Background
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death and is the main severe consequence leading to death in patients with cirrhosis and many other chronic liver diseases [
1,
2]. Despite recent progress in HCC treatment, prognosis for this refractory disease remains unsatisfactory [
3] because both solid tumours show considerable histological and functional heterogeneity [
4]. Such cellular heterogeneity is very important due to its important role in treatment resistance. Recent studies have suggested that subpopulations of cells with increased tumorigenesis capacities and self-renewal potential, termed as cancer stem cells (CSCs) [
5], exist within tumours. Persistence of CSCs is a primary cause of relapse and metastasis, which are highly resistant to chemotherapy [
6]. Therefore, more effective therapeutic strategies may be developed if the molecular mechanism underlying CSC regulation is illuminated.
The existence of CSCs has been demonstrated in a variety of solid tumours, including liver cancer [
7]. Liver CSCs can be enriched with several defined surface markers, including CD133, CD90, CD44, OV6, EpCAM, CD13, CD24, ICAM-1, CD47, Lgr5, and keratin19 [
8]. Although CSCs can be identified within the liver cancer cells, they cannot be effectively eradicated because the detailed regulatory mechanism of CSC generation and expansion remains largely unknown. Signalling pathways such as the Wnt/β-catenin, TGFβ, IL-6/STAT3, Notch and ANXA3/JNK pathways have been reported to be involved in the regulation of liver CSCs [
9‐
12]. Among these pathways, Wnt/β-catenin signalling has received increasing attention because of its important role in both normal stem cells and CSCs. Inhibition of the Wnt/β-catenin pathway has also been shown to be effective in eliminating CSCs [
13]. However, the deregulation of Wnt/β-catenin pathway in liver CSCs is not fully understood.
The phosphoinositide 3-kinase (PI3K) pathway is a very important intracellular signalling pathway, which plays crucial roles in normal cell processes and a critical role in cancers. Several studies have explored the therapeutic targeting of the PI3K pathway in cancers, and various inhibitors targeting PI3K and its isoforms have been developed [
14]; however, the clinical effect was not satisfactory. The role of the PI3K signalling pathway in CSCs has been reported, but some controversy remains [
15].
Serum and glucocorticoid-regulated kinase 3 (SGK3), an AGC protein kinase family member, has been found to play a critical role in a variety of cancers [
16]. A previous study showed that PIK3CA-mediated breast cancer cell growth and survival are dependent on the SGK3, and Akt is dispensable [
17]. SGK3 is a unique member of the SGK family because it contains an N-terminal PX domain. SGK3 binds selectively to PtdIns(3)P through its PX domain, which is required for targeting SGK3 to the endosome, where the Class III PI3K (also termed hVps34) phosphorylates PtdIns to generate a pool of PtdIns(3)P [
18,
19]. VPS34-IN1, an hVps34 inhibitor can suppress SGK3 activation by reducing PtdIns(3)P levels via lowering phosphorylation of T-loop and hydrophobic motifs [
20,
21]. Amplification and overexpression of SGK3 have been reported more frequently than those for AKT in HCC, suggesting it may have a greater functional significance in HCC [
22]. Our previous study found that SGK3 plays an important role in the invasive potential of HCC cells and epithelial-mesenchymal transition (EMT) [
23]. However, the role and mechanism of SGK3 in CSCs has not been reported.
Here, we show that SGK3 is preferentially activated in liver CSCs, and upregulated or downregulated SGK3 in HCC cells enhances or suppresses liver CSC-associated gene expression and spheroid formation via the GSK-3β/β-catenin signalling pathway. We also found that prolonged treatment of HCC cells with PI3K inhibitors leads to activation of SGK3 and expansion of liver CSCs. Additionally, our results demonstrate that inhibition of hVps34 can block SGK3 activity and suppress liver CSC expansion. Effective inhibition of SGK3 signalling may be useful in eliminating liver CSCs.
Methods
Cell lines and culture
The Huh7 cells were obtained from the Chinese Academy of Sciences Cell Bank. The MHCC-97H cells were obtained from Zhongshan Hospital of Fudan University in Shanghai, China. All cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Heclone) supplemented with 10% foetal bovine serum (FBS; Capricorn). Liver CSCs were cultured in serum-free culture medium. Serum-free culture medium was DMEM/F12 (Hyclone) consisting of 20 ng/ml basic fibroblast growth factor (bFGF; PeproTech), 20 ng/ml epidermal growth factor (EGF; PeproTech) and 20 μl/ml B27 supplement (Life Technologies).
Isolation of CD133+ cells
CD133+ cells were obtained using the CD133 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions. HCC cells were enzymatically dissociated, the cell suspension was centrifuged at 300×g for 10 min, and the supernatant was aspirated completely. The cells were resuspended in 300 μl of buffer per 108 cells, and 100 μl of FcR Blocking Reagent and 100 μl of CD133 MicroBeads were added, mixed well and incubated for 30 min at 4 °C. Then, the cells were washed by adding 1 ml of buffer, centrifuged at 300×g for 10 min, and sorted with the Mini MACS® Separator (Miltenyi Biotec). Phycoerythrin (PE)-conjugated CD133/2 antibodies (Miltenyi Biotec) were used to evaluate the efficiency of magnetic separation by flow cytometry.
RNA extraction and quantitative reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from the cells by using Trizol (Invitrogen). Complementary DNA (cDNA) synthesis was performed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara). Quantitative RT-PCR was performed using the SYBR Premix ExTaq (Takara) under standard conditions according to the manufacturer’s instructions. Quantitative RT-PCR was conducted with the CFX96 Real-Time PCR Detection System (Bio-Rad). The data were analysed using the 2
−△△Ct method. The primers are listed in Additional file
1: Table S1.
Cell siRNA transfection and inhibitors
Small interfering RNAs (siRNA) of SGK3 and negative control (NC) were designed and synthesised by RiboBio (Guangzhou, China). The cells were transfected using a ribo FECT™ CP Transfection Kit (RiboBio) according to the manufacturer’s protocol. A total of 2 × 105 cells were seeded per well and grown to 50–70% confluence. Transfection complexes were prepared according to the instructions and were added directly to the cells. The siRNA and NC were used at a final concentration of 100 nM. The mRNA level was detected by RT-PCR after incubation for 24 h, and the level of protein was determined by western blot after incubation for 48 h. Class I PI3K inhibitors ZSTK474 and LY294002, hVps34 inhibitor VPS34-IN1 and GSK 3β inhibitor AR014418 were purchased from MedChem Express (MCE).
Establishment of the SGK3 stable overexpression and knockdown cell lines
To establish stable transduction, lentiviral vectors expressing SGK3 sequence, shRNA and the control vectors were obtained from Hanbio (Shanghai, China). shRNA-mediated silencing of SGK3 required the synthesis of a set of oligonucleotides composed of a target shRNA sequence and its complement against SGK3, as previously described [
24]. Polybrene (Hanbio) was used to promote the transfection according to the manufacturer’s instruction. On the previous day, 1 × 10
5 cells were seeded per well and grown to 20–40% confluence. Then, the cells were transfected at a multiplicity of infection (MOI) of 20. After 72 h, the transfection efficiency was verified by fluorescence microscopy and RT-PCR.
The cell spheroid formation assay was performed as described previously [
10]. Briefly, single cells (1 × 10
3) were plated in a 6-well ultra-low attachment plate (Corning) or (1 × 10
2) in a 24-well ultra-low attachment plate in the serum-free culture medium. After 1–2 weeks, the number of tumour spheroids (diameter > 50 μm) was counted under an inverted microscope.
Protein extraction and western blotting
Western blot analysis to determine protein level was performed as described previously [
10]. The following antibodies were used: anti-SGK3 (sc-166,847; Santa Cruz), anti-β-actin (YT0099; Immunoway), anti-Akt1 (ab32505; Abcam), anti-Akt1 (phosphoS473; ab81283; Abcam), anti-SGK3 (phosphoThr320; #5642; CST), anti-GSK3β (ab32391, Abcam), anti-GSK3β (phosphoS9; ab75814; Abcam), anti-β-catenin (#8480, CST), anti-CD133 (YT5192; Immunoway) and anti-Nanog (YM0464; Immunoway).
Immunofluorescence
Cell slides were fixed with 4% paraformaldehyde for 20 min, and permeabilised with 0.3% Triton X-100 (Sigma-Aldrich) for 15 min. Then, the cells were blocked with normal goat serum and incubated with anti-Nanog (1:100; Immunoway) at 4 °C overnight, followed by incubated with Alex 555-conjugated goat anti-mouse antibody for 1 h. The cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min and visualised by fluorescent microscope (Nikon).
Immunohistochemical assay
Tumour tissues from the nude mice were fixed in 4% formaldehyde for 24 h, embedded in paraffin, and serially sectioned at a thickness of 6 μm. Sections were deparaffinized and stained with anti-CD133 (1:500; ab222782; Abcam) at 4 °C overnight, and incubated with the secondary antibody for 1 h at 37 °C. Reaction results were shown by incubation with 3, 3′-Diaminobenzidine (DAB; Boster). After washing with tap water to stop the chromogenic reaction, the sections were dehydrated in an ascending alcohol gradient, cleared twice with xylene and mounted in neutral balsam. Then, the sections were examined and imaged by microscope (Nikon).
Flow cytometric analysis
Cells were collected and resuspended in 100 μl of phosphate-buffered saline (PBS) containing 20 μl FcR Blocking Reagent (Miltenyi Biotec) and anti-human antibodies, PE-CD133 (Miltenyi Biotec), and then incubated for 15 min on ice in the dark. After incubating, the cells were washed twice with 1 ml of PBS. The collected cells were resuspended in 300 μl of PBS and detected using a FACSCanto II flow cytometer (BD Biosciences). Isotype-matched mouse antibodies served as controls.
In vivo xenograft experiments
All animal experiments were performed in compliance with the strict rules of the Animal Ethics Committee of Chongqing Medical University. For tumour formation assay, 1 × 104 CD133+/− cells were subcutaneously injected into 6-week-old female athymic nude mice. Tumour formation was observed every week and analysed at the sixth week. The effect of ZSTK474 was tested in vivo. MHCC97H cells were subcutaneously injected into 6-week-old female athymic nude mice (5 × 106 cells per mouse) and allowed to form tumours. Once the tumours reached 300 mm3, 6 animals were randomly divided into control and ZSTK474 groups. The ZSTK474 group was orally administered a dose of ZSTK474 (suspended in 5% hydroxypropyl cellulose) at 400 mg/kg daily for 10 days. The control group of mice was orally administered with 5% hydroxypropyl cellulose instead of ZSTK474. Tumours were measured throughout the treatment period.
Statistical analysis
Statistical analyses were performed using SPSS 21 software. All data were acquired from at least 3 independent experiments and are reported as the mean ± SD. Two independent group comparisons were analysed using Student’s t-test. P < 0.05 was considered statistically significant.
Discussion
CSCs have stem characteristics such as self-renewal, differentiation and tumourigenesis. CSCs have been recognised to contribute to cancer relapse and metastasis due to their invasive and drug-resistant capacities. It is therefore important to explore the molecular mechanism underlying liver CSC regulation so as to develop novel therapeutic strategies eliminating CSCs. In this study, we report that SGK3 plays a pivotal role in liver CSC expansion via the GSK3β/β-catenin signalling pathway, and the activation of SGK3 may contribute to the liver CSCs tolerance of PI3K inhibitor treatment.
SGK3 is emerging as a tumour oncogene in several cancers [
28,
29]. Amplification and overexpression of SGK3 was frequently detected in HCC specimens, and SGK3 can promote HCC cell survival, proliferation and tumour formation in nude mice [
22]. Our previous study found that SGK3 promotes HCC cell migration and invasive potential [
23], which confirmed its vital function in promoting HCC progression. However, whether SGK3 also plays an important role in CSCs has not yet been reported. Our data showed a high phosphorylation level of SGK3 in liver CSC-enriched spheroids and CD133+ cells. More intriguingly, we demonstrated that overexpression of SGK3 enhances the expansion of liver CSCs, while inhibition of SGK3 by shRNA had an opposite effect. These findings indicate that SGK3 is critical for liver CSC expansion.
PI3K activity is stimulated by diverse oncogenes and growth factor receptors, and elevated PI3K signalling is considered a hallmark of cancer. Many PI3K pathway-targeted therapies have been tested in oncology trials [
30]. Class I PI3K activates downstream effectors by generating phosphoinositides PtdIns-3, 4-P2 and PtdIns-3, 4, 5-P3. The shared property of these PI3K effectors is a pleckstrin homology (PH) domain selective for PtdIns-3, 4, 5-P3 or PtdIns-3, 4-P2 [
30,
31]. Arguably, the vast majority of studies have focused on the protein kinase AKT as the dominant effector of PI3K signalling associated with malignancy. SGK3 has been reported to be a critical effector of oncogenic PIK3CA mutant breast cancer cells in which Akt is dispensable [
17]. Although SGK3 lacks a PH domain, it may still be activated by Class I PI3K through PDK1 [
29]. In contrast to Akt, SGK3 possesses an N-terminal PtdIns(3)P-binding PX domain [
20,
32], which is predominantly produced at the endosome by the Class III PI3K hVps34. In the present study, we demonstrated that prolonged treatment with PI3K inhibitors triggers SGK3 activation in HCC cells, while the phosphorylation of Akt is inhibited, which is consistent with findings reported by Bago et al. [
20]. We reasoned that the activation of SGK3 may be a compensatory mechanism to substitute the blockade of the Akt signalling pathway, which causes HCC cells to tolerate the treatment of PI3K inhibitors.
In the present study, we also found that prolonged treatment with PI3K inhibitors induced expansion of liver CSCs in HCC cells. We speculated that the expansion of liver CSCs induced by prolonged treatment with PI3K inhibitors occurred via the activation of SGK3. To further confirm this hypothesis, a rescue experiment was performed using SGK3 siRNA. The results indicated that knockdown of SGK3 expression employing siRNA partially blocked prolonged PI3K inhibitor treatment from enhancing CD133 expression in both Huh7 and MHCC97H cells. Meanwhile, our in vivo models confirmed that decreased tumour growth would be achieved by ZSTK474 treatment. Consistent with the in vitro experiment, ZSTK474 treatment induced the expansion of liver CSCs and activation of SGK3 in vivo. Although ZSTK474 could inhibit the tumor growth via inhibit proliferation and promote apoptosis of non-stem cells, a small proportion of cells could survive via their owned or acquired stemness property. Inhibition of mTOR signalling has been reported to upregulate CD133 expression in gastrointestinal cancer cells [
33]. Because the PI3K signalling pathway is a complex regulatory network, other downstream signal molecules may play a role in the expansion of liver CSCs induced by prolonged treatment with PI3K inhibitors. Class I PI3K is known to negatively regulate autophagy via the AKT-MTORC1-ULK1 complex [
34]; thus, autophagy other than SGK3 may also be involved in regulating the expansion of liver CSCs after prolonged treatment with PI3K inhibitors in HCC cells.
By virtue of its PX domain, SGK3 can bind to PtdIns(3)P produced by hVps34 on the endosome; thus, hVps34 inhibitor can inhibit SGK3 activation. Our results confirmed that VPS34-IN1, an hVps34 inhibitor, induced a dose-dependent inhibition of SGK3 phosphorylation. Furthermore, the inhibition of hVps34 also causes the inhibition of liver CSC self-renewal. These results further confirm that SGK3 is critical for liver CSC expansion and that targeting SGK3 could be a promising strategy for HCC therapy.
The augmentation of Wnt/β-catenin signalling through Ser9 phosphorylation-inactivation of GSK3β is a well-recognised regulatory pathway for CSC self-renewal and cancer development [
35,
36]. Our previous study confirmed that SGK3 stimulates β-catenin signalling in HCC cells [
23]. Liu et al. reported that overexpression of SGK3 increased the phosphorylation level of GSK3-β on Ser9 and inactivates GSK3-β [
22]. In our present study, we further confirmed that SGK3 promotes β-catenin accumulation by increasing the phosphorylation level of GSK3-β on Ser9, inactivating GSK3-β and inhibiting the degradation of β-catenin. Importantly, our results demonstrated that SGK3 promotes liver CSC expansion through the GSK3β/β-catenin signalling pathway. It has been previously reported that nuclear accumulation of β-catenin confers resistance to PI3K inhibitors in colon cancer [
27]. Indeed, our data suggested that prolonged inhibition of Class I PI3K leads to significant accumulation of β-catenin. We speculated that the accumulation of β-catenin was through the activation of SGK3 in HCC cells subjected to prolonged inhibition of Class I PI3K, which led to the enhanced self-renewal of liver CSCs.