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. 2020 Apr 1:5:15.
doi: 10.1038/s41525-020-0122-7. eCollection 2020.

"V体育官网" MACROD2 deficiency promotes hepatocellular carcinoma growth and metastasis by activating GSK-3β/β-catenin signaling

Zheng-Jun Zhou #  1   2 , Chu-Bin Luo #  1   2 , Hao-Yang Xin #  1   2 , Zhi-Qiang Hu  1   2 , Gui-Qi Zhu  1   2 , Jia Li  1   2 , Shao-Lai Zhou  1   2
Affiliations

"V体育2025版" MACROD2 deficiency promotes hepatocellular carcinoma growth and metastasis by activating GSK-3β/β-catenin signaling

Zheng-Jun Zhou et al. NPJ Genom Med. .

Abstract

Structural variations (SVs) influence the development and progression of multiple types of cancer. The genes affected by SVs in hepatocellular carcinoma (HCC) and their contribution to tumor growth and metastasis remain unknown VSports手机版. In this study, through whole-genome sequencing (WGS), we identified MACROD2 as the gene most frequently affected by SVs, which were associated with low MACROD2 expression levels. Low MACROD2 expression was predictive of tumor recurrence and poor overall survival. MACROD2 expression was decreased in HCC cell lines, especially those with high metastatic potential. MACROD2 knockdown in HCC cells markedly enhanced proliferation and invasiveness in vitro and tumor progression in vivo and promoted epithelial-mesenchymal transition (EMT). By contrast, MACROD2 overexpression reversed EMT and inhibited HCC growth and metastasis. Mechanistically, MACROD2 deficiency suppressed glycogen synthase kinase-3β (GSK-3β) activity and activated β-catenin signaling, which mediated the effect of MACROD2 on HCC. In clinical HCC samples, decreased MACROD2 expression was correlated with the activation of GSK-3β/β-catenin signaling and the EMT phenotype. Overall, our results revealed that MACROD2 is frequently affected by SVs in HCC, and its deficiency promotes tumor growth and metastasis by activating GSK-3β/β-catenin signaling. .

Keywords: Cancer genomics; Gastrointestinal cancer; Metastasis. V体育安卓版.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Fig. 1
Fig. 1. MACROD2 is frequently affected by SVs in HCC, and down-regulation of MACROD2 correlates with patient poor prognosis.
a The somatic SV spectrum in 49 HCCs identified by WGS. Genes containing SVs in at least three samples are shown. b Different types of SVs affecting MACROD2 are indicated by lines with different colors (also exhibited in Table 1). c MACROD2 mRNA expression in 49 HCC tumor tissues compared with that in adjacent non-tumor tissues. d Representative MACROD2 staining in peritumor tissues and tumor tissues with no MACROD2 SV and with MACROD2 SV (25T). Scale bars = 100 μm. e The statistics of the MACROD2 staining density among different groups in 49 HCCs involved in WGS. f MACROD2 expression examined by qRT-PCR and western blot in one normal liver cell line (L0-2) and six HCC cell lines. g Representative HCC tumor and peritumor samples in the FFPE cohort showing the expression of MACROD2: patient 1, high MACROD2 expression; patient 2, low MACROD2 expression. Scale bar = 100 μm. h Kaplan–Meier survival analysis showing survival rates and cumulative recurrence rates on the basis of MACROD2 expression in the FFPE cohort. Data are shown as the mean ± standard deviation (SD) and are representative of three independent experiments.
Fig. 2
Fig. 2. MACROD2 deficiency promotes cell proliferation, colony formation, migration, and invasion in vitro, and tumor growth and metastasis in vivo.
a Proliferation of MACROD2-knockdown HepG2 and PLC/PRF/5 cells and MACROD2-overexpressing HCCLM3 and MHCC97H cells compared with that of control cells. *P < 0.05, **P < 0.01. b Colony formation activity of MACROD2-knockdown HepG2 and PLC/PRF/5 cells and MACROD2-overexpressing HCCLM3 and MHCC97H cells compared with that of control cells. The bar graphs illustrate the quantification of colony formation. **P < 0.01. c Cell monolayers were examined microscopically in wound-healing migration assays at 24 h post wounding. *P < 0.05. d Invasion of MACROD2-knockdown HepG2 and PLC/PRF/5 cells and MACROD2-overexpressing HCCLM3 and MHCC97H cells compared with that of control cells. The graphs depict the number of invasive cells after 48 h. **P < 0.01. e, f Macrograph of tumors and H&E-stained images of metastatic nodules in the selected areas of lungs in all groups. *P < 0.05, **P < 0.01. Data are shown as the mean ± SD and are representative of three independent experiments.
Fig. 3
Fig. 3. MACROD2 deficiency induces EMT of HCC cells.
a The cellular morphology of HCC cells with high or low MACROD2 expression. b Results of qRT-PCR, c western blot, and d immunofluorescent staining show changes in EMT marker (E-cadherin, vimentin, and N-cadherin) expression in HCC cell lines. e Representative images of xenograft-derived tumor sections. Scale bars = 100 μm. Data are shown as the mean ± SD and are representative of three independent experiments.
Fig. 4
Fig. 4. Activation of GSK-3β/β-catenin signaling induced by MACROD2 deficiency in HCC cells.
a Human phosphokinase array screening of changes in signaling pathways in HCC cells upon alteration of MACROD2 expression. b Western blot to validate the expression of p-GSK-3β and β-catenin in HCC cells upon alteration of MACROD2 expression. c Results of dual-luciferase assays performed 48 h after transfection of the indicated cells with TOPflash or FOPflash and Renilla pRL-TK plasmids. Reporter activity was normalized to Renilla luciferase activity. **P < 0.01. d Representative images of xenograft-derived tumor sections show p-GSK-3β and β-catenin expression. Scale bars = 100 μm. e Immunofluorescence staining showing subcellular β-catenin localization in the indicated cells. Data are shown as the mean ± SD and are representative of three independent experiments.
Fig. 5
Fig. 5. Inhibition of GSK-3β/β-catenin signaling attenuates MACROD2 deficiency-mediated HCC progression.
a Western blots showing the expression of the indicated molecules in MACROD2-deficient HCC cells treated with the GSK-3β inhibitor CHIR-99021, transfected with GSK-3βS9A, or with knockdown of β-catenin. b Proliferation of MACROD2-knockdown HepG2 cells and parental HCCLM3 cells treated with GSK-3β inhibitor, transfected with GSK-3βS9A, or with knockdown of β-catenin. *P < 0.05 compared with DMSO; #P < 0.05 compared with NC. c Colony formation activity of MACROD2-knockdown HepG2 cells and parental HCCLM3 cells treated with GSK-3β inhibitor, transfected with GSK-3βS9A, or with knockdown of β-catenin. The bar graphs illustrate the quantification of colony formation. **P < 0.01 compared with DMSO; ##P < 0.01 compared with NC. d Results of microscopic examination of cell monolayers in wound-healing migration assays at 24 h post wounding. *P < 0.05 compared with DMSO; #P < 0.05 compared with NC. e Invasion of MACROD2-knockdown HepG2 cells and parental HCCLM3 cells treated with GSK-3β inhibitor, transfected with GSK-3βS9A, or with knockdown of β-catenin. The bar graphs illustrate the quantification of colony formation. **P < 0.01 compared with DMSO; ##P < 0.01 compared with NC. Data are shown as the mean ± SD and are representative of three independent experiments.
Fig. 6
Fig. 6. Levels of MACROD2, p-GSK-3β, β-catenin, E-cadherin, vimentin, and N-cadherin in HCC tissues.
a HCC tumor samples showing MACROD2, p-GSK-3β, β-catenin, E-cadherin, vimentin, and N-cadherin expression. Patient 1: low MACROD2 expression; patient 2: high MACROD2 expression. Scale bar = 100 μm. b The bar graphs illustrate the expression levels of p-GSK-3β, β-catenin, E-cadherin, vimentin, and N-cadherin in HCC tumor sections (FFPE cohort, n = 380) from patients with low (n = 190) or high (n = 190) MACROD2 expression. **P < 0.01. Data are representative of three independent experiments.
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