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. 2020 Nov 23;147(2):499–505. doi: 10.1007/s00432-020-03422-4

Rapamycin inhibits lung squamous cell carcinoma growth by downregulating glypican-3/Wnt/β-catenin signaling and autophagy

Yanyu Bi 1,2, Yiming Jiang 2, Xia Li 2, Guoxin Hou 2, Kesang Li 1,3,4,✉
PMCID: PMC11801848  PMID: 33225417

Abstract

Purpose

There is not much progress in the treatment for lung squamous cell carcinoma LSCC in the past few years. Rapamycin Rapa, an inhibitor of mammalian target of rapamycin mTOR, has exhibited antitumor efficacy in a variety of malignant tumors. It has recently been reported that Rapamycin can induce autophagy signaling pathway in lung cancer and Glypican-3GPC3 can promote the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling pathway. The aim of this study is to investigate the mechanisms of rapamycin’s antitumor efficacy in relation to GPC3/Wnt/β-catenin pathway and autophagy in LSCC.

Methods

SK-MES-1 cells, a LSCC cell line, were treated with various concentrations of rapamycin with or without Glypican-3 GPC3-targeting siRNA. SK-MES-1 cell proliferation was determined by MTT assay. Protein expression levels of GPC3, β-catenin, Beclin-1 were checked via western blotting. We established the xenograft mice model to investigate the suppression effect of rapamycin on LSCC. In addition, we further testified the metabolism protein of autophagy process using the xenograft tumor tissue.

Results

Rapamycin could inhibit the SK-MES-1 cell proliferation in a concentration-dependent manner both in vitro and in vivo by decreasing the GPC3 expression and downregulating the glypican-3/Wnt/β-catenin signaling pathway. In addition, we found that GPC3 silencing can activate the glypican-3/Wnt/β-catenin pathway and autophagy, which contribute to the suppression of tumor growth both in vitro and in vivo.

Conclusion

Rapamycin suppresses the growth of lung cancer through down-regulating glypican-3/Wnt/β-catenin signaling, which mediates with activation of autophagy. This study suggests GPC3 is a new promising target for rapamycin in the treatment of lung cancer.

Keywords: Rapamycin, GPC3, Wnt, Lung cancer, Autophagy

Introduction

Lung cancer is the leading cause of cancer-related deaths 27% in both men and women with more than 1 million newly diagnosed cases and over 1.3 million deaths worldwide each year (Atlanta 2015; Siegel et al. 2015). In general, lung cancer can be mainly classified into small cell lung cancer SCLC and non-small cell lung cancer NSCLC. Approximately 85% of lung tumors are NSCLC. LSCC is the common histologic subtype 30% of NSCLC. However, there is no big breakthrough in LSCC therapy recently (Edelman 2017; Kim et al. 2013).

Chemotherapy and radiotherapy continue to be standard treatment of NSCLC. But even after positive clinical treatments, 5-year survival rate is still low (Baldini et al. 2020; Midthun 2014). Surgery and radiotherapy are the main treatment for early stage patients. For the local advanced NSCLC stages IIIA-B, platinum-based doublet chemotherapy and radiotherapy are the standard treatment. In addition, patients with specific genomic aberrations could benefit from molecular targeted therapies, especially for adenocarcinoma (Detterbeck et al. 2017; Postmus et al. 2017). Immunotherapy shows advantages in treating non-squamous NSCLC patients. In conclusion, there are limited strategies in treatments for LSCC. Different from other diseases, multiple gene alterations identified in LSCC show abnormalities in signaling pathways and physiological activities. As a result, much attention has recently been given to drugs related to intracellular pro-oncogenic pathways in lung cancer.

Immunotherapy shows advantages in treating non-squamous NSCLC patients. In conclusion, there are limited strategies in treatment for LSCC. Different from other disease, multiple gene alterations identified in LSCC shows abnormalities in signaling pathways and physiological activities. However, there is also natural molecular which has the ability to inhibit the progression of lung cancer. Recent studies demonstrated that Peruvoside, strophanthidin and Lanatoside C could inhibit lung cancer cell growth by modulating the proteins involved in cell cycle arrest, apoptosis, and autophagic cell death (Reddy et al. 2020; 2019; Reddy and umavath 2019). They mainly regulate the MAPK, PI3K/AKT/mTOR, and Wnt/β-catenin signaling. As a result, much attention has recently been given to drugs related to intracellular pro-oncogenic pathways in lung cancer.

Rapamycin sirilimus, Rapa is an antifungal agent with immunosuppressive properties, isolated in soil samples from Easter island in 1975 (Sehgal et al. 1975). Rapa is the target of rapamycin mTOR protein and it can inhibit the growth of various tumors in clinical research. mTOR forms two different protein complexes, mTORC1 and mTORC2. The former is acutely sensitive to Rapamycin, whereas the latter is only chronically sensitive to Rapamycin in vivo (Arriola Apelo and Lamming 2016). mTORC1 controls protein translation, autophagy and many other cellular processes through the phosphorylation of substrates. mTORC2 is a downstream effector of many oncogenic mutations that reprogram metabolic and epigenetic landscape. These changes lead to tumor cell survival and cancer resistance (Masui et al. 2020).

The autophagy pathway can be induced by the mammalian target of Rapamycin mTOR pathway. Rapamycin has been proved to be able to inhibit the growth of many tumor cells, including rhabdomyosarcoma, neu-roblastoma, small lung cancer, osteosarcoma, pancreatic cancer, breast cancer, leukemia cells and B cellular lymphoma (Euvrard et al. 2004). In addition, the mechanism of Rapamycin function has been studied in lung cancer in recent years.

GPC3 is a heparin sulfate proteoglycan and localizes to the cell surface via a glycerol-phosphatidylinositol GPI anchor. Studies has shown that GPC3 is expressed in approximately 70% of Hepatocellular carcinoma HCC and 63% of squamous non-small cell lung cancer (Gao et al. 2014). Studies has also shown that GPC3 could promote lung cancer growth by stimulating canonical Wnt/β-catenin signaling (Capurro et al. 2005a), while silencing of GPC3 could in a way inhibit the proliferation and induce apoptosis of lung cancer cells. The role of GPC3 in autophagy-mediated regulation of lung cancer growth induced by Rapamycin remains unknown. In this study, we will investigate the effects of Rapamycin on GPC3/Wnt/β-catenin signaling and the potential mechanism in LSCC.

Material and methods

Chemicals and reagents

Dulbecco’s Modified Eagle’s Medium DMEM and fetal bovine serum FBS were purchased from Thermo Fisher Scientific Waltham, MA, USA. Mouse monoclonal antibody against human c-myc was purchased from Santa Cruz Biotechnology Inc. Rabbit monoclonal antibodies against human LC3, Beclin-1, β-catenin and cyclinD1 were obtained from Cell Signaling Technology Boston, MA, USA. Immobilon Western Chemiluminescent HRP substrate was purchased from EMD Millipore Billerica, MA, USA. Mouse polyclonal antibody against human GPC3 was purchased from R&D Systems, Inc. Minneapolis, MN, USA. 3–4, 5-Dimeth-ylthiazol-2-yl-2, 5-diphenyl-2H tetrazolium bromide MTT cell proliferation and cytotoxicity assay kit was purchased from Beyotime Shanghai, China. The reverse transcription kit was purchased in Promega.

Cell culture

SK-MES-1, HepG2 and Sk-hep-1 cells were purchased from American Type Culture Collection ATCC Manassas, VA, USA. All cells were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin 10,000 U/mL penicillin and 10 mg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% of CO2.

MTT assay

SK-MES-1 cells were seeded into 96-well plates at a density of 2 × 104 cells/well and allowed to grow for 24 h. The medium was then replaced with 100 µL/well of fresh medium containing various stimuli for 24–72 h. Then, the cells were treated with 20 µL of 5 mg/mL MTT and incubated at 37 °C for 4 h. In the next step, the medium was removed and 150 µL DMSO was added to each well. Lastly, absorbance was measured at 490 nm after the crystals were fully dissolved.

Western blot analysis

The treated cells were washed twice with ice-cold PBS and harvested in RIPA lysis buffer containing protease inhibitors and then lysed for 30 min on ice. The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and then the supernatants were collected followed by protein concentration determination by bicinchoninic acid BCA analysis. Equal protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were incubated with 5% non-fat milk for 1 h and then incubated with primary antibodies overnight at 4 °C. All antibodies were diluted at a ratio of 1:1,000 with 3% non-fat milk. After washing with Tris-buffered saline with Tween-20, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Immunoreactive proteins were visualized using enhanced chemiluminescent kit according to the manufacturer’s protocol.

siRNA targeting GPC3 transfection

siRNA targeting GPC3 sense: 5′-CCAGUGGUCAGUCAAAUUATT-3′and antisense: 3′-UAAUUUGACUGACCACUGGTT-5′ and negative control siRNA were purchased from Shanghai Sangon Biotech Shanghai, China. SK-MES-1 cells were seeded into six-well plates and cultured overnight, and then transfected with siRNA at a concentration of 20 nmol/L using Lipofectamine2000 according to the manufacturer’s protocol.

Reverse transcription polymerase chain reaction RT-PCR analysis

Expression levels of GPC3 mRNA in cell lines and tissue sample were analyzed using RT-PCR analysis. The primer sequences were as follows: forward primer, 5´- CGGGGTACCGCCACCATGGCCGGGACCGTGCGCAC-3´; reverse primer, 5´-CCGGAATTCTCAGTGCACCAGGAAGAACAAGCACACCA-3´. PCR amplification conditions were as follows: 25-μL PCR mix containing 5 μL cDNA; pre-denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min; and final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 0.5 mg/mL ethidium bromide.

Tumor xenografts

Six-to-eight-week-old BALB/c nude female mice were raised and treated under specific pathogen-free conditions. All experimental animals were carried out according to the protocols approved by the Zhejiang Medical Experimental Care Commission. Single-cell suspensions of SK-MES-1 1 × 107 cells/ml were treated with intraperitoneal injection. 200 mg/kg Rapamycin was treated for Rapamycin group per day through injection with an equal volume inoculation for 1 month. Tumor dimensions were measured with calipers, and tumor volumes were calculated according to the formula V = 1/2 length × width2.

Statistics

Data were presented as mean ± standard error. Experiments were repeated at least twice. Statistical analysis was conducted using SPSS, Version22.0. p < 0.05 was considered to be statistically significant.

Results

Rapamycin inhibited SK-MES-1 cell proliferation by downregulating GPC3 expression and Wnt/β-catenin signaling

To explore the function of the Rapamycin in the growth of lung cancer cells, we evaluated the proliferation of SK-MES-1 cells under Rapamycin induction for 24–72 h by MTT assay. As shown in Fig. 1a, cell viability was significantly inhibited by Rapamycin in a dose-dependent manner, especially with 40 μg/ml Rapamycin induction. We next investigated the effect of the Rapamycin on GPC3 expression.

Fig. 1.

Fig. 1

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Rapamycin inhibited SK-MES-1 cell proliferation and induced cell apoptosis. a SK-MES-1 cells were treated with various concentration of Rapamycin for 24-72 h, and cell proliferation was determined by MTT assay n = 3. b Protein expression levels of GPC3 induced by Rapamycin were determined by western blotting. C&D, Expression of β-catenin, c-myc, cyclinD1 were verified by western blotting. E. Expression of GPC3 in SK-MES-1 and adjacent tissues were tested by RT-PCR. HepG2 was positive control and Sk-hep-1 was negative control

SK-MES-1 cells were cultured in the presence of different concentrations of Rapamycin 0, 10, 20, 40 μg/ml for 24 h. Figure 1b showed that GPC3 protein levels were significantly decreased with increasing concentrations of Rapamycin. As shown in Fig. 1c, d, after treatment with various concentrations of Rapamycin, the expression levels of Wnt/β-catenin, c-myc and cyclinD1 were also reduced.

GPC3 mRNA expression was examined by RT-PCR analysis, which showed that GPC3 was expressed in SK-MES-1 cells and tumor tissue but not in normal lung tissue Fig. 1e.

GPC3 silencing increased Rapamycin-induces SK-MES-1 cell apoptosis

To investigate the role of GPC3 in Rapamycin-induced anti-tumor effect, we transfected GPC3 siRNA into SK-MES-1 cells. Finally, the expression of GPC3 was suppressed at protein level Fig. 2a. In our study, after knockdown of GPC3, the expression levels of β-catenin, c-myc and cyclinD1 were decreased Fig. 2b. These results suggest that suppression of tumor by Rapamycin is dependent on the inhibition of GPC3 and Wnt signaling by Rapamycin. In addition, we found that combined treatment with siRNA and Rapamycin could enhance the inhibition of expression of GPC3, β-catenin, c-myc. The results indicate that the combination of GPC3 siRNA and Rapamycin treatments can exert stronger inhibition of cell proliferation.

Fig. 2.

Fig. 2

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GPC3 silencing increased Rapamycin-induced SK-MES-1 cell apoptosis. a Expression level if GPC3 in SK-MES-1 cells transfected with 20 nM siRNA for 24 h was determined by western blotting. b Expression of GPC3, β-catenin and c-myc in SK-MES-1 cells transfected with 20 nM siRNA in the presence or absence of 20 nM Rapamycin

Autophagy mediated Rapamycin-induced inhibition of GPC3 protein expression

We used western blot essay to investigate the role of autophagy in the suppression of LSCC cells. Different concentration of Rapamycin was cultured with SK-MES-1 cell for 12 h. The results showed in Fig. 3a that the biomarker of autophagy were activated by Rapamycin in a concentration-dependent manner.

Fig. 3.

Fig. 3

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Rapamycin decrease the GPC3 expression by autophagy activation. a and b SK-MES-1 cells were treated with Rapamycin at concentrations of 0, 10, 20, 40 μg/ml; expression of Beclin-1, LC3, β-actin were confirmed by western blotting

Rapamycin inhibited tumor growth by suppressing GPC3/Wnt/β-catenin signaling in vivo

To explore the function of Rapamycin in anti-tumor activities in vivo, tumor xenografts were established. Tumor-bearing animals were treated with Rapamycin by intravenous injection for 1 month. At the end of this study, the results shown in Fig. 4a revealed that Rapamycin group potently inhibited the growth of lung cancer in vivo. The protein expression levels of GPC3, β-catenin, c-myc and cyclinD1 were inhibited when treated with Rapamycin. This indicates that Rapamycin has anti-tumor effect in lung cancer by inhibiting GPC3/Wnt/β-catenin signaling.

Fig. 4.

Fig. 4

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Rapamycin inhibited tumor growth by suppressing GPC3/Wnt/β-catenin signaling in vivo. a Xenograft mice were treated with PBS or Rapamycin as control group or Rapamycin group for 1 month, tumor size were measured with calipers and calculated according to the formula V = 1/2 (length × width2). b Protein lysates extracted from tumor of xenografts. The expression of GPC3, β-catenin, c-myc and cyclinD1 were testified by Western blotting essay

Discussion

Autophagy is a survival mechanism to maintain cell activity for viability in response to cell stress and nutrient limitations by recycling subcellular organelles and proteins (Cecconi and Levine 2008; Glick et al. 2010. Given that tumorigenesis is a complex process and autophagy exert its effects in various ways (Chen and White 2011), Rapamycin has been approved by United States Food and Drug Administration for treatment in patients with metastatic renal cell carcinoma and for organ transplant rejection therapy (Yuan et al. 2009). Rapamycin functions are extensively studied in various lung cancer research in recent years (Vicary and Roman 2016).

It is not validated whether mTOR expresses in all lung cancers. There are studies showing that phosphorylated mTOR was detected in more than 70% of 110 NSCLC samples in a recent report (Balsara et al. 2004). In addition, scientists found that Cisplatin-resistant SCLC cells were susceptible to temsirolimus one of Rapamcycin analogues (Skeen et al. 2006). Studies have also shown that mTOR signal inhibition blocks tumor cell progression and angiogenesis, and induces apoptosis and autophagy. Some research groups propose that Rapamycin can be potential therapy for lung cancer because Rapamycin is capable of inhibiting the proliferation of different kinds of tumors; however, clinical trials brought up more challenges including drug adverse effect (Mita et al. 2003; Vicary and Roman 2016). The autophagy pathway can be activated by AMPK signaling, but is generally inhibited by the mTOR pathway (Kimura et al. 2003). Autophagy also participates in the inhibition of lung cancer growth. The expression of Beclin-1 is significantly decreased in lung cancer tissues, suggesting that autophagy is involved in the pathogenesis of lung cancer.

It has been elucidated that GPC3 expression in HCC tissues was positively regulated by p62 an autophagy adapter, showing a correlation between GPC3 and autophagy activation (Bao et al. 2014). GPC3 was negatively regulated by autophagy in HCC cells, and the suppression of activated autophagy can be achieved by inhibiting the GPC3/Wnt/β-catenin signaling (Capurro et al. 2005b). In this study, when lung cells were treated with Rapamycin, GPC3 expression was decreased and autophagy related molecules were increased, suggesting that there may be some association between Rapamycin-directed GPC3 negative regulation and autophagy pathway in lung cancer.

GPC3 is a membrane-bound proteoglycan that is discovered in patients with Simpson-Golabi-Behmel syndrome in 1996; (Pilia et al. 1996) GPC3 is a 66-kDa protein localized on the cell surface and the extracellular matrix (Pilia et al. 1996; Yamauchi et al. 2005). GPC3 is frequently expressed in LSCC. In contrast, its expression in normal tissues is silenced. Recent studies have reported that GPC3 is related to proliferation, differentiation, progression and metastasis of malignant tumors (Baumhoer D 2008; Cheng et al. 2008) There are also studies showing that MHCC97-H cell proliferation and invasion were effectively inhibited through knockdown of GPC3 using GPC3-targeted shRNA (Ruan et al. 2011). In our study, we speculate that depletion of GPC3 could inhibit the tumor progression through decreasing expression of β-catenin, c-myc and cyclinD1. GPC3 may be a significant target for LSCC.

In this study, we explored that whether Rapamycin, as autophagy inducer, could inhibit the growth and induce the apoptosis of LSCC. Numerous studies reported that the Wnt-signaling pathway contributes to the growth of the tumor (Reya and Clevers 2005); In the meantime, scientists showed that GPC3 promotes the growth of hepatocellular Carcinoma by stimulating canonical Wnt signaling (Capurro et al. 2005b). In addition, we conclude that Rapamycin may inhibit tumor growth by decreasing the expression of GPC3 and Wnt-signaling pathway. The regulatory association between autophagy and GPC3/Wnt/β-catenin pathway requires further investigation. The molecular basis of other autophagy effctors remains to be unconvered. Rapamycin may help overcome the challenges associated with targeted therapy for lung cancer in the future. We hope that much more study could be carried out on the lung cancer therapy in the future.

Funding

This work was supported by National Natural Science Foundation of China 81602703, the Natural Science Foundation of Ningbo2018A610382, Zhejiang Medicine and Health Sciences Research Fund 2020379758, 2019 Jiaxing Key Discipline of Medicine—OncologySupporting Subject2019-zc-11.

Compliance with ethical standards

Conflict of interest

None.

Footnotes

Publisher's Note

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