- Email: [email protected]
E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2
Accepted Manuscript E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2 Zejun Fang, Chaoju Gong, Hong Liu, Xiaomin Zhang, Lingming Mei, Mintao Song, Lanlan Qiu, Shuchai Luo, Zhihua Zhu, Ronghui Zhang, Hongqian Gu, Xiang Chen PII:
S0006-291X(15)30157-1
DOI:
10.1016/j.bbrc.2015.06.103
Reference:
YBBRC 34136
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 June 2015 Accepted Date: 15 June 2015
Please cite this article as: Z. Fang, C. Gong, H. Liu, X. Zhang, L. Mei, M. Song, L. Qiu, S. Luo, Z. Zhu, R. Zhang, H. Gu, X. Chen, E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.06.103. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT E2F1 promote the aggressiveness of human colorectal cancer by activating the
2
ribonucleotide reductase small subunit M2
3
Zejun Fang1, Chaoju Gong2, Hong Liu3, Xiaomin Zhang1, Lingming Mei1, Mintao Song4,
4
Lanlan Qiu1, Shuchai Luo1, Zhihua Zhu1, Ronghui Zhang1, Hongqian Gu1, Xiang Chen1,*
5
1
Sanmen People's Hospital of Zhejiang, Sanmen, Zhejiang, 317100, China
6
2
Department of Pathology and Pathophysiology, Zhejiang University School of Medicine,
7
Hangzhou, Zhejiang, 310058, China
8
3
9
Jinhua, Zhejiang, 321004, China
RI PT
1
SC
Zhejiang Normal University - Jinhua People's Hospital Joint Center for Biomedical Research,
10
4
11
Medical Sciences (CAMS), School of Basic Medicine, Peking Union Medical College
12
(PUMC), Beijing, 100005, China
13
*
14
Xiang Chen, Sanmen People's Hospital of Zhejiang, Renmin Road 171, Sanmen,
15
Zhejiang, 317100, China
16
Tel/Fax: +86-576-83361501, Email:[email protected]
M AN U
EP
TE D
Corresponding authors:
AC C
17
Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of
1
ACCEPTED MANUSCRIPT Abstract: As the ribonucleotide reductase small subunit, the high expression of
19
ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor
20
growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the
21
expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic
22
studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1
23
promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2
24
mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in
25
CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1
26
promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown
27
attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the
28
migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides,
29
overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells.
30
Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were
31
elevated in most cancer tissues compared to the paired normal tissues. Further analysis
32
showed that the protein expression levels of E2F1 and RRM2 were parallel with each other
33
and positively correlated with lymph node metastasis (LNM), TNM stage and distant
34
metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis
35
than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation,
36
migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the
37
role of E2F1 in activating RRM2 transcription will help to explain the relationship between
38
E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis
39
of the disease.
40
Key words: ribonucleotide reductase small subunit M2; E2F1; colorectal cancer;
41
transactivation
AC C
EP
TE D
M AN U
SC
RI PT
18
42
2
ACCEPTED MANUSCRIPT Introduction
44
Colorectal cancer (CRC) is the third most common type of cancer reported in men and is the
45
second reported in women, with 1,360,600 new cases being diagnosed and 693,900 people
46
dying of it in 2012 worldwide[1], and the incidence rates continue to increase rapidly in China
47
and other economically transitioning countries [2; 3]. In most cases, lethality in CRC patients
48
is resulted from metastasis that contributes to tumor resistance to conventional therapies and
49
an overall poor prognosis [4; 5].
50
Ribonucleotide reductase (RR) is the only rate-limiting enzyme which catalyzes the
51
conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates in the
52
metabolic process of nucleotides. RR is composed of two identical large subunits (RRM1)
53
and two identical small subunits (RRM2 or RRM2B). RRM1 contains a catalytic site and two
54
allosteric effector-binding sites. RRM2 and RRM2B contain a diiron-tyrosyl radical required
55
for the enzyme activity [6]. The RR holoenzyme constitutes two forms: RRM1-RRM2 and
56
RRM1-RRM2B which provide dNTP for DNA replication and repair, respectively [7]. The
57
balance of deoxyribonucleotide triphosphate pool is dependent on the strict regulation of RR.
58
Thus dysregulated RR is closely related with instability of genome, cancer initiation and
59
development in many types of malignancy.
60
Interestingly, the three subunits of RR display entirely different roles in the occurrence and
61
development of cancer. RRM1 functions like a cancer suppressor gene during the process of
62
cell transformation and neoplasm metastasis by activating PTEN signaling network [8; 9].
63
However, In contrast to RRM1, the high expression of RRM2 induces cancer and contributes
64
to tumor growth and invasion. Several cancers such as oral squamous cell carcinoma, cervical
65
carcinoma and gastric carcinoma have been reported to elevate the expression of RRM2[10;
66
11; 12]. RRM2 is not only an essential component in nucleotide metabolism but that it is also
67
capable of acting in cooperation with a variety of oncogenes [13]. Altered RRM2 cooperates
68
with Ras, resulting in the activation of a major Ras pathway involves the Raf-1 and MAPK2
69
which increases transformation and tumorigenic potential [14]. In pancreatic adenocarcinoma,
AC C
EP
TE D
M AN U
SC
RI PT
43
3
ACCEPTED MANUSCRIPT overexpression of RRM2 increases cellular invasiveness by promoting MMP-9 expression in
71
a NF-κB-dependent manner [15]. Furthermore, RRM2-overexpression significantly decreases
72
antiangiogenic thrombospondin-1 (TSP-1) while proangiogenic vascular endothelial growth
73
factor (VEGF) mRNA and protein increase. So cancer cells which overproduced RRM2
74
possess more angiogenic potential [16].
75
As the cell cycle-driver, E2F is a very important transcription factor in regulating RRM2 gene.
76
S-phase specific transcription of RRM2 is the result of E2F1 mediated transcriptional
77
activation and releasing E2F4 mediated transcriptional repression [17]. Compared to
78
gemcitabine-sensitive pancreatic cancer cells Panc-1, the resistant Panc-1GemR cells respond
79
to gemcitabine by increasing the expression of RRM2 through E2F1 mediated transcriptional
80
activation [18]. Several reports in colorectal carcinogenesis have shown that overexpression
81
of E2F1 is associated with the progression of adenomas to adenocarcinomas and metastasis
82
[19; 20; 21; 22]. Hence, various natural and synthetic drugs prevent colon cancer cell growth
83
by inhibiting the expression of important cell cycle protein [23].
84
In the present study, we demonstrated that E2F1 promoted the proliferation, migration,
85
invasion and metastasis of CRC cells by transactivating RRM2. Clinical specimen analyses
86
confirmed that both E2F1 and RRM2 were increased in most colorectal cancer tissues
87
compared to the paired normal tissues, while the level of E2F1 was correlated with that of
88
RRM2 in cancer tissues. Furthermore, we found that higher RRM2 and E2F1 levels correlated
89
with significantly worse survival. The possible biological significance and clinical relevance
90
of the relationship between E2F1 and RRM2 in colorectal cancer development should be
91
considered in the process of diagnosis and therapy.
92
Materials and methods
93
Cell cultures
94
DLD1, SW480, HCT116, HCT8, HT29 and RKO were cultured in RPMI 1640 supplemented
95
with 10 % fetal bovine serum (Gibco, Carlsbad, CA) at 37 °C in a humidified 5 % CO2
96
atmosphere.
AC C
EP
TE D
M AN U
SC
RI PT
70
4
ACCEPTED MANUSCRIPT Transfection and siRNA interference
98
The cmyc-E2F1 and Flag-RRM2 were transfected with X-treme GENE HP DNA
99
Transfection Reagent (Roche Applied Science, Mannheim, Germany) according to the
100
manufacturer’s protocol. E2F1 small interfering RNA (siRNA), RRM2 siRNA, and
101
scrambled siRNA (Santa Cruz Biotechnology, TX, USA) were transfected with
102
Lipofectamine™ RNAiMAX (Invitrogen, NY, USA) according to the manufacturer’s
103
instructions.
104
Quantitative real time RT-PCR
105
Total RNA was prepared using the RNAiso™ Plus reagent (Takara, Otsu, Japan) and
106
reverse-transcribed using a PrimeScript™RT reagent kit (Takara). Quantitative PCR was
107
performed with SYBR Green mix (Takara) according to the manufacturer’s instructions.
108
β-Actin was used as loading control.
109
Western blot analysis
110
The whole cell lysate were analyzed with the antibodies mouse anti-human RRM2, rabbit
111
anti-human E2F1, GAPDH (Santa Cruz Biotechnology), IRDye® 800CW- or IRDye®
112
680-conjugated secondary antibodies (LI-COR, Lincoln,NE) were used for staining and then
113
detected by an Odyssey® infrared imaging system (LI-COR).
114
EdU incorporation assay
115
The EdU (5-ethynyl-2-deoxyuridine) incorporation assay was used to represent DNA
116
synthesis in cells. Cells were transfected with the indicated siRNAs and expression plasmids.
117
Next, cells were washed 3 times with PBS, and then incubated in serum-free RPMI 1640 with
118
10 µM EdU for 2 h. After extensive washing with PBS, cells were blocked with 10% FBS in
119
PBS for 30 min. Incorporated EdU was detected by the fluorescent azide coupling reaction
120
(Invitrogen). Images of the cells were captured with a fluorescence microscope (Nikon, Tokyo,
121
Japan) and analyzed by ImageJ (National Institutes of Health, Bethesda, MD).
122
CCK-8 assay
123
Cells in logarithmic growth were plated and transfected with the indicated siRNAs and
AC C
EP
TE D
M AN U
SC
RI PT
97
5
ACCEPTED MANUSCRIPT expression plasmids. After 48h of culture, CCK-8 (Cell Counting Kit-8) (Dojindo Molecular
125
Technologies, Maryland, USA) was added, and the OD450 was measured using an automatic
126
plate reader.
127
Reporter gene assay
128
The promoter sequences of the human RRM2 gene (-2465/+23) was amplified by PCR and
129
inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). Cells were
130
plated onto 24-well plates the day before transfection. The cells were cotransfected with 0.5
131
µg of firefly luciferase reporter constructs, 0.02 µg of pRL-SV40 Renilla luciferase reporter
132
plasmids (Promega), and 0.5µg cmyc-E2F1 using the X-treme GENE HP DNA Transfection
133
Reagent. The luciferase activity was measured by a dual-luciferase reporter assay system
134
(Promega).
135
Wound healing assay
136
Cells were seeded in six-well plates. After transfection for 24 h, the monolayer was gently
137
and slowly scratched with a pipette tip across the center of the well. After scratching, the well
138
was gently washed several times with PBS to remove detached cells. The well was
139
replenished with fresh medium without serum, and the cells allowed growing for additional
140
48 h, when images of the stained monolayer were captured on a microscope. The wound was
141
evaluated using ImageJ.
142
Cell invasion assays
143
Cell invasion were assessed in Boyden chambers with Matrigel according to the
144
manufacturer’s protocol (Invitrogen). First, an 8-mm-porosity polycarbonate membrane was
145
covered with 200µL of serum-free medium containing 1×105 cells per well. The plates were
146
then incubated with 10% FBS medium for 48 h at 37°C in a 5% CO2 incubator. The invasion
147
cells on the bottom surface of the filter were fixed, stained, and counted using optical
148
microscopy.
149
Immunohistochemistry
150
A total of 154 human CRC samples were collected at the Sanmen People’s Hospital of
AC C
EP
TE D
M AN U
SC
RI PT
124
6
ACCEPTED MANUSCRIPT Zhejiang after informed consent had been given by all patients. The immunohistochemistry
152
was performed using an Envision Detection System (DAKO, Carpinteria, CA, USA)
153
according to the manufacturer's instructions. To estimate the score for each slide, at least 8
154
individual fields at 200× were chosen, and 100 cancer cells were counted in each field. The
155
immunostaining intensity was divided into four grades: 0, negative; 1, weak; 2, moderate; and
156
3, strong. The proportion of positive-staining cells was divided into five grades: 0, <5%; 1, 6
157
–25%; 2, 26 –50%; 3, 51–75%; and 4, >75%. The staining results were assessed and
158
confirmed by two independent investigators blinded to the clinical data. The percentage of
159
positivity of the tumor cells and the staining intensities were then multiplied in order to
160
generate the IHC score, and graded as low expression (score 0~6) and high expression (score
161
7~12). Cases with a discrepancy in scores were discussed to obtain a consensus.
162
Statistics
163
A database was created and transferred to SPSS 22.0 for Windows. Statistical data analysis
164
was performed using the two-tailed Student’s t-test, chi-squared, one-way ANOVA, and the
165
results are presented as the mean±SD of three separate experiments.
166
Results
167
E2F1 promotes RRM2 transactivation in CRC cells
168
To determine the relationship between E2F1 and RRM2, we tested the level of E2F1 and
169
RRM2 in several CRC cell lines. The high expression levels of E2F1 and RRM2 were
170
observed in HCT116, HT29 and SW480, while low levels of E2F1 and RRM2 were found in
171
RKO, HCT8 and DLD1 (Fig. 1a). When E2F1 was overexpressed in RKO and HCT8 cells,
172
the mRNA and protein expression of RRM2 were significantly upregulated (Fig. 1b,c,d).
173
Knocking down E2F1 expression by its specific siRNA in SW480 and HT29 cells, the mRNA
174
and protein level of RRM2 decreased as expected (Fig. 1e,f). Reporter gene analyses also
175
showed that overexpression of E2F1 induced transcriptional activity of the RRM2 promoter in
176
RKO and HCT8 cells (Fig. 1g). These results indicate that E2F1 promote RRM2
177
transactivation in CRC cells.
AC C
EP
TE D
M AN U
SC
RI PT
151
7
ACCEPTED MANUSCRIPT E2F1 promotes the proliferation of CRC cells by activating RRM2
179
Considering E2F1 and RRM2 as indispensable S phase-drivers in cell cycle, CCK-8 assay and
180
EdU incorporation assay were performed to characterize the function of E2F1 and RRM2 in
181
cell proliferation. In HT29 cells, silencing E2F1 repressed cell proliferation and additional
182
ectopic expression of RRM2 reversed the effect to some extent (Fig. 2a). Overexpression of
183
E2F1 promoted cell proliferation in the transfected RKO cells while RRM2 knockdown
184
attenuated the E2F1 induced cell proliferation (Fig. 2b). In EdU incorporation assay, E2F1
185
knockdown blocked DNA synthesis in HT29 cells whereas further overexpressed RRM2
186
increased EdU incorporation (Fig. 2c). Consistently, increased DNA synthesis was observed
187
in E2F1 transfected RKO cells, and RRM2 silencing had a destructive effect on DNA
188
synthesis stimulated by E2F1 (Fig. 2d). These results suggest that E2F1 plays a significant
189
role in boosting cell proliferation through activating RRM2 in CRC cells.
190
E2F1 promotes the migration and invasion of CRC cells by activating RRM2
191
As the high expression of RRM2 may contribute to tumor invasion and metastasis, we next
192
investigated the functional role of E2F1 and RRM2 in CRC cell migration and invasion. The
193
results showed that knockdown of either E2F1 or RRM2 weakened the acquisitive capacity of
194
migration and invasion in HT29 cells while overexpression of RRM2 rescued the wound
195
healing and invading abilities induced by siE2F1 (Fig. 3a,c). Similarly, overexpression of
196
either E2F1 or RRM2 increased migration and invasion in RKO cells, and RRM2 knockdown
197
with siRNA interference partly retarded the E2F1 effects (Fig. 3b,d). The above results
198
indicate that RRM2 transactivation by E2F1 is essential for migration and invasion in CRC
199
cells.
200
E2F1 expression is upregulated in CRC tissues and positively associated with RRM2
201
level at the advanced cancer stage
202
Immunohistochemical staining was carried out to analyze the clinical relevance of E2F1 and
203
RRM2 expressions in 154 human CRC specimens. The expression levels of RRM2 and E2F1
204
were increased in the tumor compared with the normal intestinal tissue (Fig. 4a and table 1).
AC C
EP
TE D
M AN U
SC
RI PT
178
8
ACCEPTED MANUSCRIPT Further analyses showed that the RRM2 and E2F1 staining were positively correlated with
206
lymph node metastasis (LNM), distant metastasis and advanced TNM stages (p<0.05) but not
207
with tumor size (p>0.05) (Table 2). Moreover, the E2F1 expression levels paralleled the
208
changes of RRM2 in the CRC cases as shown by immunohistochemical analyses (p<0.05)
209
(Fig. 4b). The 5-year overall survival (OS) rate of the RRM2 and E2F1 double-high
210
expression group was significantly lower than that of the RRM2 and E2F1 double-low
211
expression group (27.7% vs 55.4% , p<0.001). Of note is that patients with low levels of
212
RRM2 have a better prognosis than RRM2-high expression patients regardless of the E2F1
213
expression status (Fig. 4c). These data suggest that E2F1 is positively correlated with RRM2
214
expression, which is involved in CRC progression and metastasis.
215
Discussion
216
Abnormal expression of ribonucleotide reductase is closely relative to several types of cancer.
217
As the small subunit of RR, up-regulation of RRM2 increases RR activity, which provides
218
extra dNTPs in cancer cells. Furthermore RRM2 could serve as an independent prognostic
219
factor and predict poor survival of CRCs [24; 25]. Here we found that the increased RRM2
220
contributed to the proliferation, migration, invasion and metastasis of CRC cells. Previous
221
observation has shown that the RRM2 is not only a rate-limiting component for
222
ribonucleotide reduction, but is also capable of acting in cooperation with a variety of
223
oncogenes to promote transformation and tumorigenesis [14]. Overexpression of RRM2 in
224
KB and PC-3 cells could induce the migration ability of HUVECs (human umbilical vein
225
endothelial cells) [26]. Besides, it has been reported that RRM2 could increase the MMP-9
226
expression and enhance the cell invasion ability in cancer cell lines [15]. Therefore, dNTPs
227
pool expansion, acceleration of cell proliferation, and improvement of metastasis ability may
228
partly explain why RRM2 increases the aggressiveness and causes poor survival in CRCs.
229
The regulatory mechanisms which control the gene expression of RR subunits depend on
230
different conditions. Due to its long half-life, the level of RRM1 is virtually constant
231
throughout the cell cycle and always in excess of the level of RRM2 [27; 28]. Therefore, the
AC C
EP
TE D
M AN U
SC
RI PT
205
9
ACCEPTED MANUSCRIPT activity of RR is mainly regulated by the synthesis and degradation of the RRM2 protein. As
233
an important transcription factor, E2F1 is involved in S-phase specific expression of RRM2 in
234
physiological situations [29; 30]. On the other hand, the transcription of RRM2 was also
235
activated by E2F1 through ATM/ATR-CHK1 signal pathway in response to DNA damage [31].
236
As a potential predictor for response to fluropyrimidines in colon cancer, E2F1 controls the
237
transcription of several genes encoding proteins involved in DNA synthesis, namely
238
thymidylate synthase, dihydrofolate reductase, and thymidine kinase [29; 32]. A high
239
correlation between E2F1 and TS was recently shown in colorectal cancer metastases to the
240
lung and the liver [21]. Our results suggested that overexpressed E2F1 upregulated the
241
expression of RRM2 by promoting its transcriptional activation in CRC cell lines. Further
242
investigations showed that E2F1 promoted the proliferation, migration and invasion of CRC
243
cells which were attenuated by knockdown of RRM2. So RRM2 may function as an
244
important target gene of E2F1, which plays a vital role in the progress of CRC.
245
Analyses of clinical specimens from CRC patients showed that both E2F1 and RRM2
246
expressions were upregulated in most cancer tissues compared to paired normal tissues. The
247
high expression of E2F1 in CRC might be achieved by phosphorylation or other
248
post-translational modifications and the mechanism should be further investigated[33]. The
249
increased RRM2 protein levels were positively correlated with TNM stage and metastasis in
250
parallel with the E2F1 expression. Therefore, the clinical evidence further support the
251
hypothesis that E2F1 performs as the transcriptional activator of RRM2 in promoting CRC
252
development: the increased RRM2 protein activated by E2F1 contributes to tumor initiation
253
and metastasis. Moreover, we analyzed the survival rate of the patients with different E2F1
254
and RRM2 expression. For most patients, the expression level of RRM2 and E2F1 was
255
negatively correlated with the survival rate. However, the survival rate of some patients with
256
inverse correlation between RRM2 and E2F1 depended on RRM2 levels. So it is reasonable
257
to suggest that the role of E2F1 in the initiation and progress of CRC is dependent on the
258
expression level of RRM2. Thus, integrative assessments of RRM2 and E2F1 protein
AC C
EP
TE D
M AN U
SC
RI PT
232
10
ACCEPTED MANUSCRIPT expression in clinical samples would help reach a proper prognosis as well as the rational use
260
of different RR inhibitors: enzyme inactivators or gene expression suppressors of RRM2 for
261
personalized cancer therapy.
262
In summary, our molecular and clinical evidence demonstrated the relationship between E2F1
263
and RRM2 and their roles in promoting CRC invasion and metastasis. Still, the underlying
264
mechanism of regulating RRM2 in CRC cells has not been clearly elucidated. Further
265
understanding of the upstream regulatory pathway of RRM2 may help to provide new
266
biomarkers and therapeutic targets for human colorectal cancer.
267
Acknowledgements: This work was supported by Zhejiang Medical Association Clinical
268
Research Fund (No. 2013ZYC-A144), and Taizhou Science and Technology Plan (No.
269
1301ky55).
SC
M AN U
270
EP
TE D
References: [1] L.A. Torre, F. Bray, R.L. Siegel, et al., Global cancer statistics, 2012, CA Cancer J Clin 65 (2015)87-108. [2] M.M. Center, A. Jemal, E. Ward, International trends in colorectal cancer incidence rates, Cancer Epidemiol Biomarkers Prev 18 (2009)1688-94. [3] P. Guo, Z.L. Huang, P. Yu, et al., Trends in cancer mortality in China: an update, ANN ONCOL 23 (2012)2755-62. [4] J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, et al., Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012, EUR J CANCER 49 (2013)1374-403. [5] M. Malvezzi, P. Bertuccio, F. Levi, et al., European cancer mortality predictions for the year 2012, ANN ONCOL 23 (2012)1044-52. [6] P. Nordlund, P. Reichard, Ribonucleotide Reductases, Annu Rev Biochem 75 (2006)681-706. [7] H. Tanaka, H. Arakawa, T. Yamaguchi, et al., A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage, NATURE 404 (2000)42-9. [8] A. Gautam, Z.R. Li, G. Bepler, RRM1-induced metastasis suppression through PTEN-regulated pathways, ONCOGENE 22 (2003)2135-42. [9] H. Qi, M. Lou, Y. Chen, et al., Non-enzymatic action of RRM1 protein upregulates PTEN leading to inhibition of colorectal cancer metastasis, Tumour Biol (2015). [10] T. Morikawa, R. Hino, H. Uozaki, et al., Expression of ribonucleotide reductase M2 subunit in gastric cancer and effects of RRM2 inhibition in vitro, HUM PATHOL 41 (2010)1742-8. [11] N. Wang, T. Zhan, T. Ke, et al., Increased expression of RRM2 by human papillomavirus E7 oncoprotein promotes angiogenesis in cervical cancer, Br J Cancer 110
AC C
271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
RI PT
259
11
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
(2014)1034-44. [12] K. Iwamoto, K.I. Nakashiro, H. Tanaka, et al., Ribonucleotide reductase M2 is a promising molecular target for the treatment of oral squamous cell carcinoma, INT J ONCOL (2015). [13] H. Fan, C. Villegas, A. Huang, et al., The mammalian ribonucleotide reductase R2 component cooperates with a variety of oncogenes in mechanisms of cellular transformation, CANCER RES 58 (1998)1650-3. [14] H. Fan, C. Villegas, J.A. Wright, Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potential, Proc Natl Acad Sci U S A 93 (1996)14036-40. [15] M.S. Duxbury, E.E. Whang, RRM2 induces NF-kappaB-dependent MMP-9 activation and enhances cellular invasiveness, Biochem Biophys Res Commun 354 (2007)190-6. [16] K. Zhang, S. Hu, J. Wu, et al., Overexpression of RRM2 decreases thrombspondin-1 and increases VEGF production in human cancer cells in vitro and in vivo: implication of RRM2 in angiogenesis, MOL CANCER 8 (2009)11. [17] K. Chimploy, G.D. Diaz, Q. Li, et al., E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin, INT J CANCER 125 (2009)2086-94. [18] I.L. Lai, C.C. Chou, P.T. Lai, et al., Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells, CARCINOGENESIS 35 (2014)2203-13. [19] H. Muller, M.C. Moroni, E. Vigo, et al., Induction of S-phase entry by E2F transcription factors depends on their nuclear localization, MOL CELL BIOL 17 (1997)5508-20. [20] T. Suzuki, W. Yasui, H. Yokozaki, et al., Expression of the E2F family in human gastrointestinal carcinomas, INT J CANCER 81 (1999)535-8. [21] D. Banerjee, R. Gorlick, A. Liefshitz, et al., Levels of E2F-1 expression are higher in lung metastasis of colon cancer as compared with hepatic metastasis and correlate with levels of thymidylate synthase, CANCER RES 60 (2000)2365-7. [22] W. Yasui, J. Fujimoto, T. Suzuki, et al., Expression of cell-cycle-regulating transcription factor E2F-1 in colorectal carcinomas, PATHOBIOLOGY 67 (1999)174-9. [23] B. Yu, M.E. Lane, S. Wadler, SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells, BIOCHEM PHARMACOL 64 (2002)1091-100. [24] X. Liu, H. Zhang, L. Lai, et al., Ribonucleotide reductase small subunit M2 serves as a prognostic biomarker and predicts poor survival of colorectal cancers, Clin Sci (Lond) 124 (2013)567-78. [25] X. Liu, B. Zhou, L. Xue, et al., Ribonucleotide reductase subunits M2 and p53R2 are potential biomarkers for metastasis of colon cancer, Clin Colorectal Cancer 6 (2007)374-81. [26] X. Liu, B. Zhou, L. Xue, et al., Metastasis-suppressing potential of ribonucleotide reductase small subunit p53R2 in human cancer cells, CLIN CANCER RES 12 (2006)6337-44. [27] Y. Engstrom, S. Eriksson, I. Jildevik, et al., Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits, J BIOL CHEM 260 (1985)9114-6.
AC C
296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
12
ACCEPTED MANUSCRIPT [28] G.J. Mann, E.A. Musgrove, R.M. Fox, et al., Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation, CANCER RES 48 (1988)5151-6. [29] J. DeGregori, T. Kowalik, J.R. Nevins, Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes, MOL CELL BIOL 15 (1995)4215-24. [30] A.L. Chabes, S. Bjorklund, L. Thelander, S Phase-specific transcription of the mouse ribonucleotide reductase R2 gene requires both a proximal repressive E2F-binding site and an upstream promoter activating region, J BIOL CHEM 279 (2004)10796-807. [31] Y.W. Zhang, T.L. Jones, S.E. Martin, et al., Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response, J BIOL CHEM 284 (2009)18085-95. [32] L. Wu, C. Timmers, B. Maiti, et al., The E2F1-3 transcription factors are essential for cellular proliferation, NATURE 414 (2001)457-62. [33] C. Stevens, L. Smith, N.B. La Thangue, Chk2 activates E2F-1 in response to DNA damage, NAT CELL BIOL 5 (2003)401-9.
356
Figure legends
357
Table 1 Expression of RRM2 and E2F1 in CRC and adjacent normal tissues
358
Table 2 Correlation of the expression of RRM2 and E2F1 with clinicopathological
359
features in CRC
360
Figure 1. E2F1 promotes RRM2 transactivation in CRC cells
361
a Expression of E2F1 and RRM2 proteins in 6 CRC cell lines were analyzed by western blot.
362
b The mRNA level of RRM2 was analyzed by qRT-PCR in CRC cells after transfection with
363
the E2F1 expression plasmid at different time points. c and d The protein level of RRM2 were
364
analyzed by western blot in CRC cells after transfection with the E2F1 expression plasmid at
365
different time points. e and f The mRNA and protein levels of RRM2 in CRC cells after
366
transfected with the siRNA targeting E2F1. g Relative luciferase activity in CRC cells
367
co-transfected with EV or E2F1 expression plasmid, RRM2 promoter reporter (-2465/+23)
368
and an internal control reporter pRL-TK. *P<0.05.
369
Figure 2. E2F1 promotes the proliferation of CRC cells by activating RRM2
370
a and b At 12-60 h after the indicated siRNAs and expression plasmids transfection, the
371
viability of HT29 (a) or RKO (b) cells were determined by the CCK-8 assay. c and d DNA
372
synthesis of HT29 (c) or RKO (d) cells were measured by EdU incorporation assay after the
AC C
EP
TE D
M AN U
SC
RI PT
340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355
13
ACCEPTED MANUSCRIPT indicated transfection. *P<0.05.
374
Figure 3. E2F1 promotes the migration and invasion of CRC cells by activating RRM2
375
a and b Left panels: images from scratch assays with HT29 (a) or RKO (b) cells transfected
376
with indicated siRNAs and expression plasmids. Right panels: percentage wound closure 48 h
377
after the indicated transfection. *P<0.05. c and d Left panels: representative images of HT29
378
(c) or RKO (d) cells penetrating the Matrigel in invasion assays. Right panels: numbers of
379
invasive cells transfected with the indicated siRNAs and expression plasmids for 48 h.
380
*P<0.05.
381
Figure 4. E2F1 expression is upregulated in CRC tissues and positively associated with
382
RRM2 level at the advanced cancer stage
383
a Representative images of E2F1 and RRM2 immunostaining in cancer and adjacent
384
non-cancerous tissues. b The correlation of concurrent immunostaining scores of RRM2 and
385
E2F1 in CRC tissues. c The survival rate plot of indicated expression groups in CRC.
AC C
EP
TE D
M AN U
SC
RI PT
373
14
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Table 1 Expression of RRM2 and E2F1 in CRC and adjacent normal tissues cases CRC adjacent normal tissues *P<0.05
154 154
RRM2 expression (cancer) low high P Value cases cases 78 76 <0.001* 147 7
E2F1 expression (cancer) low high P Value cases cases 85 69 <0.001* 139 15
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
Table 2 Correlation of the expression of RRM2 and E2F1 with clinicopathological features in CRC RRM2 expression (cancer)
cases 154 Tumor location
cases
cases
78
76
male female
83 71
EP
Age
65 89
33 45
TE D
Gender
AC C
İ65 ˚65
LNM
high
P Value
low
high
cases
cases
85
69
0.988
Colon Rectum
Tumor size
low
E2F1 expression (cancer)
64 90
41 37
32 44
0.830 35 50
30 39
0.788 42 34
0.296 43 42
40 29
0.365 36 42
28 48
0.645 35 50
29 40
0.592
0.426
˘5cm
72
36
36
41
31
ı5cm
82
42
40
44
38
0.004*
0.013*
N0
87
51
36
54
33
N1/2
67
27
40
31
36
TNM
0.032*
0.014*
ĉ Ċ
23
15
8
15
8
59
29
30
32
27
ċ
60 12
32 2
28 10
35 3
25 9
Č Distant metastasis
0.002* M0 M1
LNM: lymph node metastasis;*P<0.05
142 12
76 2
66 10
P Value
0.001* 82 3
60 9
ACCEPTED MANUSCRIPT
RRM2 GAPDH
d
RKO cmyc-E2F1(h)
0
24
HCT8 48
72
0
24
48
72
cmyc-E2F1 RRM2
TE D
GAPDH
* 3
*
*
2
*
1 0
RKO
HCT8
M AN U
c
cmyc-E2F1 0hr 24hr 48hr
4
RI PT
Alternation of RRM2 mRNA levels
E2F1
Optical density of RRM2 protein (arbitrary units %)
9
48 0 D LD 1
SW
T1 1
H T2
H C
T8 H C
R K O
6
b
SC
a
250
*
cmyc-E2F1 0h 24h 48h 72h
* *
200
*
150 100
50
0
RKO
HCT8
f
0.5
0.0
SW480
RRM2 promoter relative luciferase activity
* 6 4 2 0
RKO
HCT8
-2 F1 E2 si
F1 E2 si
C tr l si
F1 E2 si
F1 E2 si
-1
-2
HT29
-1
GAPDH
EV E2F1
8
l
RRM2
HT29
*
tr
E2F1
g 10
C
*
SW480
siCtrl siE2F1
si
* 1.0
EP
1.5
AC C
Alternation of RRM2 mRNA levels
e
ACCEPTED MANUSCRIPT
b
RI PT
a HT29 siCtrl+EV
3.0
siE2F1+EV
Absorptivity
1.5
48
60
60
t/h
*
0.3
*
0.2 0.1
M 2 2F 1+ R R
si E
si E2 F1 +E V
EV tr l+ si C
E2F1+siRRM2 0.6
*
*
0.5 0.4 0.3 0.2 0.1
E2 F1 +s iR
R
tr l
M 2
0.0
E2 F1 +s iC
E2F1+siCtrl
48
0.0
%EdU incorporation
EV+siCtrl
36
0.4
AC C
HT29
DAPI
siE2F1+RRM2
EP
EdU
siE2F1+EV
24
+s iC tr l
siCtrl+EV
TE D
c
12
EV
36
%EdU incorporation
24
t/h
RKO
1.0
0.0
12
EdU
1.5
siRRM2+E2F1
0.5
0.0
d
2.0
M AN U
1.0 0.5
DAPI
siCtrl+E2F1
2.5
siE2F1+RRM2
2.0
siCtrl+EV
SC
2.5
Absorptivity
RKO
ACCEPTED MANUSCRIPT
*
0.5
si R R
si E
0.3
0.2
0.1
M 2 R
tr l E 2F 1+ si R
E 2F 1+ si C
*
200 150
*
100
2F 1+ R si E
si R
R
E V
2F 1+ E
E V
M 2
50
d
*
400
*
350 300 250 200 150 100 50
E 2F 1+ si C tr l R R M 2+ si C tr E l 2F 1+ si R R M 2
E2F1+siRRM2
+s iC tr l
RRM2+siCtrl
E V
E2F1+siCtrl
Number of invasive cells
* EV+siCtrl
RKO
M 2+ si C
tr l
0.0
si C tr l+
HT29
0.5
0.4
V
siE2F1+RRM2
*
0.6
si E
siRRM2+EV
0.7
* Number of invasive cells
siE2F1+EV
AC C
siCtrl+EV
EP
c
*
*
0.8
E V
TE D
RKO
E2F1+siRRM2
M 2
V
+E
2F 1
tr l+
si C
SC RRM2+siCtrl
+s iC tr l
E2F1+siCtrl
percent of wound closed
EV+siCtrl
M AN U
b
V
0.0
2F 1+ R R
0.1
E
0.2
*
si E
0.3
M 2+
0.4
E V
HT29
*
0.6
R M 2+
siE2F1+RRM2
R
siRRM2+EV
R
siE2F1+EV
RI PT
siCtrl+EV
percent of wound closed
a
RI PT
ACCEPTED MANUSCRIPT
normal
SC
a cancer-1
M AN U
cancer-2
TE D
RRM2
b
c
P<0.001 2 R =0.265
15
RRM2 low/E2F1 low(n=56) RRM2 low/E2F1 high(n=22)
100 Cum survival
E2F1 score
AC C
EP
E2F1
10
5
RRM2 high/E2F1 low(n=29) RRM2 high/E2F1 high(n=47)
80 60 40 20 0
0 0
5 10 RRM2 score
15
P<0.001 0
20
40 60 80 survival month
100
ACCEPTED MANUSCRIPT Highlights
2
1. E2F1 promotes RRM2 transactivation in CRC cells
3
2. E2F1 promotes the proliferation of CRC cells by activating RRM2
4
3. E2F1 promotes the migration and invasion of CRC cells by activating RRM2
5
4. E2F1 is upregulated in CRC tissues and positively associated with RRM2 level
6
5. E2F1-mediated RRM2 transcription will provide a new strategy in CRC.
AC C
EP
TE D
M AN U
SC
RI PT
1
1
S0006-291X(15)30157-1
DOI:
10.1016/j.bbrc.2015.06.103
Reference:
YBBRC 34136
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 June 2015 Accepted Date: 15 June 2015
Please cite this article as: Z. Fang, C. Gong, H. Liu, X. Zhang, L. Mei, M. Song, L. Qiu, S. Luo, Z. Zhu, R. Zhang, H. Gu, X. Chen, E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.06.103. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT E2F1 promote the aggressiveness of human colorectal cancer by activating the
2
ribonucleotide reductase small subunit M2
3
Zejun Fang1, Chaoju Gong2, Hong Liu3, Xiaomin Zhang1, Lingming Mei1, Mintao Song4,
4
Lanlan Qiu1, Shuchai Luo1, Zhihua Zhu1, Ronghui Zhang1, Hongqian Gu1, Xiang Chen1,*
5
1
Sanmen People's Hospital of Zhejiang, Sanmen, Zhejiang, 317100, China
6
2
Department of Pathology and Pathophysiology, Zhejiang University School of Medicine,
7
Hangzhou, Zhejiang, 310058, China
8
3
9
Jinhua, Zhejiang, 321004, China
RI PT
1
SC
Zhejiang Normal University - Jinhua People's Hospital Joint Center for Biomedical Research,
10
4
11
Medical Sciences (CAMS), School of Basic Medicine, Peking Union Medical College
12
(PUMC), Beijing, 100005, China
13
*
14
Xiang Chen, Sanmen People's Hospital of Zhejiang, Renmin Road 171, Sanmen,
15
Zhejiang, 317100, China
16
Tel/Fax: +86-576-83361501, Email:[email protected]
M AN U
EP
TE D
Corresponding authors:
AC C
17
Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of
1
ACCEPTED MANUSCRIPT Abstract: As the ribonucleotide reductase small subunit, the high expression of
19
ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor
20
growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the
21
expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic
22
studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1
23
promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2
24
mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in
25
CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1
26
promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown
27
attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the
28
migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides,
29
overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells.
30
Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were
31
elevated in most cancer tissues compared to the paired normal tissues. Further analysis
32
showed that the protein expression levels of E2F1 and RRM2 were parallel with each other
33
and positively correlated with lymph node metastasis (LNM), TNM stage and distant
34
metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis
35
than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation,
36
migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the
37
role of E2F1 in activating RRM2 transcription will help to explain the relationship between
38
E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis
39
of the disease.
40
Key words: ribonucleotide reductase small subunit M2; E2F1; colorectal cancer;
41
transactivation
AC C
EP
TE D
M AN U
SC
RI PT
18
42
2
ACCEPTED MANUSCRIPT Introduction
44
Colorectal cancer (CRC) is the third most common type of cancer reported in men and is the
45
second reported in women, with 1,360,600 new cases being diagnosed and 693,900 people
46
dying of it in 2012 worldwide[1], and the incidence rates continue to increase rapidly in China
47
and other economically transitioning countries [2; 3]. In most cases, lethality in CRC patients
48
is resulted from metastasis that contributes to tumor resistance to conventional therapies and
49
an overall poor prognosis [4; 5].
50
Ribonucleotide reductase (RR) is the only rate-limiting enzyme which catalyzes the
51
conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates in the
52
metabolic process of nucleotides. RR is composed of two identical large subunits (RRM1)
53
and two identical small subunits (RRM2 or RRM2B). RRM1 contains a catalytic site and two
54
allosteric effector-binding sites. RRM2 and RRM2B contain a diiron-tyrosyl radical required
55
for the enzyme activity [6]. The RR holoenzyme constitutes two forms: RRM1-RRM2 and
56
RRM1-RRM2B which provide dNTP for DNA replication and repair, respectively [7]. The
57
balance of deoxyribonucleotide triphosphate pool is dependent on the strict regulation of RR.
58
Thus dysregulated RR is closely related with instability of genome, cancer initiation and
59
development in many types of malignancy.
60
Interestingly, the three subunits of RR display entirely different roles in the occurrence and
61
development of cancer. RRM1 functions like a cancer suppressor gene during the process of
62
cell transformation and neoplasm metastasis by activating PTEN signaling network [8; 9].
63
However, In contrast to RRM1, the high expression of RRM2 induces cancer and contributes
64
to tumor growth and invasion. Several cancers such as oral squamous cell carcinoma, cervical
65
carcinoma and gastric carcinoma have been reported to elevate the expression of RRM2[10;
66
11; 12]. RRM2 is not only an essential component in nucleotide metabolism but that it is also
67
capable of acting in cooperation with a variety of oncogenes [13]. Altered RRM2 cooperates
68
with Ras, resulting in the activation of a major Ras pathway involves the Raf-1 and MAPK2
69
which increases transformation and tumorigenic potential [14]. In pancreatic adenocarcinoma,
AC C
EP
TE D
M AN U
SC
RI PT
43
3
ACCEPTED MANUSCRIPT overexpression of RRM2 increases cellular invasiveness by promoting MMP-9 expression in
71
a NF-κB-dependent manner [15]. Furthermore, RRM2-overexpression significantly decreases
72
antiangiogenic thrombospondin-1 (TSP-1) while proangiogenic vascular endothelial growth
73
factor (VEGF) mRNA and protein increase. So cancer cells which overproduced RRM2
74
possess more angiogenic potential [16].
75
As the cell cycle-driver, E2F is a very important transcription factor in regulating RRM2 gene.
76
S-phase specific transcription of RRM2 is the result of E2F1 mediated transcriptional
77
activation and releasing E2F4 mediated transcriptional repression [17]. Compared to
78
gemcitabine-sensitive pancreatic cancer cells Panc-1, the resistant Panc-1GemR cells respond
79
to gemcitabine by increasing the expression of RRM2 through E2F1 mediated transcriptional
80
activation [18]. Several reports in colorectal carcinogenesis have shown that overexpression
81
of E2F1 is associated with the progression of adenomas to adenocarcinomas and metastasis
82
[19; 20; 21; 22]. Hence, various natural and synthetic drugs prevent colon cancer cell growth
83
by inhibiting the expression of important cell cycle protein [23].
84
In the present study, we demonstrated that E2F1 promoted the proliferation, migration,
85
invasion and metastasis of CRC cells by transactivating RRM2. Clinical specimen analyses
86
confirmed that both E2F1 and RRM2 were increased in most colorectal cancer tissues
87
compared to the paired normal tissues, while the level of E2F1 was correlated with that of
88
RRM2 in cancer tissues. Furthermore, we found that higher RRM2 and E2F1 levels correlated
89
with significantly worse survival. The possible biological significance and clinical relevance
90
of the relationship between E2F1 and RRM2 in colorectal cancer development should be
91
considered in the process of diagnosis and therapy.
92
Materials and methods
93
Cell cultures
94
DLD1, SW480, HCT116, HCT8, HT29 and RKO were cultured in RPMI 1640 supplemented
95
with 10 % fetal bovine serum (Gibco, Carlsbad, CA) at 37 °C in a humidified 5 % CO2
96
atmosphere.
AC C
EP
TE D
M AN U
SC
RI PT
70
4
ACCEPTED MANUSCRIPT Transfection and siRNA interference
98
The cmyc-E2F1 and Flag-RRM2 were transfected with X-treme GENE HP DNA
99
Transfection Reagent (Roche Applied Science, Mannheim, Germany) according to the
100
manufacturer’s protocol. E2F1 small interfering RNA (siRNA), RRM2 siRNA, and
101
scrambled siRNA (Santa Cruz Biotechnology, TX, USA) were transfected with
102
Lipofectamine™ RNAiMAX (Invitrogen, NY, USA) according to the manufacturer’s
103
instructions.
104
Quantitative real time RT-PCR
105
Total RNA was prepared using the RNAiso™ Plus reagent (Takara, Otsu, Japan) and
106
reverse-transcribed using a PrimeScript™RT reagent kit (Takara). Quantitative PCR was
107
performed with SYBR Green mix (Takara) according to the manufacturer’s instructions.
108
β-Actin was used as loading control.
109
Western blot analysis
110
The whole cell lysate were analyzed with the antibodies mouse anti-human RRM2, rabbit
111
anti-human E2F1, GAPDH (Santa Cruz Biotechnology), IRDye® 800CW- or IRDye®
112
680-conjugated secondary antibodies (LI-COR, Lincoln,NE) were used for staining and then
113
detected by an Odyssey® infrared imaging system (LI-COR).
114
EdU incorporation assay
115
The EdU (5-ethynyl-2-deoxyuridine) incorporation assay was used to represent DNA
116
synthesis in cells. Cells were transfected with the indicated siRNAs and expression plasmids.
117
Next, cells were washed 3 times with PBS, and then incubated in serum-free RPMI 1640 with
118
10 µM EdU for 2 h. After extensive washing with PBS, cells were blocked with 10% FBS in
119
PBS for 30 min. Incorporated EdU was detected by the fluorescent azide coupling reaction
120
(Invitrogen). Images of the cells were captured with a fluorescence microscope (Nikon, Tokyo,
121
Japan) and analyzed by ImageJ (National Institutes of Health, Bethesda, MD).
122
CCK-8 assay
123
Cells in logarithmic growth were plated and transfected with the indicated siRNAs and
AC C
EP
TE D
M AN U
SC
RI PT
97
5
ACCEPTED MANUSCRIPT expression plasmids. After 48h of culture, CCK-8 (Cell Counting Kit-8) (Dojindo Molecular
125
Technologies, Maryland, USA) was added, and the OD450 was measured using an automatic
126
plate reader.
127
Reporter gene assay
128
The promoter sequences of the human RRM2 gene (-2465/+23) was amplified by PCR and
129
inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). Cells were
130
plated onto 24-well plates the day before transfection. The cells were cotransfected with 0.5
131
µg of firefly luciferase reporter constructs, 0.02 µg of pRL-SV40 Renilla luciferase reporter
132
plasmids (Promega), and 0.5µg cmyc-E2F1 using the X-treme GENE HP DNA Transfection
133
Reagent. The luciferase activity was measured by a dual-luciferase reporter assay system
134
(Promega).
135
Wound healing assay
136
Cells were seeded in six-well plates. After transfection for 24 h, the monolayer was gently
137
and slowly scratched with a pipette tip across the center of the well. After scratching, the well
138
was gently washed several times with PBS to remove detached cells. The well was
139
replenished with fresh medium without serum, and the cells allowed growing for additional
140
48 h, when images of the stained monolayer were captured on a microscope. The wound was
141
evaluated using ImageJ.
142
Cell invasion assays
143
Cell invasion were assessed in Boyden chambers with Matrigel according to the
144
manufacturer’s protocol (Invitrogen). First, an 8-mm-porosity polycarbonate membrane was
145
covered with 200µL of serum-free medium containing 1×105 cells per well. The plates were
146
then incubated with 10% FBS medium for 48 h at 37°C in a 5% CO2 incubator. The invasion
147
cells on the bottom surface of the filter were fixed, stained, and counted using optical
148
microscopy.
149
Immunohistochemistry
150
A total of 154 human CRC samples were collected at the Sanmen People’s Hospital of
AC C
EP
TE D
M AN U
SC
RI PT
124
6
ACCEPTED MANUSCRIPT Zhejiang after informed consent had been given by all patients. The immunohistochemistry
152
was performed using an Envision Detection System (DAKO, Carpinteria, CA, USA)
153
according to the manufacturer's instructions. To estimate the score for each slide, at least 8
154
individual fields at 200× were chosen, and 100 cancer cells were counted in each field. The
155
immunostaining intensity was divided into four grades: 0, negative; 1, weak; 2, moderate; and
156
3, strong. The proportion of positive-staining cells was divided into five grades: 0, <5%; 1, 6
157
–25%; 2, 26 –50%; 3, 51–75%; and 4, >75%. The staining results were assessed and
158
confirmed by two independent investigators blinded to the clinical data. The percentage of
159
positivity of the tumor cells and the staining intensities were then multiplied in order to
160
generate the IHC score, and graded as low expression (score 0~6) and high expression (score
161
7~12). Cases with a discrepancy in scores were discussed to obtain a consensus.
162
Statistics
163
A database was created and transferred to SPSS 22.0 for Windows. Statistical data analysis
164
was performed using the two-tailed Student’s t-test, chi-squared, one-way ANOVA, and the
165
results are presented as the mean±SD of three separate experiments.
166
Results
167
E2F1 promotes RRM2 transactivation in CRC cells
168
To determine the relationship between E2F1 and RRM2, we tested the level of E2F1 and
169
RRM2 in several CRC cell lines. The high expression levels of E2F1 and RRM2 were
170
observed in HCT116, HT29 and SW480, while low levels of E2F1 and RRM2 were found in
171
RKO, HCT8 and DLD1 (Fig. 1a). When E2F1 was overexpressed in RKO and HCT8 cells,
172
the mRNA and protein expression of RRM2 were significantly upregulated (Fig. 1b,c,d).
173
Knocking down E2F1 expression by its specific siRNA in SW480 and HT29 cells, the mRNA
174
and protein level of RRM2 decreased as expected (Fig. 1e,f). Reporter gene analyses also
175
showed that overexpression of E2F1 induced transcriptional activity of the RRM2 promoter in
176
RKO and HCT8 cells (Fig. 1g). These results indicate that E2F1 promote RRM2
177
transactivation in CRC cells.
AC C
EP
TE D
M AN U
SC
RI PT
151
7
ACCEPTED MANUSCRIPT E2F1 promotes the proliferation of CRC cells by activating RRM2
179
Considering E2F1 and RRM2 as indispensable S phase-drivers in cell cycle, CCK-8 assay and
180
EdU incorporation assay were performed to characterize the function of E2F1 and RRM2 in
181
cell proliferation. In HT29 cells, silencing E2F1 repressed cell proliferation and additional
182
ectopic expression of RRM2 reversed the effect to some extent (Fig. 2a). Overexpression of
183
E2F1 promoted cell proliferation in the transfected RKO cells while RRM2 knockdown
184
attenuated the E2F1 induced cell proliferation (Fig. 2b). In EdU incorporation assay, E2F1
185
knockdown blocked DNA synthesis in HT29 cells whereas further overexpressed RRM2
186
increased EdU incorporation (Fig. 2c). Consistently, increased DNA synthesis was observed
187
in E2F1 transfected RKO cells, and RRM2 silencing had a destructive effect on DNA
188
synthesis stimulated by E2F1 (Fig. 2d). These results suggest that E2F1 plays a significant
189
role in boosting cell proliferation through activating RRM2 in CRC cells.
190
E2F1 promotes the migration and invasion of CRC cells by activating RRM2
191
As the high expression of RRM2 may contribute to tumor invasion and metastasis, we next
192
investigated the functional role of E2F1 and RRM2 in CRC cell migration and invasion. The
193
results showed that knockdown of either E2F1 or RRM2 weakened the acquisitive capacity of
194
migration and invasion in HT29 cells while overexpression of RRM2 rescued the wound
195
healing and invading abilities induced by siE2F1 (Fig. 3a,c). Similarly, overexpression of
196
either E2F1 or RRM2 increased migration and invasion in RKO cells, and RRM2 knockdown
197
with siRNA interference partly retarded the E2F1 effects (Fig. 3b,d). The above results
198
indicate that RRM2 transactivation by E2F1 is essential for migration and invasion in CRC
199
cells.
200
E2F1 expression is upregulated in CRC tissues and positively associated with RRM2
201
level at the advanced cancer stage
202
Immunohistochemical staining was carried out to analyze the clinical relevance of E2F1 and
203
RRM2 expressions in 154 human CRC specimens. The expression levels of RRM2 and E2F1
204
were increased in the tumor compared with the normal intestinal tissue (Fig. 4a and table 1).
AC C
EP
TE D
M AN U
SC
RI PT
178
8
ACCEPTED MANUSCRIPT Further analyses showed that the RRM2 and E2F1 staining were positively correlated with
206
lymph node metastasis (LNM), distant metastasis and advanced TNM stages (p<0.05) but not
207
with tumor size (p>0.05) (Table 2). Moreover, the E2F1 expression levels paralleled the
208
changes of RRM2 in the CRC cases as shown by immunohistochemical analyses (p<0.05)
209
(Fig. 4b). The 5-year overall survival (OS) rate of the RRM2 and E2F1 double-high
210
expression group was significantly lower than that of the RRM2 and E2F1 double-low
211
expression group (27.7% vs 55.4% , p<0.001). Of note is that patients with low levels of
212
RRM2 have a better prognosis than RRM2-high expression patients regardless of the E2F1
213
expression status (Fig. 4c). These data suggest that E2F1 is positively correlated with RRM2
214
expression, which is involved in CRC progression and metastasis.
215
Discussion
216
Abnormal expression of ribonucleotide reductase is closely relative to several types of cancer.
217
As the small subunit of RR, up-regulation of RRM2 increases RR activity, which provides
218
extra dNTPs in cancer cells. Furthermore RRM2 could serve as an independent prognostic
219
factor and predict poor survival of CRCs [24; 25]. Here we found that the increased RRM2
220
contributed to the proliferation, migration, invasion and metastasis of CRC cells. Previous
221
observation has shown that the RRM2 is not only a rate-limiting component for
222
ribonucleotide reduction, but is also capable of acting in cooperation with a variety of
223
oncogenes to promote transformation and tumorigenesis [14]. Overexpression of RRM2 in
224
KB and PC-3 cells could induce the migration ability of HUVECs (human umbilical vein
225
endothelial cells) [26]. Besides, it has been reported that RRM2 could increase the MMP-9
226
expression and enhance the cell invasion ability in cancer cell lines [15]. Therefore, dNTPs
227
pool expansion, acceleration of cell proliferation, and improvement of metastasis ability may
228
partly explain why RRM2 increases the aggressiveness and causes poor survival in CRCs.
229
The regulatory mechanisms which control the gene expression of RR subunits depend on
230
different conditions. Due to its long half-life, the level of RRM1 is virtually constant
231
throughout the cell cycle and always in excess of the level of RRM2 [27; 28]. Therefore, the
AC C
EP
TE D
M AN U
SC
RI PT
205
9
ACCEPTED MANUSCRIPT activity of RR is mainly regulated by the synthesis and degradation of the RRM2 protein. As
233
an important transcription factor, E2F1 is involved in S-phase specific expression of RRM2 in
234
physiological situations [29; 30]. On the other hand, the transcription of RRM2 was also
235
activated by E2F1 through ATM/ATR-CHK1 signal pathway in response to DNA damage [31].
236
As a potential predictor for response to fluropyrimidines in colon cancer, E2F1 controls the
237
transcription of several genes encoding proteins involved in DNA synthesis, namely
238
thymidylate synthase, dihydrofolate reductase, and thymidine kinase [29; 32]. A high
239
correlation between E2F1 and TS was recently shown in colorectal cancer metastases to the
240
lung and the liver [21]. Our results suggested that overexpressed E2F1 upregulated the
241
expression of RRM2 by promoting its transcriptional activation in CRC cell lines. Further
242
investigations showed that E2F1 promoted the proliferation, migration and invasion of CRC
243
cells which were attenuated by knockdown of RRM2. So RRM2 may function as an
244
important target gene of E2F1, which plays a vital role in the progress of CRC.
245
Analyses of clinical specimens from CRC patients showed that both E2F1 and RRM2
246
expressions were upregulated in most cancer tissues compared to paired normal tissues. The
247
high expression of E2F1 in CRC might be achieved by phosphorylation or other
248
post-translational modifications and the mechanism should be further investigated[33]. The
249
increased RRM2 protein levels were positively correlated with TNM stage and metastasis in
250
parallel with the E2F1 expression. Therefore, the clinical evidence further support the
251
hypothesis that E2F1 performs as the transcriptional activator of RRM2 in promoting CRC
252
development: the increased RRM2 protein activated by E2F1 contributes to tumor initiation
253
and metastasis. Moreover, we analyzed the survival rate of the patients with different E2F1
254
and RRM2 expression. For most patients, the expression level of RRM2 and E2F1 was
255
negatively correlated with the survival rate. However, the survival rate of some patients with
256
inverse correlation between RRM2 and E2F1 depended on RRM2 levels. So it is reasonable
257
to suggest that the role of E2F1 in the initiation and progress of CRC is dependent on the
258
expression level of RRM2. Thus, integrative assessments of RRM2 and E2F1 protein
AC C
EP
TE D
M AN U
SC
RI PT
232
10
ACCEPTED MANUSCRIPT expression in clinical samples would help reach a proper prognosis as well as the rational use
260
of different RR inhibitors: enzyme inactivators or gene expression suppressors of RRM2 for
261
personalized cancer therapy.
262
In summary, our molecular and clinical evidence demonstrated the relationship between E2F1
263
and RRM2 and their roles in promoting CRC invasion and metastasis. Still, the underlying
264
mechanism of regulating RRM2 in CRC cells has not been clearly elucidated. Further
265
understanding of the upstream regulatory pathway of RRM2 may help to provide new
266
biomarkers and therapeutic targets for human colorectal cancer.
267
Acknowledgements: This work was supported by Zhejiang Medical Association Clinical
268
Research Fund (No. 2013ZYC-A144), and Taizhou Science and Technology Plan (No.
269
1301ky55).
SC
M AN U
270
EP
TE D
References: [1] L.A. Torre, F. Bray, R.L. Siegel, et al., Global cancer statistics, 2012, CA Cancer J Clin 65 (2015)87-108. [2] M.M. Center, A. Jemal, E. Ward, International trends in colorectal cancer incidence rates, Cancer Epidemiol Biomarkers Prev 18 (2009)1688-94. [3] P. Guo, Z.L. Huang, P. Yu, et al., Trends in cancer mortality in China: an update, ANN ONCOL 23 (2012)2755-62. [4] J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, et al., Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012, EUR J CANCER 49 (2013)1374-403. [5] M. Malvezzi, P. Bertuccio, F. Levi, et al., European cancer mortality predictions for the year 2012, ANN ONCOL 23 (2012)1044-52. [6] P. Nordlund, P. Reichard, Ribonucleotide Reductases, Annu Rev Biochem 75 (2006)681-706. [7] H. Tanaka, H. Arakawa, T. Yamaguchi, et al., A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage, NATURE 404 (2000)42-9. [8] A. Gautam, Z.R. Li, G. Bepler, RRM1-induced metastasis suppression through PTEN-regulated pathways, ONCOGENE 22 (2003)2135-42. [9] H. Qi, M. Lou, Y. Chen, et al., Non-enzymatic action of RRM1 protein upregulates PTEN leading to inhibition of colorectal cancer metastasis, Tumour Biol (2015). [10] T. Morikawa, R. Hino, H. Uozaki, et al., Expression of ribonucleotide reductase M2 subunit in gastric cancer and effects of RRM2 inhibition in vitro, HUM PATHOL 41 (2010)1742-8. [11] N. Wang, T. Zhan, T. Ke, et al., Increased expression of RRM2 by human papillomavirus E7 oncoprotein promotes angiogenesis in cervical cancer, Br J Cancer 110
AC C
271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
RI PT
259
11
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
(2014)1034-44. [12] K. Iwamoto, K.I. Nakashiro, H. Tanaka, et al., Ribonucleotide reductase M2 is a promising molecular target for the treatment of oral squamous cell carcinoma, INT J ONCOL (2015). [13] H. Fan, C. Villegas, A. Huang, et al., The mammalian ribonucleotide reductase R2 component cooperates with a variety of oncogenes in mechanisms of cellular transformation, CANCER RES 58 (1998)1650-3. [14] H. Fan, C. Villegas, J.A. Wright, Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potential, Proc Natl Acad Sci U S A 93 (1996)14036-40. [15] M.S. Duxbury, E.E. Whang, RRM2 induces NF-kappaB-dependent MMP-9 activation and enhances cellular invasiveness, Biochem Biophys Res Commun 354 (2007)190-6. [16] K. Zhang, S. Hu, J. Wu, et al., Overexpression of RRM2 decreases thrombspondin-1 and increases VEGF production in human cancer cells in vitro and in vivo: implication of RRM2 in angiogenesis, MOL CANCER 8 (2009)11. [17] K. Chimploy, G.D. Diaz, Q. Li, et al., E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin, INT J CANCER 125 (2009)2086-94. [18] I.L. Lai, C.C. Chou, P.T. Lai, et al., Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells, CARCINOGENESIS 35 (2014)2203-13. [19] H. Muller, M.C. Moroni, E. Vigo, et al., Induction of S-phase entry by E2F transcription factors depends on their nuclear localization, MOL CELL BIOL 17 (1997)5508-20. [20] T. Suzuki, W. Yasui, H. Yokozaki, et al., Expression of the E2F family in human gastrointestinal carcinomas, INT J CANCER 81 (1999)535-8. [21] D. Banerjee, R. Gorlick, A. Liefshitz, et al., Levels of E2F-1 expression are higher in lung metastasis of colon cancer as compared with hepatic metastasis and correlate with levels of thymidylate synthase, CANCER RES 60 (2000)2365-7. [22] W. Yasui, J. Fujimoto, T. Suzuki, et al., Expression of cell-cycle-regulating transcription factor E2F-1 in colorectal carcinomas, PATHOBIOLOGY 67 (1999)174-9. [23] B. Yu, M.E. Lane, S. Wadler, SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells, BIOCHEM PHARMACOL 64 (2002)1091-100. [24] X. Liu, H. Zhang, L. Lai, et al., Ribonucleotide reductase small subunit M2 serves as a prognostic biomarker and predicts poor survival of colorectal cancers, Clin Sci (Lond) 124 (2013)567-78. [25] X. Liu, B. Zhou, L. Xue, et al., Ribonucleotide reductase subunits M2 and p53R2 are potential biomarkers for metastasis of colon cancer, Clin Colorectal Cancer 6 (2007)374-81. [26] X. Liu, B. Zhou, L. Xue, et al., Metastasis-suppressing potential of ribonucleotide reductase small subunit p53R2 in human cancer cells, CLIN CANCER RES 12 (2006)6337-44. [27] Y. Engstrom, S. Eriksson, I. Jildevik, et al., Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits, J BIOL CHEM 260 (1985)9114-6.
AC C
296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
12
ACCEPTED MANUSCRIPT [28] G.J. Mann, E.A. Musgrove, R.M. Fox, et al., Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation, CANCER RES 48 (1988)5151-6. [29] J. DeGregori, T. Kowalik, J.R. Nevins, Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes, MOL CELL BIOL 15 (1995)4215-24. [30] A.L. Chabes, S. Bjorklund, L. Thelander, S Phase-specific transcription of the mouse ribonucleotide reductase R2 gene requires both a proximal repressive E2F-binding site and an upstream promoter activating region, J BIOL CHEM 279 (2004)10796-807. [31] Y.W. Zhang, T.L. Jones, S.E. Martin, et al., Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response, J BIOL CHEM 284 (2009)18085-95. [32] L. Wu, C. Timmers, B. Maiti, et al., The E2F1-3 transcription factors are essential for cellular proliferation, NATURE 414 (2001)457-62. [33] C. Stevens, L. Smith, N.B. La Thangue, Chk2 activates E2F-1 in response to DNA damage, NAT CELL BIOL 5 (2003)401-9.
356
Figure legends
357
Table 1 Expression of RRM2 and E2F1 in CRC and adjacent normal tissues
358
Table 2 Correlation of the expression of RRM2 and E2F1 with clinicopathological
359
features in CRC
360
Figure 1. E2F1 promotes RRM2 transactivation in CRC cells
361
a Expression of E2F1 and RRM2 proteins in 6 CRC cell lines were analyzed by western blot.
362
b The mRNA level of RRM2 was analyzed by qRT-PCR in CRC cells after transfection with
363
the E2F1 expression plasmid at different time points. c and d The protein level of RRM2 were
364
analyzed by western blot in CRC cells after transfection with the E2F1 expression plasmid at
365
different time points. e and f The mRNA and protein levels of RRM2 in CRC cells after
366
transfected with the siRNA targeting E2F1. g Relative luciferase activity in CRC cells
367
co-transfected with EV or E2F1 expression plasmid, RRM2 promoter reporter (-2465/+23)
368
and an internal control reporter pRL-TK. *P<0.05.
369
Figure 2. E2F1 promotes the proliferation of CRC cells by activating RRM2
370
a and b At 12-60 h after the indicated siRNAs and expression plasmids transfection, the
371
viability of HT29 (a) or RKO (b) cells were determined by the CCK-8 assay. c and d DNA
372
synthesis of HT29 (c) or RKO (d) cells were measured by EdU incorporation assay after the
AC C
EP
TE D
M AN U
SC
RI PT
340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355
13
ACCEPTED MANUSCRIPT indicated transfection. *P<0.05.
374
Figure 3. E2F1 promotes the migration and invasion of CRC cells by activating RRM2
375
a and b Left panels: images from scratch assays with HT29 (a) or RKO (b) cells transfected
376
with indicated siRNAs and expression plasmids. Right panels: percentage wound closure 48 h
377
after the indicated transfection. *P<0.05. c and d Left panels: representative images of HT29
378
(c) or RKO (d) cells penetrating the Matrigel in invasion assays. Right panels: numbers of
379
invasive cells transfected with the indicated siRNAs and expression plasmids for 48 h.
380
*P<0.05.
381
Figure 4. E2F1 expression is upregulated in CRC tissues and positively associated with
382
RRM2 level at the advanced cancer stage
383
a Representative images of E2F1 and RRM2 immunostaining in cancer and adjacent
384
non-cancerous tissues. b The correlation of concurrent immunostaining scores of RRM2 and
385
E2F1 in CRC tissues. c The survival rate plot of indicated expression groups in CRC.
AC C
EP
TE D
M AN U
SC
RI PT
373
14
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Table 1 Expression of RRM2 and E2F1 in CRC and adjacent normal tissues cases CRC adjacent normal tissues *P<0.05
154 154
RRM2 expression (cancer) low high P Value cases cases 78 76 <0.001* 147 7
E2F1 expression (cancer) low high P Value cases cases 85 69 <0.001* 139 15
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
Table 2 Correlation of the expression of RRM2 and E2F1 with clinicopathological features in CRC RRM2 expression (cancer)
cases 154 Tumor location
cases
cases
78
76
male female
83 71
EP
Age
65 89
33 45
TE D
Gender
AC C
İ65 ˚65
LNM
high
P Value
low
high
cases
cases
85
69
0.988
Colon Rectum
Tumor size
low
E2F1 expression (cancer)
64 90
41 37
32 44
0.830 35 50
30 39
0.788 42 34
0.296 43 42
40 29
0.365 36 42
28 48
0.645 35 50
29 40
0.592
0.426
˘5cm
72
36
36
41
31
ı5cm
82
42
40
44
38
0.004*
0.013*
N0
87
51
36
54
33
N1/2
67
27
40
31
36
TNM
0.032*
0.014*
ĉ Ċ
23
15
8
15
8
59
29
30
32
27
ċ
60 12
32 2
28 10
35 3
25 9
Č Distant metastasis
0.002* M0 M1
LNM: lymph node metastasis;*P<0.05
142 12
76 2
66 10
P Value
0.001* 82 3
60 9
ACCEPTED MANUSCRIPT
RRM2 GAPDH
d
RKO cmyc-E2F1(h)
0
24
HCT8 48
72
0
24
48
72
cmyc-E2F1 RRM2
TE D
GAPDH
* 3
*
*
2
*
1 0
RKO
HCT8
M AN U
c
cmyc-E2F1 0hr 24hr 48hr
4
RI PT
Alternation of RRM2 mRNA levels
E2F1
Optical density of RRM2 protein (arbitrary units %)
9
48 0 D LD 1
SW
T1 1
H T2
H C
T8 H C
R K O
6
b
SC
a
250
*
cmyc-E2F1 0h 24h 48h 72h
* *
200
*
150 100
50
0
RKO
HCT8
f
0.5
0.0
SW480
RRM2 promoter relative luciferase activity
* 6 4 2 0
RKO
HCT8
-2 F1 E2 si
F1 E2 si
C tr l si
F1 E2 si
F1 E2 si
-1
-2
HT29
-1
GAPDH
EV E2F1
8
l
RRM2
HT29
*
tr
E2F1
g 10
C
*
SW480
siCtrl siE2F1
si
* 1.0
EP
1.5
AC C
Alternation of RRM2 mRNA levels
e
ACCEPTED MANUSCRIPT
b
RI PT
a HT29 siCtrl+EV
3.0
siE2F1+EV
Absorptivity
1.5
48
60
60
t/h
*
0.3
*
0.2 0.1
M 2 2F 1+ R R
si E
si E2 F1 +E V
EV tr l+ si C
E2F1+siRRM2 0.6
*
*
0.5 0.4 0.3 0.2 0.1
E2 F1 +s iR
R
tr l
M 2
0.0
E2 F1 +s iC
E2F1+siCtrl
48
0.0
%EdU incorporation
EV+siCtrl
36
0.4
AC C
HT29
DAPI
siE2F1+RRM2
EP
EdU
siE2F1+EV
24
+s iC tr l
siCtrl+EV
TE D
c
12
EV
36
%EdU incorporation
24
t/h
RKO
1.0
0.0
12
EdU
1.5
siRRM2+E2F1
0.5
0.0
d
2.0
M AN U
1.0 0.5
DAPI
siCtrl+E2F1
2.5
siE2F1+RRM2
2.0
siCtrl+EV
SC
2.5
Absorptivity
RKO
ACCEPTED MANUSCRIPT
*
0.5
si R R
si E
0.3
0.2
0.1
M 2 R
tr l E 2F 1+ si R
E 2F 1+ si C
*
200 150
*
100
2F 1+ R si E
si R
R
E V
2F 1+ E
E V
M 2
50
d
*
400
*
350 300 250 200 150 100 50
E 2F 1+ si C tr l R R M 2+ si C tr E l 2F 1+ si R R M 2
E2F1+siRRM2
+s iC tr l
RRM2+siCtrl
E V
E2F1+siCtrl
Number of invasive cells
* EV+siCtrl
RKO
M 2+ si C
tr l
0.0
si C tr l+
HT29
0.5
0.4
V
siE2F1+RRM2
*
0.6
si E
siRRM2+EV
0.7
* Number of invasive cells
siE2F1+EV
AC C
siCtrl+EV
EP
c
*
*
0.8
E V
TE D
RKO
E2F1+siRRM2
M 2
V
+E
2F 1
tr l+
si C
SC RRM2+siCtrl
+s iC tr l
E2F1+siCtrl
percent of wound closed
EV+siCtrl
M AN U
b
V
0.0
2F 1+ R R
0.1
E
0.2
*
si E
0.3
M 2+
0.4
E V
HT29
*
0.6
R M 2+
siE2F1+RRM2
R
siRRM2+EV
R
siE2F1+EV
RI PT
siCtrl+EV
percent of wound closed
a
RI PT
ACCEPTED MANUSCRIPT
normal
SC
a cancer-1
M AN U
cancer-2
TE D
RRM2
b
c
P<0.001 2 R =0.265
15
RRM2 low/E2F1 low(n=56) RRM2 low/E2F1 high(n=22)
100 Cum survival
E2F1 score
AC C
EP
E2F1
10
5
RRM2 high/E2F1 low(n=29) RRM2 high/E2F1 high(n=47)
80 60 40 20 0
0 0
5 10 RRM2 score
15
P<0.001 0
20
40 60 80 survival month
100
ACCEPTED MANUSCRIPT Highlights
2
1. E2F1 promotes RRM2 transactivation in CRC cells
3
2. E2F1 promotes the proliferation of CRC cells by activating RRM2
4
3. E2F1 promotes the migration and invasion of CRC cells by activating RRM2
5
4. E2F1 is upregulated in CRC tissues and positively associated with RRM2 level
6
5. E2F1-mediated RRM2 transcription will provide a new strategy in CRC.
AC C
EP
TE D
M AN U
SC
RI PT
1
1