Biochemistry and Cell Biology
Alteration of DACH1 methylation patterns in lung cancer contributes to cell proliferation and migration
Journal: Biochemistry and Cell Biology
Manuscript ID bcb-2017-0279.R1
Manuscript Type: Article
Date Submitted by the Author: 13-Dec-2017
Complete List of Authors: Feng, Yongjie; School of Biology & Basic Medical Science, Soochow University Wang, Lin; Department of Ji Nan Children’s Hospital Wang, Mingyong;Draft College of Pharmaceutical Sciences, Soochow University
Is the invited manuscript for consideration in a Special N/A Issue? :
Keyword: DACH1, DNA methylation, NSCLC, A549 cells
https://mc06.manuscriptcentral.com/bcb-pubs Page 1 of 25 Biochemistry and Cell Biology
1 Alteration of DACH1methylation patterns in lung cancer contributes to cell proliferation and migration
2 Yongjie Feng1, Lin Wang2, Mingyong Wang3*
3
4
5
6 Affiliations:
7
8 1School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, China
9 2Department of JiNan Children’s Hospital, Jinan, 250022, China 10 3College of Pharmaceutical Sciences, SoochowDraft University, Suzhou, Jiangsu 215021, China. 11
12
13
14
15
16
17
18
19 *Correspondence to: Mingyong Wang
20 Email: [email protected]
21 Telephone: +86-0512-65881257
22 Fax: +86-0512-65881257
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 2 of 25
23 Abstract：Lung cancer is the most common cause of cancer related death. Non small cell lung cancer (NSCLC)
24 accounts for 80–85% of total lung cancer cases. Dachshund homolog 1, or DACH1, is a protein encoded by the
25 DACH1 gene in humans. DACH1 inhibits lung adenocarcinoma invasion and tumor growth, but has a lower
26 expression in NSCLC. To investigate the mechanisms of decreased DACH1 expression, its DNA methylation
27 patterns were investigated. The results showed a higher methylation rate in NSCLC compared to in adjacent
28 normal lung tissues. Cell transfection experiments showed that increased methylation impaired transcription factor
29 transactivation. In vivo demethylation treatment and overexpression DACH1 increased apoptosis and decreased
30 migration and invasion in NSCLC A549 cells. Our research provides new insight into NSCLC pathogenesis and
31 identifies a new therapeutic target. 32 Key words: DACH1; DNA methylation; NSCLC,Draft A549 cells 33
34
35
36
37
38
39
40
41
42
43
44
https://mc06.manuscriptcentral.com/bcb-pubs Page 3 of 25 Biochemistry and Cell Biology
45 1. Introduction
46 Lung cancer is the most common cause of cancer related death (TORRE et al. 2015). Lung cancer can be divided
47 into two categories: non small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of which NSCLC
48 accounts for 80–85% of total cases (STUPP et al. 2004). Mostpatients with NSCLC are diagnosed at a late stage,
49 with surgical intervention producing poor outcomes and frequent relapses (ZUGAZAGOITIA et al. 2017).
50 Radiotherapy and chemotherapy are often used as post surgical adjuvant therapies, but have undesirable side
51 effects and are largely ineffective at killing tumor cells(HALLQVIST et al. 2012). In recent years, development of
52 novel NSCLC treatments has become a hot topic of research, with efforts largely focused on elucidating NSCLC
53 pathogenesis and evolution (HALLQVIST et al. 2012; ZAPPA and MOUSA 2016). 54 Epigenetics is a branch of genetics that Draft studies heritable changes in gene expression unlinked to nucleotide
55 sequence, such as microRNA (miRNA) regulation, DNA methylation, and genomic imprinting (BROWN and
56 STRATHDEE 2002). miRNA is a class of endogenous small RNA with a length of about 20–24 nucleotides, and has
57 many important regulatory cellular roles (AMBROS 2004). miRNA regulates mRNA expression by binding to the
58 3′ UTR region of the target gene(AMBROS 2004). Research has revealed that many miRNAs regulate relative gene
59 expression in lung cancers, and hence miRNA may be useful as a therapeutic target and diagnostic marker for
60 NSCLC (YANAIHARA et al. 2006; YU et al. 2008; GAROFALO et al. 2011).
61 DNA methylation is one of the earliest discovered DNA modifications (CEDAR 1988). Methyltransferases
62 are enzymes that add methyl groups to cytosine in 5′ CG 3′ dinucleotides to form 5 methylcytosine (BESTOR
63 2000).Clusters of 5′ CG 3′sequences in the gene promoter are known as CpG Islands, which are easily methylated
64 to modulate gene expression (JABBARI and BERNARDI 2004).Many studies have shown that DNA methylation can
65 lead to changes in chromatin structure, DNA conformation and stability, and DNA protein interactions, which can
66 control gene expression (CEDAR 1988).
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 4 of 25
67 Dachshund homolog 1, or DACH1, is a protein encoded by the DACH1 gene in humans. DACH1 has been
68 shown to interact with UBE2I (RUAL et al. 2005; LIU et al. 2016), SMAD4and NCOR1 (WU et al. 2003).DACH1
69 is highly conserved across a variety of organisms. The DACH1gene is found to be critical in the development of
70 the eye, foot, and nervous system in Drosophila (BONINI et al. 1998). Knockout of DACH1 in mice leads to
71 incomplete development of the eye, limbs, and brain (BACKMAN et al. 2003). Low expression has been reported in
72 a variety of human malignancies, such as breast(POPOV et al. 2009), prostate, and endometrial cancer (NAN et al.
73 2009; KONG et al. 2016).It was found that the expression of DACH1 in breast cancer can inhibit ERB2 synthesis
74 and expression, and can also inhibit DNA synthesis (POPOV et al. 2009). A hypothetical mechanism for this may be
75 that these proteins can also function as transcription factors to affect cell function, proliferation, and migration. In
76 esophageal cancer, silencing DACH1 promotesDraft cancer growth by inhibiting the TGF β signaling pathway (POPOV 77 et al. 2009). In lung cancer, DACH1 inhibits lung adenocarcinoma invasion and tumor growth by repressing
78 CXCL5 signaling (HAN et al. 2015). Zhu et al. reported that DACH1 inhibits the proliferation and invasion of lung
79 adenocarcinoma through the downregulation of peroxiredoxin3 (ZHU et al. 2016). DACH1expression is lower in
80 lung cancers than in normal lung tissue and DACH1 is dependent on P53 for NSCLC inhibition (CHEN et al.
81 2013).
82 Although reducedDACH1 expression was reported in association with NSCLC, the underlying mechanisms
83 remain unclear. In 2015, genome wide methylation sequencing revealed that DACH1 methylation was altered in
84 lung cancer (LOKK et al. 2012), but details of the associated mechanism and its effect on NSCLC are unclear. In
85 this study, we used bisulfite sequencing PCR (BSP) technology to investigate methylation states in the DACH1
86 promoter. Our results identified two CpG islands in the DACH1promoter. BSP revealed a high methylation rate in
87 the DACH1promoter, inhibiting the activation of a candidate translational factor. Using the A549NSCLC cell line,
88 we revealed that demethylation of DACH1 increased apoptosis and decreased cell proliferation, migration, and
https://mc06.manuscriptcentral.com/bcb-pubs Page 5 of 25 Biochemistry and Cell Biology
89 invasion. Our research provides new insight into NSCLC pathogenesis and identifies a new therapeutic target.
90
91 2. Methods and materials
92 2.1 Obtaining primary human samples
93 The study was approved by the local ethical review committee at the Faculty of The Second Affiliated Hospital of
94 Soochow University, and written informed consent was obtained from all patients. A total of 35 self pairs of
95 NSCLC tumor and adjacent normal lung tissues (> 5 cm away from the tumor) were obtained from patients who
96 received radical resection of pulmonary carcinoma at the same hospital from March 2014 to June 2015. All clinical
97 specimens were collected during surgery from patients who had not undergone any preoperative treatment. 98 Pathological types were confirmed by twoDraft experienced pathologists, and the tumor stage classifications were 99 determined per the NSCLC guidelines of the National Comprehensive Cancer Network(version 7, 2015). These
100 tissues were sectioned into two, and one section was snap frozen in liquid nitrogen immediately and stored at
101 80 °C.
102 2.2 Cell culture
103 Human NSCLC A549 cell line was purchased from the Chinese Science Institute (Shanghai, China). A549cells
104 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone, Logan, UT, United States),
105 containing 10% fetal bovine serum (FBS) (Sijiqing, Hangzhou, China). The normal human bronchial epithelial
106 cells (NHBECs) were a kind gift from Yangzhou University. NHBECs were cultured in Bronchial Epithelial Cell
107 Basal Medium (Lonza Group Ltd., Basel, Switzerland). Media were supplemented with 100 U/mL penicillin and
108 100 U/mL streptomycin (Zhong Shan Golden Bridge Biological Technology CO., LTD, Beijing, China). All cells
109 were incubated at 37 °C in a humidified chamber, supplemented with 5% CO2.
110
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 6 of 25
111 2.3 Total RNA isolation and qRT-PCR
112 RNA was isolated from 100 mg of tissue(liquid nitrogen grounding method performed before RNA extraction) and
113 2×106 cells using TRIzol® Reagent(Invitrogen, Carlsbad, CA), in accordance with the manufacturers protocol.
114 RNA qualification and quantification was performed by Biotek (Winooski, VT, USA). A total of 2 µg RNA was
115 reverse transcribed to cDNA with Superscript III Reverse Transcriptase (Invitrogen). Quantitative Real Time PCRs
116 (qRT PCRs) were performed in an ABI StepOnePlus instrument, with the SYBR (TaKaRa) system, and a thermal
117 profile of 40 cycles of 95 °C for 10 s and 58 °C for 30 s. All results were standardized to the expression level of the
118 housekeeping gene,β actin. Relative mRNA expression levels were expressed as 2 ( Ct). Calculations were
119 carried out in Microsoft Office Excel. Primers are listed in the supplementary Table 1. 120 Draft 121 2.4 CpG island prediction and bisulfite sequencing PCR (BSP)
122 The human DACH1（ENST00000613252.4 ）sequence was obtained from the Ensembl genome browser
123 (http://asia.ensembl.org/index.html). MethPrimer software (http://www.urogene.org/methprimer) was used to
124 detect CpG islands, and to detect the first exon sequence,5000 bp upstream of the transcriptional start site (TSS).
125 The candidate transcription factor was predicted using JASPAR (http://jaspar.binf.ku.dk/).
126 DNA was extracted using a DNA extraction kit（Tiangen）, in accordance with the manufacturer’sprotocol.
127 DNA samples from each animal were individually processed and subjected to sodium bisulfite mediated
128 sequencing using EZ DNA Methylation Gold Kit (ZYMO), in accordance with the manufacturer’s protocol.
129 Primers were designed using online software to amplify the target sequence from the bisulfite modified DNA with
130 hot star polymerase (ZYMO).The PCR product was directly cloned into the pMD 19T vector(Takara) and
131 transformed into E.coli DH5α chemically competent cells (Takara). Ten individual clones from each sample were
132 picked and sequenced.
https://mc06.manuscriptcentral.com/bcb-pubs Page 7 of 25 Biochemistry and Cell Biology
133
134 2.5 Western-blot analyses
135 The monolayer cells were washed twice using 1×PBS. Proteins were extracted using RIPA lysis buffer(Beyotime,
136 Shanghai, China), and all specimens were centrifuged at 4 °C,10000 g for 10 min. Protein concentration was
137 measured using BCA protein assay kit(Beyotime, Shanghai, China). Proteins were resolved by 10% sodium
138 dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE, Beyotime, Shanghai, China), boiled for 5min at
139 99 °C, transferred to polyvinylidene fluoride (PVDF)membranes (Millipore, Bedford, MA, USA), and blocked
140 with 5% non fat dried milk in 1×TBS + Tween 20(TBS T). After blocking, membranes were incubated overnight
141 with anti DACH1antibody (1:1000, CST, Boston, USA) at 4 °C. PVDF membranes were then washed three times 142 with 1×TBS T for 15 min, and then incubatedDraft with secondary anti rabbit IgG antibody (1:5000; Zhong 143 Shan Golden Bridge Biological Technology Co., Ltd, Beijing, China) for 1 h. β Actin (1:1000; Zhong
144 Shan Golden Bridge Biological Technology Co., Ltd, Beijing, China) was used as an internal control. Protein
145 expression was detected and quantified using the SuperSignal West Pico chemiluminescent substrate (Pierce;
146 Thermo Fisher Scientific Inc, Waltham, MA, USA), with enhanced chemiluminescence (ECL) reagents (Millipore,
147 Bedford, MA, USA) to expose on an X ray film.
148
149 2.6 Luciferase reporter assay
150 296T cells were cultured in 24 well plates, and transfected with DACH1 luc and the corresponding transcriptional
151 expression vector, using Lipofectamine 2000 transfection reagent (Thermo Fisher, Waltham, MA, USA). After 24
152 h transfection time, luciferase activity was detected by the Dual Luciferase Reporter Assay System (Beyotime,
153 Shanghai, China), in accordance with the manufacturers protocol. Relative luciferase activity was normalized to
154 SV40 luciferase activity.
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 8 of 25
155
156 2.7 Cell proliferation assay
157 Cell proliferation was analyzed using the Cell Counting Kit 8 (Dojindo). After 2 d, A549 cells were inducible.
158 AzadC treated cells were then incubated for another 24 h. The optical density (OD) of each group was measured at
159 450 nm using a BioTek microplate reader.
160
161 2.8 Cell apoptosis assay
162 A549 cell apoptosis was analyzed by the flow cytometry method (FCM) using an Annexin V PI Apoptosis
163 Detection Kit (Abcam). Briefly, the cells were collected 2 d after AzadC treatment, washed with PBS, and 164 suspended in 500 µl binding buffer. The cellsDraft were incubated with Annexin V at room temperature for 10 min and 165 stained with propidium iodide (PI), and then analyzed by FCM for relative quantitative apoptosis.
166
167 2.9 Transwell invasion assay
168 The matrigel invasion assay was performed using 24 well transwell chambers (8 µm; Nunc, Thermo Fisher,
169 Waltham, USA). A549 cells were seeded into 6 well plates until they reached confluence, and transfected 24 h
170 prior to trypsinization, and resuspended in serum free RPMI 1640 medium. A total of 3×105cells were plated onto
171 Matrigel (1:8) invasion upper chambers(BD Biosciences, Franklin Lakes, NJ,USA).Then, 500 µl of RPMI 1640
172 medium containing 10% fetal bovine serum was placed in the lower chamber. After at least 24 h of incubation, the
173 cells on the upper surface of the matrigel (uninvaded cells) were removed with 1×PBS. Penetrated cells were fixed
174 with 95% carbinol for 20 min, stained with 0.1% crystal violet(Biosharp, Hefei, China), and scored under a light
175 microscope.
176
https://mc06.manuscriptcentral.com/bcb-pubs Page 9 of 25 Biochemistry and Cell Biology
177 2.10 Statistics
178 Data were presented as mean ± SD. Statistical differences between group pairs were determined by the student’s
179 t test. Statistical differences among groups were analyzed by a one way ANOVA, followed by Student Neuman
180 Keuls (S N K) testing. All experiments were repeated at least three times, and representative experiments were
181 shown. Differences were considered significant at p < 0.05.
182 3. Results
183 3.1 DACH1 expression is downregulated in NSCLC tissues and A549 NSCLC cell lines
184 DACH1 is a chromatin associated protein that associates with other DNA binding transcription factors to
185 regulate gene expression and cell fate determination during development (AYRES et al. 2001). To investigate 186 DACH1 expression in NSCLC tissue, NSCLCDraft tissue and the adjacent non tumor tissue from 35 patients were 187 sampled and subjected to qRT PCR analysis. mRNA levels were significantly decreased in NSCLC tissue
188 compared to in the adjacent non tumor tissue (Supplementary Fig 1A). A549 is a human NSCLC cell line, first
189 developed in 1972 by D. J. Guard, et al. through the removal and culturing of cancerous lung tissue in the
190 explanted tumor of a 58 year old Caucasian male (GIARD et al. 1973). We detected DACH1 expression in A549
191 cells. The qRT PCR results showed that DACH1 expression was significantly decreased in A549 NSCLC
192 compared to in the NHBEC cell line (Supplementary Fig 1B). H1650, SK MES 1 is another two NSCLC cell line,
193 for check DACH1 whether downregulate in the other NSCLC cell line, expression level of DACH1 was investigate
194 in H1650, SK MES 1 cell. The results showed that DACH1 expression was significantly decreased similar with
195 A549 NSCLC (Supplementary Fig 1B). These data reveal that DACH1 expression is down regulated in both
196 human NSCLC tissue and the NSCLC cell line.
197
198 3.2 Methylation levels are significantly increased in NSCLC tissues and A549 NSCLC cell lines
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 10 of 25
199 To check whetherDACH1 is downregulated by DNA methylation, DACH1 promoter methylation was
200 investigated using BSP. Because methylation often occurs at CpG islands in the gene promoter, we used online
201 software tools to locate them in the DACH1 promoter. The results identified two CpG islands within the 5 kb
202 DACH1 promoter. The first CpG island was 112 bp in length, and contained 10 CpG sites. The second CpG island
203 was 218 bp in length and contained 16 CpG sites (Figure 1A). To investigate the methylation rate of these CpG
204 islands, BSP was performed. The genomic DNA from 30 NSCLC tissue samples and 30 adjacent non tumor tissue
205 samples was treated with bisulfite, and then PCR was performed to sequence any changes at CpG sites. The results
206 showed that the DNA methylation ratio was significantly higher in the NSCLC tissue than in the adjacent
207 non tumor tissue (Figure 1B). We then checked the methylation pattern of A549 NSCLC cells. The results showed 208 that similar to the NSCLC tissue, the methylationDraft rate increased significantly in the A549 cells compared to in the 209 NHBEC cell line (Figure 1C). The same results was obtained in the H1650, SK MES 1 NSCLC cell line (Figure
210 1C). These data show that DNA methylation is increased in NSCLC tissue and the NSCLC cell line.
211
212 3.3 Analysis of theDACH1 promoter
213 The 5 kb DACH1 promoter sequence was obtained using the Ensembl genome browser. CpG islands were
214 identified using online software. In the 5 kb promoter region, two CpG islands were identified, the first stretching
215 from base 370 to base 500, and containing 10 CpG sites. The second CpG island stretched from base 40 to base
216 310, and contained 16 CpG sites. To check DACH1 gene modulation, the transcription factor binding sites of the
217 two CpG islands were predicted using online software. In the first island, transcription factors ARNT: Aryl
218 hydrocarbon receptor nuclear translocator; BCL6: B cell lymphoma 6 protein; MAFB: V maf musculoaponeurotic
219 fibrosarcoma oncogene homolog B; NKX2: Homeobox protein Nkx 2; NFIC: nuclear factor I C; SOX4:
220 Transcription factor SOX 4 and PRDM1: PR domain zinc finger protein 1 were predicted to interact with the CpG
https://mc06.manuscriptcentral.com/bcb-pubs Page 11 of 25 Biochemistry and Cell Biology
221 sites. In the second CpG island, transcription factors NFATC2: Nuclear factor of activated T cells, cytoplasmic 2;
222 NFKB1: Nuclear factor NF kappa B p105 subunit; EGR2: Early growth response protein 2; SMAD 2, 3, 4:
223 Mothers against decapentaplegic homolog 2, 3, and 4; E2F1: Transcription factor E2F1; TCF7L2: T cell
224 leukemia/lymphoma protein; ELK1: ETS domain containing protein Elk 1 were predicted to interact with the CpG
225 sites (Figure 2). The transcription factors E2F1, SOX4, ELK1, and NFKB1 have been identified in relation to
226 human NSCLC in previous research, hence we selected these four transcription factors for further investigation.
227
228 3.4 Methylated DACH1 promoter inhibits the transactivation of potential transcription factors
229 To investigate whether the transactivation of potential transcription factors was affected by DNA methylation, an 230 in vivo methylated DACH1 promoter co transfectionDraft with transcription factor luciferase assay was performed. In
231 previous experiments, E2F1 was shown to regulate small cell lung cancer invasion and metastasis (LI et al. 2014).
232 SOX4 gene mutation has been linked to the occurrence of lung cancer (CHEN et al. 2007; LI et al. 2015). Elk1
233 plays important roles in long term memory formation, drug addiction, Alzheimer's disease, Down syndrome, breast
234 cancer, and depression. In lung cancer, ELK1 is involved in the regulation of Ras active lung cancer by sensing
235 and transmitting abnormal Ras/MAPK signaling pathways (LIU et al. 2017). The study found that the recurrence of
236 metastatic lung cancer cells occurs through the NFκ B pathway to avoid EGFR targeted therapy, eventually
237 leading to the failure of targeted therapy (DEY et al. 2007). The results confirmed that transcription factors E2F1,
238 SOX4, ELK1, and NFKB1 activatedDACH1 luc expression. When DACH1 luc was methylated in vivo,
239 transcription factors E2F1, SOX4, ELK1, and NFKB1 were unable to activate DACH1 luc expression (Figure 3).
240 These data show that E2F1, SOX4, ELK1, and NFKB1 cannot activate the expression of methylated DACH1 luc,
241 revealing that the methylated DACH1 promoter affects the transactivation of potential transcription factors.
242
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 12 of 25
243 3.5 AzadC treatment increases the transactivation of potential DACH1transcription factors in A549 cells
244 We investigated whether treatment of A549 cells with the demethylation reagent AzadC would affect the
245 methylation patterns of the DACH1 promoter and DACH1 expression. BSP results showed that the methylation
246 ratio of theDACH1 promoter decreased after AzadC treatment (Figure 4A). qRT PCR revealed that DACH1
247 expression significantly increased after AzadC treatment (Figure 4B). Western blotting revealed that DACH1
248 protein levels significantly increased after AzadC treatment (Figure 4C).
249 We wanted to determine whether over expression of potential transcription factors increased demethylated
250 DACH1 gene expression. Therefore, A549 cells were co transfected with potential transcription factors. After
251 transfection, qRT PCR was performed to quantify DACH1 expression. The results showed that AzadC treatment 252 significantly increased DACH1 expressionDraft and that the transcription factors E2F1, SOX4, ELK1, and 253 NFKB1could not activate DACH1 expression alone. However, when E2F1, SOX4, ELK1, and NFKB1 were
254 treated along with AzadC, the expression of DACH1 expression increased significantly, compared to that observed
255 for AzadC alone or the transcription factor alone (Figure 4D 4G).These results revealed that demethylation
256 treatment increased the transactivation of potential transcription factors, and enabled high DACH1 expression in
257 A549 cells.
258
259 3.6 Effects of AzadC on the proliferation and apoptosis of A549 cells
260 Cellular proliferation and apoptosis have an important role in regulating cancer. We investigated
261 DACH1methylation patterns and their effect on A549 cell proliferation and apoptosis. After treatment with the
262 demethylation reagent AzadC, the CKK 8 and TUNEL experiment was performed to detectA549 cellular
263 proliferation and apoptosis. The results showed that AzadC inhibited A549 proliferation compared to that in the
264 untreated control (Figure 5A). For check the DACH1 function, overexpression was performed, the results shows
https://mc06.manuscriptcentral.com/bcb-pubs Page 13 of 25 Biochemistry and Cell Biology
265 that sole overexpression DACH obtain the same results similar AzadC treated (Figure 5A). Together DACH1
266 overexpression and AzadC treated showed more serious inhibit for cell proliferation (Figure 5A). The TUNEL
267 experiment showed that AzadC increased the apoptosis of A549 cells compared to that in the untreated control
268 (Figure 5B). The sole overexpression DACH obtain the same results similar AzadC treated (Figure 5A). Together
269 DACH1 overexpression and AzadC treated showed more serious inhibit for cell proliferation (Figure 5B).These
270 results indicate that decreased DACH1 methylation inhibits cellular proliferation and enhances apoptosis in A549
271 cells.
272
273 3.7 AzadC treatment decreases cancer cell migration and invasion in A549 cells 274 Malignant cancer cells are noted for their abilityDraft to migrate to and invade non cancerous tissue. To investigate the 275 alteration of DACH1promoter methylation patterns and its effects on A549 cell migration and invasion, a cell
276 transwell assay was performed. The results showed that demethylation using AzadC caused fewer cells to penetrate
277 through the Matrigel incomparison to the untreated control group (Figure 6A). The sole overexpression DACH
278 obtain the same results similar AzadC treated (Figure 6A). Together DACH1 overexpression and AzadC treated
279 showed more serious inhibit for cell migrate to and invade (Figure 6A).The transwell statistical data are shown in
280 Figure 6B. These results showed that decreasing DACH1methylation inhibited A549 cell migration and invasion.
281
282 4. Discussion
283 DACH1was previously observed to be less expressed in NSCLC compared to in normal tissue, but the underlying
284 mechanism was not understood (HAN et al. 2015). Here, we reported that increased methylation of the DACH1
285 promoter in patients with NSCLC maybe one reason for this. Alteration of methylation patterns in the DACH1
286 promoter disrupted the activation of candidate transcription factors and had observable effects on cell proliferation,
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 14 of 25
287 apoptosis, migration, and invasion.
288 The increased methylation rate in the DACH1 promoter causes it to be expressed less in lung cancer than in
289 normal tissue. We first checked DACH1 mRNA expression levels in lung cancer tissue and in the A549 lung cancer
290 cell line, and observed a decrease in expression. DACH1, one of the tumor suppressor genes, has been closely
291 associated with many tumors in recent years (WU et al. 2006; YAN et al. 2013; CHU et al. 2014). DACH1 is
292 involved in the transforming growth factor beta signaling pathway through the DS domain, which can regulate the
293 proliferation and differentiation of cells (WU et al. 2003). The low expression of DACH1is suggested as a
294 diagnostic tool for some cancers (ZHOU et al. 2009). The alteration of DACH1 promoter methylation has been
295 identified in breast cancers by MS‑PCR analysis (WU et al. 2006). In the DACH1 promoter, two CpG islands were 296 identified, and the methylation rate was significantlyDraft increased in the two CpG islands, which contain a total of 26 297 CpG sites. To check whether decreased mRNA expression was due to this increased DNA methylation, the
298 demethylation reagent AzadC was used to treat A549 cells. AzadC treatment decreased theDACH1promoter
299 methylation rate, and increased mRNA and protein expression. We concluded that high DACH1 promoter
300 methylation is a major contributor to decreased DACH1 expression.
301 Alteration of the DACH1 promoter disrupted the transactivation of transcription factors. DNA methylation
302 can result in changes to chromatin structure, DNA conformation, DNA stability, and DNA:protein interactions,
303 which can control gene expression (MARTINOWICH et al. 2003; JAHAN and DAVIE 2015).Using online promoter
304 analysis software, many transcription factors were predicted to transactiveDACH1 expression. To check whether
305 the alteration of DNA methylation affects the action of its transcription factors, we chose four candidate
306 transcription factors; E2F1, SOX4, ELK1, and NFKB1. These four transcription factors have been reported to
307 promote NSCLC and have predicted target binding sites located at the CpG sites in theDACH1 promoter (CHEN et
308 al. 2007; LI et al. 2014). Our in vivo cell transfection experiment showed thatE2F1, SOX4, ELK1, and NFKB1
https://mc06.manuscriptcentral.com/bcb-pubs Page 15 of 25 Biochemistry and Cell Biology
309 increased DACH1 luc expression. When treated with the demethylation reagent AzadC, E2F1, SOX4, ELK1, and
310 NFKB1 significantly upregulated DACH1 expression, compared to that in the untreated A549 cells. These data
311 revealed that alteration of the DACH1 promoter disrupted the transactivation of transcription factors.
312 DACH1has been reported to inhibit lung adenocarcinoma invasion and tumor growth (HAN et al. 2015; WU et al.
313 2015). To check for alterations in DACH1 promoter methylation and whether this affects cell behavior, we
314 performed a series of experiments. Decreased DACH1 promoter DNA methylation increased A549 cell apoptosis
315 and decreased cell proliferation, migration, and invasion. This indicates that methylation of the DACH1 promoter
316 maybe useful as a new therapeutic target and diagnostic marker for NSCLC (KONG et al. 2016).
317 We conclude that DACH1shows high methylation states in NSCLC, which may decrease its expression through 318 disrupted transactivation of candidate transcriptionDraft factors, contributing to NSCLC. Our research provides new 319 insight into NSCLC pathogenesis and identifies a potential new therapeutic target that may improve disease
320 outcomes.
321 Acknowledgments
322 This study was supported by the National Natural Science Foundation of China (Youth Science Foundation,
323 #81502500) , the Natural Science Foundation of Jiangsu Province (Youth Foundation, # BK20150293), and the
324 Postdoctoral research funding (NO:32317403) from Soochow University.
325
326 Compliance with ethical standards
327
328 Conflict of interest: The authors declare that they have no conflict of interest.
329
330
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 16 of 25
331 Ambros, V., 2004 The functions of animal microRNAs. Nature 431: 350-355. 332 Ayres, J. A., L. Shum, A. N. Akarsu, R. Dashner, K. Takahashi et al., 2001 DACH: genomic 333 characterization, evaluation as a candidate for postaxial polydactyly type A2, and 334 developmental expression pattern of the mouse homologue. Genomics 77: 18-26. 335 Backman, M., O. Machon, C. J. Van Den Bout and S. Krauss, 2003 Targeted disruption of mouse Dach1 336 results in postnatal lethality. Dev Dyn 226: 139-144. 337 Bestor, T. H., 2000 The DNA methyltransferases of mammals. Hum Mol Genet 9: 2395-2402. 338 Bonini, N. M., W. M. Leiserson and S. Benzer, 1998 Multiple roles of the eyes absent gene in 339 Drosophila. Dev Biol 196: 42-57. 340 Brown, R., and G. Strathdee, 2002 Epigenomics and epigenetic therapy of cancer. Trends Mol Med 8: 341 S43-48. 342 Cedar, H., 1988 DNA methylation and gene activity. Cell 53: 3-4. 343 Chen, K., K. Wu, S. Cai, W. Zhang, J. Zhou et al., 2013 Dachshund binds p53 to block the growth of lung 344 adenocarcinoma cells. Cancer Res 73: 3262-3274. 345 Chen, Q. L., W. L. Zheng, W. J. Yao, L. W. Nie, S. H. Cheng et al., 2007 Analysis of SOX4 gene mutation in 346 non-small cell lung cancer tissues. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 24: 505-509. 347 Chu, Q., N. Han, X. Yuan, X. Nie, H. Wu et al., 2014 DACH1 inhibits cyclin D1 expression, cellular 348 proliferation and tumor growth of renal cancer cells. J Hematol Oncol 7: 73. 349 Dey, A., E. T. Wong, P. Bist, V. Tergaonkar and D. P. Lane, 2007 Nutlin-3 inhibits the NFkappaB pathway 350 in a p53-dependent manner: implicationsDraft in lung cancer therapy. Cell Cycle 6: 2178-2185. 351 Garofalo, M., G. Romano, G. Di Leva, G. Nuovo, Y. J. Jeon et al., 2011 EGFR and MET receptor tyrosine 352 kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung 353 cancers. Nat Med 18: 74-82. 354 Giard, D. J., S. A. Aaronson, G. J. Todaro, P. Arnstein, J. H. Kersey et al., 1973 In vitro cultivation of 355 human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer 356 Inst 51: 1417-1423. 357 Hallqvist, A., B. Bergman and J. Nyman, 2012 Health related quality of life in locally advanced NSCLC 358 treated with high dose radiotherapy and concurrent chemotherapy or cetuximab--pooled 359 results from two prospective clinical trials. Radiother Oncol 104: 39-44. 360 Han, N., X. Yuan, H. Wu, H. Xu, Q. Chu et al., 2015 DACH1 inhibits lung adenocarcinoma invasion and 361 tumor growth by repressing CXCL5 signaling. Oncotarget 6: 5877-5888. 362 Jabbari, K., and G. Bernardi, 2004 Cytosine methylation and CpG, TpG (CpA) and TpA frequencies. 363 Gene 333: 143-149. 364 Jahan, S., and J. R. Davie, 2015 Protein arginine methyltransferases (PRMTs): role in chromatin 365 organization. Adv Biol Regul 57: 173-184. 366 Kong, D., Y. Liu, Q. Liu, N. Han, C. Zhang et al., 2016 The retinal determination gene network: from 367 developmental regulator to cancer therapeutic target. Oncotarget 7: 50755-50765. 368 Li, Y., L. Zu, Y. Wang, M. Wang, P. Chen et al., 2015 miR-132 inhibits lung cancer cell migration and 369 invasion by targeting SOX4. J Thorac Dis 7: 1563-1569. 370 Li, Z., Y. Guo, H. Jiang, T. Zhang, C. Jin et al., 2014 Differential regulation of MMPs by E2F1, Sp1 and 371 NF-kappa B controls the small cell lung cancer invasive phenotype. BMC Cancer 14: 276. 372 Liu, C. Y., T. T. Huang, C. T. Huang, M. H. Hu, D. S. Wang et al., 2017 EGFR-independent Elk1/CIP2A 373 signalling mediates apoptotic effect of an erlotinib derivative TD52 in triple-negative breast 374 cancer cells. Eur J Cancer 72: 112-123.
https://mc06.manuscriptcentral.com/bcb-pubs Page 17 of 25 Biochemistry and Cell Biology
375 Liu, Y., N. Han, S. Zhou, R. Zhou, X. Yuan et al., 2016 The DACH/EYA/SIX gene network and its role in 376 tumor initiation and progression. Int J Cancer 138: 1067-1075. 377 Lokk, K., T. Vooder, R. Kolde, K. Valk, U. Vosa et al., 2012 Methylation markers of early-stage non-small 378 cell lung cancer. PLoS One 7: e39813. 379 Martinowich, K., D. Hattori, H. Wu, S. Fouse, F. He et al., 2003 DNA methylation-related chromatin 380 remodeling in activity-dependent Bdnf gene regulation. Science 302: 890-893. 381 Nan, F., Q. Lu, J. Zhou, L. Cheng, V. M. Popov et al., 2009 Altered expression of DACH1 and cyclin D1 in 382 endometrial cancer. Cancer Biol Ther 8: 1534-1539. 383 Popov, V. M., J. Zhou, L. A. Shirley, J. Quong, W. S. Yeow et al., 2009 The cell fate determination factor 384 DACH1 is expressed in estrogen receptor-alpha-positive breast cancer and represses estrogen 385 receptor-alpha signaling. Cancer Res 69: 5752-5760. 386 Rual, J. F., K. Venkatesan, T. Hao, T. Hirozane-Kishikawa, A. Dricot et al., 2005 Towards a 387 proteome-scale map of the human protein-protein interaction network. Nature 437: 388 1173-1178. 389 Stupp, R., C. Monnerat, A. T. Turrisi, 3rd, M. C. Perry and S. Leyvraz, 2004 Small cell lung cancer: state 390 of the art and future perspectives. Lung Cancer 45: 105-117. 391 Torre, L. A., F. Bray, R. L. Siegel, J. Ferlay, J. Lortet-Tieulent et al., 2015 Global cancer statistics, 2012. CA 392 Cancer J Clin 65: 87-108. 393 Wu, K., A. Li, M. Rao, M. Liu, V. Dailey et al., 2006 DACH1 is a cell fate determination factor that 394 inhibits cyclin D1 and breast tumorDraft growth. Mol Cell Biol 26: 7116-7129. 395 Wu, K., Y. Yang, C. Wang, M. A. Davoli, M. D'Amico et al., 2003 DACH1 inhibits transforming growth 396 factor-beta signaling through binding Smad4. J Biol Chem 278: 51673-51684. 397 Wu, K., X. Yuan and R. Pestell, 2015 Endogenous Dach1 in cancer. Oncoscience 2: 803-804. 398 Yan, W., K. Wu, J. G. Herman, M. V. Brock, F. Fuks et al., 2013 Epigenetic regulation of DACH1, a novel 399 Wnt signaling component in colorectal cancer. Epigenetics 8: 1373-1383. 400 Yanaihara, N., N. Caplen, E. Bowman, M. Seike, K. Kumamoto et al., 2006 Unique microRNA molecular 401 profiles in lung cancer diagnosis and prognosis. Cancer Cell 9: 189-198. 402 Yu, S. L., H. Y. Chen, G. C. Chang, C. Y. Chen, H. W. Chen et al., 2008 MicroRNA signature predicts 403 survival and relapse in lung cancer. Cancer Cell 13: 48-57. 404 Zappa, C., and S. A. Mousa, 2016 Non-small cell lung cancer: current treatment and future advances. 405 Transl Lung Cancer Res 5: 288-300. 406 Zhou, J., W. Zhang, W. Dampier, M. Wang, Z. R. Yu et al., 2009 DACH1 is a cell-fate determination 407 factor that governs Forkhead protein function in tumorigenesis. Cancer Research 69. 408 Zhu, J., C. Wu, H. Li, Y. Yuan, X. Wang et al., 2016 DACH1 inhibits the proliferation and invasion of lung 409 adenocarcinoma through the downregulation of peroxiredoxin 3. Tumour Biol 37: 9781-9788. 410 Zugazagoitia, J., S. Molina-Pinelo, F. Lopez-Rios and L. Paz-Ares, 2017 Biological therapies in nonsmall 411 cell lung cancer. Eur Respir J 49.
412
413
414 Figure legends
415 Figure 1: Methylation level was significantly increased in the non small cell lung cancer (NSCLC) tissues and
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 18 of 25
416 A549 ，H1650， SK MES 1 NSCLC cells. (A) Two CpG islands were predicted in the DACH1 promoter region.
417 The first CpG Island is 112 bp and contains 10 CpG sites. The second CpG island is 218 bp and contains 16 CpG
418 sites. The methylated primer (purplearrowhead) was designed to perform methylated PCR, to check the
419 methylation rate in NSCLC tissues and NSCLC cell line A549. (B) The methylation rate was significantly
420 increased in NSCLC tissues compared to in normal tumor adjacent lung tissues (t-test, P＜0.001). (C) In the A549
421 NSCLC cell line, DACH1 promoter methylation was significantly increased compared to in normal human
422 bronchial epithelial cells (NHBECs) (t-test, P＜0.001).
423 Figure 2: DACH1 promoter analysis. Using online software, a large CpG island was predicted in the DACH1
424 promoter. This CpG island stretched from the transcriptional start site to 1038 bp (indicated in red), and contained 425 107 CpG sites (indicated in yellow). Many potentialDraft transcription factor binding sites were predicted (underlined in 426 red). The full names of the transcription factors are as follows: ARNT: Aryl hydrocarbon receptor nuclear
427 translocator; BCL6: B cell lymphoma 6 protein; MAFB: V maf musculoaponeurotic fibrosarcoma oncogene
428 homolog B; NKX2: Homeobox protein Nkx 2; NFIC: nuclear factor I C; SOX4: Transcription factor SOX 4 and
429 PRDM1: PR domain zinc finger protein 1; NFKB1: Nuclear factor NF kappa B p105 subunit; EGR2: Early
430 growth response protein 2; SMAD 2, 3, 4: Mothers against decapentaplegic homolog 2, 3, and 4; E2F1:
431 Transcription factor E2F1; TCF7L2: T cell leukemia/lymphoma protein; ELK1: ETS domain containing protein
432 Elk 1.
433 Figure 3: Candidate transcription factor transactivation of DACH1 expression in cell transfection experiment. (A)
434 The transcription factor E2F1 co transfected with DACH1 luc or methylated DACH1 luc. (B) The transcription
435 factor SOX4 co transfected with DACH1 luc or methylated DACH1 luc. (C) The transcription factor ELK1
436 co transfected with DACH1 luc or methylated DACH1 luc. (D) The transcription factor NFKB1 co transfected
437 with DACH1 luc or methylated DACH1 luc.
https://mc06.manuscriptcentral.com/bcb-pubs Page 19 of 25 Biochemistry and Cell Biology
438 Figure 4: Demethylase reagent AzadC treatment and effect on DACH1 expression and protein level in A549 cells.
439 (A) After treatment, DACH1 promoter methylation level was measured, which showed a significantly decreased
440 methylation rate. (B) After treatment, DACH1 mRNA level significantly increased. (C) After treatment, DACH1
441 protein level significantly increased. (C D). Demethylase reagent AzadC treatment increased DACH1 expression,
442 and increased transcriptional activity of transcriptional factor (E2F1, SOX4, ELK1 and NFKB).
443 Figure 5: Effects of AzadC and overexpression DACH1 on the proliferation and apoptosis of A549 cells. (A) A
444 CCK 8 experiment was performed to analyze the proliferation of AzadC treated A549 cells. AzadC treatment
445 significantly inhibited A549 cell proliferation. (B) Apoptosis was analyzed using flow cytometric analysis before
446 and after AzadC treatment and overexpression DACH1 of A549 cells. AzadC treatment, overexpression DACH 447 significantly inhibited A549 cell apoptosis, Draft Over: overexpression. 448 Figure 6: The invasion effect of A549 cells was investigated using a transwell matrigel assay. (A) Invasion effect
449 of A549 cells after AzadC treated and DACH1 overexpression. (B) Statistical data show that AzadC treatment and
450 overexpression DACH significantly inhibited the invasion effect of A549 cells. Over: overexpression.
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 20 of 25
Draft
Figure 1: Methylation level was significantly increased in the non-small cell lung cancer (NSCLC) tissues and A549 ，H1650， SK-MES-1 NSCLC cells. (A) Two CpG islands were predicted in the DACH1 promoter region. The first CpG Island is 112 bp and contains 10 CpG sites. The second CpG island is 218 bp and contains 16 CpG sites. The methylated primer (purplearrowhead) was designed to perform methylated PCR, to check the methylation rate in NSCLC tissues and NSCLC cell line A549. (B) The methylation rate was significantly increased in NSCLC tissues compared to in normal tumor-adjacent lung tissues (t-test, P＜0.001). (C) In the A549 NSCLC cell line, DACH1 promoter methylation was significantly increased compared to in normal human bronchial epithelial cells (NHBECs) (t-test, P＜0.001).
133x86mm (300 x 300 DPI)
https://mc06.manuscriptcentral.com/bcb-pubs Page 21 of 25 Biochemistry and Cell Biology
Draft
Figure 2: DACH1 promoter analysis. Using online software, a large CpG island was predicted in the DACH1 promoter. This CpG island stretched from the transcriptional start site to -1038 bp (indicated in red), and contained 107 CpG sites (indicated in yellow). Many potential transcription factor binding sites were predicted (underlined in red). The full names of the transcription factors are as follows: ARNT: Aryl hydrocarbon receptor nuclear translocator; BCL6: B-cell lymphoma 6 protein; MAFB: V-maf musculoaponeurotic fibrosarcoma oncogene homolog B; NKX2: Homeobox protein Nkx-2; NFIC: nuclear factor I C; SOX4: Transcription factor SOX-4 and PRDM1: PR domain zinc finger protein 1; NFKB1: Nuclear factor NF-kappa-B p105 subunit; EGR2: Early growth response protein 2; SMAD 2, 3, 4: Mothers against decapentaplegic homolog 2, 3, and 4; E2F1: Transcription factor E2F1; TCF7L2: T-cell leukemia/lymphoma protein; ELK1: ETS domain-containing protein Elk-1.
239x164mm (96 x 96 DPI)
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 22 of 25
Draft
Figure 3: Candidate transcription factor transactivation of DACH1 expression in cell transfection experiment. (A) The transcription factor E2F1 co-transfected with DACH1-luc or methylated DACH1-luc. (B) The transcription factor SOX4 co-transfected with DACH1-luc or methylated DACH1-luc. (C) The transcription factor ELK1 co-transfected with DACH1-luc or methylated DACH1-luc. (D) The transcription factor NFKB1 co- transfected with DACH1-luc or methylated DACH1-luc.
78x63mm (300 x 300 DPI)
https://mc06.manuscriptcentral.com/bcb-pubs Page 23 of 25 Biochemistry and Cell Biology
Figure 4: Demethylase reagent AzadC treatment and effect on DACH1 expression and protein level in A549 cells. (A) After treatment, DACH1 promoter methylation level was measured, which showed a significantly decreased methylation rate. (B) After treatment, DACH1 mRNA level significantly increased. (C) After treatment, DACH1 protein level significantly increased. (C-D). Demethylase reagent AzadC treatment increased DACH1 expression, and increased transcriptional activity of transcriptional factor (E2F1, SOX4, ELK1 and NFKB). Draft 78x33mm (300 x 300 DPI)
https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 24 of 25
Figure 5: Effects of AzadC and overexpression DACH1 on the proliferation and apoptosis of A549 cells. (A) A CCK-8 experiment was performed to analyze the proliferation of AzadC-treated A549 cells. AzadC treatment significantly inhibited A549 cell proliferation. (B) Apoptosis was analyzed using flow cytometric analysis before and after AzadC treatment and overexpression DACH1 of A549 cells. AzadC treatment, overexpression DACH significantlyDraft inhibited A549 cell apoptosis, Over: overexpression.
176x83mm (300 x 300 DPI)
https://mc06.manuscriptcentral.com/bcb-pubs Page 25 of 25 Biochemistry and Cell Biology
Figure 6: The invasion effect of A549 cells was investigated using a transwell matrigel assay. (A) Invasion effect of A549 cells after AzadC treated and DACH1 overexpression. (B) Statistical data show that AzadC treatment and overexpression DACH significantly inhibited the invasion effect of A549 cells. Over: overexpression. Draft 192x94mm (300 x 300 DPI)
https://mc06.manuscriptcentral.com/bcb-pubs