N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition

Background N-Acetyltransferase 10 (NAT10) has been reported to be expressed at high levels in hepatocellular carcinoma (HCC); however, its role in chemoresistance is unclear. This study is aimed at investigating whether NAT10 regulates the epithelial-mesenchymal transition (EMT) and chemoresistance in HCC. Methods HCC cell lines (Huh-7, Bel-7402, SNU387, and SNU449) were treated with remodelin, an inhibitor of NAT10, or transfected with small inhibitory RNAs (siRNAs) targeting NAT10 or Twist. The EMT was induced by hypoxia. The CCK-8 assay was used to quantify cell viability, the EdU incorporation assay to assess cell proliferation. siRNA knockdown efficiency and epithelial/mesenchymal marker expression were assessed by western blotting. Results Knockdown of NAT10 using siRNA or inhibition of NAT10 using remodelin increased the sensitivity of HCC cell lines to doxorubicin; similar effects were observed in cells transfected with the Twist siRNA. Inhibition of NAT10 using remodelin also reversed the ability of doxorubicin to induce the EMT in HCC cells. Furthermore, inhibiting NAT10 reversed the hypoxia-induced EMT. Finally, we confirmed that combining doxorubicin with remodelin delayed tumor growth and reduced tumor cell proliferation in a mouse xenograft model of HCC. Conclusions NAT10 may contribute to chemoresistance in HCC by regulating the EMT. The mechanism by which NAT10 regulates the EMT and doxorubicin sensitivity in HCC cells merits further investigation.


Introduction
Hepatocellular carcinoma (HCC) is the sixth most common malignant cancer worldwide. The 5-year overall survival rate for HCC is very low [1,2], and the poor prognosis is mainly attributed to acquisition of chemoresistance during therapy [3]. However, the complicated cellular and molecular mechanisms that lead to chemoresistance in HCC remain unclear [4].
The epithelial-mesenchymal transition (EMT) is a complex, reversible progress resulting in the loss of epithelial cell adhesion and acquisition of a mesenchymal phenotype that plays a critical role in tissue regeneration, embryonic development, and inflammatory response [5][6][7][8][9]. During the EMT, epithelial markers such as E-cadherin are downregulated whereas mesenchymal markers such as vimentin and Twist are upregulated [10]. The EMT is implicated in the progression of cancer, and in recent decades, the EMT has been confirmed to play a role in the chemoresistance of various carcinomas, including HCC [11,12]. The relationship between the EMT and drug resistance was first described by Mani et al., who inferred that blocking or reversing the EMT may cause chemoresistant cells to revert to chemosensitive cells [13].
We previously observed that N-acetyltransferase 10 (NAT10) is upregulated in HCC cell lines with a mesenchymal-like phenotype. Inhibition of NAT10 reduced cell migration and invasion ability and correlated with elevated E-cadherin expression and reduced vimentin expression. As E-cadherin and vimentin are canonical markers of the EMT, these data suggest that NAT10 may promote the EMT in HCC [14].
In the present study, we sought to clarify the role of NAT10 in the EMT and chemoresistance in HCC. We demonstrate that NAT10 plays a critical role in regulation of the EMT and chemoresistance in HCC; however, the underlying mechanisms require further investigation.
2.6. Immunofluorescent Staining. Huh-7, Bel-7402, SNU387, and SNU449 cells were seeded onto glass slides. At 48 h after transfection with the NAT10 siRNA or Twist siRNA or treatment with remodelin in the presence of doxorubicin or hypoxia, the cells were rinsed with PBS, fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked for 30 min in 10% BSA, and then incubated with an anti-E-cadherin monoclonal antibody (1 : 200; Cell Signaling Technology) or anti-vimentin monoclonal antibody (1 : 200; Cell Signaling Technology) overnight at 4°C. After three washes in PBS, the slides were incubated with goat anti-rabbit Cy3 as a secondary antibody (1 : 200; Cell Signaling Technology) for 1 h in the dark. After three further washes, the cells were stained with DAPI for 5 min to visualize nuclei and examined by confocal microscopy (Olympus, Tokyo, Japan).

Immunohistochemical
Staining. Sections (4 μm) from each block were deparaffinized in xylene and rehydrated in a descending alcohol series. Antigen retrieval was performed by heating in a pressure cooker in 10 mmol/L citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 0.3% H 2 O 2 for 15 min, followed by incubation with 5% serum to reduce nonspecific binding. Sections were incubated with an anti-vimentin monoclonal antibody (1 : 1000; Cell Signaling Technology), anti-E-cadherin monoclonal antibody (1 : 1000; Cell Signaling Technology), or anti-Ki-67 monoclonal antibody (1 : 500; Cell Signaling Technology) at 4°C overnight. After washing in phosphatebuffered saline (PBS), slides were incubated with horseradish peroxidase-conjugated rabbit-anti-mouse secondary antibody, developed using 3,3-diaminobenzidine (DAB) chromogen solution and counterstained with Mayer's hematoxylin.    Negative controls were performed in parallel by replacing the primary antibody with nonspecific serum.  of Health Guide for Care and Use of Laboratory Animals (NIH Publications, No. 8023, revised 1978) equal volume of diluents) intraperitoneally every 2 days. After 2 weeks of treatment, mice were euthanized by cervical dislocation and tumors were dissected and weighed. Tumor proliferation was quantified using Ki-67 immunohistochemical staining.
2.9. Statistical Analysis. All data were analyzed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± SD. Two groups were compared using the Student t-test, and multiple groups were compared using one-way analysis of variance (ANOVA). Differences were considered significant at P < 0 05.

Inhibition of NAT10 Enhances the Sensitivity of HCC Cell
Lines to Doxorubicin. First, we examined the cell viabilities of HCC cells treated with doxorubicin and remodelin, an inhibitor of NAT10, for 48 h. The CCK-8 assay revealed that remodelin increased the doxorubicin sensitivity of all four cell lines (Figures 1(a)-1(d)). The EdU incorporation assay confirmed that the inhibition of NAT10 using remodelin decreased the proliferation of all four HCC cell lines when treated with doxorubicin (Figures 1(e)-1(h) and Table 1). These data indicate that NAT10 enhances the resistance of HCC cells to doxorubicin.

Knockdown of NAT10 Increases the Sensitivity of HCC
Cell Lines to Doxorubicin. To further investigate the contribution of NAT10 to doxorubicin resistance in HCC, the four HCC cell lines were transfected with a NAT10 siRNA. Western blotting confirmed that NAT10 expression was almost completely knocked down in cells transfected with the NAT10 siRNA (Figure 2(a)). The CCK-8 assay revealed that the NAT10 siRNA had no significant effect on doxorubicin sensitivity compared to cells treated with the NAT10 inhibitor remodelin, confirming that NAT10 enhances the chemoresistance of HCC cells (Figures 2(b)-2(e)). Taken together, these data confirm that NAT10 enhances the resistance of HCC cells to doxorubicin.

NAT10 Promotes the EMT in HCC Cell
Lines. The expression of NAT10 and epithelial/mesenchymal markers were examined using western blotting to assess whether the inhibition of NAT10 using remodelin affects the EMT in HCC cells. Remodelin significantly increased E-cadherin expression and decreased NAT10 and vimentin expression in all four HCC cell lines (Figure 3(a)). The results of immunofluorescent staining were consistent with western blotting (Figure 3(b)), indicating that the inhibition of NAT10 using remodelin reversed the EMT phenotype in HCC cell lines.

Inhibition of NAT10 Using Remodelin Reverses the
Doxorubicin-Induced EMT in HCC Cell Lines. Western blotting was performed to quantify the expression of EMT markers in HCC cell lines treated with doxorubicin in the presence and absence of the NAT10 inhibitor remodelin. Doxorubicin obviously reduced E-cadherin expression and increased vimentin expression, indicating that doxorubicin promotes the EMT in HCC cell lines. However, inhibition of NAT10 using remodelin reversed the ability of doxorubicin to promote the EMT, as indicated by the upregulation of E-cadherin and the downregulation of vimentin compared to control cells (Figure 4(a)). Immunofluorescent staining provided similar results as the western blot analysis (Figure 4(b)).
The knockdown efficiency of the NAT10 siRNA was confirmed by western blotting. Moreover, we observed that the NAT10 siRNA increased E-cadherin expression and reduced vimentin expression in the HCC cell lines (Figure 4(c)). Collectively, these results indicate that inhibition of NAT10 reverses the ability of doxorubicin to induce the EMT in HCC cells.
3.5. NAT10 Induces Doxorubicin Resistance by Promoting the EMT. Twist functions as a critical transcription factor implicated in EMT and drug resistance [16]. We analyzed the effects of knocking down Twist on the sensitivity of HCC cells to doxorubicin. Western blotting confirmed that NAT10 expression was almost completely knocked down in cells transfected with the NAT10 siRNA ( Figure 5(a)). The CCK-8 assay revealed the Twist siRNA had no significant effect on the sensitivity of HCC cells to doxorubicin compared to cells treated with remodelin ( Figures 5(b)-5(e)), which confirmed that NAT10 induces doxorubicin resistance by promoting the EMT in HCC cell lines.

Inhibition of NAT10 Using Remodelin Reverses Hypoxia-Induced Doxorubicin Resistance and EMT in HCC Cell Lines.
Hypoxia can induce the EMT in HCC cells [13]. Similarly, we observed that Huh-7 and BEL-7402 cells became more resistant to doxorubicin under hypoxic conditions. However, inhibition of NAT10 using remodelin attenuated hypoxiainduced doxorubicin resistance in HCC cells (Figures 6(a) and 6(b) and Table 2). Moreover, remodelin inhibited the hypoxia-induced downregulation of E-cadherin and upregulation of vimentin ( Figure 6(c)). Immunofluorescent staining confirmed the western blot analysis. Taken together, this data indicates that NAT10 is required for the hypoxia-induced EMT and doxorubicin resistance in HCC cells.

Remodelin Enhances the Curative Efficacy of Doxorubicin in HCC In Vivo.
To investigate the efficacy of combined doxorubicin and remodelin therapy in HCC in vivo, we subcutaneously injected HuH-7 cells into nude mice to establish a xenograft model of HCC; tumor growth was monitored in each treatment group every other day. Intraperitoneal injection of doxorubicin or remodelin alone for two weeks inhibited tumor growth. Interestingly, combined treatment with doxorubicin and remodelin led to more significant inhibition of tumor growth (Figures 7(a)-7(d)). The results showed that remodelin significantly inhibited tumor cell proliferation and thus enhanced the curative efficacy of doxorubicin in HCC in vivo (Figure 7(d)).
Immunohistochemical staining was performed to quantify the expression of EMT markers in the HCC xenograft tumors treated with doxorubicin, remodelin, or doxorubicin plus remodelin. Doxorubicin downregulated E-cadherin expression and upregulated vimentin expression, suggesting that doxorubicin promoted the EMT in the mouse model of HCC. However, remodelin attenuated the doxorubicin-

Discussion
Deregulation of NAT10 has been reported in human cancer [14]. Our previous studies demonstrated that elevated NAT10 protein expression was associated with a poor prognosis in HCC [17]. Moreover, NAT10 is known to promote a more aggressive phenotype in HCC cells by inducing the EMT, as indicated by upregulation of mesenchymal markers such as E-cadherin and vimentin [14]. Chemotherapy is an effective postoperative therapy in a variety of cancers, and doxorubicin is widely used as a firstline chemotherapy agent for HCC [4]. However, acquisition of drug resistance to doxorubicin is a major factor that leads to recurrence in HCC [18]. In the present study, we investigated whether NAT10 is involved in doxorubicin resistance in HCC. Here, we report that inhibition of NAT10 using remodelin or a NAT10 siRNA increased the sensitivity of HCC cell lines to doxorubicin.
The EMT is well-recognized as an important factor associated with drug resistance in cancer [19]. We found that inhibition of NAT10 using remodelin inhibited the EMT and downregulated the expression of NAT10, E-cadherin, and vimentin in all four HCC cell lines. E-cadherin and vimentin are well-recognized markers of the mesenchymal phenotype and play key roles in the EMT by maintaining the intercellular junctions of epithelial cancer cells [20,21]. Additionally, inhibition of NAT10 using remodelin reversed the doxorubicin-induced EMT in HCC cells. In agreement with these observations, knockdown of Twist, a transcriptional repressor of E-cadherin [22], also prevented the EMT, as indicated by upregulation of E-cadherin and downregulation of vimentin. Overall, these results indicate that NAT10 confers doxorubicin resistance in HCC by promoting the EMT.
Moreover, we observed that hypoxia could induce the EMT in HCC cells, and Huh-7 and BEL-7402 cells became more resistant to doxorubicin under hypoxic conditions. Another study demonstrated that curcumin inhibits the hypoxia inducible factor-1α-induced EMT in HCC cells [23]. Interestingly, the inhibition of NAT10 using remodelin restored doxorubicin sensitivity to HCC cells exposed to hypoxic conditions. Moreover, hypoxia-induced downregulation of E-cadherin and upregulation of vimentin could be reversed by inhibition of NAT10 in Huh-7 and BEL-7402 cells. The in vivo xenograft models confirmed that remodelin significantly inhibited tumor proliferation and enhanced the curative efficacy of doxorubicin in HCC. Collectively, these data indicate that inhibition of NAT10 using the siRNA or remodelin increases doxorubicin sensitivity and prevents the EMT in HCC cells.

Conclusions
This study demonstrates that NAT10 plays important roles in the regulation of the EMT and doxorubicin sensitivity in HCC cells. These observations indicate that NAT10 represents a potential target for overcoming chemoresistance in HCC and provides a rationale for combining remodelin with doxorubicin in the treatment of HCC. The mechanism by which NAT10 regulates the EMT and doxorubicin sensitivity in HCC cells merits further investigation.