MTBP inhibits migration and metastasis of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with increasing incidence. Despite curative surgical resection and advanced chemotherapy, its survival rate remains low. The presence of microvascular invasion and occult metastasis is one of the major causes for this poor outcome. MDM2 Binding Protein (MTBP) has been implicated in the suppression of cell migration and cancer metastasis. However, clinical significance of MTBP, particularly in human cancer, is poorly understood. Specifically, clinical relevance of MTBP in human HCC has never been investigated. Here we demonstrated that expression of MTBP was significantly reduced in human HCC tissues compared to adjacent non-tumor tissues. MTBP expression was negatively correlated with capsular/vascular invasion and lymph node metastasis. Overexpression of MTBP resulted in the suppression of the migratory and metastatic potential of HCC cells, while its downregulation increased the migration. Consistent with the previous report, MTBP endogenously bound to alpha-actinin 4 (ACTN4) and suppressed ACTN4-mediated cell migration in multiple HCC cell lines. However, MTBP also inhibited migratory potential of PLC/PRF/5 HCC cells whose migration was not altered by manipulation of ACTN4 expression. These results suggest that mechanisms behind MTBP-mediated migration suppression may not be limited to the pathway involving ACTN4 in certain cellular contexts. Additionally, as a potential mechanism for reduced MTBP expression in tumors, we found that MTBP expression was increased following the treatment with histone deacetylase inhibitors (HDIs). Our study, for the first time, provides clinical relevance of MTBP in the suppression of HCC metastasis.

Fig. 1

Decreased MTBP expression in HCC tissues compared with non-tumor liver tissues. a qRT-PCR. The mRNAs from 20 pairs of non-tumor (NT) and HCC (T) tissues were extracted, and qRT-PCR was performed. Data are shown as relative values of MTBP mRNA normalized by that of GAPDH to one of the tumor samples (left). Summary of qRT-PCR and statistical analysis (right, **P < 0.01; Mann–Whitney U test). b Representative MTBP IHC results using (i) NT (scale bar 50 μm), (ii) T (scale bar 50 μm), and (iii) NT and T boundary (scale bar 200 μm) liver tissues

Fig. 2

MTBP suppresses HCC metastasis and migration. a and b SK-hep-1 HCC cells infected with either empty (control, grey) or MTBP-encoding (black) lentiviral vectors were either intravenously (tail vein, n = 10, a) or subcutaneously (s.c., n = 6, b) injected into nude mice. Seven weeks after tail vein injections, mice were examined for the number of metastatic lung nodules (arrows). Tumor sizes following s.c. injections were measured twice a week. c MTBP overexpression (OvEx) inhibits the migratory potential of Huh7 (left) and SNU-475 (right) cells by transwell migration assays. Cells infected with empty (control, grey) or different (2.5 and 5) MOIs of an MTBP-encoding adenovirus (MTBP, black) were plated on the upper chambers and migrating cells in entire fields were counted 14 h later. Relative cell migration (%) compared to the number of migrating cells in control (top) and representative staining images (middle). Western blotting results below the images (bottom). Relative MTBP protein levels to controls normalized by actin loading control were shown below the MTBP blots. d MTBP downregulation increases the migratory potential of PLC/PRF/5 (left) and SNU-475 (right) cells by transwell migration assays. One day after non-target (control, white) or MTBP-specific siRNA transfection (oblique), migration assays were performed as in c. Error bars mean ± SD from three independent experiments. *P <0.05 and **P < 0.01; Student’s t test

Fig. 3

MTBP binds to and suppresses ACTN4-mediated HCC migration. a Endogenous interactions between MTBP and ACTN4 in Huh7 and PLC/PRF/5 cell lines by co-immunoprecipitation (co-IP) studies using indicated antibodies for MTBP and ACTN4. Anti-rabbit IgG was used as an isotype control. b and c SNU-475 cells stably infected with lentiviral vectors encoding empty (control, lanes 1, 3) or ACTN4 cDNA (lanes 2, 4) were infected with empty (lanes 1, 2) or MTBP-encoding (lanes 3, 4) adenoviral vectors. b Phalloidin staining was performed 48 h later. Cells (n = 50) were examined for filopodia formation. The percentage of cells positive for filopodia formation (top), representative phalloidin staining images (middle), and representative western blotting for ACTN4 and MTBP (bottom). Grey control, black ACTN4 overexpression (OvEx), white MTBP overexpression, oblique both MTBP and ACTN4 overexpression. c Tran-swell migration assays were performed as in Fig. 2c. Graphs showing relative cell migration (%) compared to the number of migrating cells in control. Representative images are placed below the graphs. Error bars means ± SD from three independent experiments. *P < 0.05 and **P < 0.01; Student’s t test

Fig. 4

MTBP inhibits the migratory potential of PLC/ PRF/5 cells in an ACTN4–independent manner. a Transwell migration assays were performed using PLC/ PRF/5 cells overexpressed for ACTN4 and/or MTBP. Summarized graph (top), representative images (middle), and western blotting results (bottom). b and c Migration assays were performed using PLC/PRF/5 cells downregulated for ACTN4 with or without manipulation of MTBP expression (b MTBP overexpression; c MTBP downregulation). Summarized graph (top), representative staining images (middle), and western blotting results (bottom). Error bars mean ± S.D. from three independent experiments. *P <0.05 and **P < 0.01; Student’s t test. n.s. not significant

Fig. 5

MTBP expression can be transcriptionally silenced in HCC. a Results of qRT-PCR 24 h after treatment of Huh7 (grey) and SNU-475 (black) cell lines with either 5 μM of SAHA, 0.3 μM of TSA, or 10 μM of 5-aza-dC. Data are shown as relative MTBP mRNA expression to that of GAPDH. b Western blotting for MTBP and GAPDH or actin at 24 h after treatment of indicated HCC cells with SAHA (top) or TSA (bottom). c Western blotting for MTBP and β-tubulin (control) following treatment of indicated cells with proteasome inhibitors MG132 and MG115 at 30 μM for 4 h. Equivalent dilutions of dimethyl sulfoxide (DMSO) were used as controls. d Results of qRT-PCR for MDM2 (left) and MTBP (right) using Huh7 (p53Y220C) and Hep3B (p53-null) cells (grey) and those down-regulated for MDM2 (black) with or without 0.3 μM of TSA treatment for 24 h. Cont: Control. Data are shown as relative MDM2 or MTBP mRNA expression to that of GAPDH. Error bars: mean ± S.D. from three independent experiments. *P <0.05 and **P < 0.01; Student’s t test. n.s., not significant