The Decreased Expression of Fbxw7 E3 Ligase Mediated by Cancer Upregulated Gene 2 Confers Cancer Stem Cell-like Phenotypes

CUG2 유전자에 의하여 감소된 FBXW7 E3 ligase 발현이 유사-종양줄기세포 표현형을 유도

Abstract

The detailed mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. The downregulation of FBXW7 E3 ligase, a tumor suppressor known for its proteolytic regulation of oncogenic proteins such as cyclin E, c-Myc, Notch, and Yap1, has been frequently reported in several types of tumor tissues, including those in the large intestine, cervix, and stomach. Therefore, we investigated whether FBXW7 is involved in CUG2-induced oncogenesis. In this study, the decreased expression of FBXW7 was examined in human lung adenocarcinoma A549 (A549-CUG2) and human bronchial BEAS-2B cells (BEAS-CUG2) overexpressing CUG2 and compared with control cells stably expressing an empty vector (A549-Vec or BEAS-Vec). Treatment with MG132 (a proteosome inhibitor) prevented the degradation of FBXW7 and Yap1 proteins, which are substrates of the FBXW7 E3 ligase. To address the role of Fbxw7 in the development of cancer stem cell (CSC) phenotypes, we suppressed Fbxw7 protein levels using its siRNA. We observed that decreased levels of FBXW7 enhanced cell migration, invasion, and spheroid size and number in A549-Vec and BEAS-Vec cells. The enforced expression of FBXW7 produced the opposite results in A549-CUG2 and BEAS-CUG2 cells. Furthermore, the downregulation of FBXW7 elevated the activities of EGFR, Akt, and ERK1/2 and upregulated β-catenin, Yap1, and NEK2, while the enforced expression of FBXW7 generated the opposite results. We thus propose that FBXW7 downregulation induced by CUG2 confers CSC-like phenotypes through the upregulation of both the EGFR-ERK1/2 and β-catenin-Yap1-NEK2 signaling pathways.

신규 종양 유전자 Cancer Upregulated Gene (CUG) 2가 어떻게 유사-종양줄기세포 표현형을 유도하는지 잘 알려져 있지 않다. Cyclin E, c-Myc, Notch, 그리고 Yap1와 같은 종양단백질를 분해하여 그 발현을 조절하는 FBXW7 E3 ligase의 발현이 대장암, 자궁경부암, 그리고 위암 등 여러 암조직에서 낮아져 있음이 보고되고 있다. 그래서 우리는 이 FBXW7 단백질이 CUG2에 의한 종양형성에 관여할 수 있다는 가설을 세웠다. 이 연구에서 우리는 각 대조구 세포주보다 CUG2가 과발현된 A549 폐암 세포주와 BEAS-2B 기관지 세포주에서 FBXW7 단백질 발현이 낮게 나왔다. 여기서 MG132를 처리하게 되면 감소된 FBXW7과 FBXW7 기질로 알려진 Yap1 단백질 발현이 증가되는 결과를 관찰하였다. 종양줄기세포 현상에서 FBXW7의 역할을 규명하기 위하여, FBXW7 siRNA를 처리하였다. 대조구 세포주에서 감소된 FBXW7의 조건은 세포 이동 침습, 그리고 구형 형성이 증가되는 종양줄기세포 현상이 촉진되는 것을 관찰하였고, CUG2가 과발현된 두 세포주에서 FBXW7 발현 벡타 도입으로 FBXW7 발현 증가는 종양줄기세포 현상이 억제됨을 알 수 있었다. 또한 FBXW7의 감소는 EGFR-Akt-ERK1/2와 β-catenin-Yap1-NEK2 신호 경로를 활성화시키고, 반대로 FBXW7 발현 증가는 이 두 경로의 활성이 억제됨을 알 수 있었다. 이들 결과를 종합해 보면, CUG2 과발현은 FBXW7의 발현 감소로 이어지고, 이는 EGFR-Akt-ERK1/2와 β-catenin-Yap1-NEK2 신호경로를 활성화시켜 유사-종양줄기세포 현상을 촉진하는 것으로 생각된다.

Introduction

FBXW7 is a member of the F-box protein family, which is part of the Skp1-Cdc53/Cullin-F-box protein complex (SCF/b-TrCP) [16, 19]. The SCF complex functions as an E3-ubiquitin ligase, and FBXW7 protein with a WD40-repeat domain is responsible for recognizing and binding to the phosphorylated substrate, facilitating SCF complex-mediated ubiquitination and proteasomal degradation [16, 19, 20]. However, the FBXW7 gene mutation is frequently found in several cancer patients, including those with T cell-acute lymphoblastic leukemia, small and large intestine carcino- ma, and endometrial carcinoma [16, 22]. FBXW7 is considered a tumor suppressor [3, 16]. FBXW7 deficiency increases chromosomal instability and results in the development of hematopoietic cancer [22]. In particular, further studies have reported that FBXW7 substrates are proto-oncogenes, such as those encoding c-Myc, cyclinD/E, Jun, and mTOR [11, 12]. Thus, knockout of FBXW7 increases the upregulation of these proto-oncogenes, resulting in accelerated development of cancer [1, 12, 18].

The Affymetrix microarray system shows that the transcription of the cancer upregulated gene (CUG)2 is increased in various cancer tissues—including those of the lung, ovary, and colon cancer—when compared to normal tissues [9]. Faster proliferation of cancer cells and tumor formation in nude mice demonstrate the oncogenic activity of CUG2 [9]. Further studies report that CUG2 induces cancer stem cell (CSC)-like phenotypes, such as increased cell migration, in- vasion, and sphere formation, as well as resistance to anticancer drugs through TGF-β signaling [6, 7]. Both the epidermal growth factor receptor (EGFR)/Stat1-HDAC₄ signaling axis and β-catenin/yes-associated protein (Yap1)/NIMA- related kinase 2 (NEK2) signaling axis are involved in CUG2-induced CSC-like phenotypes [5, 8]. Recent studies have reported that the elevation of EGFR and β-catenin protein levels and signaling induced by CUG2 overexpression confers a decrease in the levels of the Spry2 protein, a tumor suppressor, through c-Cbl, which can explain the CUG2-in- duced CSC-like phenotypes [15].

The purpose of this study was to explore whether FBXW7, which is frequently downregulated in several tumor tissues, is involved in CUG2-induced CSC-like phenotypes. We observed that an siRNA-induced decrease in the levels of FBXW7 promoted the development of CSC-like phenotypes through the upregulation of the EGFR-Akt-ERK1/2 and β- catenin-Yap1-NEK2 signaling pathways. In contrast, FBXW7 expression inhibited the CSC phenotypes. These results indicate that enforcing FBXW7 expression is a potential therapeutic strategy for treating CUG2-induced malignant tumors.

Materials and Methods

Cell culture

Human lung adenocarcinoma A549 and immortalized human bronchial BEAS-2B cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were stably transfected with either the vector alone (A549-Vec; BEAS-Vec) or wild-type CUG2 (A549-CUG2; BEAS-CUG2) and maintained in RPMI-1640 and 50% DMEM/ 50% F12 media, respectively. The medium was supplemented with 10% FBS, 1% penicillin, 1% streptomycin, and G418 (500 μg/ml) at 37℃ and 5% CO₂.

Antibodies and reagents

Anti-FBXW7 antibody was acquired from R&D Systems (Minneapolis, MN, USA). Antibodies against β-catenin, EGFR, Akt, ERK, and their phosphorylated proteins were obtained from Cell Signaling Biotechnology (Danvers, MA, USA). Anti-NEK2 and anti-Yap1 antibodies were purchased from Abcam (Cambridge, MA, USA). Antibodies against c-Myc, cyclin D, and c-Jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Transfection

After reaching 60~70% confluence, the cells were incubated with a transfection mixture containing Lipofectamine 2, 000 (Invitrogen, Carlsbad, CA, USA) and FBXW7 siRNAs (Bioneer, Daejeon, Korea) or a negative control siRNA (Bioneer) for 24 hr. The cells were then rinsed with a medium containing 10% FBS and incubated at 37℃ for 24 hr before harvesting. For the transfection of the FBXW7 expression vector (Addgene, Watertown, MA, USA), the cells were cultured with the transfection mixture for 6 hr, rinsed with medium, incubated at 37℃ for 24 hr, and were then harvested.

Wound healing assay

The cells were cultured in 12-well plates (3×10⁵ cells/well) to achieve 80% confluence. The cells were then incubated for 24 hr to measure the closing of the scratch that was made with a pipette tip (200 μl).

Transwell invasion assay

A549-Vec and -CUG2 cells (3×10⁴ cells/well) and BEAS- Vec and -CUG2 cells (6×10⁴ cells/well) were seeded in the upper well containing RPMI-1640 and DMEM, respectively, without serum. The lower wells of the chamber were filled with 10% FBS medium and further cultured for 20 hr to allow the cells to invade the membrane coated with Matrigel (BD Bioscience, San Jose, CA, USA). Cells were fixed, stained with hematoxylin-eosin, and quantified by counting the cell number under a phase-contrast microscope (CKX31-11 PHP; Olympus, Tokyo, Japan) at 100X magnification.

Sphere forming assay

Cells were cultured in 24-well ultra-low attachment plates in a serum-free medium supplemented with 5 μg/ml insulin, 0.4% BSA, 10 ng/ml basic-FGF, and 20 ng/ml EGF for 6 days. The size and number of spheroids were analyzed using an optical microscope (CKX31-11 PHP; Olympus, Japan).

Immunoblotting

After proteins from the cell lysates were separated by 10% SDS-PAGE, the proteins on the gel were transferred to nitrocellulose membranes. The membrane was incubated with primary antibodies (1:500 dilution) at 4℃ overnight and then incubated with an appropriate secondary antibody (horser- adish peroxidase-conjugated secondary antibody, 1:2, 000) after washing. The proteins on the membrane were detected using an Image Quant LAS 4000 Mini (GE Healthcare, Tokyo, Japan).

Immunofluorescence microscopy

Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with cold acetone for 15 min, blocked with 10% goat serum for 30 min, and incubated with anti-FBXW7 antibodies overnight at 4℃. The cells on the coverslips were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody in PBS for 1 hr. For nuclear staining, the cells were incubated with 4', 6-dia- midino-2-phenylindole (DAPI) for 45 min in the dark. The stained cells were mounted on slides using PBS containing 10% glycerol, and images were acquired using a fluorescence microscope (Zeiss Axio Observer D1, Oberkochen, Germany).

Statistical analysis

Data were presented as the mean±standard deviation (SD). To compare the differences between the two groups, an unpaired t-test was used with the GraphPad Prism software. Statistical significance was set at p<0.05.

Results

CUG2 downregulates FBXW7 protein expression through the ubiquitin–proteasome pathway

As several previous studies have reported that the mutation or downregulation of FBXW7, an E3 ligase, is closely correlated with poor prognosis or metastasis in patients with esophageal squamous cell carcinoma and renal cell carcinoma [2, 17], we speculated whether CUG2 induces the down regulation of FBXW7 protein. We observed decreased protein levels of FBXW7 in A549-CUG2 and BEAS-CUG2 cells when compared to A549-Vec and BEAS-Vec cells (Fig. 1A). Immunofluorescence analysis showed the control cells to be heavily stained for FBXW7 compared with the A549-CUG2 and BEAS-CUG2 cells (Fig. 1B), confirming that CUG2 expression reduces FBXW7 levels. We further explored whether the decreased expression of FBXW7 was mediated by the ubiquitin–proteasome pathway. We treated A549-CUG2 and BEAS-CUG2 cells with MG132 (a proteasome inhibitor) and observed that the MG132 treatment inhibited the degradation of FBXW7 through the ubiquitin–proteasome pathway (Fig. 1C). We also noted that the expression of Yap1, a substrate of FBXW7, was recovered by the MG132 treatment (Fig. 1C), supporting that hypothesis that decreased levels of FBXW7 E3 ligase results in the upregulation of Yap1, which may eventually contribute to the development of cancer.

Fig. 1. Overexpression of CUG2 decreases expression of FBXW7 through ubiquitin- proteasome pathway. (A and B) FBXW7 protein levels in A549-CUG2, BEAS-CUG2 and the control cells were measured using immunoblotting assay and under immunofluorescence microscopy. DAPI was used for staining the nucleus. (C) A549-CUG2, BEAS-CUG2, and the control cells were treated with MG132 (20 mM) for 2 to 8 hr. FBXW7 and Yap1 protein levels were detected with immunoblotting using corresponding antibodies.

Suppression of FBXW7 induces cancer stem cell like phenotypes in A549-Vec and BEAS-Vec cells

Compared to the cells treated with control siRNA, A549- Vec and BEAS-Vec cells treated with FBXW7 siRNA migrated faster and then filled the gap creating by scratching using a pipette tip (Fig. 2A). In addition, A549-Vec and BEAS-Vec cells treated with FBXW7 siRNA aggressively invaded through the matrix and were abundant in the bottom well compared to the cells treated with control siRNA (Fig. 2B). Furthermore, suppression of FBXW7 in A549-Vec and BEAS-Vec cells increased the sphere size and number in the sphere formation assay compared to the results obtained for the cells treated with control siRNA (Fig. 2C). Based on these observations, we suggest that decreased levels of FBXW7 contribute to the development of CSC-like phenotypes.

Fig. 2. Treatment with FBXW7 siRNA enhances cancer stem cell phenotypes in A549-Vec and BEAS-Vec cells. (A) Cell migration from A549-Vec and BEAS-Vec cells treated with the FBXW7 siRNA or the control siRNA was measured using a wound healing assay. (B) Cell invasion from A549-Vec and BEAS-Vec cells treated with the FBXW7 siRNA or the control siRNA was measured using Transwell invasion assay. The assay was performed in triplicate, and the error bars indicate SD. (***; p 50 mm was the criterion for sphere formation. The assay was conducted in triplicate, and error bars indicate SD. (***; p<0.01, FBXW7 siRNA vs control siRNA).

Enforced FBXW7 expression diminishes CUG2-induced CSC-like phenotypes

To directly investigate the role of FBXW7 as a tumor sup- pressor, an FBXW7 expression vector was introduced into A549-CUG2 and BEAS-CUG2 cells. Enforced expression of FBXW7 lead to a decrease in the rate of cell migration compared to the observations in the cells with the control vector (Fig. 3A). In addition, A549-CUG2 and BEAS-CUG2 cells transfected with the FBXW7 expression vector showed weaker invasion into the matrix, and thus the number of invaded cells was lesser than that observed with the cells transfected with the control vector (Fig. 3B). Moreover, because sphere formation assays are considered a useful method to evaluate the self-renewal of CSCs in vitro [10], we examined the sphere-forming ability of A549-CUG2 and BEAS-CUG2 cells transfected with an FBXW7 expression vector. As expected, an enforced expression of FBXW7 reduced sphere size and number compared with the introduction of the control vector (Fig. 3C). Based on these results, we propose that increased levels of FBXW7 protein inhibit CUG2-induced CSC-like phenotypes through the tumor-suppressing action of the protein.

Fig. 3. Treatment with the FBXW7 expression vector inhibits cancer stem cell phenotypes in A549-CUG2 and BEAS-CUG2 cells. (A) Cell migration from A549-CUG2 and BEAS-CUG2 cells treated with an FBXW7 expression vector or the control vector was measured using a wound healing assay. (B) Cell invasion from A549-CUG2 and BEAS-CUG2 cells treated with the FBXW7 expression vector or the control vector was measured using Transwell invasion assay. The assay was performed in triplicate, and the error bars indicate SD. (***; p 50 mm was the criterion for sphere formation. The assay was conducted in triplicate, and error bars indicate SD. (**; p<0.05, ***; p<0.01, FBXW7 expression vector vs control vector)

EGFR-ERK and β-catenin-Yap1-NEK2 signaling are dependent on the levels of FBXW7 expression

It has been reported that EGFR-ERK1/2 and β-catenin- Yap1-NEK2 signaling are upregulated in A549-CUG2 and BEAS-CUG2 cells [4, 5]; therefore, we speculated whether these signaling pathways are influenced by FBXW7 expression levels. To answer this question, we introduced an FBXW7 expression vector into A549-CUG2 and BEAS-CUG2 cells, and FBXW7 siRNA into A549-Vec and BEAS-Vec cells. Enforced expression of FBXW7 reduced the phosphorylation levels of EGFR, Akt, and ERK1/2 (Fig. 4A) and the expression levels of β-catenin, Yap1, and NEK2 (Fig. 4A). In contrast, FBXW7 siRNA treatment upregulated the phosphorylation levels of EGFR, Akt, and ERK1/2 (Fig. 4B) and the expression levels of β-catenin, Yap1, and NEK2 (Fig. 4B). Furthermore, as c-Myc and c-Jun have been reported to be the substrates for FBXW7-mediated proteolytic degradation [11, 12], we speculated whether FBXW7 expression levels affect c-Myc and c-Jun protein levels. The enforced expression of FBXW7 decreased c-Myc and c-Jun protein levels in A549- CUG2 and BEAS-CUG2 cells, while FBXW7 siRNA treatment upregulated the protein levels of c-Myc and c-Jun (Fig. 4C). Suppression of FBXW7 with siRNA produced the opposite results (Fig. 4C). These findings suggest that the levels of FBXW7 E3 ligase affect not only EGFR-Akt-ERK1/2 and β-catenin-Yap1-NEK2 signaling, but also the levels of its substrates c-Myc and c-Jun.

Fig. 4. Levels of FBXW7 expression determines activity of EGFR-Akt-ERK1/2, expression levels of b-catein-Yap1-NEK2 signaling and its substrate levels. (A, B, C) A549-CUG2, BEAS-CUG2, and the control cells were treated with FBXW7 siRNA or the FBXW7 expression vector. Phosphorylation levels of EGFR, Akt, and ERK1/2 were measured by immunoblotting using corresponding antibodies. Protein levels of b-catenin, Yap1, NEK2, c-myc and c-jun were detected by immunoblotting using corresponding antibodies.

Discussion

As we observed decreased expression of FBXW7, an E3 ligase, under overexpression of CUG2, we inquired the fol- lowing: What is the meaning of decreased levels of FBXW7 for oncogenesis? To answer this, we introduced an FBXW7 siRNA to reduce the expression of FBXW7 in A549-Vec and BEAS-Vec cells. Because c-myc and c-jun are direct substrates of FBXW7 E3 ligase, suppression of FBXW7 increased the expression of these proteins. Additionally, we could assume that FBXW7 influences β-catenin-Yap1-NEK2 signaling because Yap1 is known to be a substrate of FBXW7. This assumption was supported by evidence showing that FBXW7 inhibits the migration and invasion of ovarian cancer cells by inactivating β-catenin [21]. However, interestingly, we observed increased phosphorylation levels of EGFR, Akt, and ERK1/2 during the suppression of FBXW7, which are not direct substrates of FBXW7. It was reported that gefitinib (an EGFR inhibitor) resistance was attributed to deletion of FBXW7 in a lung cancer mouse model [13]. Thus, these results indicate that deletion of FBXW7 contributes to CSC phenotypes, such as anti-cancer drug resistance. However, we need to resolve how deletion of FBXW7 activates the EGFR-Akt-ERK1/2 pathway.

Subsequently, we investigated how CUG2 overexpression caused a decrease in the levels of FBXW7. Several possibilities have been raised for the downregulated expression of FBXW7. Our previous study showed that CUG2 enhances histone deacetylase 4 expression through the EGFR-Stat1 signaling pathway [4, 5], which indicates that overexpression of CUG2 may affect chromosomal structure through histone modification. Instead, overexpression of CUG2 may induce methylation of the FBXW7 promoter, resulting in the suppression of FBXW7 transcription. Another suggestion is that the overexpression of CUG2 influences the levels of micro- RNAs (miRs) targeting the 3' untranslated terminal region (UTR) of FBXW7, such as miR-27, miR-223, and miR-503, leading to the downregulation of FBXW7 [16]. In our recent study, using a micro RNA array, we showed that over expression of CUG2 elevated the levels of miR-27 [14]; we further propose that CUG2-induced miR-27 targets the 3'UTR of FBXW7, resulting in the downregulation of FBXW7 expression. However, in our future studies we will investigate whether the upregulation of miR-27 induced by CUG2 overexpression decreases the levels of FBXW7 transcripts and proteins.