (Fig. 5C). Collectively, these findings TRIM65 promotes cell growth and migration in vitro. indicate that TRIM65 exerts oncogenic effects in HCC. We next tested whether TRIM65 affects tumor growth and metastasis in vivo. Tumors formed by cells with TRIM65 TRIM65 activates β-catenin signaling via ubiquitylation overexpression grew much faster than those in control groups, of Axin1 whereas tumors with TRIM65 silencing experienced growth arrest In order to unveil the underlying mechanism by which TRIM65 (Fig. 5A). The tumors in the TRIM65 overexpression groups were promotes HCC progression, we examined the role of TRIM65 in much heavier than those in the control groups, but those in the the activation of the β-catenin signaling pathway. Strikingly, the Journal of Cell Science

3111 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

Fig. 5. TRIM65 enhances tumor growth and metastasis in vivo. (A) Cells stably transfected with TRIM65 overexpression vector or shRNA were injected into the right flanks of mice for 27 days. Growth curves demonstrated that tumors with TRIM65 overexpression grew much faster than those in control groups. (B) The tumors were dissected on day 27 and weighed. Tumors with TRIM65 overexpression were much heavier than those in control groups. (C) Cells were injected through the tail vein. On day 40, the lungs were sectioned for observation of tumor metastasis by H&E staining. The indicated numbers of lung nodules suggest that TRIM65 promotes tumor metastasis. *P<0.05, **P<0.01, ***P<0.001. expression of β-catenin was upregulated by TRIM65 attention. A series of factors, including proteins and microRNAs, overexpression, leading to increased expression of c-Myc (also have been demonstrated to participate in HCC progression (Zhang known as MYC) and cyclin D1, two well-known targets of β- et al., 2016; Lu et al., 2017). In the present study, we found that catenin. However, knockdown of TRIM65 resulted in the opposite TRIM65 has prognostic implication and functions as an oncogene effect (Fig. 6A). In the nuclear fraction, the expression of β-catenin by activating β-catenin signaling via the ubiquitylation of Axin1. was dramatically elevated in cells with TRIM65 overexpression, Previous studies have demonstrated the prognostic value of TRIM and slightly reduced in cells with TRIM65 silencing (Fig. 6B). proteins in human cancers. Palmbos et al. reported that TRIM29 Immunofluorescence (IF) revealed that β-catenin translocated to the overexpression in invasive bladder cancer was associated with poor nucleus upon TRIM65 expression in Bel-7402 and QGY-7703 cells prognosis (Palmbos et al., 2015). In HCC, the expression of several (Fig. 6C). In clinical samples, TRIM65 and nuclear β-catenin TRIM proteins, such as TRIM44 and TRIM26, was able to predict the expression were positively associated (Fig. 6D). These data suggest overall survival of patients (Wang et al., 2015; Zhu et al., 2016). The that TRIM65 activates the β-catenin pathway in HCC. upregulation of TIMR65 in lung cancer was reported to be correlated To further investigate the mechanism of TRIM65-mediated β- with unfavorable outcome (Wang et al., 2016). In our study, TRIM65 catenin activation, we examined the effect of TRIM65 on the expression was significantly increased in HCC and associated with expression of Axin1 and GSK-3β, two important regulators of β- tumor grade, vascular invasion, and overall and disease-free survival catenin. Overexpression of TRIM65 noticeably downregulated in two independent cohorts containing 888 patients. These data Axin1 expression but did not affect GSK-3β expression (Fig. 7A). suggest that TRIM family proteins are promising biomarkers for Using co-immunoprecipitation (co-IP) experiments, we found that predicting the postsurgical prognosis of patients with HCC. Axin1 was detectable in the precipitate mediated by anti-TRIM65 Our data showed that the increased expression of TRIM65 was antibody, but not in IgG-precipitated compound (Fig. 7B). The partly caused by positive regulation by HMGA1, an architectural correlation between TRIM65 and Axin1 was further confirmed by chromatin factor that is capable of transcriptionally modulating gene IF, showing colocalization of the two molecules in the cytoplasm expression (De Martino et al., 2016). In both study cohorts, patients (Fig. 7C). We next examined whether TRIM65 affected the protein with high levels of HMGA1 were frequently associated with high stability of Axin1. The half-life of Axin1 protein was greatly expression of TRIM65. Furthermore, the oncogenic activity of shortened by TRIM65 overexpression (Fig. 7D). The ubiquitylation HMGA1 was consistent with the protumor activity of TRIM65. of Axin1 protein was enhanced by TRIM65 in both HCC cell lines Luciferase reporter assays demonstrated that HMGA1 affected the (Fig. 7E). Following the knockdown of TRIM65, the ubiquitylation activity of the TRIM65 promoter, indicating the regulation of of β-catenin was enhanced (Fig. S2). These findings suggest that TRIM65 by HMGA1 at a transcriptional level. Recent studies have TRIM65 triggers β-catenin signaling via Axin1 degradation in demonstrated that HMGA1 is involved in the activation of β-catenin HCC cells. signaling, and modulates the expression of DEPDC1 and KIF23 to promote cell migration (Pegoraro et al., 2013). An interaction DISCUSSION between HMGA1 and β-catenin has been proposed (Xing et al., The identification of potential biomarkers useful for the clinical 2014). Interestingly, the activation of Wnt/β-catenin resulted in the management of human cancers has been attracting increasing induction of HMGA1 in gastric cancer (Akaboshi et al., 2009). Journal of Cell Science

3112 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

as IFIH1) enhanced the antivirus activityof the innate immune system (Lang et al., 2017). Li and colleagues reported that TRIM65 regulated the microRNA profile in human cells via the ubiquitylation of TNRC6 proteins (Li et al., 2014). In lung cancer, TRIM65 exhibited oncogenic activity by reducing the protein half-life of tumor suppressor p53 (Li et al., 2016). Here, co-IP and confocal experiments confirmed the interaction between TRIM65 and Axin1. Further analyses showed that TRIM65 enhanced the ubiquitylation of Axin1. Together, these findings suggest that TRIM65 promotes HCC progression via Axin1 loss-triggered activation of β-catenin signaling, which represents a potential therapeutic target for HCC treatment.

MATERIALS AND METHODS Patients A cohort of 516 HCC cases diagnosed between January 2010 and December 2013 at Dongguan Third People’s Hospital, The Affiliated Hexian Memorial Hospital of Southern Medical University and First Affiliated Hospital of NanChang University, was recruited and named the DSN cohort. Another 48 fresh specimens for qRT-PCR and western blot analysis, and 87 HCC cases with venous metastases for IHC, were obtained from Sun Yat-sen University Cancer Center. None of the patients had received radiotherapy or chemotherapy prior to surgery. This project was approved by the Institute Research Ethics Committee of the First Affiliated Hospital of NanChang University and Sun Yat-sen University Cancer Center. All samples were anonymous. Informed consent was obtained for all patients and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. A second cohort consisting of 372 patients with HCC was obtained to verify the prognostic value of TRIM65 from The Cancer Genome Atlas (TCGA) dataset via http://www.cbioportal.org.

Cell culture and transfection HCC cells (QGY-7703 and Bel-7404) were purchased from the Cell Resource Center, Chinese Academy of Science Committee (Shanghai, China). HCC cells with high metastatic potential (MHCC-97H) were Fig. 6. TRIM65 activates β-catenin signaling. (A) The effects of TRIM65 on obtained from the American Type Culture Collection (ATCC), and the expression of β-catenin and its downstream effectors, including c-Myc maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) and cyclin D1, was determined by western blot analysis. (B) Cells were supplemented with 10% heat-inactivated fetal bovine serum (Hyclone separated into cytosolic and nuclear fractions. The expression of nuclear Laboratories) in a humidified incubator at 37°C and 5% CO2. All the cell β-catenin was increased by TRIM65 overexpression. Lamin B was used as lines have been tested for contamination. The cells were transiently or stably a loading control. (C) IF was used to indicate the cellular localization of transfected with TRIM65 overexpression vector and siRNAs with β-catenin in cells with TRIM65 overexpression. (D) The coexpression of Lipofectamine 2000, according to the manufacturer’s instructions. Stable TRIM65 and nuclear β-catenin as shown by IHC staining in a cohort of 516 cell line with TRIM65 overexpression were established by G418 screening patients with HCC. for 2 weeks. The siRNAs used in this study were as follows: siTRIM65-1: ′ ′ ′ β 5 -GAUUAUCGCAAUCUGACCU-3 ; siTRIM65-2: 5 -UCGGUUCGG- Whether a positive feedback loop of HMGA1/TRIM65/ -catenin ACACCUGAAU-3′; siHMGA1-1: 5′-UGGCCUCCAAGCAGGAAAA- affects HCC progression requires further investigation. 3′; siHMGA1-2: 5′-CACAACUCCAGGAAGGAAA-3′; negative control Axin1 and GSK-3β are the two core regulators involved in the Wnt/ siRNA: 5′-UUGUACUACACAAAAGUACUG-3′. β-catenin pathway (Clevers and Nusse, 2012). Overexpression of Axin1 facilitated the formation of the Axin1–GSK-3β–β-catenin qRT-PCR and western blot analysis degradation complex, resulting in a decrease in β-catenin (Clevers and qRT-PCR and western blot analyses were performed according to our Nusse, 2012). A potential strategy to trigger the β-catenin pathway is previous study (Zhang et al., 2012). The sequences of the PCR primers were inhibition of Axin1. Picco et al. showed that targeting Axin1 with the as follows: TRIM65, forward: 5′-CCTTCCATGCCCTCTTCAAC-3′ and porcupine inhibitor LGK974 was a potential approach for the reverse: 5′-CTCCATCCCATGCCTTCTTC-3′; β-actin, forward: 5′-TGG- ′ ′ treatment of colorectal cancer (Picco et al., 2017). Chimge and CACCCAGCACAATGAA-3 and reverse: 5 -CTAAGTCATAGTCCGC- ′ colleagues reported that RUNX1-mediated Axin1 suppression led to CTAGAAGCA-3 . The antibodies used in western blotting were as follows: β TRIM65 (1:1000, SAB1408433, Sigma-Aldrich), HMGA1 (1:1000, 120- the activation of -catenin (Chimge et al., 2016). Mah et al. β γ 94, Cell Signaling Technology), -catenin (1:1000, 9582S, Cell Signaling demonstrated that the stabilization of Axin1 by the -protocadherin- Technology) and GAPDH (1:1000, Santa Cruz Biotechnology). C3 isoform inhibited Wnt signaling (Mah et al., 2016). Strikingly, mice with conditional disruption of Axin1 developed liver cancer, β IHC partly resulting from the constitutive activation of -catenin (Feng IHC for TRIM65 (1:1000, HPA021575, Sigma-Aldrich), HMGA1 and β- et al., 2012). In our study, overexpression of TRIM65 triggered the catenin was performed on a HCC tissue microarray. Expression levels were activation of β-catenin via the ubiquitylation of Axin1. The effect of scored as a proportion of immunopositive staining area (0%, 0; 1–25%, 1; TRIM65 on protein stability has been shown previously. Lang et al. 26–50%, 2; 51–75%, 3; 76%–100%, 4) multiplied by the intensity of showed that TRIM65-catalyzed ubiquitylation of MDA5 (also known staining (0, negative; 1, weak; 2, moderate; 3, intense). The scores were Journal of Cell Science

3113 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

Fig. 7. TRIM65 triggers β-catenin signaling via ubiquitylation of Axin1. (A) The effects of TRIM65 on Axin1 and GSK-3β expression were examined. (B) Co-IP experiments showed the coexistence of TRIM65 and Axin1 in the precipitant. (C) IF revealed the colocalization of TRIM65 and Axin1 in HCC cells. (D) The overexpression of TRIM65 affected the half-life of Axin1 (left). The related protein levels at the indicated treatment times (right). (E) Cells were transfected with HA-Ubiquitin and/or TRIM65 for 36 h. The ubiquitylation of Axin1 was enhanced by the overexpression of TRIM65. independently assessed by two pathologists (Y.-F.Y. and M.-F.Z.). The At day 27, tumors were dissected and weighed. For the metastasis model, median IHC score (4.44) was selected as the cut-off value for defining high approximately 5×105 cells were injected via the tail vein. After 6 weeks, the and low expression. mice were killed. The lungs were fixed in 4% paraformaldehyde and stained with H&E. Lung metastases were counted and quantified in a random Colony formation and transwell assays selection of high-power fields. All animal studies were approved by the Experiments were performed as described in our previous study (Zhang Medical Experimental Animal Care Commission of Sun Yat-sen University et al., 2016). For colony formation assays, stable cells were constructed. Cells Cancer Center. were collected and seeded in six-well plates at a density of 1.0×103 per well and then incubated at 37°C for 10 days. Colonies were fixed with methanol, Statistical analysis stained with 0.1% Crystal Violet and counted. For transwell assays, cells The Student’s t-test was used for comparisons between groups. resuspended in 200 μl serum-free medium were placed in the upper Kaplan–Meier analyses were used for survival analysis. P<0.05 was compartment of an uncoated transwell chamber (Corning; 24-well insert; considered significant. Data from three separate experiments are pore size, 8 mm). The lower chamber was filled with 15% fetal bovine serum presented as mean±s.e.m. as a chemoattractant and incubated for 48 h. The cells on the lower surface were fixed with methanol, stained with 0.1% Crystal Violet and counted. Competing interests The authors declare no competing or financial interests.

Luciferase reporter assay Author contributions For the luciferase reporter assay, Bel-7402 cells were co-transfected with Conceptualization: Y.-F.Y., C.Z.Z.; Methodology: Y.-F.Y., M.-F.Z., Q.-H.T., C.Z.Z.; HMGA1 overexpression vector, siRNAs or the negative control and 500 ng Validation: M.-F.Z., C.Z.Z.; Investigation: Y.-F.Y., M.-F.Z., Q.-H.T., C.Z.Z.; psiCHECK-2-TRIM65-3′-UTR reporter. Cells were collected 48 h after Resources: Y.-F.Y., Q.-H.T.; Writing - original draft: Y.-F.Y., M.-F.Z., C.Z.Z.; Writing - transfection and analyzed with a Dual-Luciferase Reporter Assay System review & editing: Y.-F.Y., C.Z.Z.; Supervision: C.Z.Z.; Funding acquisition: C.Z.Z. (Promega). Funding The study was supported by the National Natural Science Foundation of China Animal model (81572405). Male BALB/c-nude mice (aged 4 weeks, six mice per group) were 7 subcutaneously injected with 1×10 cells with TRIM65 overexpression or Supplementary information depletion into the left flanks. Tumors were measured with calipers and Supplementary information available online at calculated by the formula: volume (mm3)=[width2 (mm2)×length (mm)]/2. http://jcs.biologists.org/lookup/doi/10.1242/jcs.206623.supplemental Journal of Cell Science

3114 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3108-3115 doi:10.1242/jcs.206623

References Mah, K. M., Houston, D. W. and Weiner, J. A. (2016). The gamma-Protocadherin- Akaboshi, S., Watanabe, S., Hino, Y., Sekita, Y., Xi, Y., Araki, K., Yamamura, K., C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at Oshima, M., Ito, T., Baba, H. et al. (2009). HMGA1 is induced by Wnt/beta- the membrane. Sci. Rep. 6, 31665. catenin pathway and maintains cell proliferation in gastric cancer. Am. J. Pathol. Monga, S. P. (2015). beta-catenin signaling and roles in liver homeostasis, injury, 175, 1675-1685. and tumorigenesis. Gastroenterology 148, 1294-1310. Andreozzi, M., Quintavalle, C., Benz, D., Quagliata, L., Matter, M., Calabrese, D., Nusse, R. and Clevers, H. (2017). Wnt/beta-catenin signaling, disease, and Tosti, N., Ruiz, C., Trapani, F., Tornillo, L. et al. (2016). HMGA1 expression in emerging therapeutic modalities. Cell 169, 985-999. human hepatocellular carcinoma correlates with poor prognosis and promotes Palmbos, P. L., Wang, L., Yang, H., Wang, Y., Leflein, J., Ahmet, M. L., tumor growth and migration in in vitro models. Neoplasia 18, 724-731. Wilkinson, J. E., Kumar-Sinha, C., Ney, G. M., Tomlins, S. A. et al. (2015). Chimge, N.-O., Little, G. H., Baniwal, S. K., Adisetiyo, H., Xie, Y., Zhang, T., ATDC/TRIM29 drives invasive bladder cancer formation through miRNA- O’Laughlin, A., Liu, Z. Y., Ulrich, P., Martin, A. et al. (2016). RUNX1 prevents mediated and epigenetic mechanisms. Cancer Res. 75, 5155-5166. oestrogen-mediated AXIN1 suppression and beta-catenin activation in ER- Pegoraro, S., Ros, G., Piazza, S., Sommaggio, R., Ciani, Y., Rosato, A., Sgarra, positive breast cancer. Nat. Commun. 7, 10751. R., Del Sal, G. and Manfioletti, G. (2013). HMGA1 promotes metastatic Clevers, H. and Nusse, R. (2012). Wnt/beta-catenin signaling and disease. Cell processes in basal-like breast cancer regulating EMT and stemness. Oncotarget 149, 1192-1205. 4, 1293-1308. Coleman, M. P. (2014). Cancer survival: global surveillance will stimulate health Picco, G., Petti, C., Centonze, A., Torchiaro, E., Crisafulli, G., Novara, L., policy and improve equity. Lancet 383, 564-573. Acquaviva, A., Bardelli, A. and Medico, E. (2017). Loss of AXIN1 drives De Martino, M., Forzati, F., Arra, C., Fusco, A. and Esposito, F. (2016). HMGA1- acquired resistance to WNT pathway blockade in colorectal cancer cells carrying pseudogenes and cancer. Oncotarget 7, 28724-28735. RSPO3 fusions. EMBO Mol. Med. 9, 293-303. Dükel, M., Streitfeld, W. S., Tang, T. C. C., Backman, L. R. F., Ai, L., May, W. S. Reymond, A., Meroni, G., Fantozzi, A., Merla, G., Cairo, S., Luzi, L., Riganelli, D., and Brown, K. D. (2016). The breast cancer tumor suppressor TRIM29 is Zanaria, E., Messali, S., Cainarca, S. et al. (2001). The tripartite motif family identifies cell compartments. EMBO J. 20, 2140-2151. expressed via ATM-dependent signaling in response to hypoxia. J. Biol. Chem. Sumter,T.F.,Xian,L.,Huso,T.,Koo,M.,Chang,Y.-T.,Almasri,T.N.,Chia,L., 291, 21541-21552. Inglis, C., Reid, D. and Resar, L. M. S. (2016). The High Mobility Group A1 Feng, G. J., Cotta, W., Wei, X. Q., Poetz, O., Evans, R., Jardé, T., Reed, K., (HMGA1) transcriptome in cancer and development. Curr. Mol. Med. 16, Meniel, V., Williams, G. T., Clarke, A. R. et al. (2012). Conditional disruption of 353-393. Axin1 leads to development of liver tumors in mice. Gastroenterology 143, Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., Lortet-Tieulent, J. and Jemal, A. 1650-1659. (2015). Global cancer statistics, 2012. CA Cancer J. Clin. 65, 87-108. Groner, A. C., Cato, L., de Tribolet-Hardy, J., Bernasocchi, T., Janouskova, H., Wang, Y., He, D., Yang, L., Wen, B., Dai, J., Zhang, Q., Kang, J., He, W. and Ding, Melchers, D., Houtman, R., Cato, A. C. B., Tschopp, P., Gu, L. et al. (2016). Q. (2015). TRIM26 functions as a novel tumor suppressor of hepatocellular TRIM24 is an oncogenic transcriptional activator in prostate cancer. Cancer Cell carcinoma and its downregulation contributes to worse prognosis. Biochem. 29, 846-858. Biophys. Res. Commun. 463, 458-465. Lang, X., Tang, T., Jin, T., Ding, C., Zhou, R. and Jiang, W. (2017). TRIM65- Wang, X.-L., Shi, W.-P., Shi, H.-C., Lu, S.-C., Wang, K., Sun, C., He, J.-S., catalized ubiquitination is essential for MDA5-mediated antiviral innate immunity. Jin, W.-G., Lv, X.-X., Zou, H. et al. (2016). Knockdown of TRIM65 inhibits lung J. Exp. Med. 214, 459-473. cancer cell proliferation, migration and invasion: a therapeutic target in human Lau, K.-M., Chan, Q. K. Y., Pang, J. C. S., Ma, F. M.-T., Li, K. K. W., Yeung, W. W., lung cancer. Oncotarget 7, 81527-81540. Cheng, A. S. L., Feng, H., Chung, N. Y. F., Li, H.-M. et al. (2012). Overexpression Xian, L., Georgess, D., Huso, T., Cope, L., Belton, A., Chang, Y.-T., Kuang, W., of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion Gu, Q., Zhang, X., Senger, S. et al. (2017). HMGA1 amplifies Wnt signalling and through a cdc25A-independent pathway in medulloblastoma. Acta Neuropathol. expands the intestinal stem cell compartment and Paneth cell niche. Nat. 123, 553-571. Commun. 8, 15008. Li, S., Wang, L., Fu, B., Berman, M. A., Diallo, A. and Dorf, M. E. (2014). TRIM65 Xing, J., Cao, G. and Fu, C. (2014). HMGA1 interacts with beta-catenin to positively regulates microRNA activity by ubiquitination of TNRC6. Proc. Natl. Acad. Sci. regulate Wnt/beta-catenin signaling in colorectal cancer cells. Pathol. Oncol. Res. USA 111, 6970-6975. 20, 847-851. Li, Y., Ma, C., Zhou, T., Liu, Y., Sun, L. and Yu, Z. (2016). TRIM65 negatively Zhang, C. Z. Y., Chen, G. G., Merchant, J. L. and Lai, P. B. S. (2012). Interaction regulates p53 through ubiquitination. Biochem. Biophys. Res. Commun. 473, between ZBP-89 and p53 mutants and its contribution to effects of HDACi on 278-282. hepatocellular carcinoma. Cell Cycle 11, 322-334. Liang, L., Li, X., Zhang, X., Lv, Z., He, G., Zhao, W., Ren, X., Li, Y., Bian, X., Liao, Zhang, C. Z., Cao, Y., Fu, J., Yun, J.-P. and Zhang, M.-F. (2016). miR-634 exhibits W. et al. (2013). MicroRNA-137, an HMGA1 target, suppresses colorectal cancer anti-tumor activities toward hepatocellular carcinoma via Rab1A and DHX33. Mol. cell invasion and metastasis in mice by directly targeting FMNL2. Oncol. 10, 1532-1541. Gastroenterology 144, 624-635.e4. Zhu, X., Wu, Y., Miao, X., Li, C., Yin, H., Yang, S., Lu, X., Liu, Y., Chen, Y., Shen, Lu, S. X., Zhang, C. Z., Luo, R. Z., Wang, C. H., Liu, L. L., Fu, J., Zhang, L., Wang, R. et al. (2016). High expression of TRIM44 is associated with enhanced cell H., Xie, D. and Yun., J. P. (2017). Zic2 promotes tumor growth and metastasis via proliferation, migration, invasion, and resistance to doxorubicin in hepatocellular PAK4 in hepatocellular carcinoma. Cancer Lett. 402, 71-80. carcinoma. Tumour Biol. 37, 14615-14628. Journal of Cell Science

3115