TRIM65 triggers β-catenin signaling via ubiquitylation of Axin1 to promote hepatocellular carcinoma Free

The outcomes of hepatocellular carcinoma (HCC) remain extremely poor. Although efforts have been made to develop new strategies for clinical intervention, the 5 year survival of patients with HCC has barely improved (Coleman, 2014; Torre et al., 2015). The identification of potential biomarkers for disease diagnosis and prognostic prediction is a priority in precision medicine, especially for tumors lacking targeted therapy. As a result, identifying proteins that promote the malignant progression of HCC has been attracting increasing attention.

Deregulation of ubiquitin ligase is responsible for many human diseases. Tripartite motif-containing (TRIM) proteins, containing a RING, a B box type 1 and B box type 2, and a coiled-coil region, have been identified as E3 ubiquitin ligases (Reymond et al., 2001). TRIM proteins have been shown to have important roles in various cellular processes, including cell development, proliferation and differentiation. Several TRIM members, such as TRIM24 and TRIM29, participate in the progression of solid tumors by inducing ubiquitylation of their downstream targets (Dükel et al., 2016; Groner et al., 2016). For example, TRIM65 is upregulated in lung cancer and promotes tumor growth via ubiquitylation of p53 (also known as TP53) to inhibit its antitumor activity (Li et al., 2016; Wang et al., 2016). Li et al. reported that TRIM65 was capable of modulating the microRNA profile in lung and cervical cancer cells (Li et al., 2014). However, the role of TRIM65 in HCC remains unclear.

Aberrant activation of β-catenin signaling contributes to the initiation and progression of HCC. The activation of β-catenin signaling is dependent on two protein families: Wnt and Akt (Monga, 2015). Canonically, β-catenin is activated by Wnt/Frizzled signaling. Cytoplasmic β-catenin is phosphorylated by a complex with glycogen synthase kinase-3β (GSK-3β), Axin1/2 and adenomatosis polyposis coli (APC), and is subsequently targeted for proteosomal degradation. Once Wnt signaling is activated by Frizzled, β-catenin is dissociated from the degradation complex, and accumulates in the nucleus to form a transcriptional complex with TCF/LEF proteins to induce the transcription of downstream genes implicated in carcinogenesis (Nusse and Clevers, 2017). Previous studies have reported that Wnt/β-catenin signaling could be triggered by HMGA1 (Akaboshi et al., 2009; Xian et al., 2017), an architectural transcriptional factor. HMGA1 is a nonhistone nuclear protein involved in genetic recombination, chromosomal modulation, DNA repair and cell apoptosis (Sumter et al., 2016). Overexpression of HMGA1 in human cancers, such as medulloblastoma, colorectal cancer and HCC, promotes malignant progression via transcriptional regulation of several oncogenes or tumor suppressor genes (Andreozzi et al., 2016; Lau et al., 2012; Liang et al., 2013).

In this study, we evaluated the expression of TRIM65 and its clinical significance in two independent cohorts of 888 patients with HCC. We investigated the effects of TRIM65 on HCC progression and the underlying mechanisms. Our data suggest that TRIM65, regulated by HMGA1, exerts oncogenic activity in HCC through ubiquitylation of Axin1 to activate β-catenin signaling.

To determine the expression of TRIM65 in HCC, 48 pairs of fresh tissue were collected. We observed that TRIM65 mRNA was overexpressed in HCC tissues, compared with the corresponding adjacent nontumorous tissues (Fig. 1A). Consistently, the level of TRIM65 protein was much higher in HCC tissues than in nontumorous tissues (Fig. 1B). To validate the observation of increased expression of TRIM65 in HCC, a cohort of 516 patients was recruited. Results from tissue microarray-based immunohistochemistry (IHC) showed that TRIM65 mainly localized in the cytoplasm. In 56.6% (292/516) of the cases, TRIM65 expression was upregulated in HCC (Fig. 1C). In another cohort of patients with venous metastasis, TRIM65 expression was higher in the portal vein embolus than in the primary tumor in 83.9% (73/87) of the cases (Fig. 1C).

To reveal the clinical implications of TRIM65 in HCC, the relationship between TRIM65 and pathological features of the disease was evaluated. Results showed that the IHC score for TRIM65 steadily increased from G1 to G4 in the DSN cohort (see Materials and Methods) (Fig. 2A). Patients with tumor vascular invasion had increased TRIM65 expression (Fig. 2B). These findings were further confirmed in the TCGA cohort (see Materials and Methods) (Fig. 2A,B). According to the median IHC score (DSN cohort) and mRNA expression value (TCGA cohort) for TRIM65, patients were separated into two groups: high or low TRIM65. High TRIM65 expression was significantly associated with tumor size (P=0.022), tumor differentiation (P=0.004), clinical stage (P=0.001) and vascular invasion (P=0.024) in the DSN cohort (Table S1). Kaplan–Meier analyses indicated that HCC patients with high expression of TRIM65 were associated with shorter overall and disease-free survival in both the DSN and TCGA cohorts (Fig. 2C,D). Multivariate analysis revealed that TRIM65 was an independent factor for overall survival (hazard ratio, 1.354; 95% confidence interval, 1.099–1.668; P=0.004) (Table S2), but not for disease-free survival (data not shown).

Using bioinformatics programs, HMGA1 was predicted to be an upstream regulator of TRIM65. The in vitro experiments showed that the expression of TRIM65 was increased in QGY-7703 and Bel-7402 cells with HMGA1 overexpression, but decreased in MHCC-97H cells with HMGA1 silencing (Fig. 3A). Luciferase reporter assays confirmed that HMGA1 was capable of modulating the activity of the TRIM65 promoter. The related luciferase activity of the TRIM65 promoter was enhanced by HMGA1 overexpression, but attenuated by HMGA1 depletion, in Bel-7402 cells (Fig. 3B). In clinical samples, the expression of TRIM65 was positively correlated with HMGA1 expression. According to the TCGA data, TRIM65 mRNA coexpressed with HMGA1 mRNA in 423 cases (Fig. 3C). In 19 pairs of fresh HCC specimens, TRIM65 mRNA expression was associated with HMGA1 protein expression (Fig. 3D). As indicated by IHC staining, patients with high TRIM65 expression frequently expressed more HMGA1 than those with low TRIM65 expression (Fig. 3E).

Because TRIM65 expression correlated with poor outcome, we next investigated whether TRIM65 was involved in the malignant progress of HCC. According to the expression of TRIM65 mRNA (Fig. S1), TRIM65 was induced in QGY-7703 and Bel-7402 cells, and reduced in MHCC-97H cells (Fig. 4A). As shown by MTT assays, cell growth rates were increased in cells with TRIM65 overexpression, but decreased in cells with TRIM65 depletion (Fig. 4B). The effect of TRIM65 on cell proliferation was further confirmed by colony formation assays, which showed that more foci were formed by cells with TRIM65 overexpression than those with TRIM65 depletion (Fig. 4C). Transwell assays were performed to assess the impact of TRIM65 on cell migration. Exogenous TRIM65 remarkably enhanced cell movement, resulting in greater numbers of migrating cells (Fig. 4D). These data indicated that TRIM65 promotes cell growth and migration in vitro.

We next tested whether TRIM65 affects tumor growth and metastasis in vivo. Tumors formed by cells with TRIM65 overexpression grew much faster than those in control groups, whereas tumors with TRIM65 silencing experienced growth arrest (Fig. 5A). The tumors in the TRIM65 overexpression groups were much heavier than those in the control groups, but those in the TRIM65 depletion group were lighter (Fig. 5B). For detection of tumor metastasis, a caudal vein injection model was used. On day 42, lungs were sectioned and stained with Hematoxylin–Eosin (H&E). H&E staining of lung nodules was often observed in the TRIM65 overexpression groups, but rarely in the TRIM65-deficient group. Statistically, the frequency of lung metastasis was significantly higher in the TRIM65 overexpression groups than in the TRIM65-silenced groups (Fig. 5C). Collectively, these findings indicate that TRIM65 exerts oncogenic effects in HCC.

In order to unveil the underlying mechanism by which TRIM65 promotes HCC progression, we examined the role of TRIM65 in the activation of the β-catenin signaling pathway. Strikingly, the expression of β-catenin was upregulated by TRIM65 overexpression, leading to increased expression of c-Myc (also known as MYC) and cyclin D1, two well-known targets of β-catenin. However, knockdown of TRIM65 resulted in the opposite effect (Fig. 6A). In the nuclear fraction, the expression of β-catenin was dramatically elevated in cells with TRIM65 overexpression, and slightly reduced in cells with TRIM65 silencing (Fig. 6B). Immunofluorescence (IF) revealed that β-catenin translocated to the nucleus upon TRIM65 expression in Bel-7402 and QGY-7703 cells (Fig. 6C). In clinical samples, TRIM65 and nuclear β-catenin expression were positively associated (Fig. 6D). These data suggest that TRIM65 activates the β-catenin pathway in HCC.

To further investigate the mechanism of TRIM65-mediated β-catenin activation, we examined the effect of TRIM65 on the expression of Axin1 and GSK-3β, two important regulators of β-catenin. Overexpression of TRIM65 noticeably downregulated Axin1 expression but did not affect GSK-3β expression (Fig. 7A). Using co-immunoprecipitation (co-IP) experiments, we found that Axin1 was detectable in the precipitate mediated by anti-TRIM65 antibody, but not in IgG-precipitated compound (Fig. 7B). The correlation between TRIM65 and Axin1 was further confirmed by IF, showing colocalization of the two molecules in the cytoplasm (Fig. 7C). We next examined whether TRIM65 affected the protein stability of Axin1. The half-life of Axin1 protein was greatly shortened by TRIM65 overexpression (Fig. 7D). The ubiquitylation of Axin1 protein was enhanced by TRIM65 in both HCC cell lines (Fig. 7E). Following the knockdown of TRIM65, the ubiquitylation of β-catenin was enhanced (Fig. S2). These findings suggest that TRIM65 triggers β-catenin signaling via Axin1 degradation in HCC cells.

The identification of potential biomarkers useful for the clinical management of human cancers has been attracting increasing attention. A series of factors, including proteins and microRNAs, have been demonstrated to participate in HCC progression (Zhang et al., 2016; Lu et al., 2017). In the present study, we found that TRIM65 has prognostic implication and functions as an oncogene by activating β-catenin signaling via the ubiquitylation of Axin1.

Previous studies have demonstrated the prognostic value of TRIM proteins in human cancers. Palmbos et al. reported that TRIM29 overexpression in invasive bladder cancer was associated with poor prognosis (Palmbos et al., 2015). In HCC, the expression of several TRIM proteins, such as TRIM44 and TRIM26, was able to predict the overall survival of patients (Wang et al., 2015; Zhu et al., 2016). The upregulation of TIMR65 in lung cancer was reported to be correlated with unfavorable outcome (Wang et al., 2016). In our study, TRIM65 expression was significantly increased in HCC and associated with tumor grade, vascular invasion, and overall and disease-free survival in two independent cohorts containing 888 patients. These data suggest that TRIM family proteins are promising biomarkers for predicting the postsurgical prognosis of patients with HCC.

Our data showed that the increased expression of TRIM65 was partly caused by positive regulation by HMGA1, an architectural chromatin factor that is capable of transcriptionally modulating gene expression (De Martino et al., 2016). In both study cohorts, patients with high levels of HMGA1 were frequently associated with high expression of TRIM65. Furthermore, the oncogenic activity of HMGA1 was consistent with the protumor activity of TRIM65. Luciferase reporter assays demonstrated that HMGA1 affected the activity of the TRIM65 promoter, indicating the regulation of TRIM65 by HMGA1 at a transcriptional level. Recent studies have demonstrated that HMGA1 is involved in the activation of β-catenin signaling, and modulates the expression of DEPDC1 and KIF23 to promote cell migration (Pegoraro et al., 2013). An interaction between HMGA1 and β-catenin has been proposed (Xing et al., 2014). Interestingly, the activation of Wnt/β-catenin resulted in the induction of HMGA1 in gastric cancer (Akaboshi et al., 2009). Whether a positive feedback loop of HMGA1/TRIM65/β-catenin affects HCC progression requires further investigation.

Axin1 and GSK-3β are the two core regulators involved in the Wnt/β-catenin pathway (Clevers and Nusse, 2012). Overexpression of Axin1 facilitated the formation of the Axin1–GSK-3β–β-catenin degradation complex, resulting in a decrease in β-catenin (Clevers and Nusse, 2012). A potential strategy to trigger the β-catenin pathway is inhibition of Axin1. Picco et al. showed that targeting Axin1 with the porcupine inhibitor LGK974 was a potential approach for the treatment of colorectal cancer (Picco et al., 2017). Chimge and colleagues reported that RUNX1-mediated Axin1 suppression led to the activation of β-catenin (Chimge et al., 2016). Mah et al. demonstrated that the stabilization of Axin1 by the γ-protocadherin-C3 isoform inhibited Wnt signaling (Mah et al., 2016). Strikingly, mice with conditional disruption of Axin1 developed liver cancer, partly resulting from the constitutive activation of β-catenin (Feng et al., 2012). In our study, overexpression of TRIM65 triggered the activation of β-catenin via the ubiquitylation of Axin1. The effect of TRIM65 on protein stability has been shown previously. Lang et al. showed that TRIM65-catalyzed ubiquitylation of MDA5 (also known as IFIH1) enhanced the antivirus activity of 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.

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.

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 obtained from the American Type Culture Collection (ATCC), and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories) in a humidified incubator at 37°C and 5% CO₂. All the cell lines have been tested for contamination. The cells were transiently or stably transfected with TRIM65 overexpression vector and siRNAs with Lipofectamine 2000, according to the manufacturer's instructions. Stable cell line with TRIM65 overexpression were established by G418 screening for 2 weeks. The siRNAs used in this study were as follows: siTRIM65-1: 5′-GAUUAUCGCAAUCUGACCU-3′; siTRIM65-2: 5′-UCGGUUCGGACACCUGAAU-3′; siHMGA1-1: 5′-UGGCCUCCAAGCAGGAAAA-3′; siHMGA1-2: 5′-CACAACUCCAGGAAGGAAA-3′; negative control siRNA: 5′-UUGUACUACACAAAAGUACUG-3′.

qRT-PCR and western blot analyses were performed according to our previous study (Zhang et al., 2012). The sequences of the PCR primers were as follows: TRIM65, forward: 5′-CCTTCCATGCCCTCTTCAAC-3′ and reverse: 5′-CTCCATCCCATGCCTTCTTC-3′; β-actin, forward: 5′-TGGCACCCAGCACAATGAA-3′ and reverse: 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The antibodies used in western blotting were as follows: TRIM65 (1:1000, SAB1408433, Sigma-Aldrich), HMGA1 (1:1000, 12094, Cell Signaling Technology), β-catenin (1:1000, 9582S, Cell Signaling Technology) and GAPDH (1:1000, Santa Cruz Biotechnology).

IHC for TRIM65 (1:1000, HPA021575, Sigma-Aldrich), HMGA1 and β-catenin was performed on a HCC tissue microarray. Expression levels were scored as a proportion of immunopositive staining area (0%, 0; 1–25%, 1; 26–50%, 2; 51–75%, 3; 76%–100%, 4) multiplied by the intensity of staining (0, negative; 1, weak; 2, moderate; 3, intense). The scores were independently assessed by two pathologists (Y.-F.Y. and M.-F.Z.). The median IHC score (4.44) was selected as the cut-off value for defining high and low expression.

Experiments were performed as described in our previous study (Zhang et al., 2016). For colony formation assays, stable cells were constructed. Cells 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, stained with 0.1% Crystal Violet and counted. For transwell assays, cells resuspended in 200 μl serum-free medium were placed in the upper compartment of an uncoated transwell chamber (Corning; 24-well insert; pore size, 8 mm). The lower chamber was filled with 15% fetal bovine serum 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.

For the luciferase reporter assay, Bel-7402 cells were co-transfected with HMGA1 overexpression vector, siRNAs or the negative control and 500 ng psiCHECK-2-TRIM65-3′-UTR reporter. Cells were collected 48 h after transfection and analyzed with a Dual-Luciferase Reporter Assay System (Promega).

Male BALB/c-nude mice (aged 4 weeks, six mice per group) were subcutaneously injected with 1×10⁷ cells with TRIM65 overexpression or depletion into the left flanks. Tumors were measured with calipers and calculated by the formula: volume (mm³)=[width² (mm²)×length (mm)]/2. At day 27, tumors were dissected and weighed. For the metastasis model, approximately 5×10⁵ cells were injected via the tail vein. After 6 weeks, the mice were killed. The lungs were fixed in 4% paraformaldehyde and stained with H&E. Lung metastases were counted and quantified in a random selection of high-power fields. All animal studies were approved by the Medical Experimental Animal Care Commission of Sun Yat-sen University Cancer Center.

The Student's t-test was used for comparisons between groups. Kaplan–Meier analyses were used for survival analysis. P<0.05 was considered significant. Data from three separate experiments are presented as mean±s.e.m.