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Introduction

Reactive oxygen species (ROS) are crucial cellular secondary messengers involved in diverse biological processes in cancer cells (1–7). ROS are oxygen-containing species, including superoxide (O2•−), hydroxyl (•OH), and hydrogen peroxide (H2O2). The majority of ROS are produced as side products of oxidative phosphorylation in mitochondria (4,5,8,9), but they are also induced by NADPH oxidase (NOX), growth factors, and cytokines (2,4). ROS are increased in many types of cancers and these elevated ROS are highly involved in tumor development and progression (2,6,10). High levels of ROS exert cytotoxic effects by inducing DNA damage or cell death, thereby limiting cancer progression (2,4,5,7). On the other hand, at low levels, ROS function as signaling molecules that can induce nucleic acid and protein synthesis, cell cycle progression, the expression of numerous genes involved in cell proliferation and survival, the activation of redox-sensitive transcription factors [such as p53, hypoxia inducible factor (HIF)-1, and NF-κB], epigenetic alterations, and tumorigenesis (2,4,5,7). In addition, ROS have been shown to induce epithelial-mesenchymal transition (EMT) in cancer cells (11–14). However, the precise mechanism of ROS-induced EMT remains to be elucidated.

EMT plays critical roles in embryogenesis as well as in tumor invasion and metastasis. Epithelial cells undergoing EMT lose their polarization and acquire mesenchymal-like morphology. EMT is also characterized by decreased expression of epithelial markers, such as E-cadherin and induction of mesenchymal markers, such as vimentin. Loss of E-cadherin is considered a hallmark of EMT (15–19). EMT is regulated by several transcription factors, including Snail, Slug, Twist-related protein (Twist)1, Twist2, zinc finger E-box binding homeobox (ZEB)1, ZEB2, and E12/E47. Snail, a key transcription factor in EMT, promotes invasion and metastasis in response to several oncogenic signaling pathways (20–22). Recently, Snail has been implicated in metabolic alterations, such as glycolytic switch (21,23). Glycolytic switch is a phenomenon whereby cancer cells rely on glycolysis more than mitochondrial oxidative phosphorylation for ATP production, even when sufficient oxygen is available (24–28). Recently, we showed that distal-less homeobox-2 (Dlx-2) induces EMT and glycolytic switch by activating Snail (29,30). Dlx-2 is one of the human distal-less (Dlx) gene family proteins and is involved in embryonic development (31,32) and tumor progression (33–36). Dlx-2 is known to play an important role in shifting the activity of TGF-β from tumor suppression to tumor promotion (34).

In this study, we show that ROS induce EMT through Snail and Dlx-2 cascades. We also show that Dlx-2/Snail signaling plays a critical role(s) in ROS-induced glycolytic switch, inhibition of mitochondrial respiration, and cytochrome c oxidase (COX) inhibition. These results clarify the mechanism by which ROS promotes tumor progression through Dlx-2/Snail axis-dependent EMT and glycolytic switch in MCF-7 cells.

Materials and methods

Cell culture and ROS treatment

MCF-7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; authenticated by short tandem repeat profiling). MCF-7 cells were cultured in Eagle's minimal essential medium (EMEM; Hyclone; GE Healthcare, Logan, UT, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Hyclone; GE Healthcare) and 1% penicillin-streptomycin (PS, Hyclone; GE Healthcare). Cells were cultured at 37°C in a humidified incubator with 5% CO2 and were passaged two times per week. Low-passage cultures (passage 5–25) were used for all experiments. Cells were routinely checked for mycoplasma contamination using the Mycoplasma PCR Detection kit (iNtRON Biotechnology). MCF-7 cells were treated with ROS [200 µM H2O2 (Sigma-Aldrich, St. Louis, MO, USA) and 10 µM menadione (an O2− generator; Sigma)].

Transfection and short hairpin RNA (shRNA) interference

pCR3.1-Snail-Flg (provided by J.I. Yook, Yonsei University, Korea) was transfected into MCF-7 cells using jetPEI (Polyplus-transfection SA, New York, NY, USA). pSUPER vectors with control shRNA and shRNAs against Dlx-2 and Snail were produced and transfected as described previously (23). shRNA target sequences have also been described previously (23,29).

Immunoblotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Immunoblotting was performed using the following antibodies: anti-Dlx-2 (EMD Millipore, Billerica, MA, USA); anti-Snail (Abgent, San Diego, CA, USA); anti-E-cadherin, anti-COXVIIc, anti-COX15, and anti-COX19 (Santa Cruz Biotechnology, Dallas, TX, USA); anti-COXVIc, and anti-COXVIIa (MitoSciences, Eugene, OR, USA); anti-COX18 (Abcam, Cambridge, UK); anti-α-tubulin (Biogenex, Fermont, CA, USA).

Total mRNA was isolated from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the supplier's instructions. Transcript levels were assessed with reverse transcription-quantitative polymerase chain reaction using primers for EMT-inducing transcription factors (including Snail, Slug, Twist1, Twist2, ZEB1, ZEB2, E12/E47, Dlx-2), E-cadherin, N-cadherin, vimentin, fibronectin, cluster of differentiation 44 (CD44), COX subunits and assembly factors, and β-actin. Primer sequences described previously were used (23,29,37), along with the following additional primers: Twist1 sense, 5′-CAGACCCTCAAGCTGGCGGC-3′; Twist1 antisense, 5′-CCAGGCCCCCTCCATCCTCC-3′; Twist2 sense, 5′-TGGGCACCAGCGAGGAGGAG-3′; Twist2 antisense, 5′-CTGGGGCTGCCCTTCTTGCC-3′; ZEB1 sense, 5′-GGGAGGATGACAGAAAGGAAGG-3′; ZEB1 antisense, 5′-TGCCTCTGGTCCTCTTCAGGTGC-3′; ZEB2 sense, 5′-CAGAAGCCACGATCCAGACCGC-3′; ZEB2 antisense, 5′-GTGCCAAGGCGAGACAGCTCC-3′; E12/E47 sense, 5′-GCCGGGCACATGTGAAAGTAAACAA-3′; E12/E47 antisense, 5′-CAGGTTTCCACAGCATCCCCCTT-3′; N-cadherin sense, 5′-AGCCAACCTTAACTGAGGAGT-3′; N-cadherin antisense, 5′-GGCAAGTTGATTGGAGGGATG-3′; vimentin sense, 5′-TGAAGGAGGAAATGGCTCGTC-3′; vimentin antisense, 5′-GTTTGGAAGAGGCAGAGAAATCC-3′; fibronectin sense, 5′-CAGTGGGAGACCTCGAGAAG-3′; fibronectin antisense, 5′-TCCCTCGGAACATCAGAAAC-3′; CD44 sense, 5′-TGCCGCTTTGCAGGTGTATT-3′; CD44 antisense, 5′-CCGATGCTCAGAGCTTTCTCC-3′. Values were normalized to those of β-actin.

Immunofluorescence (IF) staining

Cells were fixed with 3.7% formaldehyde, permeabilized in 0.2% Triton X-100, and blocked with 2% BSA. Cells were then incubated overnight with anti-E-cadherin antibody and immunostained with AlexaFluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen). Hoechst 33342 (Invitrogen) was used to stain cell nuclei. Cells were viewed under a fluorescence microscope.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed using a ChIP assay kit (EMD Millipore) according to the manufacturer's instructions. Isotype control IgG, anti-Dlx-2, and anti-Snail (Santa Cruz Biotechnology) were used to immunoprecipitate DNA-containing complexes. ChIP-enriched DNA was analyzed by PCR using primers complementary to promoter regions containing Dlx-2 or Snail binding sites, as described previously (23,29).

Assays for mitochondrial respiration, COX activity, glucose consumption, lactate production, and ATP production

Mitochondrial respiration and COX activity were measured as described previously (23,38). For the mitochondrial respiration assay, exponentially growing cells (1.5×106) were washed with TD buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM Tris-HCl; pH 7.4), collected, and resuspended in complete medium without phenol red. Cells (5×105) were transferred to a Mitocell chamber equipped with a Clark O2 electrode (782 O2 Meter; Strathkelvin Instruments, Glasgow, UK). O2 consumption rates were measured after adding 30 µM DNP to obtain the maximum respiration rate and specificity for mitochondrial respiration was confirmed by adding 5 mM KCN. COX activity was determined by measuring the KCN-sensitive, COX-dependent O2 consumption rate by adding 3 mM TMPD in the presence of 30 µM DNP and 20 µM antimycin A.

Glucose, lactate, and intracellular ATP levels in the media were determined using a glucose oxidation assay kit (Sigma-Aldrich), a colorimetric and fluorescence-based lactate assay kit (BioVision, Inc., Milpitas, CA, USA), and an ATP Bioluminescence Assay kit (Roche Diagnostics, Basel, Switzerland), respectively, according to the manufacturers' instructions. Levels of glucose, lactate, and intracellular ATP were normalized to protein concentrations. Levels of ATP produced by aerobic respiration and glycolysis were determined by measuring lactate production and O2 consumption (23,39).

Measurement of circularity

Circularity assays were performed as described previously (29). Briefly, microscopic images were analyzed with AxioVision LE software (version 4.8). Circularity was calculated using the formula, 4π (area/perimeter2). Values closer to 1.0 indicate a more circular cell morphology.

Statistical analysis

Reverse transcription-quantitative polymerase chain reaction, mitochondrial respiration, glucose consumption, lactate production, and ATP production assays were performed at least in triplicate. Most experiments were repeated more than twice. Data were analyzed by Student's t-test (unpaired, two-tailed) for comparison between two groups, one-way ANOVA with Tukey's multiple-comparisons test for comparison between three groups, and two-way ANOVA for comparison between three groups with multiple factors and results were expressed as mean ± SE. P<0.05 was considered to indicate a statistically significant difference.

Results and Discussion

Dlx-2/Snail cascade is implicated in ROS-induced EMT

It has been shown that ROS induces EMT by increasing Snail expression (11–14). We examined the EMT-inducing effects of H2O2 and O2− in MCF-7 cells, a non-invasive luminal A subtype breast cancer cell line. MCF-7 cells have low levels of Snail, but various stimuli including TGF and Wnt can induce Snail expression, and subsequently, EMT in MCF-7 cells; thus, MCF-7 cells are widely used for studying breast cancer, particularly EMT (40–42). ROS induced a phenotypical change to an elongated morphology with pseudopodia, which is a characteristic of mesenchymal cells, and decreased the expression of an epithelial marker, E-cadherin, indicating that ROS induce EMT (Fig. 1A and B). We further examined the effects of ROS on the expression of mesenchymal markers, such as N-cadherin, vimentin, and fibronectin. In addition, we also examined the expression of CD44, a glycoprotein receptor for various extracellular matrix (ECM) ligands, such as hyaluronic acid (HA); HA-CD44 interaction promotes EMT and CSC self-renewal through Nanog expression (43,44). ROS were shown to increase the expression of mesenchymal markers including N-cadherin, vimentin, and fibronectin (Table I and Fig. 1B). ROS also upregulated the CD44 expression (Table I and Fig. 1B). We investigated which EMT-inducing transcription factors (Snail, Slug, Twist1, Twist2, ZEB1, ZEB2, E12/E47, and Dlx-2) were involved in ROS treatment-induced EMT using reverse transcription-quantitative polymerase chain reaction (Table I). ROS induced Snail and Dlx-2 expression, but had no effect on the other EMT-inducing transcription factors (Table I and Fig. 1B). Therefore, we examined if the Dlx-2/Snail cascade was involved in ROS-induced EMT. Dlx-2 and Snail shRNAs appeared to block the EMT phenotype and downregulation of E-cadherin induced by ROS (Fig. 1C-E). We also found that Dlx-2 and Snail shRNAs prevented the ROS-induced expression of N-cadherin, vimentin, fibronectin, and CD44 (Fig. 1D). Because Dlx-2 has been shown to be an upstream regulator of Snail (29,30), we examined the effects of Dlx-2 shRNA on ROS-induced Snail expression. In the immunoblotting of Dlx-2, anti-Dlx-2 antibody recognized 2 bands (33). The lower band (indicated by arrowhead, 34 kDa) is Dlx-2; the higher band is non-specific band. Expectedly, Snail shRNA suppressed ROS-induced Snail expression and Dlx-2 shRNA suppressed ROS-induced Dlx-2 expression. In addition, Dlx-2 shRNA also prevented Snail induction by ROS, whereas Snail shRNA did not affect Dlx-2 induction by ROS (Fig. 1D and E), indicating that Dlx-2 consistently acts upstream of ROS-induced Snail. A ChIP assay showed that ROS increased Dlx-2 binding at the Snail promoter, which was detected at an early time point (3 h) after ROS treatment (Fig. 1F). Dlx-2 appeared to act as a mediator for ROS-Snail-induced EMT. These results suggest that the Dlx-2/Snail axis are implicated in ROS-induced EMT.

Figure 1. The Dlx-2/Snail cascade is implicated in ROS-induced EMT. (A and B) MCF-7 cells were treated with H2O2 (200 µM) or menadione (O2−; 10 µM) for 2 or 3 days. (C-E) MCF-7 cells were transfected with Dlx-2 shRNA or Snail shRNA and then treated with H2O2 (200 µM) or menadione (O2−; 10 µM) for 3 days. Cells were analyzed by phase-contrast and fluorescence microscopy for cell morphology and E-cadherin expression (fluorescence in green; left) and circularity (right). Borders were drawn along the cell edges for quantification of circularity. (A and C) Results (123–603 cells in each group) are represented as mean ± SE. (B and D) Cells were analyzed by reverse transcription-quantitative polymerase chain reaction using the indicated primers. *P<0.05 and **P<0.01 as indicated; ##P<0.01 as indicated. (E) Cells were also analyzed by immunoblotting using the indicated antibodies. Quantification of the intensity of Dlx-2 and Snail bands was performed using Image J software (NIH, Bethesda, MD, USA) and normalized to the signal of tubulin. (F) MCF-7 cells were treated with H2O2 (200 µM) for the indicated times. ChIP assays were performed using IgG or anti-Dlx-2 antibodies and ChIP-enriched DNA was analyzed by polymerase chain reaction using primers complementary to the Dlx-2-binding region. All scale bars represent 100 µm. EMT, epithelial-mesenchymal transition; ROS, reactive oxygen species; Dlx, distal-less; ChIP, chromatin immunoprecipitation; CD44, cluster of differentiation 44.

Table I. Regulation of EMT markers and EMT-inducing transcription factors by ROS.

Table I.

Regulation of EMT markers and EMT-inducing transcription factors by ROS.

	H2O2			O2−
Genes	24 h	48 h	72 h	24 h	48 h	72 h
EMT markers
E-cadherin	0.980	0.670b	0.665b	0.963	0.496b	0.430b
N-cadherin	1.612a	3.382b	5.719b	1.644b	2.043b	2.135b
Vimentin	1.028	1.226	1.894b	0.975	1.537b	1.628b
Fibronectin	1.390	1.527b	1.945b	1.249	1.353	1.651b
CD44	1.551b	1.738b	2.034b	1.129	2.208b	2.068b
EMT-inducing transcription factors
Snail	1.254	1.243	1.724b	1.209	2.183b	2.553a
Slug	1.210	0.835	0.940	0.935	0.705	1.080
Dlx-2	1.573b	1.564b	1.573b	1.615b	3.457b	2.383b
Twist1	1.240	1.080	1.255	1.150	1.025	1.270
Twist2	1.065	1.070	1.155	0.895	1.110	1.360
ZEB1	0.955	0.940	1.100	1.315	1.260	1.245
ZEB2	0.950	1.050	1.500	0.835	0.960	1.435
E12/E47	0.834	0.960	0.934	1.061	0.877	0.937

{ label (or @symbol) needed for fn[@id='tfn1-mmr-20-03-2339'] } MCF-7 cells were treated with H₂O₂ (200 µM) or menadione (O₂⁻, 10 µM) for the indicated times and then analyzed by reverse transcription-quantitative polymerase chain reaction with the indicated primers. The data were normalized against an internal control β-actin and relative expression levels were expressed as fold changes.

a P<0.05

b P<0.01 vs. untreated group. EMT, epithelial-mesenchymal transition; ROS, reactive oxygen species; CD44, cluster of differentiation 44; Dlx, distal-less; ZEB, Zinc finger E-box binding homeobox; Twist, Twist-related protein.

Dlx-2/Snail signaling is implicated in ROS-induced glycolytic switch and inhibition of mitochondrial respiration

Cancer cells exhibit glycolytic switch as well as EMT during tumor development and progression (24–28). Thus, we examined the effects of ROS on glycolytic switch and mitochondrial respiration. ROS significantly increased glucose consumption and lactate production (Fig. 2A). In addition, ROS reduced O2 consumption (Fig. 2B). Although total ATP concentrations remained the same in all cells, ROS increased the ATP ratio produced by glycolysis versus aerobic respiration (Fig. 2C), indicating that ROS induced glycolytic switch. Previously, we showed that the Dlx-2/Snail cascade induces glycolytic switch and inhibits mitochondrial respiration (29,30). We further examined whether the Dlx-2/Snail cascade affected ROS-induced glycolytic switch and inhibition of mitochondrial respiration. Dlx-2 and Snail shRNAs impaired the effects of ROS on glucose consumption, lactate production, and O2 consumption (Fig. 2A-C), indicating that the Dlx-2-Snail axis is involved in ROS-induced glycolytic switch and inhibition of mitochondrial respiration.

Figure 2. The Dlx-2/Snail cascade is implicated in ROS-induced glycolytic switch, inhibition of mitochondrial respiration, and COX inhibition. MCF-7 cells were transfected with Dlx-2 shRNA or Snail shRNA and treated with H2O2 (200 µM) or menadione (O2−; 10 µM) for 3 days. Cells were analyzed for (A) glucose consumption and lactate production and (B) mitochondrial respiration and (C) total ATP concentration. The amount of ATP produced by aerobic respiration (black bars) and glycolysis (gray bars) was calculated by measuring O2 consumption and lactate production in the cells (right panels in C). (D) COX activity was measured. (E) MCF-7 cells were transfected with Dlx-2 shRNA or Snail shRNA and then treated with H2O2 (200 µM) or menadione (O2−; 10 µM) for 3 days and analyzed by immunoblotting with the indicated antibodies. (F) MCF-7 cells were transfected with Dlx-2 shRNA or Snail shRNA and then treated with H2O2 (200 µM) for 2 days and analyzed by real-time reverse transcription-quantitative polymerase chain reaction using the indicated primers. *P<0.05 and **P<0.01 as indicated; #P<0.05 and ##P<0.01 as indicated. Bars in the graph represent mean ± SE. (G) MCF-7 cells were transfected with a Snail expression vector for 2 days. ChIP assays were performed using IgG or anti-Snail antibodies and ChIP-enriched DNA was analyzed by polymerase chain reaction using primers complementary to the Snail binding region. Dlx, distal-less; ROS, reactive oxygen species; COX, cytochrome c oxidase; ChIP, chromatin immunoprecipitation.

Dlx-2/Snail signaling is implicated in ROS-induced COX activity repression

Inhibition of mitochondrial respiratory activity is closely related to changes in the activity of COX, the terminal enzyme of the mitochondrial respiratory chain. We found that ROS repressed COX activity (Fig. 2D). Thus, we further examined the involvement of the Dlx-2/Snail cascade in ROS-induced downregulation of COX activity. Dlx-2 and Snail shRNAs suppressed ROS-induced downregulation of COX activity (Fig. 2D), implicating the Dlx-2/Snail cascade in ROS-induced COX activity repression.

Dlx-2/Snail signaling is implicated in ROS-induced COX activity repression by downregulating multiple COX subunits and assembly factors

Eukaryotic COX is composed of 13 different subunits in the inner mitochondrial membrane and the sequential action of several assembly factors regulates its assembly (Table II). We further examined the effects of ROS on the expression of COX subunits and assembly factors. ROS downregulated the expression of COXVIc, COXVIIa, COXVIIc, COX15, COX18, and COX19 (Table II). Note that ROS decreased the mRNA levels of COX15 and COX18, but not their protein levels (Fig. 2E and F; Table II). We previously found that Snail decreased expression of COXVIc, COXVIIa, and COXVIIc (23) and Dlx-2 decreased expression of COXVIc and COX19 (29). Snail shRNA suppressed the ROS-induced reduction in the levels of COXVIc, COXVIIa, and COXVIIc, but not COX19 (Fig. 2E and F). Dlx-2 shRNA suppressed the ROS-induced reduction in the levels of COXVIc and COX19, but not COXVIIa and COXVIIc (Fig. 2E and F).

Table II. Regulation of gene expression of COX subunits and assembly factors by ROS.

Table II.

Regulation of gene expression of COX subunits and assembly factors by ROS.

	H2O2 (n=2-3)	O2− (n=4)
Genes	48 h	48 h
COX subunits
COXIV	1.067	1.138
COXVa	1.047	1.018
COXVb	0.978	1.214
COXVIa	1.021	1.047
COXVIb	0.853	1.425
COXVIc	0.728b	0.704b
COXVIIa	0.826b	0.431b
COXVIIb	1.188	1.045
COXVIIc	0.804a	0.778b
COXVIII	1.072	1.053
Assembly factors
COX10	0.942	0.918
COX11	1.103	1.028
COX15	0.776a	0.653b
COX17	1.016	0.939
COX18	0.836a	0.865b
COX19	0.742b	0.849b
LRPPRC	1.139	1.051
SURF1	0.773	1.274
SCO1	1.036	1.170
SCO2	1.058	1.055

{ label (or @symbol) needed for fn[@id='tfn4-mmr-20-03-2339'] } MCF-7 cells were treated with H₂O₂ (200 µM) or menadione (O₂⁻, 10 µM) for 48 h and then analyzed by reverse transcription-quantitative polymerase chain reaction with the indicated primers. The data were normalized against an internal control β-actin and relative expression levels were expressed as fold changes.

a P<0.05

b P<0.01 vs. untreated group. COX, cytochrome c oxidase; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; SCO, synthesis of cytochrome c oxidase; SURF1, surfeit locus protein 1; ROS, reactive oxygen species.

Several Snail-binding sites (E-box) were previously found in the promoters of COX subunits (23)

To examine whether the expression of the COX subunits was regulated by Snail, we conducted a ChIP assay. Snail bound to the COXVIc, COXVIIa, and COXVIIc promoters (Fig. 2G), which is consistent with previous observations (23). Among the COX subunits, COXVIc acted as a common target of ROS, Dlx-2, and Snail. Therefore, COXVIc may play an important role in the repression of COX activity via the ROS-Dlx-2/Snail-mediated pathway. For judging the transformation of the proteome accurately, the proteome needs to be analyzed by mass spectrometry-based methods, such as liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), together with bioinformatics analyses (45,46). Thus, proteome transformation is yet to be examined in further studies, but cumulatively, our findings suggest that the ROS-Dlx-2/Snail axis plays a crucial role in breast tumor progression by regulating EMT, mitochondrial repression, and glycolytic switch.

NF-κB has been shown to be involved in ROS-induced EMT (13)

NF-κB induces EMT-inducing transcription factors such as Snail, Slug, ZEB1, ZEB2, and Twist; NF-κB/p65 transcriptionally regulates the expression of these transcription factors, which in turn represses the epithelial marker E-cadherin and activates the mesenchymal marker N-cadherin, thereby resulting in the induction of EMT (47–49). Additionally, NF-κB stimulates the expression of HIF-1α, also contributing to EMT (48). Furthermore, NF-κB activation induces matrix-degrading enzymes such as MMP9, thus contributing to EMT (50). NF-κB is known to be involved in ROS-induced EMT via Snail induction (13). Because we found that ROS induce EMT through the Dlx-2/Snail cascade, we think that an interplay between NF-κB and Dlx-2 may possibly exist for Snail activation in ROS-induced EMT; this interplay between NF-κB and Dlx-2 is yet to be elucidated in further studies.

It has been shown that ROS are involved in many aspects of cellular signaling

TGF-β1 has been shown to activate the ROS-NFκB pathway, which plays an important role in TGF-β1-induced EMT, cell migration, and invasion (51). We have also shown that Dlx-2/Snail signaling is involved in TGF-β-induced EMT (29,30). Thus, the ROS-Dlx-2/Snail cascade may be involved in TGF-β-induced EMT. In addition, it was recently reported that ROS are involved in EMT and cancer metastasis induced by chemotherapeutics, such as 5-fluorouracil (5-FU), gemcitabine (GEM), and oxaliplatin (52–54). EMT contributes to chemoresistance in cancer cells. Dual oxidase 2 (DUOX2)-induced ROS promote the induction of EMT in 5-FU-resistant colon cancer cells (52). In addition, in GEM-treated pancreatic ductal adenocarcinoma (PDAC) patients, the levels of glutathione peroxidase-1 (GPx1), an antioxidant enzyme, are negatively regulated. GPx1 inhibits ROS-mediated Akt/GSK3β/Snail signaling, thereby suppressing EMT and chemoresistance in PDAC (53). ROS have been shown to mediate oxaliplatin-induced EMT and invasive potential in colon cancer (54). Our results suggest that ROS-induced Dlx-2/Snail signaling may be involved in EMT and may be induced by these chemotherapeutics. Note that we used only one cell line in this study, thus our results are limited to MCF-7 cells. Therefore, the Dlx-2/Snail axis is a potential therapeutic target for the prevention of metastasis and tumor progression in MCF-7 cells; this mechanism may be the case for breast cancer in general.

Acknowledgements

Not applicable.

Funding

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (grant nos. 2015M2B2A9028108, 2015R1A2A2A01004468, 2017R1A2B4010411 and 2017R1A6A3A11030673).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

SYL, MKJ, HMJ and HSK conceived and designed the project. SYL, MKJ and HMJ performed the experiments. SYL, MKJ, HMJ, CHK, HGP, SIH, YJL and HSK analyzed and interpreted the data. SYL, MKJ, HMJ, YJL and HSK wrote the manuscript. HSK supervised the project. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References