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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)

International Journal of Oral Biology Vol.43 No.3 pp.161-169
DOI : https://doi.org/10.11620/IJOB.2018.43.3.161

Beta-Catenin Downregulation Contributes to Epidermal Growth Factor-induced Migration and Invasion of MDAMB231 Cells

Arang Kwon, Hyun-Jung Park, Jeong-Hwa Baek*

Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea

†

Present address: Tung Wah Group of Hospitals, Hong Kong; Faculty of Dentistry, The University of Hong Kong, Hong Kong

Correspondence to: Jeong-Hwa Baek, Department of Molecular Genetics, School of Dentistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea Tel: +82-2-880-2322 E-mail: baekjh@snu.ac.kr

Received September 5, 2018 Review September 20, 2018 Accepted September 23, 2018

Abstract

We previously demonstrated that epidermal growth factor (EGF) enhances cell migration and invasion of breast cancer cells in a SMAD ubiquitination regulatory factor 1 (SMURF1)-dependent manner and that SMURF1 induces degradation of β-catenin in C2C12 cells. However, the relationship between EGF-induced SMURF1 and β-catenin expression in breast cancer cells remains unclear. So, we investigated if EGF and SMURF1 regulate β-catenin expression in MDAMB231 human breast cancer cells. When MDAMB231 cells were incubated with EGF for 24, 48, and 72 hours, EGF significantly increased expression levels of SMURF1 mRNA and protein while suppressing expression levels of β-catenin mRNA and protein. Overexpression of SMURF1 downregulated β-catenin mRNA and protein, whereas knockdown of SMURF1 increased β-catenin expression and blocked EGF-induced β-catenin downregulation. Knockdown of β-catenin enhanced cell migration and invasion of MDAMB231 cells, while β-catenin overexpression suppressed EGF-induced cell migration and invasion. Furthermore, knockdown of β-catenin enhanced vimentin expression and decreased cytokeratin expression, whereas β -catenin overexpression decreased vimentin expression and increased cytokeratin expression. These results suggest that EGF downregulates β-catenin in a SMURF1-dependent manner and that β-catenin downregulation contributes to EGF-induced cell migration and invasion in MDAMB breast cancer cells.

Key Words : beta-catenin , epidermal growth factor , SMURF1 , migration , invasion

초록

키워드 :

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Funding:

National Research Foundation of Korea
2010-0021044

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Metastasis of cancer cells is one of the most significant concerns for cancer treatment because it increases the mortality rate in cancer patients [1,2]. Breast cancer commonly metastasizes to lung, liver, brain, lymph nodes and bone. Breast cancer can be treated by surgery, radiation, chemotherapy and biological/hormonal therapy, but there is still not a cure [3].

Epidermal growth factor (EGF) plays multiple regulatory roles in cancer cells. EGF receptor (EGFR) overexpression in breast cancer increases tumor size with poor differentiation and increases risk of metastasis, resulting in poor clinical outcomes [4]. EGF-induced signaling is associated with tumor invasion and metastasis [4]. Enhanced EGFR signaling also promotes epithelial-mesenchymal transition (EMT), which plays an important role in migration and invasion of carcinoma cells by changing a polarized epithelial phenotype to a mesenchymal phenotype [5,6].

We previously reported that EGF-induced SMAD ubiquitination regulatory factor 1 (SMURF1) plays a role in breast cancer cell migration and invasion through the downregulation of RhoA [7]. SMURF protein controls cell polarity, migration, and adhesion through the regulation of target proteins, including RhoA, Rap1B, and Talin [8-10]. SMURF1 expression was increased in breast cancer cells, and knockdown of SMURF1 reduced cell migration by inducing lamellipodia formation and RhoA signaling [11-13]. SMURF1 also has a role in controlling cell polarity through non-canonical WNT signaling in embryonic development and tumor cell invasion [14].

Beta-catenin, a key mediator of the canonical WNT signaling pathway, plays an important role in embryogenesis and tumorigenesis [15]. Stabilized β-catenin in the cytoplasm translocates to the nucleus, binds to the LEF/TCF transcription factor, and activates transcription of WNT target genes, such as cyclin D1, matrix metallopeptidase 7, and c-myc to contribute to tumor formation [16,17]. In addition, β-catenin functions in the regulation of cell-cell adhesion: β-catenin binds to E-cadherin in the cytoplasmic membrane and helps to connect E-cadherin with the intracellular cytoskeleton through the interaction with α-catenin and γ-catenin [18]. Cell-cell adhesion is frequently disturbed in cancer cells either by downregulation of or mutations in the E-cadherin or β-catenin genes, or tumor suppressor gene product APC [18,19].

We have previously demonstrated that EGF-induced SMURF1 inhibits WNT/β-catenin signaling in osteoblastic cells by promoting β-catenin degradation [20]. However, the effect of EGF on the expression levels of β-catenin in breast cancer cells and the regulatory role of β-catenin in cancer cell migration and invasion remains elusive. In the present study, we demonstrate that EGF downregulates β-catenin expression levels in a Smurf1-dependent manner in MDAMB231 human breast cancer cells and down-regulation of β-catenin enhances EMT, migration, and invasion of MDAMB231 cells.

Materials and Methods

Cell culture

MDAMB231 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin. When indicated, recombinant human EGF (Sigma-Aldrich; St. Louis, MO, USA) was added to culture medium at the concentration of 10 ng/ml.

Gene knockdown using small interfering RNA

Small interfering RNAs (siRNA) for SMURF1 and β-catenin and non-targeting control siRNA were purchased from Dharmacon (Chicago, IL, USA). Transient transfection of siRNA into MDAMB231 cells was performed using Dharmafect (Dharmacon) according to the manufacturer’s instructions.

Western blot analysis

Whole cell lysates were prepared by lysing cells in buffer [10 mM Tris (pH 8.0), 1 mM EDTA, 0.1% SDS, 1% deoxycholate, 1% NP40, 0.14 M NaCl, and a protease inhibitor mixture (Roche Diagnostics; Indianapolis, IN, USA)], and 20 μg protein of each sample was used for SDS-PAGE using 4– 12% Nu-PAGE gels (Invitrogen Life Technologies; Carlsbad, CA, USA), followed by transfer to PVDF membrane. Immunoblotting and detection of immune complexes were performed as described previously [7]. Antibodies were purchased from vendors as follows: SMURF1 (Invitrogen), β -catenin (Santa Cruz Biotechnology; Santa Cruz, CA, USA), actin (Santa Cruz Biotechnology), VIMentin (Cell Signaling Technology, Danvers, MA, USA), cytokeratin 19 (Santa Cruz Biotechnology), N-cadherin (Santa Cruz Biotechnology), actin (Santa Cruz Biotechnology), and HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total mRNA was extracted using easy-BLUE RNA extraction reagents (iNtRON Biotechnology, Korea). Complementary DNA synthesis and quantitative RP-PCR were performed using the AccuPower RT PreMix (Bioneer, Korea) and SYBR premix EX Taq (Takara; Otsu, Japan), respectively. The primer sequences used for quantitative RT-PCR are as follows: 5′-GTC CAG AAG CTG AAA GTC CTC AGA-3′ and 5′-CACGG AATTTCACCATCAGCC-3′;β-catenin (CTNNB1) (f) 5′-GTT CGT GCA CAT CAG GAT AC-3′ and (r) 5′-CGA TAG CTA GGA TCA TCC TG-3′; cytokeratin 19 (KRT19) (f) 5′-GAT GAG CAG GTC CGA GGT TA-3′ and (r) 5′- TCT TCC AAG GCA GCT TTC AT-3′; VIMentin (VIM) (f) 5′-CTG GAT TTC CTC TTC GTG GA-3′ and (r) 5′-CGA AAA CAC CCT GCA ATC TT-3′; N-cadherin (CDH2) (f) 5′-AGC TTC TCA CGG CCA TAC ACC-3′ and (r) 5′-GTG CAT GAA GGA CAG CCT CT-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (f) 5′-CCA TCT TCC AGG AGC GAG ATC-3′ and (r) 5′-GCC TTC TCC ATG GTG GTG AA-3′.

Migration and invasion assays

Cell migration and invasion were assessed using transwells with 8-μm pore size (Corning Life Sciences; Tewksbury, MA, USA) as described previously [7]. The MDAMB231 cells were seeded in the upper chamber of a 24-well transwell culture plate, incubated overnight, and serum-starved for 24 hours. Then, the upper chambers were filled with serum-free DMEM, and the lower chambers were filled with serum-free DMEM with or without EGF (10 ng/ml). After incubation for 48 hours, the cells that had migrated to the underside of the inserts were fixed with 4% paraformaldehyde and stained with H&E solution. Migrated cells were counted using a microscope and Image J program in three randomly selected fields per insert.

Immunofluorescence staining

MDAMB231 cells were seeded in a chamber slide and treated as indicated. At the end of the culture, the cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS, and blocked with 5% normal serum. The cells were then incubated with the indicated primary antibody overnight at 4°C, rinsed with 0.1% Triton X-100/PBS, and then incubated with an alex-fluor 594 (red)- or alex-fluor 488 (green)-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 hours at room temperature in dark. Subsequently, the cells were thoroughly washed with 0.1% Triton X-100/PBS, mounted with Vectashield (Vector Laboratories; Burlingame, CA, USA), which contains DAPI in the mounting solution, and subjected to confocal microscopy (Carl Zeiss LSM700; Oberkochen, Germany).

Statistical analysis

Each experiment was performed with a sample number of 3 to 4 and repeated at least twice. Statistical significance was analyzed by Student’s t-test. A p-value less than 0.05 was considered statistically significant.

Results

EGF inversely regulates SMURF1 and β-catenin expression in MDAMB231 cells

To examine whether β-catenin expression is regulated by EGF in breast cancer cells, MDAMB231 cells were incubated for 24, 48, or 72 hours in the presence or absence of EGF. Quantitative RT-PCR and Western blot analyses showed that EGF significantly suppressed both mRNA and protein levels of β-catenin (Fig. 1A and 1B). We also examined expression levels of SMURF1. Similar to the results obtained in the previous study [7], EGF increased SMURF1 mRNA and protein expression (Fig. 1A and 1B). These results indicate that EGF inversely regulates expression levels of SMURF1 and β-catenin in MDAMB human breast cancer cells.

Smurf1 downregulates β-catenin expression

To investigate whether SMURF1 is involved in EGF-induced downregulation of β-catenin, β-catenin expression level was observed under Smurf1 overexpression or knockdown conditions in MDAMB231 cells. In the cells transiently transfected with SMURF1 expression plasmids, expression levels of SMURF1 mRNA and protein were similar to those in EGF-treated cells (Fig. 2A and 2B). Similarly to the EGF treatment, overexpression of SMURF1 significantly reduced β-catenin expression both in mRNA and protein levels (Fig. 2A and 2B).

To further confirm the role of Smurf1 in β-catenin regulation, Smurf1 knockdown was induced and β-catenin expression levels were examined in the presence or absence of EGF treatment. Consistent with the results from SMURF1 overexpressing cells, reduction of SMURF1 level by SMURF1 siRNA significantly increased β-catenin mRNA and protein levels (Fig. 2C and 2D). β-Catenin mRNA levels in SMURF1-silenced cells under EGF treatment conditions did not increase to the same level as those in vehicle-treated SMURF1-silenced cells (Fig. 2C). However, immunofluorescence staining of β-catenin clearly showed that EGF decreased β-catenin protein expression, whereas SMURF1 knockdown strongly increased β-catenin protein expression both in the presence and absence of EGF (Fig. 2D). These results indicate that SMURF1 is a negative regulator of β -catenin mRNA and protein expression levels in MDAMB231 cells and that SMURF1 plays an important role in EGFmediated downregulation of β-catenin in these cells.

Beta-catenin negatively regulates breast cancer cell migration and invasion

Because EGF-induced SMURF1 contributes to enhanced cell migration and invasion of MDAMB231 cells [7], we next examined whether β-catenin downregulation by EGF/SMURF1 exerted any effect on cancer cell migration and invasion. Similar to EGF treatment, knockdown of β-catenin itself significantly increased the number of migrated/invaded cells (Fig. 3A). On the contrary, overexpression of β-catenin itself significantly reduced breast cancer cell migration and invasion (Fig. 3B). Furthermore, β-catenin overexpression significantly inhibited EGF-induced migration and invasion of MDAMB231 cells (Fig. 3B). These results suggest that β-catenin is a negative regulator of breast cancer cell migration and invasion and that EGF/SMURF1 increases cancer cell migration and invasion partly through the downregulation of β-catenin in breast cancer cells.

Beta-catenin inhibits EMT marker expression in MDAMB231 cells

In order for carcinoma cells to migrate and invade the surrounding matrix, tight cell-to-cell interaction needs to be disrupted, which is enhanced by EMT [5,6]. Therefore, to understand how β-catenin downregulates cell migration and invasion of breast cancer cells, we examined the regulatory effect of β-catenin on EMT of MDAMB231 cells.

We first investigated whether EMT marker expression is regulated by EGF and SMURF1. Quantitative RT-PCR, Western blot and immunofluorescence staining data confirmed that EGF significantly increased expression levels of mesenchymal phenotype markers such as VIMentin and N-cadherin, while decreasing expression of epithelial marker cytokeratin (Fig. 4). However, SMURF1 knockdown inhibited EGF-induced changes in EMT marker expression (Fig. 4). These results suggest that SMURF1 plays a regulatory role in EGF-induced EMT.

We next examined whether β-catenin knockdown mimics EGF-induced EMT phenotypic change in MDAMB231 cells. β-Catenin knockdown significantly increased VIMentin mRNA and protein expression, while decreasing cytokeratin mRNA and protein expression (Fig. 5A and 5B). On the contrary, β-catenin overexpression suppressed VIMentin expression and increased cytokeratin expression (Fig. 5C and 5D). Furthermore, β -catenin overexpression rescued mRNA expression levels of VIMentin and cytokeratin in the presence of EGF (Fig. 5C). These results suggest that β-catenin downregulation contributes for EGF to induce EMT in MDAMB231 cells.

Discussion

Breast cancer primarily metastasizes to bone in the body and EGF/EGFR is involved in breast cancer and prostate cancer bone metastasis [21-24]. EGFR overexpression causes poor prognosis in oral cancer, and EGFR activation is associated with a malignant phenotype, apoptosis inhibition, and increased metastatic potential [25]. Therefore, analysis of EGF-mediated cancer cell migration and invasion is necessary to understand more about cancer metastasis.

Previous studies have demonstrated that EGFR signaling induces phosphorylation of β-catenin in the cadherin-catenin complex, leading to dissociation of β-catenin from adhesion complex and increase in transcriptional activity of β-catenin [26-28]. These reports suggest that a shift of β-catenin in cell adhesion complex to nuclear transcriptional complex contributes to EGF-induced EMT and migration/invasion. However, the data from the present study demonstrated that EGF decreased total β-catenin protein levels and that β-catenin knockdown significantly enhanced EMT marker expression and cell migration/invasion of MDAMB231 cells. The reason for the discrepancy is not clear. However, the differences in the experimental conditions may provide small clues. Dissociation of β-catenin from the adhesion complex starts to appear within 10 minutes of exposure to EGF in serum-free medium, and there was no change in β-catenin protein levels during those time periods [29]. However, we observed the downregulation of β-catenin expression after incubation of cells with EGF for 24, 48, and 72 hours in growth medium containing of 10% serum. Therefore, the regulatory effect of EGF on β-catenin may be different depending on the time periods and presence or absence of serum. However, in the present study, the negative effect of β-catenin on cell migration and invasion was observed in serum-free conditions. Therefore, further study is necessary to clarify the reason/mechanism for the contradictory effect of β-catenin on EMT and cell migration/invasion in breast cancer cells.

SMURF1 targets substrate proteins for ubiquitination and proteasomal degradation. Accumulating evidence suggests that SMURF1 is an oncogenic factor in human malignancies and regulates cancer cell motility [12,30,31]. Consistent with the results from the previous study [7], EGF increased SMURF1 expression in MDAMB231 cells in the present study. Because SMURF1 induces ubiquitination and proteasomal degradation of β-catenin in C2C12 cells [20], we examined whether SMURF1 regulates β-catenin level in cancer cells. Overexpression and knockdown of SMURF1 resulted in a decrease and increase in β-catenin mRNA and protein levels, respectively. Although SMURF1 did not regulate β-catenin mRNA levels in C2C12 cells [20], it downregulated β-catenin mRNA in MDAMB231 cells. These results suggest that in addition to direct regulation by ubiquitination, SMURF1 may indirectly regulate β-catenin expression in breast cancer cells.

EMT is associated with cancer aggressiveness, invasiveness, metastatic behavior, and chemotherapeutic resistance [32]. EMT plays a role in the metastatic progression of epithelial cancer cells from the primary tumor site to the surrounding tissues or distant organ. In the present study, SMURF1 knockdown significantly suppressed EGF-induced changes in expression of EMT markers such as VIMentin, N-cadherin, and cytokeratin. These results are consistent with those from previous reports showing that EGF enhances cancer cell migration and invasion in a SMURF1-dependent manner [7]. Consistent with the pro-EMT effect of SMURF1, β-catenin knockdown also increased VIMentin expression and decreased cytokeratin expression in MDAMB231 cells. These results suggest that β -catenin downregulation contributes to the pro-EMT effect of EGF/SMURF1, at least in part.

In conclusion, in the present study we demonstrated that EGF downregulates β-catenin expression through SMURF1 induction and β-catenin negatively regulates EMT and cell migration/invasion in MDAMB231 cells. Further studies are needed to understand how β-catenin downregulation enhances EMT marker gene expression, cell migration and invasion in breast cancer cells.

Acknowledgements

This work was supported by the National Research Foundation of Korea grant (NRF-2010-0021044).

Figure

Fig. 1.EGF inversely regulates SMURF1 and β-catenin expression in MDAMB231 breast cancer cells.

MDAMB231 cells were incubated in the presence or absence of EGF (10 ng/ml) for the indicated periods. Beta-catenin and SMURF1 expression levels were evaluated by quantitative RT-PCR (A) and Western blot analyses (B). Quantitative data are presented as the mean ± SD of triplicates. # p < 0.05 for the indicated pairs. CTNNB1, β-catenin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase

Fig. 2.SMURF1 downregulates β-catenin expression in MDAMB231 cells.

(A, B) MDAMB231 cells were incubated with EGF treatment or SMURF1 overexpression for 48 hours, followed by quantitative RT-PCR (A) and Western blot analysis (B). (C, D) MDAMB231 cells were transiently transfected with non-targeting control siRNA (si con) or with SMURF1 siRNA (si SMURF1), followed by incubation for 48 hours in the presence or absence of EGF. Beta-catenin expression levels were examined by quantitative RT-PCR (C) or by immunofluorescence staining (D). The merged image of DAPI (blue) and β-catenin (red) staining is presented. *p < 0.05 compared to the control cells; #p < 0.05 for the indicated pairs. C, vehicle-treated control; E, 10 ng/ml EGF; S, SMURF1 overexpression; ns, not significant

Fig. 3.Beta-catenin negatively regulates breast cancer cell migration and invasion.

MDAMB231 cells were transiently transfected with pcDNA, β-catenin expression plasmid, control siRNA (si con), or β-catenin siRNA (si β-cat) and seeded in the upper compartment of transwell plates coated with type I collagen (migration assay) or matrigel (invasion assay). After serum-starvation for 24 hours, the cells were incubated in the presence or absence of EGF for 48 hours. The cells that migrated or invaded to the undersurface of the transwell were H&E stained and observed under microscope (100×). The number of cells was counted by Image J. *p < 0.05 compared to the vehicle-treated pcDNA or si control cells; #p < 0.05 for the indicated pairs.

Fig. 4.SMURF1 partly mediates EGF-induced epithelial-mesenchymal transition marker expression in MDAMDB231 cells.

MDAMB231 cells were transfected with control siRNA, or SMURF1 siRNA (si SMURF1) and incubated in the presence or absence of EGF for 48 hours. The expression levels of VIMentin, β-catenin, and cytokeratin 19 were evaluated by quantitative RT-PCR (A), Western blot (B), and immunofluorescence staining (C). DAPI (blue), VIMentin (red), N-cadherin (green), cytokeratin (red). *p < 0.05 compared to the vehicle-treated si control cells; #p < 0.05 for the indicated pairs. VIM, VIMentin; CDH2, N-cadherin; KRT19, cytokeratin 19

Fig. 5.Decrease in β-catenin expression contributes to EGF-induced epithelial-mesenchymal transition marker expression in MDAMDB231 cells.

MDAMB231 cells were transfected with pcDNA, β-catenin expression plasmid, control siRNA (si con), or β -catenin siRNA (si β-cat) and incubated in the presence or absence of EGF for 48 hours. The expression levels of VIMentin, β-catenin, and cytokeratin 19 were evaluated by quantitative RT-PCR (A, C) and Western blot (B, D) analyses. *p < 0.05 compared to the pcDNA or si control cells; #p < 0.05 for the indicated pairs.

Table

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