TMEM220 is downregulated in human HCC and associated with poor clinical outcomes
We noticed the presence of high expression levels of TMEM220 in healthy human liver as compared to the other tissues or organs in the body (Fig. S1a), whereas TMEM220 expression was significantly decreased in tumor when compared with normal liver tissue (Fig. 1a, b). Moreover, TMEM220 gene copy number decreased and methylation of TMEM220 gene increased in HCC tissues (Fig. 1c, d and Fig. S1b). On this basis, we analyzed the expression of TMEM220 in 120 pairs of collected HCC clinical samples. Consistent with the findings above, the expression level of TMEM220 in tumor was much lower than adjacent normal livers (ANLs) (Fig. 1e), and 91.7% (110/120) of them showed under expression (Fig. 1f, g). Most importantly, by grouping the samples with TMEM220 expression level, we found that the overall survival curve of the high expression group was significantly better than that of the low expression group through 5-year follow-up (Fig. 1h and Fig. S1c). In addition, TMEM220 mRNA levels weremuch lower in SMMC7721, MHCC97H and HCCLM3 HCC cell lines compared with normal hepatic cell line (Fig. S1d), therefore we choose these 3 cell lines for subsequent research. These data suggest that the expression of TMEM220 is significantly decreased in HCC tissues, and its low expression is closely related with poor prognosis in HCC patients.
Overexpression of TMEM220 blocks HCC cell growth and decreases metastasis
Since the subcellular localization of TMEM220 is yet to be reported, using immunofluorescence, we clearly identified that TMEM220 mainly localized on the plasma membrane in HCC cells (Fig. 2a and Fig. S2).
To study the effect of TMEM220 on HCC cells, we generated HCC cell lines that overexpress TMEM220. First, cell cycle analysis by flow cytometry showed that the percentage of TMEM220 over-expressed cells at G1 stage was significantly higher than the control cells (Fig. 2b). Subsequent cell proliferation assay and colony formation assay indicated that the growth of TMEM220 overexpressed cells was slowed down markedly (Fig. 2c, d). Moreover, tumor xenograft study in nude mice showed that the tumor growth of overexpression cell was blocked (Fig. 2e), and accordingly, these mice survived longer than those of the control group (Fig. 2f). Taken together, data here suggested that overexpression of TMEM220 inhibited the growth of HCC cells both in vitro and in vivo.
In addition to growth, metastasis is another important indicator of HCC malignancy. The migration assay, invasion assay and wound healing assay results showed that the migratory and invasive capacity of TMEM220 overexpression HCC cells were decreased in vitro (Fig. 3a, b). At the same time, in vivo metastasis assay showed that the liver metastasis (Fig. 4c) and lung metastasis (Fig. 4d) of TMEM220 overexpressed cells were significantly reduced. These data suggested that overexpression of TMEM220 suppressed metastasis in HCC cells.
Signaling downstream of TMEM220 in HCC
To further explore the role of TMEM220 in HCC, we performed Reverse Phase Protein Array (RPPA) in TMEM220 overexpression SMMC7721 cell lines and control cell lines (Table S2). Based on the RPPA results, we found 131 altered proteins (P < 0.05; more than 1.5-fold change), 58 of which were upregulated or phosphorylation levels were increased, while 73 were downregulated or phosphorylation levels were decreased (Fig. S3a). Some of the altered proteins (Fig. 4a) were verified by Western blot in MHCC97H and HCCLM3 (Fig. 4b). Furthermore, the protein-protein interaction (PPI) network is established to investigate the signal network affected by TMEM220 (Fig. 4c and Fig. S3b, c). The proteins that were upregulated or had increased phosphorylation levels in TMEM220 over expression cells were significantly enriched in FOXO signaling pathway (Fig. 4d upper panel), whereas the proteins that were downregulated or had decreased phosphorylation levels were highly enriched in PI3K-Akt signaling pathway (Fig. 4d lower panel). CDKN1A (p21), PTEN and FOXO3 (FOXO3-pS318/321 inactive form decreased) were involved in the activated PPI subset (Fig. S3b), while PCNA, AKT, MAPK and β-catenin (β-catenin pT41/S45 inactive form increased) were present in the inactivated subset (Fig. S3c). Data reported here indicated that TMEM220 might be involved in HCC progression through PI3K-Akt and FOXO3 pathways.
TMEM220 affect downstream gene expression by altering β-catenin and FOXO3 subcellular localization
The RPPA result of over-expressed TMEM220 suggested that TMEM220 might function via regulating two hub transcription factors, β-catenin and FOXO3. RPPA results also implicated that TMEM220 overexpression also can inhibit AKT and GSK3β phosphorylation. So we proposed that TMEM220 could regulate the activity of these two transcription factors.
We first examined the effect of TMEM220 on subcellular localization of β-catenin. When transfected with TMEM220, β-catenin was translocated from the nucleus to the cytoplasm; and if the cells were treated with BIO (GSK3β inhibitor), the rate of nuclear-cytoplasmic shuttling of β-catenin could be blocked efficiently (Fig. 5a-c). Meanwhile, TOPFALSH/FOPFLASH reporter assay showed that the transcription activity of β-catenin induced by Wnt3a or β-catenin was gradually suppressed by TMEM220 over expression (Fig. S4a, b). β-catenin S33Y, which is insensitive to GSK-3β, abrogated the inhibition due to TMEM220 (Fig. S4c). Moreover, TMEM220 overexpression could inhibit Snail expression, which is a β-catenin downstream gene related to epithelial-to-mesenchymal transition (EMT) in HCC (Fig. S4d). Collectively, these results suggested that TMEM220 might negatively regulates β-catenin mediated EMT acts through AKT-GSK3β cascade.
On the other hand, we found that overexpression of TMEM220 also changed the sub cellular localization of FOXO3. However, FOXO3 is transferred from the cytoplasm to the nucleus (Fig. 5e, f). Consistently, the phosphorylation level of FOXO3, its inactivated form, was decreased with TMEM220 overexpression (Fig. 4b). Correspondingly, TMEM220 overexpression increased FOXO3 binding on p21 (FOXO3 target gene) promoter and activated its expression (Fig. 5g, h). Consistent with critical cyclin-dependent kinase (Cdk) inhibitory roles for p21, p21 arrests cell cycle by blocking the G1 phase (Fig. 2b). Collectively, our data implicated that TMEM220 could promote FOXO3 nuclear accumulation and increase p21 to inhibit HCC cell growth.