Purmorphamine

Involvement of PI3K/Akt pathway in the inhibition of hepatocarcinoma cell invasion and metastasis induced by SASH1 through downregulating Shh-Gli1 signaling

Changyu Sun*, Zhihao Zhang, Ping He, Yan Zhou, Xuhua Xie

Abstract

The SASH1 gene is discovered as a tumor suppressor recently. However, the molecular mechanisms of SASH1 in hepatocarcinoma (HCC) remain unclear. In present studies, we investigated the molecular mechanisms of SASH1 on cell invasion and metastasis of hepatocarcinoma in vivo and in vitro. In this study, SASH1 overexpression HCC cell lines were treated with purmorphamine (0, 0.5, 1, 2 μmol/l). Western blot and qRT-PCR were used to examine the related gene expression of EMT markers and the Shh-Gli1 and PI3K/Akt-dependent pathway. Cell migration and invasion were assessed by Transwell assay. In addition, a mice SASH1 overexpression HCC orthotopic xenograft model was established and treated with purmorphamine or 740Y-P or PDGF. Tumor volume was assessed, and H&E staining was applied to histopathologic analysis. The results showed that purmorphamine exposure significantly increased the mRNA and protein expression levels of Shh and Gli1 in a dose-dependent manner in the SASH1 overexpression HepG2 and HCCLM3 cells.
Besides, purmorphamine promoted the migration and invasion of SASH1 overexpression HCC cells, as well as the EMT progress. Moreover, purmorphamine significantly increased the synthesis of PI3K and pAkt in a dose-dependent manner. Furthermore, the invasion and migration abilities were also improved by treatment with 740Y-P or PDGF in the SASH1 overexpression HCC cells. Additionally, the agonists promoted tumor growth and intrahepatic and pulmonary metastasis of the orthotopic transplantation tumors grown from SASH1 overexpression HCC cells in vivo. In conclusion, SASH1 may inhibit hepatocarcinoma cell invasion and metastasis through down-regulating the Shh-Gli1 and PI3K-AKT pathways in vivo and in vitro.

Key words: SASH1; Hepatocarcinoma; Invasion; Metastasis; Shh-Gli1; PI3K-AKT

1. Introduction

Hepatocarcinoma (HCC) is the most common primary liver malignancy (Ferlay et al., 2015). The high incidence makes HCC become the second most common cause of cancer-related deaths worldwide (Dhanasekaran et al., 2013). Although the diagnosis and treatment of HCC has been improved, the patients still have a median survival of only a few months (Baek et al., 2011). And the high recurrent and metastatic rates cause the poor prognosis (Baek et al., 2011). Thus, the suppression of tumor metastasis is of vital importance in clinical practice.
The SASH1 (SAM- and SH3-domain containing 1) gene, which is a member of the SLY family of signal adapter proteins, is discovered as a tumor suppressor recently (Zeller et al., 2003). Increasing evidences indicate that SASH1 may inhibit cancer cell proliferation, migration and invasion in various cancer cells (Ren et al., 2016; Rimkus et al., 2006; Sun et al., 2015; Yang et al., 2012). In previous studies, we have demonstrated that overexpression of SASH1 can inhibit the proliferation, invasion and epithelial mesenchymal transition (EMT) in hepatocarcinoma cells (He et al., 2016). Thus further studies on the mechanism of SASH1 in the invasion and metastasis of hepatocarcinoma are required.
Sonic hedgehog (Shh) is a member of the hedgehog family morphogens and plays critical roles in the development of many tissues and organs (Wang et al., 2008). In addition, when Shh binds to its receptor Patched1, it suppressed the associated signaling receptor Smoothened (Smo) by releasing inhibitor, leading to nuclear translocation and up-regulate Gli transcription factors, Gli1, Gli2 and Gli3 (Jacob and Briscoe, 2003). Moreover, Shh signaling is involved in a number of tumors, such as prostate cancer (Sanchez et al., 2004), ovarian cancer (Ke et al., 2015), gastric cancer (Chen et al., 2016) and so on. Besides, we found Shh signaling was essential for the invasion and metastasis of HCC cells (He et al., 2016). Here we further tested the involvement of Shh-Gli1 signaling in hepatocarcinoma.
PI3K/Akt pathway is an intracellular signaling pathway, which transduces signals from cell membrane receptors to the cytoplasm (Vita et al., 2014). With the comprehensive research of the molecular biology of cancer, PI3K/Akt signal pathways are closely related to proliferation, growth, expansion, and metastasis of malignant cells (Yang et al., 2015). Dysregulation of PI3K/Akt pathway has been found in many cancers such as squamous cell carcinoma(Wang et al., 2014), breast cancer (Suter and Marcum, 2007), prostate cancer (Mckenzie and Kyprianou, 2006) and hepatoblastoma cancer (Gao et al., 2014). However, the role of the PI3K/Akt-dependent pathway in SASH-induced cell invasion and metastasis of HCC has never been elucidated. The aim of our studies was to investigate the relevant mechanisms of SASH1 on cell invasion and metastasis of hepatocarcinoma.

2. Materials and methods

2.1 Cell Culture

Hepatocarcinoma (HCC) cell lines HepG2 and HCCLM3 were purchased from American Type Culture Collection (ATCC) and were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Biochrom, Germany) in 5% CO2 at 37oC.

2.2 Overexpression of SASH1 gene in HepG2 and HCCLM3 cells

The construction of SASH1 overexpression vector was performed according to our previous studies (He et al., 2016). For transfection, the cells were seeded in six well plates at a density of 1.0 × 105 cells/well. Then the cells were transfected with pcDNA3.1-SASH1 using Lipofectamine 2000™ (Invitrogen, USA) according to the manufacturer’s instructions. And the stably transfected colonies were selected by using 1 μg/ml G418 (Sigma-Aldrich, USA).

2.3 Pharmacological Treatments

After transfection, SASH1 overexpression HepG2 and HCCLM3 cells were treated with different concentration of Shh-Gli1 signals agonist purmorphamine (0, 0.5, 1, 2 μmol/l; TESTMART, China)) for 48 h. To investigate the PI3K/Akt signaling pathway, SASH1 overexpression HepG2 and HCCLM3 cells were incubated with PI3K agonist 740Y-P (50 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 90 min or incubated with Akt agonist PDGF (100 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. And each treatment repeated six times. Then all the cells were harvested for further use.

2.4 Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from HCC cells with Trizol reagent (Invitrogen) according to the manufacturer’s protocols. The RNA was reverse transcribed to cDNA using a PrimeScript™ 1st Strand cDNA Synthesis kit (Takara, Dalian, China). qRT-PCR was performed in a final volume of 10 μl reaction mixture, which contained 5 μl of SsoFast™ EvaGreen Supermix (Bio-Rad Laboratories), 0.5 μl of each primers (Gli1, forward: 5’-TCTGCCCCCATTGCCCACTTG-3’; reverse: 5’-TACATAGCCC CCAGCCCATACCTC-3’; Shh, forward: 5’-CGGAGCGAGGAAGGGAAAG-3’; reverse: 5’-TTGGGGATAAACTGCTTGTAGGC-3’; GAPDH, forward: 5’-ACGGAT TTGGTCGTATTGGG-3’; reverse: 5’-TGGAAGATGGTGATGGGATT-3’), 1 μl of the cDNA template and 3 μl ddH2O. The standard PCR conditions: 50oC (2 min), 95oC (10 min), and 35 cycles of 95oC (45 s) and 60oC (30 s), and 72oC (30 s), finally, 72oC (5 min), followed by the standard denaturation curve. GAPDH was used as an internal control. The relative expression of mRNA was calculated by the 2-ΔΔCt method.

2.5 Western blot analysis

Western blot analysis was performed as described previously (Hamid et al., 2009). In brief, proteins were extracted from HCC cells using RIPA buffer (Invitrogen), and the protein concentration was measured by using a BCA protein assay kit (Beyotime, Nantong, China). A total of 20 μg of protein was separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham, UK). Then the membranes were blocked with 5% nonfat milk powder and then incubated with primary antibodies (Santa Cruz Biotechnology, USA) at 4oC overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The proteins were then detected by enhanced chemiluminescence (Pierce, USA).

2.6 Cell migration and invasion assays

Cell migration and invasion assays were performed as described previously (Wang et al., 2014). For cell migration assay, cell migration was determined using the Transwell assay (Boyden chambers; Corning, Cambridge, MA, USA). Cells were added in serum-free medium in the upper chamber and 500 μl of DMEM containing 10% FBS were added in the lower chamber. After 48 h migration, cells that had migrated through and adhered to the underside of the membrane were counted at × 200 magnification. In addition, the cell invasion assay was performed similarly, except that 50 μl Matrigel (BD Biosciences, USA) was added to each well overnight before cells were seeded onto the membrane.

2.7 Animal models and treatments

Six-week-old healthy C57BL/6 male mice were purchased from the Animal Center of the Chinese Academy of Sciences (Beijing, China). All the experiments with live animals were approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University and were conducted according to the Guide for Care of Laboratory Animals by the National Ministry of Science. The mice were anaesthetized with chloral hydrate before any invasive operations. Mice were randomly and equally separated into purmorphamine-treated (n = 6), 740Y-P-treated (n = 6), PDGF-treated (n = 6) and control group (n = 6). Tumors were generated by subcutaneous injection into the right anterior flank with 1 × 106 SASH-overexpression HCCLM3 cells suspended in 100 μl PBS (control group) or the cells incubated with purmorphamine (1 μmol/l) or 740Y-P (50 μg/ml) or PDGF (100 ng/ml). After cell inoculation for two weeks, when palpable tumors were established, all of the mice were sacrificed. Tumors were excised and the largest (a) and smallest (b) diameters of tumors were measured to calculate tumor volume (V=ab2/2) (Wang et al., 2014). The organs lung and liver were removed and examined by hematoxylin and eosin staining (H&E).

2.8 Histopathology analysis

All the tissue samples were processed and embedded in paraffin. Then organs were sectioned with a steel knife mounted in a microtome to obtain 5 μm thick tissue sections. Then the sections were mounted on a microscope slide individually and treated with H&E stain. After that, the samples were examined under an optical microscope.

2.9 Statistical analysis

The data were expressed as the mean ± S.D. All analyses of the results were performed using the GraphPad Prism software version 6.0 (GraphPad Software, San Diego, CA, USA). Statistical analyses were performed using the Student’s t-test. Statistical significance was set at P < 0.05. 3. Results 3.1 Purmorphamine increased the expression levels of Shh and Gli1 in the SASH1 over-expression HCC cell lines In order to elucidate the detailed relationship between Shh-Gli1 signaling and SASH1, HepG2 and HCCLM3 cells transfected with SASH1 overexpression vectors were adopted to do the following experiments. Besides, we used Shh-Gli1 signals agonist purmorphamine to treat SASH1 overexpression HepG2 and HCCLM3 cells for 48 h. As shown in Figure 1A and 1C, purmorphamine exposure significantly increased the mRNA expression levels of Shh and Gli1 in a dose-dependent manner in the SASH1 overexpression HepG2 and HCCLM3 cells. In addition, the results of western blot were similar to those detected by qRT-PCR (Figure 1B and 1D). As a significant increase of the expression of Shh and Gli1 was observed in the HCC cells treated with 1 μmol/l purmorphamine, we chose this concentration for the following experiments. 3.2 The effect of SASH1 on EMT process and cell invasion and migration was mediated via Shh-Gli1 signaling in the HCC cell lines As EMT is an important step for cancer cells in promoting metastasis capability (Ke et al., 2015). We investigated the molecular pathways of SASH1 on the EMT process of HCC cells. As shown in Figure 2A and 2C, purmorphamine reduced the protein expression of the epithelial marker E-cadherin in the SASH1 overexpression HepG2 and HCCLM3 cells. In contrast, the protein expression of mesenchymal markers, snail，vimentin，N-cadherin was up-regulated in the SASH1 overexpression HepG2 and HCCLM3 cells treated with purmorphamine. Next, by using Transwell assay, we found that purmorphamine led to an increased invasion and migration ability of the SASH1 over-expression HepG2 and HCCLM3 cells compared to those in control groups (Figure 2B and 2D). These results showed that SASH1 might regulate the EMT process and invasion and metastasis abilities of HCC cells via Shh-Gli1 signaling. 3.3 Shh-Gli1 signaling might activate PI3K-Akt signaling pathway To verify the cross talk between Shh-Gli1 and PI3K-Akt pathway in the HCC cells, we detected the PI3K, Akt, pAkt, mTOR and p-mTOR proteins in the SASH1 overexpression HepG2 and HCCLM3 cells treated with different concentration of purmorphamine. As shown in Figure 3A, the results of western blot clearly showed that purmorphamine significantly promoted the synthesis of PI3K, pAkt and p-mTOR in a dose-dependent manner. These results revealed that the Shh-Gli1 signaling pathway might activate PI3K-Akt signaling pathway. 3.4 Employment of PI3K/Akt agonists on the effect of SASH1 on cell invasion and migration in the HCC cell lines To further explore the mechanism of SASH1 on cell invasion and migration, SASH1 overexpression HepG2 and HCCLM3 cells were incubated with PI3K agonist 740Y-P and Akt agonist PDGF, respectively. Treatments with the agonists significantly increased PI3K, pAkt and p-mTOR protein expression levels in the SASH1 overexpression HepG2 and HCCLM3 cells than that in the control group. However, no significant differences were observed in the Akt, mTOR, Shh and Gli1 proteins (Figure 3B). Moreover, the invasion and migration abilities were also improved by treatment with the agonists in the SASH1 overexpression HepG2 and HCCLM3 cells (Figure 3C and 3D). These results told us that SASH1 might modulate the invasion and metastasis ability of HCC cells via PI3K/Akt signaling pathway. 3.5 The effect of SASH1 on tumor growth and intrahepatic and pulmonary metastasis of hepatic tumors through the Shh-Gli1 and PI3K-AKT pathways in vivo To investigate the molecular mechanisms of SASH1 on tumor growth and intrahepatic and pulmonary metastasis of hepatic tumors in vivo, mice were implanted with SASH1 overexpression HCCLM3 cells incubated with purmorphamine or 740Y-P or PDGF. Our results indicated that tumor volume was significantly increased in the purmorphamine and 740Y-P and PDGF-treated mice (Figure 4A). Besides, the purmorphamine and 740Y-P and PDGF-treated mice exhibited more intrahepatic and pulmonary metastatic lesions and irregular tumor margins detected by H&E staining. Furthermore, a higher ratio of intrahepatic metastasis and pulmonary metastasis was detected (Figure 4B and 4C). 4. Discussion The incidence of HCC has been up-regulated in the last two decades and the prognosis is still dismal (Belghiti and Fuks, 2012). In addition, metastasis from the primary tumor to other organs is prevalent among patients with HCC, which accounts for 90% deaths (Qiao et al., 2009). Thus, understanding the genetic programs and how they affect cellular signaling pathways is of vital significance to uncover the complex process of metastasis. Previous studies demonstrated that SASH1 is a tumor suppressor gene, which is located on chromosome 6q24.3 (Zeller et al., 2003). Chen et al. reported overexpression of SASH1 may be related to the decreased migration of A549 human lung cancer cells (Chen et al., 2012). Zong et al. indicated overexpression of SASH1 inhibited TGF-β1-induced EMT in gastric cancer cells (Zong et al., 2016). Meng et al. found that overexpression of SASH1 significantly reduced osteosarcoma cell viability and invasive ability (Meng et al., 2013). Most recently, our studies demonstrated that overexpression of SASH1 might inhibit the migration and invasion by preventing EMT in hepatocarcinoma cells (He et al., 2016). In this study, we show the molecular mechanisms of SASH1 on cell invasion and metastasis of hepatocarcinoma in vivo and in vitro. Increasing evidences support the critical roles of Shh-Gli1 signaling pathway during the progression of different kinds of cancers (Oue et al., 2013; Yanai et al., 2008). Moreover, EMT is one of the crucial events regulating hepatocarcinoma cell invasion and metastasis (Ma et al., 2014). Typically, cells undergoing EMT exhibit decreased E-cadherin expression (Hazan et al., 2004) and increased expression of mesenchymal biomarkers, such as N-cadherin, vimentin and snail (Peinado et al., 2007). In our studies, we detected that Shh-Gli1 signaling agonist purmorphamine might suppress the E-cadherin protein levels but increase the levels of N-cadherin, vimentin and snail and enhance the cell migration and invasion abilities in SASH1 overexpression HCC cells. Based on previous studies and our data, we found overexpression of SASH1 inhibited the migration and invasion by suppressing EMT through downregulating Shh-Gli1 signaling in hepatocarcinoma cells. Subsequently, we demonstrated that Shh-Gli1 signaling might activate PI3K/Akt pathway by increasing the protein expression of P13K and pAkt. The cross talk between Shh-Gli1 signaling pathway and PI3K/Akt pathway has also been demonstrated in ovarian cancer (Ke et al., 2015). Additionally, accumulating genetic and cancer biology evidences illustrate that SASH1 inhibited cancer cell growth and invasion and EMT process through PI3K/Akt pathway (Sun et al., 2015; Zong et al., 2016). In our studies, we observed that the invasion and migration abilities were improved by treatment with the PI3K and Akt agonists in the SASH1 overexpression HCC cells, suggesting overexpression of SASH1 inhibited the migration and invasion through down-regulating PI3K/Akt pathway in hepatocarcinoma cells. At last, we demonstrated that SASH1 might inhibit tumor growth and intrahepatic and pulmonary metastasis in mouse models of hepatocarcinoma through the Shh-Gli1 signaling and PI3K/Akt pathway. 5. Conclusions In conclusion, the present study demonstrated that SASH1 inhibited cell invasion and metastasis through down-regulating the Shh-Gli1 and PI3K-AKT pathways in vivo and in vitro. Therefore, SASH1 represents an effective drug candidate for molecular-targeted therapy for hepatocarcinoma. Reference Baek, K.K., Kim, J.H., Uhm, J.E., Park, S.H., Lee, J., Park, J.O., et al., 2011. 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