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Abstract. – OBJECTIVE: Non-small cell lung cancer (NSCLC) is one of the most ordinary can-cers worldwide. Recent studies have discovered many oncogenes play vital roles in the tumor-igenesis of malignant tumors. The purpose of our study was to uncover the role of GBP1 in NS-CLC and the underlying mechanism.

PATIENTS AND METHODS: GBP1 expres-sion in NSCLC samples was detected by Real Time quantitative-Polymerase Chain Reaction (RT-qPCR). Function assays were performed in NSCLC cells transfected with GBP1 shRNA. Furthermore, RT-qPCR and Western blot assay were conducted to explore the target signaling pathway of GBP1.

RESULTS: GBP1 expression was significant-ly upregulated in NSCLC tissue samples com-pared with adjacent normal tissues. Function assays showed that the proliferation of NSCLC cells was significantly inhibited via knockdown of GBP1, while cell apoptosis was promoted. Re-sistance to paclitaxel was reversed after GBP1 knockdown in paclitaxel resistance A549 cells (A549/Taxol). In addition, Wnt/β-catenin signal-ing pathway was repressed via knockdown of GBP1 in NSCLC cells and A549/Taxol cells.

CONCLUSIONS: In our study, GBP1 was first-ly identified as a novel oncogene in NSCLC. Fur-thermore, it could promote NSCLC development and paclitaxel resistance by inducing Wnt/β-cat-enin signaling pathway.

Key Words:GBP1, Development, Chemotherapy, Wnt/β-cat-

enin signaling pathway, Non-small cell lung cancer (NSCLC).

Introduction

Lung cancer is one of the most common can-cers globally both in terms of morbidity and mor-tality1. Non-small cell lung cancer (NSCLC) is

the major subtype of lung cancer. Current studies have indicated that NSCLC contributes to 85% of all lung cancer cases. It has been reported that the incidence and mortality of NSCLC will continue to increase for the next several decades due to the characteristics of cell migration and invasion2,3. Most patients have already been in an advanced stage at the time of diagnosis. Moreover, the median survival time of these patients barely exceeds 18 months4. Therefore, there is an urgent need to elucidate the underlying mechanism of NSCLC and improve the poor prognosis for un-fortunate patients.

Chemoresistance remains one of the major obstacles to the success of cancer therapies. In the past several decades, chemoresistance has been found closely associated with the expression of many genes and complex biological mech-anisms, including epithelial-mesenchymal tran-sition (ETM), enrichment of cancer stem cells (CSCs), autophagy, drug efflux mechanism, and so on5,6. Consistently, the MDR1 gene is one of the determinants involved in the chemoresistance of ovarian cancer7. CSCs contribute to chemo-resistance in breast cancer. Furthermore, it may lead to a novel therapy to prevent the relapse and improve the prognosis of breast cancer patients8. By interacting with Notch1, DDR1 induces inef-fective induction of cell death, eventually leading to a chemo-resistant phenotype9. Paclitaxel, as a clinically common chemotherapy drug, cannot avoid drug resistance during the treatment. How-ever, the mechanism of paclitaxel resistance in malignancies remains unclear.

Recently, GBP1 has been reported as a novel oncogene that promotes the development of mul-tiple cancers. However, the role of GBP1 in NS-CLC development and chemotherapy resistance has not been fully elucidated. In our study, we

European Review for Medical and Pharmacological Sciences 2020; 24: 5465-5472

J. SONG1, Q.-Y. WEI2

1Department of Geriatrics, DaLian Friendship Hospital, Dalian, China2Department of Allergy, General Hospital of Northern Theater Command, Shenyang, China

Corresponding Author: Qingyu Wei, Ph.D; e-mail: weiqingyu1964@163.com

GBP1 promotes non-small cell lung carcinoma malignancy and chemoresistance via activating the Wnt/β-catenin signaling pathway

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discovered that GBP1 was involved in cell prolif-eration, metastasis, and chemotherapy resistance by inducing Wnt/β-catenin signaling pathway in NSCLC. Our findings might offer new insight into the therapy of NSCLC.

Patients and Methods

Clinical SamplesTotally, 56 paired NSCLC tissues and matched

paracancerous tissues were collected from pa-tients who underwent surgical resection at Gener-al Hospital of Northern Theater Command from June 2016 to December 2018. Collected tissues were quickly placed in liquid nitrogen and im-mediately stored in -80°C cryogenic refrigerator. This study was approved by the Ethics Commit-tee of the General Hospital of Northern Theater Command. All the enrolled patients were diag-nosed with NSCLC by two independent patholo-gists without any controversial. Written informed consent was obtained from each patient before surgery.

Cell CultureFour NSCLC cell lines (A549, SPCA1, PC-9,

and H1299) and one normal human bronchial epithelial cell line (16HBE) were purchased from the Institute of Biochemistry and Cell Biolo-gy, Shanghai Institutes for Biological Sciences (Shanghai, China). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) in an incubator with 5% CO2 at 37°C. A549/Taxol cells were cultured in DMEM medi-um added with 10% FBS.

Cell TransfectionFor transfection, lentivirus expressing

short-hairpin RNA (shRNA) targeting GBP1 was compounded and cloned to pLenti-EF1a-EGFP-F2A-Puro vector (Biosettia Inc., San Diego, CA, USA). GBP1 shRNA or negative control shRNA (NC) was transfected into NSCLC cells accord-ing to the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Real Time-Quantitative Polymerase Chain Reaction (RT-qPCR)

The total RNA from tissues and cells was extracted through TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by measurement

of RNA concentration using an ultraviolet spec-trophotometer (Hitachi, Tokyo, Japan). SYBR green (Roche, Basel, Switzerland) was conducted to measure the relative expression levels of genes. β-actin was used as an internal reference. The primers used in this study were shown as fol-lows: GBP1, forward: 3’-ACTTCAGGAACAG-GAGCAAC-5’ and reverse: 3’-TATGGTACAT-GCCTTTCGTC-5’; β-actin, forward: 5′-GATG-GAAATCGTCAGAGGCT-3′ and reverse: 5′-TG-GCACTTAGTTGGAAATGC-3′. The specific thermal cycle was as follows: 30 sec at 95°C, 5 sec for 40 cycles at 95°C, and 35 sec at 60°C. The relative expression was calculated by the 2-∆∆CT

method.

Methyl Thiazolyl Tetrazolium(MTT) Assay

2 × 103 transfected cells were seeded into 96-well plates containing 100 μL medium with-out 10% FBS. This was to ensure that the number of cells per well was about 2000. 10 μL MTT reagent (Roche, Basel, Switzerland) was added to each well at 0 h, 24 h, 48 h, and 72 h, respectively, followed by incubation in an incubator. Optical density (OD) value at 490 nm was measured by an ELISA reader system (Multiskan Ascent, Lab-Systems, Helsinki, Finland).

Colony Formation Assay1.5 × 103 of cells were seeded into 6-well plates

and cultured. 10 day later, the formed colonies were fixed with 10% formaldehyde for 30 min and stained with 0.5% crystal violet for 5 min. Nikon camera was used for taking a photograph of colonies. Finally, the number of formed colo-nies was counted and compared.

Flow Cytometry Assay1-5 × 105 cells in logarithmic growth phase were

digested with trypsin and made into cell suspen-sion. After washing twice with Phosphate-Buffer Saline (PBS) and centrifugation at 1000 rpm/min for 5 min), 100 μL Binding Buffer was added to suspend the cells. Later, 5 μL of 7-aad and 5 μL of PE dye were added to each tube, followed by mixing with 400 μL of Binding Buffer. At the same time, blank control (no dye) and single dye (5 μL 7-aad and 5 μL PE respectively) were set for 15 min at room temperature. Flow cytometry was performed, and the results were expressed as the percentage of total apoptosis.RE

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Western Blot AnalysisThe cells were first washed with pre-cooled

PBS and lysed with cell lysis solution (RIPA; Beyotime, Shanghai, China). The protein con-centration was detected by the bicinchoninic acid (BCA; Thermo Fisher Scientific Inc., Waltham, MA, USA). The total proteins were separated and transferred on polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Af-ter blocking in Tris-Buffered Saline and Tween 20 (TBST; 25 mM Tris, 140 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% skimmed milk for 2 h, the membranes were incubated with pri-mary antibodies of Wnt3a, β-catenin, C-myc, and Survivin (Abcam Inc., Cambridge, MA, USA) in Wnt/β-catenin signaling pathway and β-actin (Abcam Inc., Cambridge, MA, USA) at 4°C over-night. After washing (3 × 10 min) with TBST, the membranes were incubated with corresponding secondary antibody at room temperature for 1 h. The immunoreactive band were exposed and an-alyzed by Image J software (Media Cybernetics, Silver Springs, MD, USA).

Statistical AnalysisThe Statistical Product and Service Solutions

(SPSS) 20.0 (IBM Corp., Armonk, NY, USA) was

used for all statistical analyses. Independent-sam-ple t-test was selected when appropriate. p<0.05 was considered statistically significant.

Results

GBP1 Expression in NSCLC Tissues and Cells

GBP1 expression was first detected in 56 pairs of NSCLC tissues and 4 NSCLC cells. As shown in Figure 1A, GBP1 was significantly upregulated in NSCLC tissues when compared with adja-cent normal tissues through RT-qPCR detection. Western blot assay indicated that the protein level of GBP1 was significantly upregulated in NSCLC tissues as well (Figure 1B). Moreover, the expres-sion level of GBP1 in four NSCLC cell lines was shown in Figure 1C.

GBP1 Knockdown Repressed Cell Growth Ability and Promoted Cell Apoptosis in NSCLC

To determine whether GBP1 exerted a vital function in NSCLC, A549 cells were chosen for knockdown of GBP1. GBP1 shRNA and nega-tive control shRNA were synthesized and trans-

Figure 1. Expression level of GBP1 in NSCLC tissues and cell lines. A, GBP1 expression was significantly upregulated in NSCLC tissues compared with adjacent tissues. B, The protein level of GBP1was significantly upregulated in NSCLC tissues through Western blot. C, Expression levels of GBP1 relative to β-actin in human NSCLC cell lines were determined by RT-qPCR. Data were presented as mean ± standard error of the mean. *p<0.05.RE

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duced into SW780 cells. Transfection efficien-cy was verified by RT-qPCR (Figure 2A). As shown in Figure 2B, the MTT assay showed that the knockdown of GBP1 significantly inhibited the viability of NSCLC cells. To further confirm the effect of GBP1 on the apoptosis of NSCLC cells, the flow cytometry assay was performed. As shown in Figure 2C, the percentage of cell apoptosis rate remarkably increased after GBP1 was knocked down.

Knockdown of GBP1 Reversed Paclitaxel Resistance of A549/Taxol NSCLC Cells

To investigate the effect of GBP1 on the pa-clitaxel resistance of A549/Taxol NSCLC cells, MTT, and colony formation assay were applied in A549/Taxol NSCLC cells after transfection of GBP1 shRNA. Transfection efficiency was monitored by RT-qPCR (Figure 3A). MTT assay found that paclitaxel sensitivity increased re-markably through knockdown of GBP1 in A549/Taxol cells (Figure 3B). The colony formation assay demonstrated that the colony formation

ability was significantly suppressed after the knockdown of GBP1 in A549/Taxol cells (Fig-ure 3C).

Knockdown of GBP1 Inhibited Development and Reversed Paclitaxel Resistance Through Wnt/β-Catenin Signaling Pathway in NSCLC

To explore the underlying mechanism of GBP1 function in the development of NSCLC, RT-qP-CR and Western blot assays were conducted. The mRNA and protein expressions of Wnt3a, β-cat-enin, C-myc, and Survivin were determined, which were the target proteins of the Wnt/β-cat-enin signaling pathway. As shown in Figure 4A and Figure 4B, the mRNA and protein expres-sions of the above molecules were remarkably downregulated via knockdown of GBP1 in A549 cells. Conversely, as shown in Figure 4C and Figure 4D, the expressions of these proteins were significantly downregulated via knockdown of GBP1 in A549/Taxol cells at both mRNA and protein levels. These results suggested that the

Figure 2. Knockdown of GBP1 inhibited NSCLC cell proliferation and promoted cell apoptosis. A, GBP1 expression in NSCLC cells transduced with negative control shRNA (NC) or GBP1 shRNA (shRNA) was detected by RT-qPCR. β-actin was used as an internal control. B, MTT assay showed that the viability of NSCLC cells was significantly inhibited by the knockdown of GBP1. C, Flow cytometry assay results showed that the percentage of cell apoptosis rate increased remarkably after GBP1 was knocked down. The results represented the average of three independent experiments (mean ± standard error of the mean). *p<0.05. RE

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knockdown of GBP1 participated in the regula-tion of Wnt/β-catenin signaling pathway, further inhibiting NSCLC development and reversing paclitaxel resistance.

Discussion

Guanine nucleotide-binding protein 1 (GBP1) belongs to the large GTPase family, which is encoded by a gene cluster located on chromo-some10. As an IFN-gamma-response mediator, GBP-1 inhibits cell proliferation by suppressing the Hippo signaling transcription factor TEAD11. Via mediating EGFR-vIII, GBP-1 enhances the progression of glioblastoma in vivo12. The knock-down of GBP1 affects the growth of triple-neg-ative breast cancer, which serves as a novel po-tential therapeutic target13. The overexpression of GBP1 is significantly associated with resistance to paclitaxel and poor prognosis of ovarian cancer patients14. In our study, we explored the function of GBP1 in the proliferation and apoptosis of

NSCLC cells. The results showed that GBP1 ex-pression was significantly upregulated in NSCLC tissues. After GBP1 was knocked down, NS-CLC cell proliferation was suppressed, while cell apoptosis was promoted. These data indicated that GBP1 functioned as an oncogene in NSCLC and promoted its tumorigenesis.

Although chemotherapy is effective for NS-CLC patients at an early stage of treatment, many patients develop resistance to chemotherapeu-tic drugs during the period of treatment. This brings a huge burden to the patient’s family and the society. The possible mechanisms underlying drug resistance include DNA damage repair, drug transport system error, anti-apoptosis, self-pro-tection of thiol molecules inside the cells, etc. All of them are related to dysregulated genes and activation or inhibition of signaling pathways in NSCLC cells. Paclitaxel, as a clinically common chemotherapy drug, cannot avoid drug resistance during the treatment. However, the paclitaxel resistance remains unclear currently. In the pres-ent study, the target genes and related signaling

Figure 3. Knockdown of GBP1 reversed paclitaxel resistance of A549/Taxol NSCLC cells. A, 48 h after A549/Taxol NSCLC cells were transfected with GBP1 shRNA, transfection efficiency was verified by RT-qPCR. GAPDH was used as an internal control. B, Paclitaxel sensitivity of A549/Taxol NSCLC cells transfected with control or GBP1 shRNA was analyzed by MTT assay. C, Colony formation assay showed that the colony formation ability of cells was significantly suppressed after the treatment of paclitaxel and GBP1 shRNA (magnification × 40). The results represented the average of three independent experiments. *p<0.05.

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pathways associated with paclitaxel resistance were explored. Our findings showed that cell proliferation was significantly suppressed after knockdown of GBP1 in A549/Taxol cells.

Canonical Wnt/β-catenin signaling functions as an important pathway involved in phenotype and chemoresistance of cancer-initiating cells. Wnt/β-catenin signaling pathway plays an ex-tremely important role in embryonic develop-ment, tissue proliferation, cell differentiation, cancer, etc. Wnt gene was originally named Int-1 gene in 1982. At present, it has been identified as an oncogene by numerous researches15,16. There

are 19 members in the Wnt gene family. Many previous studies have demonstrated that the Wnt signaling pathway plays an extremely important role in the development of various malignant can-cers17,18. The most common mechanism of activa-tion of the Wnt signaling pathway is through reg-ulating β-catenin. Briefly, β-catenin excessively accumulates in the cytoplasm and activates TCF/LEF inside the nucleus, further regulating the re-lated pathways. Dysregulated β-catenin is discov-ered in different types of human tumors. Whether the Wnt/β-catenin signaling pathway plays a key role in the drug resistance of NSCLC cells still

Figure 4. Knockdown of GBP1 inhibited development and reversed paclitaxel resistance through Wnt/β-catenin signaling pathway in NSCLC. A, RT-qPCR results revealed that the mRNA expression of target proteins in Wnt/β-catenin signaling pathway was downregulated in A549 cells of shRNA group compared with NC group. B, Western blot assay revealed that the expression of the target proteins in Wnt/β-catenin signaling pathway was downregulated in A549 cells of shRNA group compared with NC group. C, RT-qPCR results revealed that the mRNA expression of target proteins in Wnt/β-catenin signaling pathway was downregulated in A549/Taxol cells of shRNA group compared with NC group. D, Western blot assay revealed that the expression of the target proteins in Wnt/β-catenin signaling pathway was downregulated in A549/Taxol cells of shRNA group compared with the NC group. The results represented the average of three independent experiments. Data were presented as mean ± standard error of the mean. *p<0.05.

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requires more comprehensive researches19,20. In our study, we explored the association between Wnt/β-catenin pathway and GBP1. GBP1 knock-down significantly downregulated the mRNA and protein expressions of target proteins in Wnt/β-catenin signaling pathway in vitro. These results above suggested that the knockdown of GBP1 might inhibit tumorigenesis and reverse paclitaxel resistance of NSCLC via regulating Wnt/β-catenin signaling pathway.

Conclusions

GBP1 served as a novel biomarker in the development of NSCLC. Furthermore, it could enhance NSCLC tumorigenesis and chemore-sistance through activation of the Wnt/β-catenin signaling pathway.

Conflict of InterestThe Authors declare that they have no conflict of interests.

Funding AcknowledgementsNatural Science Research Fund Guidance Program of Lia-oning Province, China.

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