SGK3 is preferentially activated in liver CSCs
It has been confirmed that the liver cancer cell spheroids cultured in serum-free culture medium are highly enriched in liver CSCs. To explore the relevance between SGK3 and liver CSCs, we detected expression levels of SGK3 mRNA in CSC-enriched hepatoma spheroids compared with monolayer-attached cells. The results showed that the mRNAs of several stem cell markers, including CD90, CD133, Oct4, Nanog, Bmi-1, and SOX2 were all upregulated in the spheroids compared to the monolayer-attached cells, and moderately elevated expression of SGK3 mRNA was observed in the spheroids (Fig. 1a). We next analysed SGK3 protein levels and phosphorylated levels in spheroids, and we found elevated SGK3 phosphorylation in spheroids, while no difference was observed between the expression of the total SGK3 protein in spheroids and attached cells. Consistently, the phosphorylation of Akt rather than total Akt proteins was elevated in spheroids (Fig. 1b).
CD133 is widely used as liver CSCs markers [25]. We then sorted CD133+ cells from MHCC97H HCC cells. After sorting, the purity of the selected CD133+ population was monitored by flow-cytometric analysis to ensure the positivity of CD133 was over 85% (Fig. 1c). After cultured in serum-free culture medium for 7 days, the number of spheroids (diameter > 50 μm) in the CD133+ cells was significantly higher than that in the CD133- cells (Fig. 1d). Furthermore, CD133+ cells displayed much higher tumour-initiating capacity in in vivo experiments (Fig. 1e). To further investigate the role of SGK3 in liver CSCs, we determined the phosphorylation of SGK3 in CD133+ and CD133- cells. As expected, phosphorylation of SGK3 in CD133+ HCC cells was significantly higher than that in CD133- cells (Fig. 1f). In addition, the phosphorylation of Akt rather than total Akt proteins was elevated in CD133+ HCC cells (Fig. 1f), which is consistent with findings reported by Ma et al. [26]. Taken together, these results indicate that SGK3 may play a crucial role in the expansion of liver CSCs.
SGK3 enhances the expansion of liver CSCs
To assess the role of SGK3 in liver CSC regulation, we established a lentivirus-mediated stable SGK3 or negative control (NC) expression cell lines using MHCC-97H and Huh7. Notably, SGK3 overexpression significantly enhanced the mRNA expression of CSC-related genes CD133, CD90, Oct4, Nanog, Bmi-1 and Sox2 compared with NC cells (Fig. 2a and b). Consistently, the expression of CD133 and Nanog proteins increased with the increase of SGK3 (Fig. 2c). To further elucidate the role of SGK3 in CSCs, we examined spheroid-forming ability (a surrogate marker of CSCs self-renewal). We found that SGK3 overexpression increased spheroid formation ability compared with NC cells (Fig. 2d). These results suggest that SGK3 enhances the expansion of liver CSCs.
Inhibition of SGK3 attenuates liver CSCs expansion
To further confirm the involvement of SGK3 in liver CSC expansion, 2 lentivirus vectors were designed to express shRNA for SGK3 knockdown (SGK3 shRNA1 and SGK3 shRNA2). After SGK3 inhibition, stemness genes were significantly suppressed in HCC cell lines (Fig. 3a and b). The protein levels of CD133 and Nanog were decreased in HCC cells after knockdown of SGK3, determined by western blot (Fig. 3c). In addition, SGK3 inhibition resulted in smaller spheroids, and they were significantly decreased in numbers (Fig. 3d). To confirm the result, flow cytometric analysis revealed a diminished proportion of liver CSCs in SGK3 shRNA1 stably transfected HCC cells (Fig. 3e).
Prolonged treatment of HCC cells with PI3K inhibitors leads to activation of SGK3 and expansion of liver CSCs
Because SGK3 can be activated by PI3K independent of AKT, we tested whether the phosphorylation of SGK3 can be blocked by PI3K inhibitors. We treated Huh7 cells with PI3K inhibitors (LY294002 and ZSTK474) for 24 h with increasing doses. Interestingly, the results showed that both inhibitors induced a dose-dependent increase in SGK3 phosphorylation, while the phosphorylation of Akt was inhibited with increasing concentration (Fig. 4a). In addition, after PI3K inhibitor treatment, we observed a dose-dependent increase in the expression of CSC-relative genes CD133 and Nanog (Fig. 4a, b and c). A recent study reported that prolonged treatment of breast cancer cells with class I PI3K or Akt inhibitors leads to increased expression and activation of SGK3 [20]. To investigate whether prolonged treatment of liver cancer cells with PI3K inhibitors could promote expression and activation of SGK3, we treated cells with ZSTK474 in a time gradient (0 h, 4 h, 8 h, 24 h, 48 h, and 72 h). As expected, prolonged ZSTK474 treatment over 24 to 72 h led to a time-dependent increase of SGK3 phosphorylation under conditions in which the CSC-relative genes CD133 and Nanog were induced (Fig. 4d).
Flow cytometric analysis revealed an enlarged proportion of CD133+ cells after Huh7 cells were treated with ZSTK474 for 72 h (Fig. 4e). Interestingly, knockdown of SGK3 expression employing siRNA partially blocked prolonged PI3K inhibitor treatment from enhancing CD133 expression in both Huh7 and MHCC97H cells (Fig. 4f). These results indicate that prolonged treatment with PI3K inhibitors induced expansion of CD133+ cells via enhancing SGK3 phosphorylation.
We next assessed the effect of ZSTK474 on tumour growth in an in vivo experiment. After the tumours formed (300–500 mm3), the mice were orally administered with 400 mg/kg of ZSTK474 for 10 days. The results indicate that ZSTK474 showed significant anti-tumour activity in the treatment period as compared to control mice (Fig. 4g). Western blot analysis of ZSTK474-treated tumours exhibited increased expression of Nanog and CD133, and the increased phosphorylation of SGK3 was validated (Fig. 4h). The increased mRNA level of CD133 was also confirmed in vivo (Fig. 4i). Immunohistochemical assay showed that tumours treated with ZSTK474 have higher levels of CD133 than control tumours (Fig. 4j).
The inhibitor of hVps34 can block SGK3 activity and suppress liver CSC expansion induced by PI3K inhibitors
It has been reported that SGK3 can be activated by PtdIns(3)P produced by hVps34, and hVps34 inhibitor VPS34-IN1 can inhibit SGK3 activation [20, 21]. Our results confirmed that VPS34-IN1 induced a dose-dependent inhibition of SGK3 phosphorylation in MHH97H and Huh7 cells (Fig. 5a). In addition, treatment of Huh7 and MHCC97H cells with VPS34-IN1 also reduced the expression of CSC-related markers CD133 and Nanog (Fig. 5a and c). Consistently, hVps34 inhibitor VPS34-IN1 suppressed the spheroid formation ability tested by spheroid formation assay (Fig. 5b). Flow cytometric analysis revealed a diminished proportion of CD133+ cells after Huh7 cells were treated with VPS34-IN1 for 24 h (Fig. 5d). Furthermore, the increased expression of CD133 and Nanog induced by prolonged PI3K inhibitor treatment can be depleted by VPS34-IN1 (Fig. 5e). Collectively, these results indicated that the inhibitor of hVps34 can block liver CSC expansion after prolonged treatment of HCC cells with PI3K inhibitors via the inhibition of SGK3.
SGK3 promotes liver CSCs through β-catenin accumulation by GSK3β
Our previous study confirmed that SGK3 stimulates β-catenin signalling in HCC cells [23]. It is well established that SGK3 promotes the inactivation of GSK-3β by phosphorylation of GSK-3β on Ser9 [22]. To investigate whether SGK3 can promote liver CSC expansion via GSK-3β/β-catenin signalling, we first detected the expression of GSK-3β and β-catenin in spheroids and attached cells. The western blot analysis revealed that the phosphorylation of GSK-3β on Ser9 and expression of β-catenin was significantly increased in spheroid cells (Fig. 6a). We next analysed the phosphorylation of GSK-3β on Ser9 after SGK3 overexpression or knockdown. Our results indicated that overexpression of SGK3 increased the phosphorylation level of GSK-3β on Ser9 (Fig. 6b). Conversely, knockdown of SGK3 reduced the phosphorylation level of GSK-3β on Ser9 (Fig. 6c). Furthermore, overexpression of SGK3 increased β-catenin levels, whereas knockdown of SGK3 reduced β-catenin expression (Fig. 6b and c), indicating a canonical regulation of β-catenin stability by SGK3. Treatment of MHCC97H cells with AR-A014418, a selective GSK-3β inhibitor reduced the expression of CD133 (Fig. 6d). To further determine the role of SGK3 in liver CSC self-renewal via the GSK-3β/β-catenin signalling pathway, SGK3 overexpression and control cells were treated with AR-A014418. Spheroid formation analysis showed that AR-A014418 weakened the SGK3-enhanced self-renewal of liver CSCs (Fig. 6e).
Prolonged treatment of HCC cells with PI3K inhibitors stimulates the β-catenin signalling pathway via activation of SGK3
Nuclear accumulation of β-catenin has been reported to confer resistance to PI3K inhibitors in colon cancer [27]. To explore the β-catenin levels changed under prolonged treatment of HCC cells with PI3K inhibitors, we treated MHCC97H cells with PI3K inhibitors for 0 to 5 days and detected β-catenin expression. The results showed that prolonged PI3K inhibitor ZSTK474 treatment over 1 to 5 days led to a time-dependent accumulation of β-catenin (Fig. 7a). To study the effect that PI3K inhibitors had on β-catenin accumulation via SGK3, SGK3 was knocked down in MHCC97H cells by siRNAs and subjected to treatment with ZSTK474. As expected, inhibition of SGK3 decreased the accumulation of β-catenin after treatment with ZSTK474 for 72 h (Fig. 7b). Taken together, these data suggest that prolonged treatment of HCC cells with inhibitors of PI3K leads to marked accumulation of β-catenin via the activation of SGK3 (Fig. 7c).