CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation

Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction.


Introduction
Cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and shares enzymatic activities with two other CYP1 family members, CYP1A1 and CYP1A2 [1]. It primarily acts as a GAPDH; mouse monoclonal antibody for ZEB2 and c-Myc; Texas Red-conjugated goat antirabbit IgG; and UltraCruz TM Mounting Medium were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit IgG and DyLight 1 594-conjugated goat anti-mouse were obtained from Bethyl (Montgomery, TX, USA) and mouse monoclonal antibody for PCNA was purchased from Cell Signaling Technology (Beverly, MA, USA). Other chemicals and reagents were of the highest quality commercially available.
Cell culture MCF-7, MDA-MB-231, and HeLa cells were obtained from the Korean Society Cell Bank (KCLB), and MCF-10A cells were kindly provided by Dr. Aree Moon (Duksung Women's University, Seoul, Korea). Authentication of cells has been performed by KCLB based on DNA fingerprinting analysis using short tandem repeat analysis. MCF-7 and MDA-MB-231 cells were cultured in RPMI medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. HeLa cells were cultured in MEM medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. MCF-10A cells were cultured in monolayer as described previously [30]. For treatment of MCF-7 cells with 4-OHE 2 or 2-OHE 2, 1×10 6 cells were seeded in growth media as a monolayer onto 100-mm dish plates and maintained at 37°C in a humidified atmosphere with 5% CO 2. After 24 h, the media was changed to phenol red-free RPMI (Thermo Scientific, IL, USA) with 10% (v/v) charcoal-stripped FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained for 72 h and were subsequently provided fresh media containing designated concentrations of 4-OHE 2 or 2-OHE 2 . After 48 h, cells were harvested and processed for further studies.
Transient transfection of plasmid DNA and siRNA CYP1B1-specific siRNA (target sequence: CAGCATGATGCGCAACTTCTT, Qiagen) and the overexpression vector pcDNA 3.1/Zeo containing the CYP1B1-encoding sequence were used in transfections. Cells were transfected at room temperature with 37.5 nM siRNA or 8 μg plasmid with the Neon Transfection System (Invitrogen, Carlsbad, CA, USA) and cultured in 100-mm dishes in antibiotic-free RPMI with 10% FBS for 48 h.

Adenovirus infection
The infection of adenovirus carrying CYP1B1-ORF genes (ViGene Biosciences Inc., Rockville, MD, USA) was performed in serum-free media at an m.o.i. of 750 vp (virus particles)/cell for MCF-7 cells. After 24 h, media change was carried out with serum-containing fresh media. Cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 for 24 h and harvested or fixed for further studies. Under these circumstances, the transduction efficiency of the CYP1B1 gene carrying adenovirus reached almost 100%.
Cell viability assay CYP1B1-overexpressed cells (1×10 4 cells/well) were plated onto 96-well plates and incubated in 37°C. After stabilization for 48 h, 10 μl EZ-CyTox (Daeil Lab Service, Seoul, Korea) was added to each well and incubated for 2 h at 37°C. Formazan formation was quantified by spectrophotometry at 450 nm using a Sunrise™ microplate reader (Tecan, Männedorf, Switzerland). Each experiment was performed at least three times independently.

Subcellular fractionation
Subcellular fractionation was performed using the NE-PER 1 Nuclear and Cytoplasmic Extraction kit from Thermo Scientific. Western blot analyses were carried out using antibodies against the following control marker proteins: β-actin for the cytosolic fraction and Hsp70 for the nuclear fraction.

Invasion assay
Cell invasion was measured using the QCM™ 24-well Cell Invasion Assay Kit (Millipore), according to the manufacturer's instructions. Briefly, cells were seeded onto insert chambers containing a collagen-coated polycarbonate membrane with 8-μm pores. Cells that invaded the ECM layer were stained with 4 0 ,6-diamidino-2-phenylindole (DAPI). Invading cells in five fields per chamber were visualized and counted under the LSM700 Confocal Laser Scanning Microscope (Carl Zeiss, Jena, Germany). Each experiment was performed three times independently.

Wound healing assay
Cells (1×10 6 cells/well) were cultured in 6-well culture plates. After 24 h, cells with 90% confluence were washed with PBS and treated with mitomycin C (25 μg/ml) for 30 min. After washing, a single wound per monolayer was created using sterile pipette tips. Plates were photographed after the indicated time. Each experiment was performed at least three times independently.

Quantitative PCR (qPCR)
Total RNA was extracted using Ribospin™ (GeneALL, Seoul, Korea). Total RNA (500 ng) was reverse transcribed at 37°C for 1 h in 20 μl total volume containing 5× RT buffer, 10 mM dNTPs, 40 U RNase inhibitor, 200 U Moloney murine leukemia virus reverse transcriptase, and 100 pmol oligo-dT primer. Quantitative PCR (qPCR) was performed using the Rotor-Gene SYBR 1 PCR Kit, as recommended by the manufacturer, and analyzed using QIAGEN Rotor-Gene Q Series software. Each reaction contained 12.5 μl 2× SYBR 1 Green PCR Master Mix, 1 μM oligonucleotide primers, and 2 μl cDNA in a final volume of 25 μl. Amplification was conducted as follows: one cycle at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing/extension at 60°C for 10 seconds. Primer sequences are listed in S1 Table. Western blot Whole cells were harvested by scraping and lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1% nonidet P-40, 1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin for 30 min followed by centrifugation at 22000×g for 15 min at 4°C. Protein concentrations were measured using BCA Protein Assay Reagents (Thermo). Extracted proteins (20 μg) were separated by SDS-PAGE on 10%-12% polyacrylamide gels and electrophoretically transferred onto PVDF membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at 4°C, and then incubated overnight with specific antibodies. After incubating with secondary antibodies for 2 h, proteins were visualized using enhanced chemiluminescence reagents (Thermo). Quantitative data were obtained using Quantity One software (Bio-Rad, Hercules, CA, USA).

Immunofluorescence
Cells grown on coverslips were treated with the indicated reagent concentrations, rapidly washed with PBS, and fixed with 3.7% (w/v) paraformaldehyde for 30 min at room temperature. After washing with PBS, the cells were blocked for 30 min in PBS containing 5% goat serum and 0.2% Triton X-100, and then incubated with specific primary antibodies overnight. Next, the cells were washed extensively and stained with Texas Red-conjugated goat anti-rabbit IgG or DyLight 1 594-conjugated goat anti-mouse IgG (1:500) for 2 h. After additional washes, the coverslips were mounted onto glass slides using UltraCruz™ Mounting Medium containing DAPI. Fluorescence signals were analyzed using an LSM700 Confocal Laser Scanning Microscope (Carl Zeiss).

7-Ethoxyresorufin-O-Deethylation (EROD) assay
Cells (5×10 5 ) were plated in 2 ml of culture medium and incubated for 48 h. After incubation, the cells were harvested by scrapping in ice-cold 0.1 M potassium phosphate buffer (pH 7.4). Cells were centrifuged at 1000×g for 5 min at 4°C and the pellets were resuspended in the same buffer. The cells were sonicated for 30 seconds at 4°C. The reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.4), 2 mg/ml bovine serum albumin, 50 pM rabbit NAPDH-P450 reductase, 2 μM ethoxyresorufin, and cellular sonicates. The reaction mixtures were pre-incubated at 37°C for 3 min and the reaction was initiated by addition of 120 μM NADPH. After 20 min of incubation at 37°C in a shaking water bath, the reaction was terminated by addition of 1 ml of ice-cold methanol. The formation of resorufin was determined fluorometrically with FlexiStation 3 (Molecular Devices), with excitation and emission wavelengths of 544 nm and 590 nm, respectively. Protein concentrations were estimated using the BCA Protein Assay Reagents (Thermo) according to the supplier's recommendations.

Statistical analysis
Statistical analyses were performed using one-way analysis of variance and Dunnett's Multiple Comparison t-test on Graph-Pad Prism Software (GraphPad Software Inc., San Diego, CA). The difference was considered statistically significant when p 0.05.

CYP1B1 induces cell proliferation and metastasis
To explore the role of CYP1B1 in cancer progression, its effects on cell proliferation, migration, and invasion were investigated. CYP1B1 overexpression significantly increased cell proliferation in MCF-7 cells (Fig 1A).
PCNA (Proliferating cell nuclear antigen) has been widely used as a marker for cell proliferation [31]. Accordingly, PCNA protein was upregulated by CYP1B1 overexpression (Fig 1B), while CYP1B1 knockdown had the opposite effect ( Fig 1C). Confocal microscopic analysis likewise indicated that DMBA, a CYP1B1 inducer, increased PCNA expression while TMS, a CYP1B1-specific inhibitor, decreased PCNA levels ( Fig 1D). These data suggest that CYP1B1 enhances cell proliferation through PCNA expression.
To identify whether CYP1B1 induces EMT-related cell morphology, we observed morphological changes in MCF-10A cells subsequent to CYP1B1 overexpression. In the models of CYP1B1 upregulation, cells acquired mesenchymal morphologies (Fig 2A). To investigate whether CYP1B1 also induces cell migration and invasion, we performed wound healing and transwell invasion assays. In wound healing assays, DMBA-treated MCF-10A cells demonstrated 1.7-fold higher migration rates compared to controls; however, this effect was abrogated when cells were co-treated with DMBA and TMS ( Fig 2B). Cell invasion by DMBA-treated MCF-10A cells increased 1.4-fold and DMBA-treated MCF-7 cells increased 1.7-fold compared to controls, but again, this effect was negated in cells treated with both DMBA and TMS ( Fig 2C).
Matrix metalloproteinases (MMPs) are established markers of cellular invasion. Therefore, we measured MMP1, MMP9, MMP13, and MMP14 levels following CYP1B1 modulation and found that CYP1B1 upregulated these MMP transcripts ( Fig 2D).  demonstrated that DMBA treatment and CYP1B1 overexpression caused β-catenin to localize to the nucleus, while co-treatment with both DMBA and TMS failed to induce this effect ( Fig  3K-3O). CYP1B1 increased mRNA and protein levels of c-Myc and cyclin D1, widely known Wnt/β-catenin target proteins (Fig 3). Furthermore, CYP1B1 enhanced the promoter activity of β-catenin/TCF/LEF (Fig 3F). These results suggest that CYP1B1 promotes cell proliferation via Wnt/β-catenin signaling activation following β-catenin upregulation and nuclear localization.

CYP1B1 enhances cell invasion through EMT induction
We observed mesenchymal characteristics in MCF-10A cells with increased CYP1B1 expression (Fig 2A). Generally, the loss of E-cadherin expression during EMT allows cells to break tight junctions and become motile, thus permitting metastasis [32]. To elucidate whether CYP1B1 induces mesenchymal-like phenotypes by initiating EMT, we measured the expression of multiple EMT-related factors in MCF-7 and MCF-10A cells. CYP1B1 induction by overexpression increased mRNA expression of mesenchymal markers including N-cadherin, α-SMA, vimentin, fibronectin, and integrin α5. Transcriptional suppressors of E-cadherin including ZEB1/2, SNAI1, and TWIST1 were also induced by CYP1B1. However, CYP1B1 decreased the expression of epithelial markers such as E-cadherin and α-catenin ( Fig 4A). These effects were reversed when we decreased CYP1B1 levels by treating cells with TMS or CYP1B1-specific siRNA (Fig 4B and 4C). ZEB1 promoter activity was increased in CYP1B1-overexpressing cells and was inhibited in cells following CYP1B1 knockdown, while CDH1 promoter activity showed the opposite effect (Fig 4D and 4E). We further measured multiple EMT-related factors by western blot, which consistently demonstrated that CYP1B1 induces EMT (Fig 4F-4I). Confocal microscopic analyses of ZEB2, SNAI1, and vimentin also confirmed that CYP1B1 promotes EMT, while these effects were inhibited by TMS (Fig 4J-4N). Furthermore, we found that CYP1B1 considerably decreased E-cadherin expression (Fig 4O).

CYP1B1-mediated Wnt/β-catenin activation and EMT are regulated by Sp1
To identify the key regulator of CYP1B1-mediated EMT and Wnt/β-catenin signaling activation, we considered Sp1, because it is widely known as a transcription factor involved in cell proliferation and metastasis. Moreover, Sp1 was recently implicated in ZEB2-induced EMT [33,34]. Therefore, we investigated whether CYP1B1 regulates Sp1 expression by measuring its expression subsequent to CYP1B1 induction or inhibition in MCF-7, MCF-10A, and MDA-MB-231 cells. Sp1 mRNA and protein was upregulated in CYP1B1-overexpressing cells (Fig 5A, 5B, 5E and 5F). This effect was reversed when CYP1B1 expression was suppressed by TMS or siRNA (Fig 5C, 5D, 5G and 5H; S2 Fig). Confocal microscopic analysis confirmed that CYP1B1 overexpression and DMBA increased Sp1 expression while TMS treatment blocked this effect (Fig 5I-5k). These data indicate that CYP1B1 positively regulates Sp1 expression.
To clarify whether Sp1 DNA-binding plays a role in CYP1B1-mediated transcriptional regulation, we co-treated cells with DMBA and mithramycin A, an inhibitor of Sp1 DNA-binding, and measured the expression of multiple key proteins. The upregulation of β-catenin, c-Myc, cyclin D1, ZEB2, vimentin, and SNAI1 induced by DMBA was suppressed by mithramycin A in a concentration-dependent manner (Fig 6I and 6J; S4 Fig). These data suggested that Sp1 serves as the transcription factor that facilitates CYP1B1-mediated oncogenesis.
In confocal microscopic and subcellular fractionation analyses, DMBA likewise increased PCNA, ZEB2, and β-catenin. Interestingly, when cells were treated with both DMBA and mithramycin A (100 nM), the induction of PCNA, ZEB2, and β-catenin was almost completely blocked and β-catenin failed to localize to the nucleus (Fig 6K-6N). Similarly, the enhanced promoter activities of β-catenin/TCF/LEF, ZEB1, and TWIST1observed with DMBA treatment were suppressed in the presence of mithramycin A (Fig 6O). These results suggest that Sp1 directly regulates the transcriptional activities of β-catenin, ZEB1, and TWIST1 and initiates EMT and Wnt/β-catenin signaling.

4-Hydroxyestradiol (4-OHE 2 ) may play an important role in CYP1B1-mediated oncogenesis
To clarify whether CYP1B1-mediated EMT and Wnt/β-catenin signaling activation are initiated by CYP1B1 activity, we examined the enzyme activity of CYP1B1 following CYP1B1 overexpression (Fig 7A). The significant increase of CYP1B1 enzymatic activity could suggest that the oncogenic events occurred by CYP1B1 overexpression may be the results of CYP1B1 activity. To identify whether our hypothesis is valid, the expression levels of key proteins following treatment of the enzymatic products of CYP1B1, 4-OHE 2 or 2-OHE 2 . CTNNB1 and MYC mRNA levels were upregulated whereas CDH1 was suppressed in 4-OHE 2 -treated cells (Fig 7B). Sp1, a key regulator for CYP1B1-mediated effects, was induced by 4-OHE 2 in a concentration-dependent manner (Fig 7C). β-catenin protein also increased with 4-OHE 2 treatment, and we found that βcatenin in 4-OHE 2 -treated cells localized to the nucleus, as was observed in DMBA-treated or CYP1B1-overexpressing cells (Fig 7D). To compare the effects of estrogen metabolites produced by CYP1B1, cells were treated with 4-OHE 2 or 2-OHE 2 (Fig 7E and 7F; S5A and S5B Fig).  4-OHE 2 significantly increased Sp1, β-catenin, c-Myc, cyclin D1, PCNA, ZEB2, SNAI1, and vimentin expression and decreased E-cadherin levels, while 2-OHE 2 did not demonstrate any significant effects (Fig 7G and 7H; S5C Fig). The allelic variants of CYP1B1 gene having higher or lower enzymatic activity have been reported previously and CYP1B1 L432V and N203S have been reported to have markedly higher and lower enzymatic activity, respectively [35][36][37]. To elucidate whether the enzymatic activity of CYP1B1 is a major cause of EMT induction and Wnt/β-catenin signaling activation, the expression levels of Wnt/β-catenin signaling target proteins and Sp1 were determined following overexpression of CYP1B1 L432V or N203S polymorphic genes and showed to be positively regulated by CYP1B1 enzymatic activity. E-cadherin, however, showed the opposite result ( Fig 7I). These data indicate that the activity of CYP1B1 with generation of 4-OHE 2 , a major metabolite produced from estrogen by CYP1B1, may play a crucial role in CYP1B1-mediated EMT and Wnt/β-catenin signaling activation through induction of Sp1.

Discussion
Increased cell proliferation, migration, and invasion are widely considered as cancer hallmarks and key processes for tumor progression. To the best of our knowledge, the current study represents the first evidence that CYP1B1 enhances EMT and activates Wnt/β-catenin signaling by upregulating Sp1. Sp1 expression was promoted in cells treated with 4-OHE 2 and mediated the upregulation of EMT-inducing factors. This cascade of events inhibited E-cadherin expression and simultaneously increased Wnt/β-catenin signaling through the upregulation and nuclear localization of β-catenin. These results demonstrate that Sp1 mediates the downstream transcriptional effects associated with elevated CYP1B1 and is essential for EMT and Wnt/βcatenin signaling.
Up to this point, the relationship between Sp1 and Wnt/β-catenin signaling has been unclear. Several studies have reported that Sp1-related transcription factors might act as activators of Wnt/β-catenin target genes during cell development [38,39]. Importantly, we show that CYP1B1-induced cell proliferation in MCF-7 and MCF-10A cells is caused by PCNA upregulation. PCNA acts as an auxiliary component of the DNA polymerase-δ complex and plays an important role in DNA replication [40]. Recently, the relationship between PCNA and Wnt/βcatenin signaling became clearer with the report that PAF (PCNA-associated factor) can dissociate from PCNA complexes and bind to β-catenin, which enhances Wnt/β-catenin target gene expression upon Wnt signaling activation [41]. Based on these data, we suggest that PCNA mediates CYP1B1-induced Wnt/β-catenin signal activation, and that the relationship between PCNA and Sp1 be investigated in detail.
In this study, we found that Sp1 upregulates E-cadherin repressors like ZEB1/2, SNAIL, and TWIST1, which subsequently induce EMT. Recently, it has been reported that Sp1 induces cell migration and invasion in cooperation with ZEB2 [34]. Moreover, Sp1 has been shown to inhibit miR-200a expression; this subsequently allows HDAC4-mediated promoter diacetylation at ZEB1/2, which inhibits their expression [42,43]. The relationship between Sp1 and SNAIL is not fully understood, although it has been shown that Sp1 directly binds to the SNAIL promoter and thus upregulates SNAIL during EMT [44]. Moreover, SNAIL can induce Sp1 by suppressing an inhibitor of Sp1, miR-128 [45]. These data suggest that Sp1 and SNAIL mutually upregulate one another. Finally, Sp1 upregulation of TWIST1 expression by associating with CCT repeats in the TWIST1 promoter has been suggested; however, this process requires further investigation [46].
During invasion, cancer cells secrete MMPs to induce extracellular matrix (ECM) degradation. Sp1 has been reported to regulate the expression of multiple MMPs. For example, the promoters of MMP1, MMP9, and MMP14 contain Sp1 binding sites, and transcriptions from these loci are directly upregulated by Sp1 [47,48]. MMP1 and MMP14 have been reported to induce cancer cell invasion, and MMP14 is further recognized in the activation of MMP2 and MMP9 [49,50]. MMP13 expression is also increased by Sp1, and both MMP13 and MMP9 are implicated in the progression of various tumors [51][52][53][54]. In this study, we show that CYP1B1 upregulated the transcripts for all of these MMPs. Therefore, Sp1 is likewise assumed to play an important role in cell invasion, since CYP1B1 increases Sp1 expression and DNA binding.
There are several studies that have been reported the effects of CYP1B1 knockout in vivo models. The Cyp1b1(-/-) mice represented the elevated protection against DNA adduct formation induced by carcinogenic agents like DMBA or benzo[a,l]pyrene in tumors [55][56][57] and also showed the blocking effect on tumor tissue metastasis induced by benzo[a]pyrene [58]. Based on these previously reported in vivo data, the novel mechanism of CYP1B1-induced cell proliferation, migration, and invasion might have the preclinical significance but the in vivo experiments such as transplantation assay should be investigated in further study.
Although CYP1B1 upregulation via Sp1 binding in the CYP1B1 promoter has been reported, the reciprocal effect of CYP1B1 on Sp1 expression has not yet been described [59]. Recently, estrogens have been reported to regulate microRNA expression [60]. Among the estrogen-dependent microRNAs, miR-375 is generally suppressed in multiple cancers, including gastric, cervical, liver, lung, and esophageal cancer. This downregulation has recently been attributed to hypermethylation of its promoter in cancer cells [61][62][63][64][65]. These findings suggest that miR-375 may act as a tumor suppressor. As miR-375 directly binds the 3 0 UTR of Sp1 and thereby negatively regulates Sp1 expression, this microRNA might suppress cell migration and invasion [61]. Furthermore, miR-375 downregulation accompanies tamoxifen resistance and EMT in tamoxifen-resistant breast cancer cells [66]. Since CYP1B1 overexpression and 4-OHE 2 treatment induce Sp1 expression, 4-OHE 2 might be responsible for the suppression of miR-375 or other microRNAs, which subsequently promotes Sp1 expression.
In summary, to the best of our knowledge, our present study is the first report to identify the molecular mechanism underlying CYP1B1-mediated cancer progression. Our results demonstrate that CYP1B1 enhances cell proliferation via Wnt/β-catenin signaling activation by inducing the expression and nuclear localization of β-catenin. Moreover, EMT induction by CYP1B1 was mediated by the upregulation of E-cadherin transcriptional repressors. Our results further indicate that CYP1B1 enzymatic activity is essential for CYP1B1-mediated EMT and Wnt/β-catenin signaling activation, because 4-OHE 2 treatment was sufficient to induce Sp1 and other key proteins in EMT and Wnt/β-catenin signaling. The scheme in Fig 8   cells. (E) Protein levels of Wnt/β-catenin signaling target proteins and (F) EMT-related factors were determined using western blot in 4-OHE 2 -treated MCF-7 cells. All western blots were performed independently three times and the bands were quantified using Quantity One software program. (G) Wnt/β-catenin signaling target proteins in 4-OHE 2 -treated cells, and (H) EMT-related factors in 4-OHE 2 -treated cells. The results were from three independently quantified experiments. (*p 0.05) (I) Wnt/β-catenin signaling target proteins, Sp1, and E-cadherin proteins were measured by western blot following overexpression of CYP1B1 polymorphic genes in MCF-10A cells.
doi:10.1371/journal.pone.0151598.g007 CYP1B1 Induces EMT and Wnt/β-Cat Signaling via Sp1 summarizes these novel findings revealing CYP1B1-induced oncogenic mechanisms. Since CYP1B1 is implicated as a significant factor in the development of various cancers, a more detailed understanding of the precise mechanisms underpinning CYP1B1-mediated cancer progression may facilitate the development of new strategies for cancer treatment.