COMMD10 inhibits tumor progression and induces apoptosis by blocking NF-κB signal and values up BCLC staging in predicting overall survival in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Currently, there is limited knowledge of dysregulation of cellular proliferation and apoptosis that contribute to the malignant phenotype in HCC. Copper metabolism gene MURR1 domain 10 (COMMD10) is initially identified as a suppressor gene in the pathogenesis of HCC in our observations. Here we aimed to explore its function and prognostic value in the progression of HCC.

Methods: Functional experiments were performed to explore the role of COMMD10 in HCC. The molecular mechanisms of COMMD10 were determined by luciferase assay, immunofluorescence, and immunoprecipitation. The nomogram was based on a retrospective and multicenter study of 516 patients who were pathologically diagnosed with HCC from three Chinese hospitals. The predictive accuracy and discriminative ability of the nomogram were determined by a C-index and calibration curve and were compared with COMMD10 and the Barcelona Clinic Liver Cancer (BCLC) staging system. The primary endpoint was overall survival (OS).

Results: COMMD10 expression was significantly lower in HCC than that in normal liver tissues. In vitro and in vivo experiments revealed that COMMD10 suppressed cell proliferation and induced apoptosis in HCC. Mechanistically, COMMD10 inhibits TNFα mediated ubiquitination of IκBα and p65 nuclear translocation through the combination of COMMD10-N terminal to the Rel homology domain of p65, which inhibited NF-κB activity and increased expression of cleaved caspase9/3 in HCC. Clinically, COMMD10 stratifies early-stage HCC patients into two risk groups with significantly different OS. Additionally, the nomogram based on COMMD10 and BCLC stage yielded more accuracy than BCLC stage alone for predicting OS of HCC patients in three cohorts.

Conclusions: COMMD10 suppresses proliferation and promotes apoptosis by inhibiting NF-κB signaling and values up BCLC staging in predicting OS, which provides evidence for the identification of potential therapeutic targets and the accurate prediction of prognosis for patients with HCC.

Keywords: Barcelona Clinic Liver Cancer (BCLC); copper metabolism MURR1 domain-containing 10 (COMMD10); hepatocellular carcinoma; proliferation and apoptosis.

FIGURE 1

COMMD10 suppresses proliferation of HCC in vitro and in vivo. (A) Representative images of the clone formation assay (upper); the percentage of colonies was counted in graphs (bottom). (B) Growth curves for the indicated HCC cells (SMMC‐7721, HepG2, QGY‐7703) were evaluated by CCK‐8 assay. (C) NF‐κB luciferase‐reporter activity in COMMD10 overexpressing SMMC7721 and COMMD10‐deficient HepG2 cells under TNFα (10 ng/ml) treatment. (D) Overexpressing IκBα‐mut inhibited COMMD10‐deficiency induced proliferation as evaluated by CCK‐8 assay in HepG2 cells. (E) Tumor growth of HepG2 cells expressing vector or shCOMMD10 or shCOMMD10+IκBα‐mut in nude mice (n = 6, left). Representative images of IHC staining of the proliferation marker Ki67 in the indicated group, and quantification of the positive nuclei for this marker (n = 6, right). COMMD10 content of tumor xenografts in each group was detected by Western blot (right). GAPDH was used as loading control. Each bar represents the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001

FIGURE 2

COMMD10 inhibits TNFα mediated ubiquitination and degradation of IκBα and suppresses the NF‐κB signaling pathway. (A) Effect of COMMD10 deficiency on the level of IκBα, p‐IκBα in HepG2/Vector and HepG2/siCOMMD10 cells exposed to TNFα (10 ng/ml) as illustrated. Line graph showed the gray scale value of IκBα at indicated time in HepG2/Vector and HepG2/siCOMMD10 cells. (B) Effect of COMMD10 overexpression on the level of IκBα, p‐IκBα in SMMC7721/Mock and SMMC7721/COMMD10 cells exposed to TNFα (10 ng/ml) as illustrated. Line graph showed the gray scale value of IκBα at indicated time in SMMC7721/Mock and SMMC7721/COMMD10 cells. (C) Effect of COMMD10 deficiency on the level of IκBα, p‐IκBα in HepG2/Vector and HepG2/siCOMMD10 cells exposed to MG132 (40μM) and TNFα (10 ng/ml) as illustrated. Line graph showed the gray scale value of IκBα at indicated time in HepG2/Vector and HepG2/siCOMMD10 cells. (D) Effect of COMMD10 overexpression on the level of IκBα, p‐IκBα in SMMC7721/Mock and SMMC7721/COMMD10 cells exposed to MG132 (40μM) and TNFα (10 ng/ml) as illustrated. Line graph showed the gray scale value of IκBα at indicated time in SMMC7721/Mock and SMMC7721/COMMD10 cells. (E) Effect of COMMD10 deficiency on the level of IκBα ubiquitination in HepG2/Vector and HepG2/siCOMMD10 cells exposed to TNFα (10 ng/ml)

FIGURE 3

COMMD10 interacts with RHD of p65 and suppresses the nuclear translocation of p65. (A) Endogenous COMMD10 was immunoprecipitated (IP) from HepG2 cell lysates. (B) Immunofluorescence staining of COMMD10 and NF‐κB p65 colocalization in the cytoplasm. (C) Translocation of NF‐κB p65 in HepG2 cells by Immunofluorescence staining. (D) Western blot of nuclear, cytoplasmic, and total NF‐κB p65 expression in HCC cells. H2AX (H2AX histone family member) was used as the nuclear loading control; GAPDH was used as the cytoplasmic loading control. (E) Schematic structure of COMMD10 and its truncations (top) and GST‐Pull down assay showed domains of COMMD10 involved in the interaction with p65 (bottom). (F) Schematic structure of p65 and its truncations (top) and GST‐pull down assay showed domains of p65 involved in the interaction with COMMD10 (bottom). Abbreviation: PD, pull down.

FIGURE 4

COMMD10/NF‐κB/Bcl‐2/Bax/Caspase9/3 axis associates with apoptosis. (A) Annexin V‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining of indicated cells treated with irradiation (6 Gy). (B) A series of apoptosis assay induced by cisplatin (10ug/ml). Representative images of AOEB staining (upper), TUNEL assay (middle) and caspase3/7 activation assay (bottom) were showed from top to bottom, and the corresponding statistical analysis chart arranged from left to right. (C) Western blot of Bcl2/, Bax, cleaved‐caspase3, cleaved‐caspase9, and cleaved‐PARP expression in the indicated cells exposed to cisplatin (100μg/ml). GAPDH was used as a loading control. (D) Analysis of mRNA level (left) and correlation (right) of COMMD10 with BAX, BCL2, CASP3, and CASP9 in 10 freshly collected human HCC samples. The data are shown from a single representative experiment of three repeats. Each bar represents the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001

FIGURE 5

The association between COMMD10 expression and the clinical features in HCC. (A) Relative COMMD10 mRNA levels in 48 paired primary HCC tissues and matched normal liver tissues by real‐time PCR analysis. GAPDH mRNA was used as internal control. The data are shown from a single representative experiment of three repeats. (B) COMMD10 protein expression in 20 paired primary HCC tissues (T) and matched normal tissues (N); GAPDH was used as loading control. (C) Strong expression of COMMD10 protein in paraffin‐embedded normal, peritumorial liver tissues, and weak expression of COMMD10 in paraffin‐embedded HCC tissues by IHC staining analysis. (D) COMMD10 protein expression in well, medium, and poor differentiated paraffin‐embedded HCC tissues by IHC staining analysis in NF training cohort. Statistical quantification graph was shown in the right panel. Each bar represents the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001

FIGURE 6

COMMD10 is a predictor of stratification and prognosis in HCC. (A‐C) Prediction of HCC patients’ OS based on COMMD10 expression in NF training cohort (A), NF internal validation cohort (B), and ZY external validation cohort (C) using Kaplan‐Meier survival analysis. (D‐F) Prediction of early HCC (BCLC stage 0 and A) patients’ OS based on COMMD10 expression in NF training cohort (D), NF internal validation cohort (E), and ZY external validation cohort (F) using Kaplan‐Meier survival analysis

FIGURE 7

Development and evaluation of COMMD10‐based nomogram in HCC cohorts. (A) Nomogram, including patient age, ALB (g/L), tumor size (cm), embolus, and COMMD10 expression for 1‐, 2‐, 3‐year overall survival in HCC patients. The nomogram allows the user to obtain the probability of 1‐, 2‐, 3‐year overall survival corresponding to a patient's combination of covariates. (B‐D) The calibration curve of nomogram for predicting overall survival at 1‐, 2‐, 3‐year in NF training cohort (B), NF validation cohort (C) and ZY external validation cohort (D). Actual overall survival is plotted on the y‐axis; nomogram predicted probability of overall survival is plotted on the x‐axis. (E‐G) Time dependent ROC curve areas of nomogram, BCLC stage system, and COMMD10 expression in NF training cohort (E), NF internal validation cohort (F), and ZY external validation cohort (G)