A splicing variant of Merlin promotes metastasis in hepatocellular carcinoma

Abstract

Merlin, which is encoded by the tumour suppressor gene Nf2, plays a crucial role in tumorigenesis and metastasis. However, little is known about the functional importance of Merlin splicing forms. In this study, we show that Merlin is present at low levels in human hepatocellular carcinoma (HCC), particularly in metastatic tumours, where it is associated with a poor prognosis. Surprisingly, a splicing variant of Merlin that lacks exons 2, 3 and 4 ((Δ2-4)Merlin) is amplified in HCC and portal vein tumour thrombus (PVTT) specimens and in the CSQT2 cell line derived from PVTT. Our studies show that (Δ2-4)Merlin interferes with the capacity of wild-type Merlin to bind β-catenin and ERM, and it is expressed in the cytoplasm rather than at the cell surface. Furthermore, (Δ2-4)Merlin overexpression increases the expression levels of β-catenin and stemness-related genes, induces the epithelium-mesenchymal-transition phenotype promoting cell migration in vitro and the formation of lung metastasis in vivo. Our results indicate that the (Δ2-4)Merlin variant disrupts the normal function of Merlin and promotes tumour metastasis.

Figure 1. Low expression of Merlin in HCC and PVTT is associated with a cacoethic prognosis.

(a) Merlin expression was decreased in HCC and PVTT in HCC tissue microarray sections. Merlin was stained in adjacent-tumour tissues, tumour tissues without PVTT, tumour tissues with PVTT and PVTT tissues (magnification: × 100; Small square magnification: × 400). (b) Merlin staining scores in the TMA (0–5 denote different degrees of IHC staining; 5 is the maximum and 0 is the minimum degree). The data are shown as the mean±s.d., 148 HCC with PVTT, 37 HCC without PVTT and 29 tumour-adjacent tissues specimens *P<0.05; **P<0.01, based on one-way ANOVA. (c) Western blots of Merlin proteins from eight pairs of HCC tumours with PVTT and tumour adjacent tissues using Merlin antibody. The quantification of the bands relative to GADPH is shown below the panels. (d) The disease-free and overall survival rates were obtained from a 5-year follow-up of 148 HCC patients based on their expression of Merlin. **P<0.01, based on the student's t-test.

Figure 2. Interfering with the expression of Merlin promotes metastasis in HCC cell lines.

(a) Lysates of HCCLM3 cells transfected with Merlin shRNAs were immunoblotted with the indicated antibodies. (b) Generation of shMerlin stable HCCLM3, Huh7 and L02 cell lines. (c) Transwell and (d) invasion assays were used to test the migration and invasion abilities of HCC cells. The respective infiltrated cell numbers are shown in the lower panes. (Scale bar, 50 μM). Results are shown as the mean±s.d., n=3. **P<0.01, ***P<0.001, based on the student's t-test. (e) Representative lung tissue sections of BALB/C mice killed at 13 weeks from each group are shown (H&E; Magnification : × 100, Scale bar, 300 μM ). The arrows indicate lung metastatic tumours (lower images). The average number of lung metastatic tumours per lung in each group (n=5) was calculated (upper panel). Results are shown as the mean±s.d., n=5. **P<0.01, based on the student's t-test. (f) Huh7 cells were treated in vitro with GTPγS or GDP to activate Rho and Cdc42. Cell lysates (50 μl at 1 mg ml⁻¹) in Western blotting showed active Rho and Cdc42 levels in the shMerlin cell line and the control.

Figure 3. The alternative splicing of Merlin in HCC is determined by PCR and western blotting.

(a) Expression of the Nf2 gene in HCC, HCC without PVTT and HCC with PVTT tissues. (b) Detection of the sizes of NF2 from HCC, adjacent and PVTT tissues by primers designed from the Nf2 exon 1 and 5. (c) The splicing patterns of Nf2 in normal liver, HCCLM3, Huh-7, CSQT2 and L02 cells. (d) Detection of expression of Merlin and its mutant in HCC cell lines (CSQT2, Huh-7, HCCLM3 and HepG2 cells), and hepatocyte L02 cells by antibodies (C-18 and C-19). (e) Detection of ^wtMerlin and ^Δ2–4Merlin by different antibodies. (f) Detection of the expression of Merlin and its mutant in HCC, PVTT and tumour adjacent tissues by antibody C-18. (g) Detection of ^Δ2–4Merlin by specific antibody for ^Δ2–4Merlin (Merlin-N15). (h) Detection of expression of ^Δ2–4Merlin in HCC, PVTT and tumour adjacent tissues by Merlin N15 antibody. T: HCC tumour; N: non-tumour adjacent tissues; P: PVTT; M/G: Merlin/GAPDH density.

Figure 4. ^Δ2–4Merlin expression promotes cell migration and induces EMT.

(a) ^Δ2–4Merlin promotes HCCLM3 migration (Magnification: × 200; Scale bar, 50 μm). The number of migrated cells was counted (Mean±s.d., n=3) and is shown to the right of the panels. *P<0.05; **P<0.01, based on the student's t-test. (b) EMT-related genes were detected in Huh-7 and HCCLM3 cells with knockdown of Merlin. (c) EMT-related genes were detected in CSQT2 cells. (d) EMT-related genes were detected in ^wtMer (^wtMerlin) or ^Δ2–4Merlin expressing HCCLM3 cells. (e) Western analysis for EMT-related gene expression in HCCLM3 cells with knockdown of Merlin after treatment with TGF-β. (f) TGF-β induced expression of EMT-related genes in the background of the overexpression of ^wtMerlin and ^Δ2–4Merlin in HCCLM3 cells. (g) Western analysis showed β-catenin and N-cadherin expression in ^wtMerlin, ^Δ2–4Merlin or ^wtMerlin/^Δ2–4Merlin transiently transfected HCCLM3 cells with the indicated antibodies. *P<0.05; **P<0.01, based on the student's t-test.

Figure 5. The silencing of Merlin or the expression of ^Δ2–4Merlin promotes stemness activity.

(a) shMerlin-expressing Huh-7 cells formed a greater number of and larger spheroids than scrambled-expressing Huh-7 cells in ultralow attachment plates without serum media. The quantities and sizes of the spheroids were measured and are shown under the each panel. Scal bar, 100 μm. (b) EMT-related genes were detected in Huh7-scrambled and Huh7-shMerlin cells. (c) The quantities and sizes of spheroids were measured in shMerlin-expressing and scrambled-expressing CSQT2. The knockdown of Merlin increased the number of spheroids in Huh-7 cells. Scal bar, 100 μm. (d) The expression of EMT-related genes was detected in CSQT2-scrambled and CSQT2-shMer cells. (e) Both ^Δ2–4Mer type I and type II promoted the formation of spheroids of HCCLM3 cells compared with ^wtMer-expression cells. Scal bar, 100 μm. (f) Stemness genes were detected in ^wtMer-expressing and ^Δ2–4Mer-expressing HCCLM3 cells. The above assays in a–f were repeated three times, respectively. The average values express as mean ±s.d. Statistic analysis in this figure was all based on the student's t-test. *P<005; **P<0.01; ***P<0.001. (g) The proportion of side population cells in empty vector, ^wtMerlin, and ^Δ2–4Merlin expressing HCCLM3 cells. (h) The proportion of side population cells in scrambled and shMerlin expressing CSQT2 cells.

Figure 6. ^Δ2–4Merlin loses its functions of anchoring the membrane and binding to ERM and β-catenin.

(a) Myc-tagged ^wtMer or Myc-tagged ^Δ2–4Mer was expressed in HCCLM3 cells, and an anti-Myc antibody was used for immunofluorescence staining. (Scale bar, 20 μm). (b) Immunofluorescence analysis of β-catenin in HCCLM3 cells expressing shMerlin. DAPI was used for nuclear staining. Scale bar, 20 μm; Magnification for small square: × 400) (c) Confocal analysis for ^wtMer and ^Δ2-4Mer in HCCLM3 cells. Myc-tagged ^wtMer or myc-tagged ^Δ2-4Mer was expressed in HCCLM3 cells. The cells were stained with antibodies for β-catenin or Myc-tag. The nuclei were stained with DAPI. (Scale bar, 20 μm; Magnification for small square: × 400). (d) Immunoprecipitation assay for ^wtMer or ^Δ2–4Mer with ERM and β-catenin. (e) The fractionation of ^wtMer and ^Δ2–4Mer expressing HCCLM3 cells was performed and analysed by immunoblotting for β-catenin expression with the indicated antibodies. (f) ^Δ2-4Mer- or ^wtMer-expressing HCCLM3 cells were injected into the caudal veins of BALB/C nude mice. The lung metastatic tumours were stained by H&E; Scale bar, 100 μm. The tumour numbers were counted and are shown to the right of the graphs (five mice per group). *P<0.05; **P<0.01, based on the student's t-test.