Expression of C1orf61 promotes human liver cancer cell migration
To evaluate the effect of C1orf61 on cancer cell metastasis, we first examined the relationship between the expression level of C1orf61 and HCC migration. Wound-healing assays and transwell assays showed that the expression level of C1orf61 positively correlated with cell migration in human liver cancer cells. HCCLM9 cells that expressed the highest level of C1orf61 possessed the strongest migration ability compared to other cell lines (Fig. 1a and Fig. S1a, b). Furthermore, we found that the up-regulation of C1orf61 promoted migration in BEL7402 and L02 cells; in contrast, C1orf61 knock-down inhibited migration in HCCLM9 and Huh7 cells (Fig. 1b, c and Fig. S1c, d). Similarly, knock-out of C1orf61 using CRISPR/Cas9 also significantly inhibited cell migration in HCCLM9 cells (Fig. 1d and Fig. S1e, f). Anoikis and soft agar colony formation suggested that multicellular survival and anchorage-independent growth occurred when cells were removed from inappropriate cell/ECM interactions; [20] these abilities were closely related to the metastasis potential of cells. Figure 1e and f show that C1orf61 expression inhibited anoikis in cells and promoted anchorage-independent growth without correct attachment in HCC. Taken together, these data suggest that the intracellular level of C1orf61 was associated with metastatic potential and the high expression of C1orf61 promoted cellular migration in human liver cancer cells.
C1orf61 regulates EMT-related gene expression and induces cellular EMT
To investigate the underlying molecular signaling events involved in C1orf61-mediated cell migration, we first performed gene expression profiling analysis using L02 (low C1orf61 expression), L02-C1orf61 (C1orf61 overexpression) and Huh7 cells (high C1orf61 expression). The results showed that C1orf61 regulated the expression of a diverse set of genes associated with biological function, cellular components and molecular activity (Fig. 2a and Fig. S2a). Further comparison and analysis revealed that some genes were related to cell growth, migration, invasion and EMT (Fig. 2b). RT-PCR results for a number of genes were all consistent with the microarray data, suggesting that the gene expression profiling array was correct (Fig. 2c). EMT is a key process in the metastatic cascade in tumors. It is regulated by multiple genes and complex interactions within the tumor microenvironment. Next, we examined whether C1orf61-induced cell migration was associated with EMT. Microscopy showed that the morphology of highly expressed C1orf61 cells changed from tightly packed colonies into spindle-shaped cells; the latter morphology is a typical feature of cells undergoing EMT (Fig. 2d). Moreover, isothiocyanate-conjugated phalloidin staining indicated that C1orf61 expression promoted BEL7402 and HCCLM9 cells to reorganize F-actin into parallel bundles and form lamellipodia. These findings were consistent with the cellular morphology results (Fig. 2e). To further confirm above observation, we examined the status of epithelial and mesenchymal cell markers by Western blotting and immunofluorescence analyses. As shown in Fig. 2f and Fig. S2b, overexpression of C1orf61 in L02 and BEL7402 cells resulted in a decrease in the protein levels of the epithelial cell markers E-cadherin and occludin and the up-regulation of the mesenchymal cell markers N-cadherin, Vimentin and Snail. Correspondingly, C1orf61 knock-down in HCCLM9 and Huh7 cells resulted in a decrease in the protein levels of the mesenchymal cell markers and increased epithelial cell markers (Fig. 2f and Fig. S2b). Similar results were observed when C1orf61 was knocked-out in HCCLM9 (Fig. S2c, d). Therefore, we propose that C1orf61-induced cell migration is associated with the promotion of EMT in human liver cells.
Activating STAT3 is essential for C1orf61-induced cellular EMT and migration
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in a major cascade responsive to stimulation by many growth factors and cytokines. [21] STAT3 is involved in the regulation of cell proliferation, apoptosis, cell invasion, angiogenesis and EMT processes. [22–24] Hence, we determined the levels of STAT3 and phosphorylated-STAT3. As shown in Fig. 3a, cells with high levels of C1orf61 protein expressed high levels of the phosphorylated-STAT3. These proteins were also translocated into the nucleus, where STAT3 regulates the expression of other genes (Fig. 3b and Fig. S3a). The STAT3-specific inhibitor S3I-201 suppressed endogenous STAT3 phosphorylation and blocked the transfer of STAT3 into the nucleus (Fig. S3b). Correspondingly, the suppression of STAT3 with S3I-201 also impaired EMT in cells and attenuated cell migration in L02-C1orf61 but not L02 cells (Fig. 3c, d and Fig. S3c, d). Ectopic expression of STAT3 in L02 cells increased the STAT3 protein level and facilitated the transfer of STAT3 into the nucleus as expected (Fig. S3e). In this scenario, the protein levels of epithelial cell markers (E-cadherin and Occludin) were reduced and mesenchymal cell markers (N-cadherin, Vimentin) were up-regulated; cell migration was also promoted (Fig. 3e, f and Fig. S3f, g). The overexpression of STAT3-Y705F, which is an artificially generated STAT3 containing a point mutation in tyrosine 705 resulting in a phenylalanine substitution, resulted in a pronounced dominant-negative effect on the activation of wild-type STAT3. STAT3-Y705F overexpression also inhibited cellular EMT and prevented migration (Fig. 3e, f and Fig. S3f, g). These results suggested that the activation of STAT3 has a significant promoting effect on C1orf61-induced cellular EMT and migration.
Akt activation is also involved in C1orf61-induced cellular EMT and migration
It has been shown that Akt is a key mediator of tumor metastasis and EMT induction in the progression of many human tumors. [25, 26] Next, we evaluated the role of Akt in C1orf61-mediated cellular EMT and migration. As shown in Fig. 4a, ectopic C1orf61 expression in L02 and BEL7402 cells remarkably increased the level of phosphorylated Akt. C1orf61 silencing in HCCLM9 and Huh7 cells robustly suppressed Akt phosphorylation. A luciferase assay reflecting PI3K/Akt pathway activation also confirmed that high C1orf61 expression significantly enhanced the PI3K/Akt activity (Fig. 4b). This finding was consistent with the phosphorylation level. To examine whether Akt activation plays a role in C1orf61-induced cellular EMT and migration, we treated cells with the specific PI3K/Akt inhibitor LY294002. The results showed that Akt inactivation by LY294002 increased E-cadherin expression and suppressed the level of N-cadherin in L02-C1orf61 cells. Moreover, wound-healing and transwell assays indicated that LY294002 significantly decreased cell migration in L02-C1orf61 cells; cell migration did not decrease in L02 cells where C1orf61 expression was low (Fig. 4c, d and Fig. S4a, b). In contrast, the ectopic expression of continually activated Akt (Akt CA) induced EMT in L02 cells and facilitated migration; the migration ability of these cells approached that of L02-C1orf61 cells (Fig. 4e, f and Fig. S4c, d). These results revealed that Akt was involved in C1orf61-induced cellular EMT and migration.
C1orf61 Promoted Tumor Growth And Facilitated Metastasis In Vivo
To evaluate the effects of C1orf61 on tumor growth and metastasis in vivo, we established subcutaneous tumor xenograft models and an experimental metastasis assay (intravenous tumor cell inoculation) in athymic nude mice using HCCLM9 wild type and HCCLM9 C1orf61 knock-out cells, respectively. As shown in Fig. 5a and b, subcutaneous tumors containing HCCLM9 wild type cells grew dramatically faster than those containing C1orf61 knock-out cells after 28 days later. This was reflected in both tumor volume and weight. These observations revealed that C1orf61 facilitated tumor growth in vivo. To determine whether C1orf61 knock-out in HCCLM9 cells modulated the development of metastasis, we examined the formation of lung metastases in subcutaneous and intravenous tumor models. As shown in Fig. 5c and d, C1orf61 knock-out resulted in a significant reduction in metastatic animals and decreases in the number of metastatic nodules in the lung. Further examination of the expression of some related proteins by Western blot and immunohistochemistry indicated that C1orf61 knock-out inhibited cellular EMT, decreased STAT3 and Akt activity; in vivo results were consistent with in vitro findings (Fig. 5e and f). These data suggested that the up-regulation of C1orf61 promoted tumor growth and metastasis in vivo. The potential molecular mechanism may be related to the induction of cellar EMT, STAT3 and Akt regulation.
C1orf61 is involved in HBV infection-induced cell migration in HCC
Hepatitis B virus (HBV) is a major etiological factor for HCC and is closely associated with regulating liver cell malignancy, proliferation, metastasis and apoptosis. [27, 28] Here, we determined whether C1orf61 modulated HBV infection-induced cell migration. HepG2.2.15 is a liver cancer cell line integrated with HBV; these cells exhibited increased EMT and migration compared to HepG2 cells without HBV infection. However, C1orf61 knock-down remarkably impaired HBV infection-induced cell migration (Fig. 6a, b and Fig. S5a). To further confirm the role of C1orf61 in HBV-induced cell migration, L02-PHAGE cells were infected with HBV; EMT and migration potential were then examined. As shown in Fig. 6c and Fig. S5b, c, C1orf61 levels were positively correlation with HBV infection-induced EMT and migration. These findings were consistent with that of HepG2 cells. The HBV genome encodes some primary proteins associated with infection and virus replication. These proteins include HBe, HBs, HBc, HBp and HBx, which regulate multiple HCC processes. Next, we determined which elements play critical roles in C1orf61-mediated tumor metastasis. Real-time PCR and Western blot analyses indicated that the transient expression of HBe and HBc remarkably increased C1orf61 mRNA and protein levels (Fig. 6d). Importantly, HBe and HBc induced C1orf61 expression in a transfection dose-dependent manner and facilitated EMT (Fig. 6e). Further wound-healing and transwell assays revealed that C1orf61-knockdown inhibited cell migration by HBe and HBc (Fig. 6f and Fig. S5d,e). These results showed that HBV, particularly the HBe and HBc proteins, promoted the migration of HCC cells via the up-regulation of C1orf61 expression.
C1orf61 improves the therapeutic response to sorafenib in HCC cells
Sorafenib, an oral small molecule multikinase inhibitor, has been recently approved for the treatment of hepatocellular carcinoma. Functionally, sorafenib inhibits tumor growth and angiogenesis, induces apoptosis and remodels the tumor microenvironment. [29, 30] We next examined whether C1orf61 regulated cancer treatment in HCC. As shown in Fig. 7a and Fig. S6a, HCCLM9 cells highly expressing C1orf61 exhibited a more sensitive response to sorafenib treatment than other cells lines in regard to cell proliferation inhibition and induced cell death. BEL7402 cells, which express C1orf61 at a low level, showed relative resistance. We speculated that C1orf61 was involved in the regulation of sorafenib treatment. To further confirm this hypothesis, the same dose of sorafenib was used to treat BEL7402-PHAGE and BEL7402-C1orf61 or HCCLM9 and HCCLM9 C1orf61-/- cells. The results indicated that cells highly expressing C1orf61 performed better in response to sorafenib treatment (Fig. 7b). FACS and Western blot analyses of apoptosis revealed that the capacity of sorafenib to induce apoptosis was closely related the expression level of C1orf61 in human liver cells (Fig. 7c and Fig. S6b). Moreover, we also found that 4 µM sorafenib inhibited migration and repressed EMT in cells with a high level of C1orf61 but not in cells with low level of C1orf61 (Fig. 7d, e and Fig. S6c, d). Altogether, these findings strongly suggest that C1orf61 improved the efficacy of sorafenib anticancer therapy against HCC.