bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 DNMT family induced down-regulation of NDRG1 via DNA methylation and
2 clinicopathological significance in gastric cancer
3 Xiaojing Chang1*, Jinguo MA2*, Xiaoying Xue1, Guohui Wang1, Linlin Su1, Xuetao
4 Han1, Huandi Zhou3, Liubing Hou3.
5 1 Department of Radiotherapy, The Second Hospital of Hebei Medical University,
6 Shijiazhuang , China.
7 2 Hulun Buir People’s hospital, Hulun Buir Medical school in nationalities University
8 of inner Mongolia, Hulun Buir, China.
9 3Department of Central Laboratory, The Second Hospital of Hebei Medical
10 University, Shijiazhuang, China
11 *Xiaojing Chang and Jinguo MA have contributed equally to this manuscript.
12 Corresponding author: Xiaoying Xue, MD, PhD, Professor, Department of
13 Radiotherapy, The Second Hospital of Hebei Medical University, NO.215 Heping
14 West Road, Shijiazhuang, China. E-mail:[email protected]. 15 E-mail addresses of authors:
16 Xiaojing Chang: [email protected] 17 Jinguo MA: [email protected] 18 Xiaoying Xue: [email protected]/[email protected] 19 Guohui Wang: [email protected] 20 Linlin Su:[email protected] 21 Xuetao Han :[email protected] 22 Huandi Zhou: [email protected] 23 Liubing Hou: [email protected]
24 Running title:
25 The relationship of DNA methylation of NDRG1and DNMT family in gastric cancer .
26 Funding statement: The authors received funding through the National Natural
27 Science Foundation of Hebei province of China(#H2018206180).
28 Conflict of interest statement: No conflicts of interest are declared by the authors.
29 Ethical approval statement: Our study did not require an ethical board approval
30 because it did not contain human or animal trials.
1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
31 DNMT family induced down-regulation of NDRG1 via DNA methylation and
32 clinicopathological significance in gastric cancer
33 Abstract Background Aberrant DNA methylation of tumor suppressor genes is a
34 common event in the development and progression of gastric cancer(GC). Our
35 previous study showed NDRG1, which could suppress cell invasion and migration,
36 was frequently down-regulated by DNA methylation of its promoter in GC. Purpose
37 and Methods To analyze the relationship between the expression and DNA
38 methylation of NDRG1 and DNA methyltransferase (DNMT) family. We performed
39 a comprehensive comparison analysis using 407 patients including sequencing
40 analysis data of GC from TCGA. Results NDRG1 was negatively correlative to
41 DNMT1（p =0.03）, DNMT3A（p =0.01）, DNMT3B（p =0.88）, respectively.
42 Whereas, the DNA methylation of NDRG1 was positively correlative to DNMT
43 family(DNMT1 p<0.01, DNMT3A p<0.001 ， DNMT3B p=0.57, respectively).
44 NDRG1 expression was significantly inverse correlated with invasion depth (p
45 =0.023), and DNMT1 was significantly positive correlated with the degree of tumor
46 cell differentiation (p =0.049). DNMT3B was significantly correlated with tumor cell
47 differentiation (p =0.030). However, there was no association between the expression
48 of DNMT3A and clinicopathological features. The univariate analysis showed that
49 NDRG1and DNMTs had no association with prognosis of GC patients. But,
50 multivariate analysis showed DNMT1 was significantly correlated with prognosis of
51 GC patients. Conclusion These data suggest that down-regulation of NDRG1 in
52 gastric cancer is due to DNA methylation of NDRG1 gene promoter via DNMT
2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
53 family. The demethylating agent maybe a potential target drug for GC patients.
54 Keywords: Gastric carcinoma, tumor suppressor gene, NDRG1, DNMT, DNA
55 methylation, metastasis
56 Introduction
57 Gastric carcinoma (GC) is one of the most common digestive malignancies
58 worldwide with the third most common cause of cancer-related death1, 2. The poor
59 prognosis and high recurrence rate are mainly due to lymph node metastasis in its
60 early stage.
61 To date, a number of laboratories have shown that the occurrence of GC is usually
62 caused by oncogenes activation and tumor suppressor genes(TSGs) inactivation.
63 TSGs could suppress tumor cells migration and invasion. Studies have shown that the
64 inactivation of most TSGs are related to DNA methylation of CpG, while the
65 silencing of these genes possibly contributes to the development and progression of
66 tumors3-5. There is increasing evidence that aberrant DNA promoter methylation of
67 TSGs is a major event in the development and progression of gastric cance6. Our
68 previous studies showed NDRG1(N-myc downstream-regulated gene 1), a
69 differentiation-related and tumor suppressor gene, which could suppress cancer cells
70 invasion and migration, was frequently down-regulated by DNA methylation of its
71 promoter in GC，but there were not any mutations in NDRG1 cDNA, and also not
72 significant correlation with histone acetylation 7, 8.
73 DNA methylation of CpG islands, which is carried out
74 by DNA methyltransferase (DNMT) enzymes, is the most widely and the best well
75 studied epigenetic modification event, and leads to transcriptional gene silencing9.
76 DNA methyltransferase (DNMT) enzymes include DNMT1, DNMT2, DNMT3A,
77 DNMT3B, and DNMT3L, among which DNMT1(a maintenance DNMT), DNMT3A
78 and DNMT3B(de novo methyltransferases) are the most important10. In the current
79 study, we focused on the relationship between NDRG1 and DNMT family(DNMT1,
80 DNMT3A and DNMT3B) and their clinical significance in GC by analyzing
81 high-throughput data obtained from TCGA. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
82 Materials and methods
83 The data that we investigated the mRNA expression of NDRG1 and DNMTs, and
84 the DNA methylation of NDRG1 in GC was obtained from the data portal of TCGA
85 （https://portal.gdc.cancer.gov/）, which is a well-known cancer research project that
86 collects and analyzes high-quality tumor samples and makes the related data available
87 to researchers. We obtained the GC data set (March 1st, 2018 updated), which
88 encompasses 407 gastric tumor samples, and of which 338 cases encompasses the
89 data of DNA methylation of NDRG1. Total of 315 cases contain the complete
90 clinicopathological information.
91 Gastric Cells and Culture
92 Human gastric cancer cell lines, SGC7901 and MKN45 were obtained from the
93 Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai,
94 China), and one immortalized normal gastric cell line, GES1 was obtained from the
95 Oncology Institute of China Medical University. Cells were cultured with RPMI 1640
96 (Invitrogen/Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum
97 (Invitrogen/Gibco) in a humidified atmosphere of 5 % CO2 at 37 C.
98 Protein Isolation and Western Blot
99 Total cellular protein was extracted from cultured cells and western bolt was
100 performed as previous description7. Protein bands were scanned and quantified using
101 densitometric software (Bio-Rad, California, USA). A rabbit monoclonal
102 anti-NDRG1 antibody was at a dilution of 1:1,000 (Cell Signaling Technology,
103 Danvers, MA, USA). DNMT1, DNMT3A and DNMT3B antibody at a dilution of
104 1:500 were purchased from Bioss, Beijing, China.
105 Statistics
106 All statistical analyses were conducted using SPSS 24.0 (Chicago, IL, USA) and R
107 3.6.1 (https://www.r-project. org/). R language and GraphPad Prism 7 (San Diego,
108 CA, USA) were performed to draw plots. X2 test and Fisher’s exact test were used to
109 generate P values, Values with p < 0.05 are considered as statistically significant.
110 Results
4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
111 NDRG1 was negatively correlative to DNMT family
112 Total of 407 gastric cancer cases （ 375 was gastric cancer tissues and 32 was
113 nonmalignant gastric tissues ） which included sequencing analysis data were
114 downloaded form TCGA, then we performed a comprehensive comparison analysis
115 about the mRNA expression of NDRG1 and DNMTs and their clinicopathological
116 features. As shown in Figure 1A-C, NDRG1 mRNA expression was negatively
117 correlative to DNMT1（r=-0.1067, p =0.03）, DNMT3A（r=-0.1015, p =0.01）,
118 DNMT3B（r=-0.0069, p =0.88）. Noteworthy, NDRG1 was significantly associated
119 with DNMT1(p =0.03) and DNMT3A （ p =0.01 ） , and DNMT3A showed the
120 strongest association. However, there was no significant association between NDRG1
121 and DNMT3B (p=0.8885).
122 To confirm these results, we further assessed the protein levels of NDRG1 and
123 DNMTs in one immortalized normal gastric cell line, GES1 and two gastric cell lines,
124 SGC7901 and MKN45 by western blot. Results showed DNMT1, DNMT3A and
125 DNMT3B were up-regulated in SGC7901 and MKN45 cells compared with GES1,
126 while NDRG1 expression was inversed (p < 0.05, Fig. 2). This was in accordance to
127 the results of the relationship between NDRG1 and DNMT family mRNA expression
128 from TCGA.
129 Methylation of NDRG1 gene promoter was positively correlative to the
130 expression DNMT family
131 Among all of 407 GC cases, 338 cases encompass the data of DNA methylation of
132 NDRG1. Results showed DNA methylation of NDRG1 gene promoter was positively
133 correlative to the expression DNMT family, and was significantly associated with
134 DNMT1 (p =0.002, Fig.1D) and DNMT3A (p <0.001, Fig.1E). However, no
135 statistical significance was found between NDRG1 and DNMT3B (p =0.57, Fig. 1F).
136 The association of NDRG1 and DNMT family with the clinicopathological
137 parameters and prognosis of GC
138 The association of NDRG1 and DNMT family with the clinicopathological
139 parameters of gastric cancer patients were shown in Table 1. Total of 315 cases which
5 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
140 contain the complete clinicopathological and survival information were analyzed. The
141 high and low expression of NDRG1 and DNMTs was based on the median value of
142 their mRNA levels. We found NDRG1 expression was significantly inverse correlated
143 with invasion depth (p =0.023), whereas DNMT1 was statistically significantly
144 positive correlated with invasion depth (p = 0.049), DNMT3B was significantly
145 positive correlated with the degree of tumor cell differentiation (p =0.030). There was
146 no significant association between DNMT3A and the clinicopathological parameters
147 of the GC patients (Table 1).
148 Univariate analysis of prognosis showed that age, invasion depth, lymph metastasis
149 and stage were significantly inverse correlated with prognosis of GC patients. But
150 NDRG1and DNMTs showed no association with prognosis of GC patients(Fig. 3).
151 Whereas, Multivariate analysis of prognosis showed age, stage and DNMT1 were
152 significantly correlated with prognosis of GC patients. But NDRG1, DNMT3A
153 andDNMT3B showed no association with prognosis(Fig. 4).
154 Discussion
155 NDRG1, a differentiation-related gene, which belongs to NDRG family, is
156 down-regulated in many tumors, and proposed as a metastasis suppressor gene11-14.
157 Our previous studies confirmed the anti-cancer effect of NDRG1 in GC7, 8, 15. Gene
158 network analysis shows that the 5 'end of the NDRG1 gene contains a large CpG
159 island. Some studies have confirmed the downregulation of NDRG1 caused by DNA
160 methylation of CpG islands of its promoter in breast cancer, prostate cancer cells and
161 pancreatic cancer16-18. In GC, the down-regulation of NDRG1 was also found to be
162 regulated by DNA methylation of its promoter in our previous study. DNA
163 methylation is carried out by DNA methyltransferase (DNMT) enzymes. To date, not
164 any studies reported the relationship of NDRG1 and DNMTs in GC.
165 In the current study, total 407 GC cases which included sequencing analysis data
166 of NDRG1 mRNA were analyzed. We found that NDRG1 mRNA level was
167 negatively correlative to DNMT1, DNMT3A and DNMT3B, and DNMT3A showed
168 the strongest association. No significant association was found between NDRG1 and
6 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
169 DNMT3B. The results of western blot analysis confirmed these data from TCGA.
170 This is the first time for analyzing the relationship between NDRG1 and DNMT
171 family. NDRG2, another member of NDRG family which includes four members
172 NDRG1-4, was reported to be significantly decreased in human cancers, and in GC, H.
173 pylori silenced Ndrg2 by activating the NF-κB pathway and up-regulating DNMT3b,
174 promoting gastric cancer progression19. Our current data were similar to those of
175 NDRG2. Noteworthy, our data showed that aberrant DNA methylation of NDRG1was
176 mainly due to DNMT3A.
177 We further investigate the association of DNA methylation of NDRG1 and DNMTs.
178 Total 338 of all 407 GC cases encompasses the data of DNA methylation of NDRG1.
179 Conversely, DNA methylation of NDRG1 was positively correlative to the expression
180 of DNMTs, and was significantly associated with DNMT1 and DNMT3A.These
181 results once again confirmed the relationship of NDRG1 mRNA expression and
182 DNMTs in GC. Whereas, NDRG1 expression was significantly inverse correlated
183 with invasion depth, and DNMT1 was positively correlated with invasion depth.
184 DNMT3B was significantly positive correlated with the degree of tumor cell
185 differentiation. But, the univariate analysis of prognosis showed no association
186 between NDRG1and DNMTs with the prognosis of GC patients. While, multivariate
187 analysis of prognosis showed only DNMT1 was significantly correlated with
188 prognosis of GC patients. These results were mainly based on the data of
189 bioinformatics, the current data remain suggest that high NDRG1 expression and low
190 DNMTs expression could suppress cell invasion and migration, may improved the
191 rate of recurrence and metastasis. Our previous study had showed GC patients with
192 high expression of NDRG1 had better overall survival rate than those with low
193 NDRG1 expression8. So large sample data of immunohistochemistry, western blot
194 and RT-PCR of GC tissues were urgently needed.
195 DNMT inhibitor, including azacitidine, 5-aza-2′-deoxycytidine (5-Aza-Dc,
196 decitabine) , guadecitabine, hydralazine, procaine, MG98 and/or zebularine, among
197 which 5-Aza-Dc is the most common used, could cause demethylating and reactivate
7 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
198 the expression of TSGs, then suppress the metastasis of tumor cell20-22. NDRG1
199 expression was found to be increased after treatment with 5-Aza-Dc in breast cancer,
200 prostate cancer and pancreatic cancer16-18. Lee E et al23 found that the epidrug 5-Aza
201 could up-regulated NDRG1 expression by reduction of suppressive histone marks,
202 H3K9me3 and H3K27me3 on NDRG1 promoter in prostate cancer.
203 In the current study, the aberrant DNA methylation of NDRG1was found to be
204 significantly associated with DNMT family, especially DNMT1 and DNMT3A. Our
205 previous study showed that 5-Aza-Dc could up-regulate the expression of NDRG1 in
206 GC cells. It suggests that the demethylating agent maybe a potential target drug in GC.
207 Up to now, demethylating treatment has been used for the treatment of myeloid
208 leukemias24, 25. In vitro studies of solid tumors, demethylating agents combined
209 chemotherapy and/or immunotherapy could enhances the therapeutic effect in cancer
210 cells26-29. In a case report, azacitidine was used to treat a 57-year-old woman newly
211 diagnosed with MDS during palliative chemotherapy for metastatic breast cancer,
212 azacitidine showed promising effects for MDS, and also stabilized the patient's lung
213 and lymph node metastases without any major toxicity30. Now, An increasing number
214 of clinical trials of demethylating treatment is ongoing，some have presented exciting
215 effects31-34（Table 2）. A Phase I study showed the security of decitabine treated by
216 hepatic arterial infusionin, and a dose level of 20 mg/m2/day on five consecutive days
217 every 4 weeks could be considered for further investigation in combinatorial
218 immunotherapy regimens in patients with Unresectable Liver-predominant Metastases
219 From Colorectal Cancer33. In a phase I/II clinical trial, 15 patients with metastatic
220 castration-resistant prostate cancer (mCRPC) were enrolled in phase I which is dose
221 climbing experiment of 5-Aza-Dc, and 7 patients were enrolled in phase II. In phase I,
222 no dose-limiting toxicity (DLT) was observed, and most patients were well tolerated.
223 The highest level reached was 5-Aza-Dc with 150 mg/m2 daily for 5 days followed
224 by docetaxel with 75 mg/m2 on day 6, which was considered as the recommended
225 phase II dose. In phase II, 6 patients received the recommended phase II dose during
226 46 cycles. Two episodes of Grade 3 hematologic and three Grade 3 nonhematologic
8 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
227 toxicities were observed, and one patient died from neutropenic sepsis. Subsequently,
228 5-Aza was reduced to 75 mg/m2, and no treatment-related>Grade 3 toxicities was
229 observed in one patient. In this clinical trial, PSA response was observed in 10 of 19
230 (52.6 %) patients, and the median duration of response was 20.5 weeks.
231 Kaplane-Meier estimate of median PFS was 4.9 months, and median OS was 19.5
232 months , which were both favorable34. It suggests that the combination of azacitidine
233 and chemotherapy is active in mCRPC patients, and it maybe a new treatment in
234 future for tumors.
235 This study is mainly based on the data of bioinformatics, large sample data of
236 immunohistochemistry, western blot and RT-PCR of GC tissues were urgently
237 needed.
238 We will further detect and confirm the relationship of NDRG1 and DNMTs in GC
239 tissues.
240 In conclusion, the current data showed that aberrant DNA methylation of NDRG1
241 which could suppress cell invasion and migration, was regulated by DNMT family in
242 GC, especially DNMT1 and DNMT3A. Furthermore, the demethylating agent
243 5-Aza-Dc, a DNMT inhibitor, maybe a potential target drug. Therefore, further
244 clinical studies are warranted to evaluate and confirm the effect of demethylating
245 treatment in GC.
246 Acknowledgments This study was supported in part by a grant from the National
247 Natural Science Foundation of Hebei province of China(#H2018206180).
248 Disclosures No conflicts of interest are declared by the authors.
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383 12 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
384 Caption：
385 Figure.1 Association between NDRG1 and DNMTs. A-C, NDRG1 mRNA expression
386 was negatively correlative to DNMT1, DNMT3A and DNMT3B. D-F, DNA
387 methylation of NDRG1 gene promoter was positively correlative to the expression
388 DNMT family.
389 Figure.2 The protein levels of NDRG1 and DNMTs in GES1 and two gastric cell
390 lines, SGC7901 and MKN45.A, the protein level of NDRG1 and DNMTs; B.
391 Histogram of the protein level.
392 Figure.3 Univariate analysis of prognosis of GC patients.
393 Figure.4 Multivariate analysis of prognosis of GC patients.
394 395 Table.1 Clinicopathological parameters of NDRG1 and DNMTs mRNA expression 396 from TCGA cohort 397 Table.2 Ongoing clinical trials evaluating decitabine drug in solid tumors 398
13 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.433329; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.