Suppression of CUL4A attenuates TGF-β1-induced epithelial-to-mesenchymal transition in breast cancer cells

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1 1114 Suppression of CUL4A attenuates TGF-β1-induced epithelial-to-mesenchymal transition in breast cancer cells YUNSHAN WANG 1,2*, XIAOYAN LIU 1*, HUI ZHENG 1, QIN WANG 3, LI AN 4 and GUANGWEI WEI 1 1 Department of Anatomy and Key Laboratory of Experimental Teratology, Ministry of Education, Shandong University School of Medicine, Jinan, Shandong ; 2 Department of International Biotechnology R&D Center, Shandong University School of Ocean, Weihai, Shandong ; 3 Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan, Shandong ; 4 Department of Science and Education, Taian Maternal and Child Health-Care Hospital, Taian, Shandong , P.R. China Received January 31, 2016; Accepted July 21, 2017 DOI: /ijmm Abstract. Transforming growth factor-β1 (TGF-β1) plays a vital role in the process of epithelial-to-mesenchymal transition (EMT) in breast cancer and the cullin 4A (CUL4A) gene is overexpressed in primary breast cancer. However, whether TGF-β1 signaling can induce CUL4A expression has not been investigated to date, at least to the best of our knowledge. In this study, using breast cancer cell lines, we found that the CUL4A expression level was increased following EMT induced by TGF-β1. Silencing CUL4A expression or CUL4A inhibition by thalidomide suppressed the EMT process induced by TGF-β1. We also found that CUL4A was associated with the expression of zinc finger E-box-binding homeobox 1 (ZEB1) which was induced by TGF-β1. These results suggest that CUL4A is upregulated in TGF-β1-induced EMT, and has a regulatory function in this process. The identification of CUL4A as a downstream target of TGF-β1 represents a critical pro-survival mechanism in breast cancer progression and provides another point for therapeutic intervention in breast cancer. Introduction Metastasis, a process by which tumor cells disseminate from the site of the primary tumor and establish secondary tumors in distant organs, is one of the 8 distinct hallmarks of cancer (1). Clinically defined, metastasis is the major cause of lethality among cancer patients, including those with breast cancer (2,3). Correspondence to: Professor Guangwei Wei, Department of Anatomy and Key Laboratory of Experimental Teratology, Ministry of Education, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong , P.R. China gwweisdu@gmail.com * Contributed equally Key words: CUL4A, epithelial-to-mesenchymal transition, transforming growth factor-β1 Epithelial-to-mesenchymal transition (EMT) has been implicated in embryonic development, fibrosis and tumor metastasis. An essential outcome of EMT is the migration of cells due to increased motility (4). Transforming growth factor-β1 (TGF-β1) is a pleiotropic factor that plays a physiological role in regulating proliferation, differentiation, development, wound healing and angiogenesis (5). In addition, the process of EMT induced by TGF-β1 is well established as a critical mechanism of breast cancer progression (6-8). However, TGF-β1-induced EMT is a very complex process, and its precise role in the stimulation of EMT is poorly understood. Thus, further studies on its regulatory mechanisms are of utmost importance. Cullin 4A (CUL4A), a member of the cullin family of proteins that composes the multifunctional ubiquitin ligase E3 complex, is essential for the ubiquitination of several welldefined tumor suppressor genes, such as p21 and p27, p53, p73 and DNA damage-binding protein 2 (DDB2) (9-13). Alterations in CUL4A expression potentially lead to a pleiotropic effect that alters cellular functions, including proliferation, differentiation and apoptosis (14). A recent analyses in our laboratory established a key role for CUL4A in the process of EMT in breast cancer cells (15). However, whether CUL4A facilitates the potential pathophysiological activities between TGF-β1 and EMT remains unknown, at least to the best of our knowledge. In this study, we examined whether CUL4A mediates the TGF-β1-induced EMT properties of breast cancer cells. We found that the CUL4A expression level was increased following EMT induced by TGF-β1. The knockdown of CUL4A or the use of CUL4A inhibitor inhibited the TGF-β1- induced EMT process. We also found that CUL4A was closely associated with the expression of zinc finger E-box-binding homeobox 1 (ZEB1) induced by TGF-β1. These results thus suggest a critical role for CUL4A in the enhancement of malignancy by TGF-β1 in breast cancer. Materials and methods Reagents and antibodies. Lipofectamine 2000 transfection reagent and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). Thalidomide was purchased from Sigma-Aldrich

2 WANG et al: SUPPRESSION OF CUL4A ATTENUATES TGF-β1-INDUCED EMT 1115 (St. Louis, MO, USA). Human psuper puro-shcul4a was kindly provided by Professor J.H. Mao (16). RPMI-1640 medium and penicillin-streptomycin were from Invitrogen. Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). Protease inhibitor cocktail was from Roche Molecular Biochemicals (Mannheim, Germany). All antibodies used are listed in Table I. HRP-conjugated sheep anti-mouse (cat. no. DPAB1253), sheep anti-rabbit (cat. no. MBS5731) and the enhanced chemiluminescence detection reagent were purchased from Amersham Biosciences (Uppsala, Sweden). Unless otherwise stated, all other chemicals were from Sigma- Aldrich. Cell lines and culture. The human breast cancer cell lines, MDA-MB-468, MDA-MB-231, BT549 and MCF7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. All the cell lines were grown at 37 C with 5% CO 2 /95% air atmosphere in a humidified incubator. MDA-MB-468 or BT549 cells were stimulated with TGF-β1 (2 ng/ml or 100 µg/ ml) for the corresponding periods of time. Subsequently, the cell properties were measured by different methods. The cells were also transfected with shcul4a as descibed below or co-incubated with thalidomide (100 µg/ml), an inhibitor of the ubiquitin ligase. Knockddown of CUL4A using CUL4A-specific short hairpin RNA (shrna). To knockdown CUL4A expression in the cells, shrna against CUL4A expressed in the psuper-puro vector were prepared as previously described (15). The cells were grown in dishes until they reached 75% confluence, at which point they were transfected for 24 h with psuper-puro-shrna specific to CUL4A or empty vector using Lipofectamine 2000, according to the manufacturer's instructions. Following transfection, the cells were trypsinized and used in various experiments. Western blot analysis. Briefly, the cells were lysed in RIPA buffer containing protease inhibitor. Equal amounts of protein lysate were electrophoretically separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dried milk for 2 h at room temperature, and then incubated with primary antibodies in phosphatebuffered saline (PBS). Following incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system. The membranes were stripped and probed with an anti-β-actin mouse monoclonal antibody to confirm equal loading of the samples. RNA extraction and reverse transcription-pcr. Total RNA from the different cells was extracted using TRIzol reagent. RNA (1 µg) was reverse transcribed using the First Strand kit (Fermentas, Waltham, MA, USA) following manufacturer's instructions (20 µl reaction) and diluted with water. The diluted cdna was used to perform PCR assay. The PCR products were obtained after cycles of amplification with an annealing temperature of C. The PCR primers used are listed in Table II. Table I. List of antibodies used in this study. Antigen Catalog no. Source Application CUL4A ab92554 Abcam IB E-cadherin Ab1012 Abcam IB, IF N-cadherin MAB4304 Millipore IB, IF α-catenin MAB1637 Millipore IB, IF Vimentin #3932 Cell Signaling Technology IB, IF ZEB1 Ab5694 Abcam IB, IF Snail #3895 Cell Signaling Technology IB Slug #9585S Cell Signaling Technology IB NANOG #4893 Cell Signaling Technology IB SOX2 #3579 Cell Signaling Technology IB OCT4 #2890 Cell Signaling Technology IB β-actin A2172 Sigma-Aldrich IB Wound healing assay. The cells were seeded in 6 cm culture plates, and the cell monolayers were wounded by scratching with sterile plastic 200 µl micropipette tips and photographed using a phase-contrast microscope (IX51; Olympus, Beijing, China) immediately, and 24 h after wounding. The assays were independently performed in triplicate. The migration distance of each cell was measured after the photographs were converted to Photoshop files. Cell invasion and motility assay. The invasion of the cells was measured by Boyden's chamber in Matrigel (BD Falcon, Franklin Lakes, NJ, USA)-coated Transwell inserts (6.5 mm; Costar, Cambridge, MA, USA) containing polycarbonate filters with 8 µm pores. Twenty thousand cells were seeded into Transwell inserts. After h, the cells on the upper surface of the filters were removed with a cotton swab. For visualization, cells on lower filter surfaces were fixed and stained with 0.5% crystal violet. Three to five fields per filter were counted. Data are presented as migrated cells per field. Methods used in cell migration assay were similar to Matrigel invasion assay except that the Transwell insert was not coated with Matrigel. Confocal immunofluorescence microscopy. The cells were plated on culture slides (Costar). After 24 h, the cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS, and the cell membrane was permeabilized using 0.5% Triton X-100. These cells were then blocked for 30 min in 10% BSA in PBS and then incubated with primary monoclonal antibodies in 10% BSA overnight at 4 C. Following 3 washes in PBS, the slides were incubated for 1 h in the dark with FITC-conjugated secondary goat anti-mouse (ab6785), or goat anti-rabbit (ab6717) antibodies (both from Abcam). Following 3 further washes, the slides were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min to visualize the nuclei, and examined using an Carl Zeiss confocal imaging system (LSM 780; Carl Zeiss, Jena, Germany). Statistical analysis. Data are presented as the means ± SD and analyzed by a Student's two-tailed t-test. The limit of statistical

3 1116 Table II. List of primers used in this study. Primer Sequence (5' to 3') Applications hgapdh-s370 GCT GGC GCT GAG TAC GTC GT GAPDH RT-PCR hgapdh-as821 ACG TTG GCA GTG GGG ACA CG hcul4a-s84 CAG CGG CTC TGA TTA CAG ACC TCG CUL4A RT-PCR hcul4a-as285 GTC TTC ACA GGC CTG ACG CAG T hzeb1-s358 ATT GAG CTG TTG CCG CTG TTG CTG ZEB1 RT-PCR hzeb1-as614 GCC CTT CCT TTC CTG TGT CAT CCT C hnanog-s569 AAT ACC TCA GCC TCC AGC AGA TG NANOG RT-PCR hnanog-as716 TGC GTC ACA CCA TTG CTA TTC TTC hoct4-s1106 AGT GAG AGG CAA CCT GGA GAA OCT4 RT-PCR hoct4-as1215 ACA CTC GGA CCA CAT CCT TC hsox2-s667 TAC AGC ATG TCC TAC TCG CAG SOX2 RT-PCR hsox2-as776 GAG GAA GAG GTA ACC ACA GGG he-cadherin-s1117 TGG GCT GGA CCG AGA GAG TTT C E-cadherin RT-PCR he-cadherin-as1562 ATC CAG CAC ATC CAC GGT GAC G hn-cadherin-s1152 CCG GTT TCA TTT GAG GGC ACA TGC N-cadherin RT-PCR hn-cadherin-as1562 GCC GTG GCT GTG TTT GAA AGG C hvimentin-s83 AAC TTA GGG GCG CTC TTG TC Vimentin RT-PCR hvimentin-as518 GGT GGA CGT AGT CAC GTA GC hα-catenin-s961 TCA TTG TGG ACC CCT TGA GC α-catenin RT-PCR hα-catenin-as1168 TTA CGT CCA GCA TTG CCC AT hsnail-s1276 AAT ACT GCA ACA AGG AAT ACC TCA GCC TGG Snail RT-PCR hsnail-as981 GGA CAG GAG AAG GGC TTC TCG CCA GTG TG hslug-s632 CGG ACC CAC ACA TTA CCT TGT GTT T Slug RT-PCR hslug-as391 CAC AGC AGC CAG ATT CCT CAT GTT T significance was P<0.05. Statistical analysis was carried out using SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA). Results TGF-β1 stimulation induces the upregulation of CUL4A. Firstly, we examined the endogenous expression of CUL4A in the MDA-MB-468, MDA-MB-231, MCF7 and BT549 cells. We then selected the MDA-MB-468 and BT549 cell lines to investigate the role of CUL4A in TGF-β1-induced EMT in breast cancer. These cells were selected as they had the lowest and highest expression of CUL4A, respectively among the 4 cell lines. RT-PCR and western blot analysis were used to examine the endogenous expression of CUL4A in the breast cancer cell lines. RT-PCR analysis revealed a low expression of CUL4A in the MDA-MB-468 cells (Fig. 1A), and western blot analysis yielded the same results regarding the protein levels (Fig. 1B). The acquisition of a fibroblastic morphology and mesenchymal markers suggested that the MDA-MB-468 and BT549 cells had undergone an EMT following stimulation with TGF-β1. As already mentioned before, TGF-β1 is believed to play a major role in EMT. In this study, the MDA-MB-468 and BT549 cells were stimulated with 2 ng/ml of TGF-β1 to observe the changes in cell morphology and in the expression of EMT markers. Due to the effect of TGF-β1, a significant change in cell morphology was observed under a phase-contrast microscope, with transition from typical a cobblestone morphology to a mesenchymal spindle-shaped one with fusiform features (Fig. 2). As detected by confocal immunofluorescence microscopy, the expression levels of the epithelial markers, E-cadherin and α-catenin (Fig. 2), were downregulated following TGF-β1 stimulation. On the contrary, TGF-β1 significantly stimulated the expression of the mesenchymal markers, N-cadherin and vimentin (Fig. 2). Recently, we found that CUL4A plays an essential role in regulating EMT in breast cancer cells (15). Due to the association of CUL4A with EMT, in this study, we investigated whether the expression of CUL4A is upregulated by TGF-β1 in the MDA-MB-468 and BT549 cells. Compared to the untreated cells, TGF-β1 increased CUL4A mrna expression at 6 h after treatment, and reached the highest level at 48 h in the time points we analyzed (Fig. 3A). The promoting effects of TGF-β1 on the CUL4A protein levels were confirmed by western blot analysis (Figs. 3B and 4A). In addition, we confirmed the increased expression of CUL4A by stimulation with TGF-β1 in a concentration-dependent manner, with the increasing level of TGF-β1, the level of CUL4A increased gradually both at the mrna and protein level (Figs. 3C, D and 4B). The mrna levels in the BT549 cells are not presented (data not shown).

4 WANG et al: SUPPRESSION OF CUL4A ATTENUATES TGF-β1-INDUCED EMT 1117 Figure 1. The endogenous mrna and protein expression of CUL4A in MDA-MB-468, MDA-MB-231, BT549 and MCF7 measured by (A) RT-PCR and (B) western blot analysis, respectively. Suppression of CUL4A attenuates TGF-β1-induced EMT in MDA-MB-468 cells. Since TGF-β1 stimulation can induce EMT and upregulate CUL4A expression in MDA-MB-468 and BT549 cells. To further determine the specific biological functions that CUL4A exerts during TGF-β1-induced EMT, we knocked down CUL4A expression in the MDA-MB-468 and BT549 cells by transfection with a CUL4A-targeting shrnaexpression plasmid. Firstly, we observed the morphological changes in the TGF-β1-stimulated breast cancer cells after silencing CUL4A expression. Compared with the psupercontrol-transfected cells, the psuper-shcul4a-transfected cells exhibited a more epithelial-like morphology (Fig. 5A). The expression of CUL4A was then observed in the transfected cells following stimulation with TGF-β1 by western blot analysis and RT-PCR. As shown in Figs. 5B and C, and 6, the expression of CUL4A in the psuper-shcul4a transfected cells was downregulated significantly, compared with the psuper-control transfected cells. Our results indicated that the CUL4A levels were decreased in the psuper-cul4a-shrnatransfected cells. Thus, shrna against CUL4A effectively reduced the expression of CUL4A. At the same time, the silencing of CUL4A resulted in a decrease in the levels of the mesenchymal markers, N-cadherin and vimentin, and an increase in the levels of the epithelial markers, E-cadherin and α-catenin (Figs. 5B and C and 6). The silencing of CUL4A expression thus reversed the TGF-β1 induced morphological transition. Suppression of CUL4A effectively suppresses the TGF-β1- induced migration of breast cancer cells. We identified an association between CUL4A expression and the EMT phenotype. We then examined whether CUL4A modulates the migratory and invasive capacities of TGF-β1-stimulated breast cancer cells. To illustrate the changes in the behavior of the breast cancer cells that occurred following the suppression of CUL4A, the effect of CUL4A on TGF-β1-induced cell migration was first assessed by a wound healing assay. After waiting for the cells to uniformly cover the 6 cm culture plates, we scraped some cells to produce a wound. Stimulation of the cells with TGF-β1 induced the migration of the cells to close the wound, while transfection with shcul4a or co-incubation with thalidomide (100 µg/ml), an inhibitor of the ubiquitin ligase (17), reversed the TGF-β1 induced migration. Treatment with the vector or inhibitor alone did not markedly affect cell migration (Fig. 7A). This result was confirmed using Boyden's chamber assay, in which the cells exhibited decreased migration through the Transwell membranes than the TGF-β1-stimulated cells after 24 h of incubation. Moreover, the suppression of CUL4A markedly reduced the capacity of the cells to invade through the Matrigel (Figs. 7B and 8). Taken together, these results indicated that the suppression of CUL4A effectively suppressed the TGF-β1-induced migration of thye breast cancer cells. CUL4A modulates ZEB1 expression induced by TGF-β1. ZEB1, Snail and Slug have been identified as inducers of EMT (18) by suppressing E-cadherin and other epithelial genes, and inducing the expression of mesenchymal genes in epithelial cells of diverse origin (19). Therefore, in this study, we examined the expression levels of these genes in relation to CUL4A in TGF-β1 induced EMT. The expression levels of ZEB1, Snail and Slug were increased by stimulation with TGF-β1 compared to the untreated cells. Transfection with CUL4A shrna or pre-treatment with thalidomide significantly decreased ZEB1 expression. However, the expression levels of Snail and Slug were not significantly altered at both the mrna and protein level (Figs. 9 and 10). These results indicated that ZEB1 may function as an effective mediator of CUL4A in modulating TGF-β1-induced EMT of breast cancer cells. Discussion It has been demonstrated that breast cancer cells stimulated with TGF-β1 undergo EMT phenotypic changes (5). The induction of EMT in cancer cells confers these cells with the ability to become more motile and invasive with an increased tumori-

5 1118 Figure 2. Epithelial-to-mesenchymal transition (EMT) in (A) MDA-MB-468 and (B) BT549 cells was induced by transforming growth factor-β1 (TGF-β1). MDA MB 468 and BT549 cells were grown on glass coverslips for 24 h, starved for 24 h, and then incubated for 6, 12 and 24 h with 2 ng/ml TGF β1. Compared with the untreated cells, the stimulated cells displayed an altered mesenchymal morphology as shown by phase-contrast microscopy, with a decreased expression of E-cadherin and α-catenin, and an increased expression of N-cadherin and vimentin, as shown by immunofluorescence microscopy. The green and red signal represents the staining of the corresponding protein, and the blue signal represents nucleur staining by DAPI.

6 WANG et al: SUPPRESSION OF CUL4A ATTENUATES TGF-β1-INDUCED EMT 1119 Figure 3. Transforming growth factor-β1 (TGF-β1) promotes the expression of CUL4A. (A) RT-PCR and (B) western blot analysis evaluation of CUL4A in MDA-MB-468 cells after 0, 3, 6, 12, 24 and 48 h of stimulation with TGF-β1 (2 ng/ml). (C) RT-PCR and (D) western blot analysis evaluation of CUL4A expression in MDA-MB-468 cells after 24 h of stimulation with TGF-β1 at 0, 1, 2, 4, 8 and 10 ng/ml. * P<0.05 and ** P<0.01 compared to 0 h. All results are from 3 independent experiments. Error bars indicate standard deviation. Figure 4. Transforming growth factor-β1 (TGF-β1) promotes the expression of CUL4A. (A) Western blot analysis evaluation of the expression of CUL4A in BT549 cells after 0, 3, 6, 12, 24 and 48 h of stimulation with TGF-β1 (2 ng/ml). (B) Western blot analysis evaluation of the expression of CUL4A in BT549 cells after 24 h of stimulation with TGF-β1 at 0, 1, 2, 4, 8 and 10 ng/ml. * P<0.05 and ** P<0.01 compared to 0 h. All results are from 3 independent experiments. Error bars indicate standard deviation.

7 1120 Figure 5. Suppression of CUL4A attenuates transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in MDA-MB-468 cells. (A) Inverted microscopic analysis of MDA-MB-468 cells cultured under different conditions. (B) mrna levels of CUL4A, E-cadherin, α-catenin, N-cadherin and vimentin were analyzed by RT-PCR in MDA-MB-468 cells cultured under different conditions. (C) Protein levels of CUL4A, E-cadherin, α-catenin, N-cadherin and vimentin were analyzed by western blot analysis in MDA-MB-468 cells cultured with different conditions. Original magnification, x100. ** P<0.01 compared with controls. All results are from 3 independent experiments. Error bars indicate standard deviation. Figure 6. Suppression of CUL4A attenuates transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in BT549 cells. Protein levels of CUL4A, E-cadherin, α-catenin, N-cadherin and vimentin were analyzed by western blot analysis in BT549 cells cultured under different conditions. ** P<0.01 compared to control. All results are from 3 independent experiments. Error bars indicate standard deviation.

8 WANG et al: SUPPRESSION OF CUL4A ATTENUATES TGF-β1-INDUCED EMT 1121 Figure 7. Suppression of CUL4A effectively suppresses the transforming growth factor-β1 (TGF-β1)-induced migration and invasion of MDA-MB-468 cells. (A) Cell migration was assayed by wound healing assay. (B) Cell migration and invasion were assayed by Transwell and Matrigel assays. Original magnification: (A) x40; (B) x100. ** P<0.01 compared to psuper + TGF-β1. All results are from 3 independent experiments. Error bars indicate standard deviation. genic potential. Furthermore, the EMT phenotype seems to be associated with resistance to therapeutic drugs (20). Therefore, the inhibition of EMT may provide a novel method with which to advance the effects of conventional therapeutics. The amplification of 13q34 is found in 5% of all human breast cancers and as high as 20% of basal-type breast cancer (21), the subtype of breast cancer most often associated with aggressive growth and poor prognosis (21,22). Several candidate genes, including CUL4A have been proposed for the region of 13q34 (21,23,24). Considering that TGF-β1 promotes EMT and the invasiveness of tumor cells, it is important to discover the mechanisms through which TGF-β1 controls intracellular signaling in transformed cells and whether CUL4A regulates TGF-β1-induced EMT and the invasiveness of breast cancer cells. In the present study, we proved that breast cancer cells stimulated with TGF-β1 underwent EMT phenotypic changes. Our data also indicated the increased ability of TGF-β1-stimulated breast cancer cells to migrate and invase and to acquire an enhanced tumorigenic potential compared to the untreated cells. The results obtained are in agreement with those previously reported (5,8). Of note, we also found that the TGF-β1-stimulted

9 1122 Figure 8. Suppression of CUL4A effectively suppressed the transforming growth factor-β1 (TGF-β1)-induced migration and invasion of BT549 cells. Cell migration and invasion were assayed by Transwell and Matrigel. Original magnification, x100. ** P<0.01 compared to psuper + TGF-β1. All results are from 3 independent experiments. Error bars indicate standard deviation. Figure 9. CUL4A modulates ZEB1 expression induced by transforming growth factor-β1 (TGF-β1) in MDA-MB-468 cells. (A) mrna levels of ZEB1, Snail and Slug levels were analyzed by RT-PCR in MDA-MB-468 cells cultured under different conditions. (B) Protein levels of ZEB1, Snail and Slug were analyzed by western blot analysis in MDA-MB-468 cells cultured under different conditions. β-actin was used as a loading control. ** P<0.01 compared to psuper + TGF-β1. All results are from 3 independent experiments. Error bars indicate standard deviation. a, no treatment (control); b, stimulation with TGF-β1; c, transfection with psuper (control vector); d, transfection with shcul4a.

10 WANG et al: SUPPRESSION OF CUL4A ATTENUATES TGF-β1-INDUCED EMT 1123 Figure 10. CUL4A modulates ZEB1 expression induced by transforming growth factor-β1 (TGF-β1) in BT549 cells. Protein levels of ZEB1, Snail and Slug were analyzed by western blot analysis in BT549 cells cultured under different conditions. β-actin was used as a loading control. ** P<0.01 compared to psuper + TGF β1. All results are from 3 independent experiments. Error bars indicate standard deviation. a, no treatment (control); b, stimulation with TGF-β1; c, transfection with psuper (control vector); d, transfection with shcul4a. MDA-MB-468 and BT549 cells exhibited a high expression of CUL4A compared to the untreated cells. Our finding is of interest, not only as it connects two very important molecules, such as TGF-β1 and CUL4A in cells with an aggressive phenotype, but it is also consistent with the role of EMT in tumor aggressiveness and metastasis in published studies (5,8,15). Our findings also suggest that the activation of the CUL4A signaling pathway in cancer epithelial cells could lead to the acquisition of an aggressive phenotype of cancer cells. Our data clearly suggest that the activation of CUL4A by TGF-β1 leads to the increased tumor cell migration, invasion and tumorigenic potential of breast cancer cells, as documented by our mechanistic experiments using the knockdown approach and by using chemical inhibitors of CUL4A. Our results revealed that the TGF-β1-stimulated cells were able to maintain the EMT phenotype due to the sustained activation of CUL4A. Of note, the inhibition of the TGF-β1-induced expression of CUL4A by an inhibitor or by shrna decreased the ability of the TGF-β1- stimulated breast cancer cells to migrate and invade. We further demonstrated the importance of CUL4A in the EMT phenomenon, wherein the inhibition of CUL4A signaling by thalidomide was able to downregulate the levels of mesenchymal markers, such as N-cadherin and vimentin, which was consistent with the upregulation of the epithelial marker, E-cadherin. These results suggest that the attenuation of CUL4A signaling could reverse the EMT phenotype to mesenchymal-to-epithelial transition; cells appeared more annular following transfection with CUL4A shrna, resulting in decreased cell migration and invasion. More importantly, to the best of our knowledge, our data indicate for the first time that TGF-β1-induced EMT is mediated through the upregulation of CUL4A as the knockdown of CUL4A by CUL4A-specific shrna significantly attenuated the induction of EMT by TGF-β1. The oncogenic role of CUL4A in cancer development is evidenced by studies indicating that CUL4A is overexpressed in various malignant tumors and the demonstrations that CUL4A ubiquitinates and degrades some well-known tumor suppressor genes, including p21, p27 and p53 (13,25-27). Beyond that, our study sheds light onto a new function of CUL4A in breast cancer progression by regulating the TGF-β1-induced migration and invasion of breast cancer cells. It has been demonsrated that several transcription factors, including ZEB1, Snail, Slug and Twist are EMT regulators (28). In our aim to elucidate the mechanisms through which CUL4A modulates TGF-β1-induced EMT in breast cancer cells, we identified that ZEB1 may an effective mediator. Stimulation with TGF-β1 markedly increased ZEB1 expression, whereas the silencing of CUL4A expression mrkedly decreased ZEB1 expression at both the protein and mrna level. However, additional studies are warranted in order to determine whether other EMT regulators are associated with the function of CUL4A in TGF-β1-induced EMT. In conclusion, based on available evidence in the literature and our present data, we propose a mechanism through which epithelial tumor cells can be exposed to TGF-β1 secreted by either stromal cells, immune cells or the tumor cells within the tumor microenvironment, leading to an increase in CUL4A levels, and consequently resulting in the activation of CUL4A signaling and the acquisition of the EMT phenotype, which is responsible for tumor cell aggressiveness and metastasis. Therefore, the inhibition of CUL4A signaling may be a useful method with which to reduce breast cancer cell invasiveness. Acknowledgements This study was supported by the National Natural Science Foundation of China to [G.W. ( , and ), to Y.W. ( ), and to.qw. ( )] and the Natural Science Foundation of Shandong Province [to Q.W. (BS2015YY005)]. References 1. Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. Cell 144: , Mego M, Mani SA and Cristofanilli M: Molecular mechanisms of metastasis in breast cancer--clinical applications. Nat Rev Clin Oncol 7: , 2010.

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