SGK2 promotes hepatocellular carcinoma progression and mediates GSK-3β/β-catenin signaling in HCC cells

Abstract

The phosphoinositide 3-kinase pathway is one of the most commonly altered pathways in human cancers. The serum/glucocorticoid-regulated kinase (SGK) family of serine/threonine kinases consists of three isoforms, SGK1, SGK2, and SGK3. This family of kinases is highly homologous to the AKT kinase family, sharing similar upstream activators and downstream targets. Few studies have investigated the role of SGK2 in hepatocellular carcinoma. Here, we report that SGK2 expression levels were upregulated in hepatocellular carcinoma tissues and human hepatoma cell lines compared to the adjacent normal liver tissues and a normal hepatocyte line, respectively. We found that downregulated SGK2 inhibits cell migration and invasive potential of hepatocellular carcinoma cell lines (SMMC-7721 and Huh-7).We also found that downregulated SGK2 suppressed the expression level of unphosphorylated (activated) glycogen synthase kinase 3 beta. In addition, SGK2 downregulation decreased the dephosphorylation (activation) of β-catenin by preventing its proteasomal degradation in the hepatocellular carcinoma cell lines. These findings suggest that SGK2 promotes hepatocellular carcinoma progression and mediates glycogen synthase kinase 3 beta/β-catenin signaling in hepatocellular carcinoma cells.

Introduction

Hepatocellular carcinoma (HCC) is a common malignancy and the third leading cause of cancer-related deaths worldwide.^1,2 In patients with advanced HCC, death is usually caused by tumor cell invasion and metastasis. ³ Moreover, conventional treatments are not effective in these patients, ⁴ and thus, novel HCC therapies are urgently sought. HCC development has been linked to several well-researched risk factors,^5,6 such as cirrhosis, hepatitis B, and hepatitis C. In addition, a growing number of molecular signaling pathways^7–9 have been suggested to be implicated in hepatocarcinogenesis, for example, Wnt/β-catenin, phosphoinositide 3-kinase (PI3K), AKT, and glycogen synthase kinase 3 β (GSK-3 β). These molecular pathways represent potential targets for novel HCC therapies.

The abnormal activation of the Wnt/β-catenin pathway has been linked to the development of carcinomas of the colon, breast, liver, pancreas, and other organs.^10,11 Aberrant activation of this pathway can be induced by genetic mutations (such as activating mutations of CTNNB1, which encodes β-catenin, and inactivating mutations of AXIN1 or adenomatous polyposis coli (APC)) ¹² as well as by cross talk with other signaling pathways (such as the transforming growth factor β pathway). ¹³ GSK-3 has been shown to be involved in the Wnt/β-catenin signaling pathway. ¹⁴ Mice with a homozygous deletion of the GSK-3β gene experience massive hepatocyte apoptosis during embryogenesis, leading to premature death. ¹⁵

Serum/glucocorticoid-regulated kinases (SGKs) exhibit structural similarity with Akt1–3,^16–18 suggesting that SGKs may have similar functions to Akt in cancer development. SGK2 is the most poorly studied member of the SGK family. Unlike SGK1, SGK2 messenger RNA (mRNA) is not induced by stimulation with serum or glucocorticoid and is present at significant levels only in the liver, kidney, and pancreas and at low levels in the brain. ¹⁹ However, similar to SGK1 and SGK3, SGK2 also activates certain potassium and sodium channels, suggesting an involvement in the regulation of processes such as cell survival, neuronal excitability, and renal sodium excretion.^20,21 SGK3 has been shown to inactivate GSK-3β in HCC cells. ²² As both SGK2 and SGK3 belong to the AGC protein kinase family, we hypothesized that SGK2 could act on GSK-3β and β-catenin signaling pathway. This study aimed to determine whether or not SGK2 contributes to HCC development and to characterize its relationship with GSK-3β and the β-catenin pathway in HCC.

Materials and methods

HCC samples

HCC specimens were obtained from 20 HCC patients who were treated at the Department of Hepatobiliary Surgery, Zhoukou Central Hospital, Zhoukou, China, during 2014–2016. All patients had consented to tissue collection for research at the time of surgery. All the tumor specimens were confirmed to be HCCs by the Department of Pathology, Zhoukou Central Hospital. None of the patients had received chemotherapy, immunotherapy, or radiotherapy prior to specimen collection. The patients’ clinical and pathological records were retrieved, and the following information was noted: age at initial diagnosis, sex, tumor size, American Joint Committee on Cancer (7th edition) tumor–node–metastasis stage, follow-up duration, and disease-free and overall survival. Tissue samples were frozen at −80°C. This research was approved by the ethics committee of Zhoukou Central Hospital.

Cell lines and culture

The normal human hepatocyte cell line, L02, and the human HCC cell lines, Huh-7 and SMMC-7721, were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China, in October 2014. All cell lines had been authenticated using DNA fingerprinting in the Cell Bank and tested for mycoplasma using the Hoechst staining method, which confirmed that the cells were negative for mycoplasma. In addition, we did not find any other microorganisms in these cell lines. We used less than five generations of cells for the experiments in this study. All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Los Angeles, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Los Angeles, CA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained in an incubator at 37°C in 5% CO₂.

SGK2 knockdown

SGK2 small interfering RNA (siRNA) and a negative control were designed and synthesized by Shanghai Genechem Co., Ltd (Shanghai, China). SMMC-7721/Huh-7 or L02 cells were transfected using siRNA-Mate^™ (Shanghai GenePharma Co., Ltd, Shanghai, China) according to the manufacturer’s protocol. In brief, the cells were seeded at 2–3 × 10⁵ cells per well and grown in DMEM to 30%–50% confluence. Transfection complexes were prepared according to the manufacturer’s instructions and were added directly to the cells. The cells were harvested 72 h after the transfection, washed, and stored at −80°C until they were used in the subsequent experiments. The knockdown efficiency was assessed by western blotting.

Real-time fluorescence quantitative polymerase chain reaction

Total RNA was extracted from HCC specimens or human cell lines using TRIzol^® reagent (Thermo Fisher Scientific, Los Angeles, CA, USA) according to the manufacturer’s instructions. For the analysis of mRNA expression, complementary DNA was synthesized using the PrimeScript^™ RT reagent kit with gDNA Eraser (Takara Biomedical Technology (Beijing) Co., Ltd, Beijing, China). Quantitative real-time fluorescence polymerase chain reaction (RT-qPCR) was performed using the SYBR Premix ExTaq (Takara Biomedical Technology (Beijing) Co., Ltd) under standard conditions according to the manufacturer’s instructions. RT-qPCR was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as the internal control, and the results were analyzed using the 2^−ΔΔCt method. The SGK2 primers were 5′-GCAGGAACCTGGAGTTTTCA-3′ (forward) and 5′-CTCCCCCGTTGACATAGTCG-3′ (reverse). The GAPDH primers were 5′-GGGAAGCTTGTCATCAATGG-3′ (forward) and 5′-CATCGCCCCACTTGATTTTG-3′ (reverse).

Western blot analysis

Total protein was extracted from the cells by radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentration was determined using a bicinchoninic acid (BCA) kit (Beyotime Biotechnology). Equal amounts of the proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h and incubated overnight with the primary antibodies at 4°C, followed by incubation with the secondary antibodies for 1 h at 37°C. The following antibodies were used: mouse anti-human SGK2 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-β-catenin (#8480; Cell Signaling Technology, Denver, MA, USA), rabbit anti-non-phospho (active) β-catenin (Ser33/37/Thr41; #8814; Cell Signaling Technology, Denver, MA, USA), rabbit anti-phospho-GSK-3β (Ser9; #5558; Cell Signaling Technology), rabbit anti-GSK-3β (#12456; Cell Signaling Technology, Denver, MA, USA), and rabbit/mouse anti-β-actin (YT0099/YM1207; ImmunoWay, Plano, TX, USA). Protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified using the Quantity one software package (Bio-Rad, Hercules, CA, USA).

Transwell migration assay and Matrigel invasion assay

For migration assay, approximately 1 × 10⁴ cells/well were seeded. Cells were resuspended in 200 µL of serum-free DMEM and placed in the upper chamber of a 24-well Transwell^® plate with an 8.0-µm pore polycarbonate membrane insert (Corning, NY, USA). A total of 700 µL of cell culture medium containing 10% FBS was added to the lower chamber to create a gradient. The inserts were maintained for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. Next, the non-invading cells on the upper chamber were scraped off carefully using a moist cotton swab, and the cells that migrated through the polycarbonate membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 20 min. The numbers of migrated cells were calculated under a microscope in five randomized fields. For cell invasion assay, approximately 3 × 10⁴ cells were placed in each well, and the procedures were similar to the migration assay, except that the polycarbonate membrane was coated with Matrigel (BD Biosciences, San Jose, CA, USA), and the inserts were maintained at 37°C in an atmosphere containing 5% CO₂ for 48 h.

Wound-healing assay

After the cells were cultured to reach approximately 100% confluency in a six-well cell culture cluster, wound-healing assay was performed. In brief, a scratch on the confluent cell monolayer was created with a new 200 µL sterile pipette tip, after which the medium was removed and subsequently replaced with fresh serum-free cell culture medium. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂, and five marked fields were observed under the microscope at 0 and 24 h time points to assess the rate of gap closure.

Statistical analysis

Statistical analysis was performed using SPSS for Windows version 19.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation (SD). The Student’s t test was used to analyze the raw data, and a value of p < 0.05 was accepted as statistically significant.

Results

SGK2 overexpression in HCC

Few studies have previously documented the expression of SGK2 in HCC. Here we selected 20 cases of human liver cancer that were diagnosed as HCC with the paracancer normal liver tissues. SGK2 mRNA and protein expression were upregulated in HCC specimens as compared to the adjacent normal liver tissues (Figure 1(a) and (b)). Consistent with this, SGK2 protein levels were significantly (p < 0.01) higher in the two human hepatoma cell lines (Huh-7 and SMMC-7721) than in the normal human hepatocyte line (L02; Figure 1(c)).

Figure 1.

SGK2 overexpression in HCC. (a) The SGK2 mRNA expression level in 20 HCC specimens was higher than that in the adjacent non-cancerous liver tissues. The SGK2 mRNA level was quantified by RT-qPCR. (b) The SGK2 protein expression level in 7 HCC specimens was higher than that in the adjacent non-cancerous liver tissues. The SGK2 protein level was determined by Western bloting. (c) Western blot analysis of SGK2 protein expression in the normal human hepatocyte line L02 and two hepatoma cell lines (Huh-7 and SMMC-7721). All experiments were performed in triplicate.

SGK2 knockdown inhibits cell migration and invasive potential in HCC cells

Studies suggest that SGK3 has strong oncogenic potential and is amplified and hyperactivated in breast cancer and HCC.^22,23 Because both the SGK2 and SGK3 belong to SGKs kinase family, we hypothesized that SGK2 has a role in the cell migration and has invasive potential in HCC cells. We tested the effect of SGK2 depletion on cell migration and invasion ability by performing Transwell migration and invasion assays. Silencing the expression of SGK3 decreased the invasion (p < 0.02) and migration ability (p < 0.02) in both SMMC-7721 and Huh-7 cells (Figure 2(a) and (b)). We also performed a scratch assay to determine the role of SGK2 in wound-healing capacity and therefore the migration ability of HCC cells. Downregulated SGK2 increased the wound-healing capacity in both the SMMC-7721 cells and Huh-7 cells (Figure 2(c); p < 0.01).

Figure 2.

SGK2 knockdown inhibits cell migration and invasive potential in HCC cells. (a) Control and SGK2-depleted SMMC-7721 and Huh-7 cells were subjected to a Transwell and Matrigel invasion assays. (b) Control and SGK2-depleted SMMC-7721 and Huh-7 cells were subjected to a Transwell and migration assays. (c) A wound-healing assay was performed to determine the migratory capacity of SMMC-7721 cells transfected with SGK2 siRNA or Control. (d) A wound-healing assay was performed to determine the migratory capacity of Huh-7 cells transfected with SGK2 siRNA or Control. All experiments were performed in triplicate.

SGK2 knockdown decreases GSK-3β expression in HCC cells

To elucidate the underlying mechanism of SGK2 in HCC, we transfected SGK2 siRNA in both SMMC-7721 and Huh-7 cells (Figure 3(a)). Next, western blotting was used to determine the expression level of GSK-3β phosphorylated at Thr9 (inactivated form) and the total GSK-3β level in the SGK knockdown cells. The downregulation of SGK2 was associated with a decrease in the expression of phosphorylated GSK-3β (p-GSK-3β) as compared to the negative control (Figure 3(b)).

Figure 3.

SGK2 knockdown decreases GSK-3β expression in HCC cells. (a) Depletion of SGK2 in SMMC-7721 and Huh-7 cells. Cells were transfected with SGK2 siRNA and a negative control and then subjected to western blotting with an anti-SGK3 antibody. β-actin was used as the loading control. (b) GSK-3β levels in SGK2-depleted SMMC-7721 and Huh-7 cells. The expression levels of active GSK-3β and total GSK-3β were determined using western blot analysis. β-actin was used as the loading control. All experiments were performed in triplicate.

SGK2 knockdown decreases β-catenin expression in HCC cells

In the Wnt signaling pathway, GSK-3β forms a destruction complex with AXIN and APC for the phosphorylation and subsequent degradation of β-catenin. However, a direct relationship between SGK2 and β-catenin has not been demonstrated. Therefore, we sought to elucidate a potential role for SGK2 in β-catenin signaling. In SMMC-7721 and Huh-7 cells, the downregulated SGK2 was associated with a marked reduction in the level of the active form of β-catenin (dephosphorylated at Ser37 and Ser41; Figure 4(a)). This effect of SGK2 depletion on β-catenin was blocked in the presence of the proteasome inhibitor MG132 (Figure 4(b)).

Figure 4.

SGK2 knockdown decreases β-catenin expression in HCC cells. (a) β-catenin protein levels in SGK2-depleted SMMC-7721 and Huh-7 cells. The expression levels of active β-catenin and total β-catenin were determined using western blot analysis. β-actin was used as the loading control. (b) In the presence of the proteasome inhibitor MG132 (10 µM; 8 h), the degradation of β-catenin induced by SGK2 depletion was blocked in SMMC-7721 and Huh-7 cells. All experiments were performed in triplicate.

Discussion

SGKs are involved in the regulation of cell growth, proliferation, survival, and migration.^24,25 The SGK family contains three highly homologous isoforms—SGK1, SGK2, and SGK3—that share a similar domain structure. Furthermore, they have been reported to share ~55% sequence identity with the catalytic domains of Akt1–3.^16–18 Akt plays important roles in cell survival and proliferation and is the best-known effector of the PI3K pathway, which is one of the most important pathways in cancer metabolism and growth. ²⁶ The results of this study indicated that SGK2 expression is increased in HCC. We found that SGK2 mRNA and protein expression levels were higher in HCC tissues than in the adjacent normal liver tissues and that SGK2 protein levels were higher in the HCC cell lines SMMC-7721 and Huh-7 than in the normal liver cell line L02. In addition, downregulated SGK2 inhibits cell migration and invasive potential in HCC cell lines (SMMC-7721 and Huh-7)

Recent study indicated that SGK3 can contribute to breast cancer through an Akt-independent mechanism, promote cell proliferation and survival in HCC,^22,23,27 and inactivate GSK-3β in HCC cells. ²² In this study, we found that SGK2 knockdown inhibits invasion and migration ability of HCC cells. SGK2 knockdown was also associated with a decreased protein expression level of inactivated GSK-3β (phosphorylated at Ser9), implying that SGK2 contributes to GSK-3β inactivation.

GSK-3β forms a destruction complex with AXIN and APC for the phosphorylation and degradation of β-catenin. ²⁸ Our data also showed that the downregulated SGK2 was associated with a decreased protein expression level of active β-catenin (dephosphorylated at Ser37 and Ser41). However, in the presence of the proteasome inhibitor MG132, this effect of SGK2 on β-catenin was blocked. This suggests that SGK2 can stabilize β-catenin by inhibiting its proteasomal degradation. Both SGK2 and SGK3 affect GSK-3β and β-catenin signaling pathway, but the difference in their effects is unclear, and would be an interesting topic for further research. The limitations of this study are the lack of animal experiments and the small sample size, owing to which we could not fully verify our conclusion. We intend to improve this deficiency in a future study.

In conclusion, our study showed that SGK2 was expressed at a higher level in HCC tissues and cells than in normal tissues/cells. SGK2 knockdown inhibits invasion and migration ability of HCC cells. Furthermore, the results of this study indicated that SGK2 may inactivate GSK-3β by promoting the phosphorylation of the latter at Ser9. In addition, GSK-3β inactivation was associated with an increase in the expression level of activated β-catenin. This provides an alternative mechanism for the PI3K signaling axis to drive cancer progression under conditions in which Akt is inactive. These findings advocate for diagnosis of cancer and the development of small-molecule inhibitors targeting SGK2 for PIK3CA-addicted tumors resistant to Akt inhibition.