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High expression of CREPT promotes tumor growth and is correlated with poor prognosis in colorectal cancer
Biochemical and Biophysical Research Communications 480 (2016) 436e442
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
High expression of CREPT promotes tumor growth and is correlated with poor prognosis in colorectal cancer Guoxu Zheng a, 1, Weimiao Li b, 1, Baile Zuo a, Zhangyan Guo a, Wenjin Xi a, Ming Wei c, Peng Chen d, Weihong Wen a, *, An-Gang Yang a, ** a
State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi'an, 710032, China Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, China Department of Urology, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, China d Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2016 Accepted 19 October 2016 Available online 20 October 2016
CREPT (cell cycle-related and expression elevated protein in tumor) is highly expressed in many kinds of cancer, and has been shown to be prognostic in certain cancers. However, the clinical significance of CREPT in colorectal cancer (CRC) has not been sufficiently investigated. In this study, we examined the CREPT expression in 225 clinical CRC tissues and paired adjacent normal tissues, and analyzed the correlation between CREPT expression and other clinicopathological features. We also evaluated the biological function of CREPT both in vitro and in vivo using knockdown or overexpressing CRC cells. Our results showed that CREPT expressed in 175 of 225 (77.8%) CRC patients and the CREPT expression was significantly associated with tumor differentiation (P ¼ 0.000), Dukes' stages (P ¼ 0.013) and metastasis (P ¼ 0.038). Patients with high CREPT expression tended to have shorter survival time. Multivariate analysis showed that positive CREPT expression can be used as an independent predictor for CRC prognosis. CREPT knockdown cells showed inhibited cell proliferation and arrested cell cycle, while CREPT overexpressing cells showed increased proliferation and promoted cell cycle. In addition, CREPT overexpression significantly promoted tumor growth in vivo. Mechanism study showed that CREPT may regulate cell proliferation and cell cycle through the regulation on cyclin D3, CDK4 and CDK6. © 2016 Elsevier Inc. All rights reserved.
Keywords: CREPT Cell proliferation Cell cycle Colorectal cancer Prognosis
1. Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with an estimated over 1 million new cases and more than half million deaths in 2012 [1]. Surgery is the most effective treatment for CRC, but most patients have already in the late stage at diagnosis because of the lack of effective diagnostic markers. In these cases, effectiveness of surgery is limited. Therefore, the identification of new accurate biological markers for diagnosis and prognosis are urgently required [2]. CREPT (cell cycle-related and expression elevated protein in
* Corresponding author. Department of Immunology, Fourth Military Medical University, 169 Changle West Road, Xi'an, 710032, China. ** Corresponding author. Department of Immunology, Fourth Military Medical University, 169 Changle West Road, Xi'an, 710032, China. E-mail addresses: [email protected] (W. Wen), [email protected] (A.-G. Yang). 1 Contributed equally. http://dx.doi.org/10.1016/j.bbrc.2016.10.067 0006-291X/© 2016 Elsevier Inc. All rights reserved.
tumor), also called RPRD1B (regulation of nuclear pre-mRNA domain containing protein 1B), is a novel protein that belongs to a new protein family with the RPR domain. It has been shown that CREPT is highly expressed in many kinds of cancers, such as lung cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer et al., and CERPT can promote tumor growth through binding with and recruiting RNA polymerase II (RNAPII) to the promoter of CYCLIN D1 [3]. CREPT was highly expressed in retroperitoneal leiomyosarcoma and its expression was significantly correlated with poor prognosis [4]. In endometrial cancer, the overexpression CREPT was correlated with tumor stage, histology type and depth of myometrial invasion. And CREPT can promote cell proliferation and accelerate cell cycle by upregulating cyclin D1, CDK4 and CDK6 in endometrial cancer cells, and can accelerate tumor growth in vivo [5]. However, the function of CREPT and the relationship between CREPT expression and prognosis in CRC remains unclear. In this study, we detected the CREPT expression in 225 CRC tissues and paired adjacent normal tissues and evaluated the
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predictive value of CREPT in the prognosis of CRC patients. We also examined the function of CREPT in CRC cells both in vitro and in vivo. 2. Materials and methods 2.1. Cell culture and tissue preparation Seven colorectal cancer cell lines (Caco-2, HCT115, HCT116, SW480, SW620, Lovo and HT29) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The SW480 and SW620 cells were cultured in L-15 medium (Macgen, China) supplemented with 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin-streptomycin, and cultured at 37 C and with 5% CO2 in a humidified incubator. Tumor tissues with paired adjacent normal tissues were obtained from 225 CRC patients at the Department of Colorectal Surgery, Tianjin Union Medical Center from 2004 to 2005. All the patients underwent surgery without chemotherapy or radiotherapy before operation and all the histology and clinical stages of the patients were classified according to the criteria of UICC (Union for International Cancer Control). The final patient follow-up date was January 1st, 2009, and the overall median survival time was 48 months (range: 4e61 months). This study was approved by the Ethics Committee of the Fourth Military Medical University (Xi'an, China), and all the patients gave their written informed consent.
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2.4. Cell cycle analysis Cell cycle distribution was analyzed by flow cytometry after PI staining. Cells were collected 48 h after transfection, and fixed overnight with 70% ice-cold ethanol, then stained with 50 mg/ml propidium iodide (MP Biomedicals, USA), and examined using a FACS scan flow cytometer (BD Biosciences, USA) at 488 nm. The results were analyzed by FlowJo 6.1 software. 2.5. Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The RNA was reverse transcribed using the Prime-Script RT Reagent Kit Perfect Real Time (TaKaRa, Japan). The qPCR was performed using a CFX96™ Real-Time PCR system (BioRad, USA) with SYBR Green reagents (TaKaRa, Japan). Gene expression was normalized to GAPDH. The primers used for real-time PCR were the following: CREPT, 50 -GCTGGAGAAGAAGCTCTCGG-3’ (forward) and 50 -ATTTGATTTGGCTTTGCGGAG-3’ (reverse); Cyclin D3, 50 -GACCATCGAAAAACTGTGCATCTA-3’ (forward) and 50 -CCCACTTGAGCTTCCCTAGGA-3’ (reverse); CDK4, 50 TGAGGGGGCCTCTCTAGCTT-30 (forward) and 50 -CAAGGGAGACCCTCACGCC-30 (reverse); CDK6, 50 -TGCACAGTGTCACGAACAGA3’ (forward) and 50 -ACCTCGGAGAAGCTGAAACA-30 (reverse); GAPDH, 50 -GGTGAAGGTCGGTGTGAACG-3’ (forward) and 50 CTCGCTCCTGGAAGATGGTG-3’ (reverse). 2.6. Western blot
2.2. Immunohistochemistry (IHC) Paraffin-embedded tissue sections were stained by immunohistochemistry (IHC) to detect the expression of CREPT in CRC tissues and adjacent normal tissues. IHC staining was performed as previously described with minor modifications [6]. Briefly, the slides were deparaffinized in xylene and rehydrated in a graded alcohol series and the endogenous peroxidase activity was blocked with 3% H2O2. Pre-immune rabbit serum was used to block nonspecific binding before the sections were incubated overnight with anti-CREPT primary antibody (GTX119969, GeneTex, USA) in a humidity chamber at 4 C. Slides were then washed three times with phosphate-buffered saline (PBS), and incubated with a biotinylated secondary antibody for 30 min at room temperature. The signal visualization was performed using 3, 30 -diaminobenzidine (DAB) chromogen for 2e3 min. The IHC evaluation was performed according to the intensity and extent of staining as previously described [7]. 2.3. siRNA and plasmids transfection The siRNAs targeting CREPT were synthesized by GenePharma (Shanghai, China). The CREPT expressing plasmid (pcDNA3.1CREPT) and the empty plasmid were obtained from the Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University. The siRNAs and plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. The siRNA duplexes (siCREPT-#1:50 GCAAGAACGAAGUGUUAUTT-30 , siCREPT-#2:50 -GUCUGUUACUAGCAGAAUATT-30 and siCREPT-#3:50 -ATGTCTGTTACTAGCAGAA30 ) target CREPT gene. The negative control duplex (50 -UUCUCCGAACGUGUCACGUTT-30 ) target a nonspecific sequence. For in vitro study, cells were harvested 48 h after transfection for analysis. To generate stable CREPT overexpressing cell lines, cells were screened by 800 mg/ml G418 after transfection and cells stably expressing CREPT were maintained in medium with 200 mg/ml G418.
Cells were harvested 48 h after transfection in lysis buffer (50 mM NaCl, 50 mM EDTA, 1% Triton X-100) containing protease inhibitor cocktail (Roche, USA). Cell lysates were separated by SDSPAGE and transferred onto nitrocellulose membranes [6]. The membranes were blocked with 5% nonfat milk diluted in PBS for 2 h at room temperature before being incubated with primary antibodies: anti-Cyclin D3, CDK4 and CDK6 (#2936, #12790, #3136, Cell Signaling Technology, USA); anti-CREPT (GTX119969, GeneTex, USA); and anti-b-actin (A4700; Sigma-Aldrich, USA). The membranes were then washed with PBS containing 0.05% Tween and incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The bands were visualized using a chemiluminescence reagent (New England Nuclear, USA). 2.7. MTT assay Cell proliferation was analyzed in vitro using tetrazolium salt 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent. Cells were transfected with specific siRNA or plasmid for 48 h before analysis. Briefly, 4000 cells from each group were plated in five 96-well plates in 200 mL of medium. For analysis, 20 mL of the MTT substrate (2.5 mg/mL) in PBS was added to each well. The plates were then returned to the incubater for an additional 4 h. The medium was then removed, and the cells were solubilized in 150 mL dimethylsulfoxide before colorimetric analysis (wavelength, 490 nm). Then, one plate was analyzed immediately after the cells adhered (approximately 4 h after plating), and the remaining plates were analyzed for the next 4 consecutive days. 2.8. Tumor growth analysis The protocol for animal study was approved by the Ethics Committee of the Fourth Military Medical University (Xi'an, China). Ten athymic Balb/c nude mice (5e6 weeks old, weighing 18 ± 0.39 g) were randomly assigned into two groups, with 5 mice per group (control and pcDNA3.1-CREPT group). CREPT
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overexpressing SW480 cells and control cells and were washed and resuspended to 5 106 cells/mL in PBS, and 0.2 mL was subcutaneously injected into the right back near the thigh of nude mice. Tumor diameter was measured with a Vernier caliper five days after injection and recorded every other five days until the 30th day. Tumor volume was calculated as the length width2/2, where the length and width are the longest and shortest axes in millimeters. At the end of the experiment (the 30th day), following euthanasia, tumors were excised for further analysis. 2.9. Statistical analysis Statistical analysis was performed using IBM SPSS statistical software (version 20.0). The Kaplan-Meier method was used to estimate survival curves, and the long-rank test was used for comparison. Cox regression was used for univariate and multivariate analysis. The differences in characteristics between the 2 groups were examined using Pearson's c2 test. All the P values were determined using 2-sided tests, and statistical significance was based on a P value of 0.05. 3. Results 3.1. CREPT is highly expressed in human colorectal cancer and is correlated with tumor stage and metastasis To evaluate the CREPT expression in clinical CRC tissues, we
performed IHC staining using 225 CRC tumor tissues and adjacent normal tissues. Results showed that 175 of 225 (77.8%) CRC tissues have positive CREPT expression, while CREPT expression was not detectable in all adjacent normal tissues (Fig. 1AeE). We also detected the expression of CREPT in seven colorectal cancer cell lines by qRT-PCR and Western blot. Results showed that the SW620 cells have high CREPT expression, while SW480 cells have pretty low CREPT expression in both mRNA and protein level (Fig. 1F), thus the SW620 and SW480 cells were used for later function study. We then analyzed the relationship between the CREPT expression and other clinicopathologic characteristics using Pearson chi-square test. We found that positive CREPT expression was significantly associated with tumor differentiation (P ¼ 0.000), Dukes' stages (P ¼ 0.013) and metastasis (P ¼ 0.038). However, CREPT expression was not correlated with gender, age and tumor site (Table 1). The results show that CREPT is highly expressed in CRC tumor tissues, and the CREPT level is correlated with tumor stage and metastasis. 3.2. High CREPT expression is associated with poor overall survival Kaplan-Meier analysis and log-rank test were used to analyze the relationship between CREPT expression and overall survival. Significant difference in overall survival was found between the CREPT-positive (þ, þþ, þþþ) and CREPT-negative groups (Fig. 1G, log-rank test: P ¼ 0.0024). Patients with positive CREPT expression tended to have a higher risk. In a multivariate analysis, we found
Fig. 1. CREPT is highly expressed in human colorectal cancer and high CREPT expression is correlated with poor overall survival. (AeE). Representative immunohistochemical staining of CREPT in clinical CRC tumor tissues and adjacent normal tissues. A. CREPT negative adjacent normal tissue. B. CREPT negative tumor tissue. C-E. CREPT positive tumor tissues: C, weak (þ); D, moderate (þþ); E, strong (þþþ). F. QRT-PCR and Western blot to show the CREPT expression in seven colorectal cancer cell lines. G. Patients with lower CREPT expression level showed better overall survival.
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Table 1 The correlation between clinicopathological features and CREPT expression of 225 CRC patients. Clinicopathological features
Age(years) ＜60 60 Gender Female Male Tumor site Colon Rectum Tumor differentiation Poorly/moderately Well Dukes' stages A-B C-D Metastasis No Yes
No. of cases 225
CREPT expression e
þ
þþ
þþ
104 121
17 33
36 40
33 28
18 20
106 119
22 28
35 41
29 32
20 18
73 152
18 32
25 51
18 43
12 26
191 34
34 16
64 12
58 3
35 3
125 100
34 16
45 31
28 33
18 20
130 95
34 16
46 30
31 30
19 19
c2
P value
2.671
0.012
0.601
0.438
0.180
0.671
14.504
0.000*
6.201
0.013*
4.293
0.038*
*P < 0.05 was considered statistically significant.
that positive CREPT expression is associated with decreased overall survival. The adjusted HR was 2.060 (95% CI: 1.081e3.926; p ¼ 0.028), indicating that CREPT could be used as an independent prognostic factor (Table 2). 3.3. Knockdown of CREPT induces cell cycle arrest and inhibits cell proliferation in SW620 cells To examine the function of CREPT in CRC cells, siRNAs targeting CREPT were transfected into SW620 cells to inhibit the CREPT expression. Compared with the control group, CREPT expression was significantly inhibited in cells transfected with three different kinds of CREPT-targeting siRNAs, both in mRNA and protein level (Fig. 2A). Cell cycle and MTT analysis were performed to examine the cell cycle progression and cell proliferation. Results showed that knockdown of CREPT led to obvious cell cycle arrest and significantly inhibited cell proliferation (Fig. 2B, C). We examined the expression level of several cell cycle related protein, including cyclin D3, CDK4 and CDK6, and found that the knockdown of CREPT can obviously inhibit their expression (Fig. 2D).
overexpression of CREPT can obviously accelerate cell cycle and significantly promote cell proliferation in SW480 cells (Fig. 3B, C). We also examined the expression level of cell cycle related protein, and found that the overexpression of CREPT obviously induced the expression of cyclin D3, CDK4 and CDK6 (Fig. 3D). These results indicate that CREPT may regulate regulated cell cycle progression and cell proliferation through the regulation of these cell cycle related protein in CRC cells. 3.5. Overexpression of CREPT promotes tumor growth in vivo To analysis whether overexpression of CREPT can promote tumor growth in vivo, we established CRC xenograft model by inoculating control SW480 cells and SW480 cells stably overexpressing CREPT into nude mice. Results showed that the CREPT overexpressing xenograft grew much faster than the control group (Fig. 4A, B). And the tumor weight in CREPT overexpressing group was significantly higher than the control group (Fig. 4C). These results indicate that CREPT has an oncogenic function in CRC. 4. Discussion
3.4. Overexpression of CREPT accelerates cell cycle and promotes cell proliferation in SW480 cells To obtain CREPT overexpressing cells, the pcDNA3.1-CREPT plasmid or control empty plasmid was transiently transfected into SW480 cells, which has relatively low CREPT expression. The overexpression of CREPT was confirmed in both mRNA and protein level (Fig. 3A). Cell cycle and MTT analysis were performed and results showed that, compared with the control group,
CREPT is a novel gene that was identified during homolog searching for new genes that are related to tumorigenesis. CREPT can promote cell proliferation by enhancing the transcription of Cyclin D1. CREPT prevents RNAPII from ‘‘reading through’’ and possibly promotes the recycling of RNAPII to the promoter of Cyclin D1 via the formation of a chromatin loop [3]. Recent study showed that Wnt/b-catenin signaling pathway was involved in CREPTmediated gene transcription activation. CREPT can bind with both
Table 2 Univariate and multivariate analysis of the overall survival in colorectal cancer. Univariate analysis
CREPT expression Age Gender Tumor site
Multivariate analysis
P value
HR (95% CI)
0.025* 0.411 0.654 0.122
2.074 0.779 1.091 0.682
HR: hazard ratio; CI: confidence interval. *P < 0.05 was considered statistically significant.
(1.096, (0.494, (0.692, (0.421,
3.924) 1.228) 1.719) 1.107)
P value
HR (95% CI)
0.028* 0.173 0.686 0.827
2.060 1.014 0.910 1.033
(1.081, (0.994, (0.578, (0.754,
3.926) 1.034) 1.434) 1.389)
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Fig. 2. Knockdown of CREPT induces cell cycle arrest and inhibits cell proliferation in SW620 cells. A. The mRNA and protein level of CREPT in SW620 cells after the transfection of siRNA-CREPT for 48 h; B. Downregulation of CREPT resulted in G1 phase arrest in SW620 cells. C. The proliferation of CREPT knockdown SW620 cells was inhibited; D. The expression of cyclin D3, CDK4 and CDK6 decreased in CREPT knockdown cells. *P < 0.05 versus NC group; **P < 0.01 versus NC group.
b-catenin and TCF4, and enhances the association of b-catenin with TCF4. Over-expression of CREPT enhances the activity of the bcatenin/TCF4 complex to initiate the transcription of Wnt target genes, thus promote cell proliferation and tumor invasion [8]. CREPT is highly expressed in many kinds of human cancers and is correlated with short survival time of cancer patients. CREPT has been found to be correlated with the histology type, tumor differentiation and tumor stage in endometrial cancer [5]. In non-small cell lung cancer, it has been shown that the CREPT expression was significantly increased in NSCLC compared with adjacent lung tissues. And the CREPT expression was correlated with histological type, differentiation, and pTNM stages of NSCLC [9]. In retroperitoneal leiomyosarcoma, it has also been shown that patients with high CREPT expression had significantly shorter overall postoperative survival [4]. Therefore, CREPT is an important factor in tumorigenesis and tumor progression in certain cancers. Previous study has shown that CRC tissues have high CREPT expression, but the sample number is limited, and the correlation between CREPT expression and clinicopathological features has not been analyzed. In this study, we evaluated the CREPT expression in 225 clinical CRC samples, and found that CREPT expressed in 175 of 225 (77.8%) CRC patients, while no CREPT expression was observed in paired adjacent normal tissues. These findings confirmed the oncogenic function of CREPT in CRC. We also studied the relationship between CREPT expression and clinicopathological features and found that positive CREPT expression was significantly
associated with tumor differentiation, Dukes' stages and metastasis. In addition, we found that the expression level of CREPT is significantly associated with overall survival of CRC patients. Our results indicated that CREPT expression is correlated with tumor progression, and positive CREPT expression can be used as an independent predictive marker for poor prognosis in CRC patients. Abnormal cell proliferation is an important factor for tumorigenesis, and cell proliferation is controlled by cell cycle machinery. The cell cycle progression is delicately controlled by the activity of different cyclin-dependent kinases (CDKs) and their regulatory subunits known as cyclins. Specific cyclin-CDK complexes are needed to ensure the cell cycle progresses in an ordered way [10e12]. Cyclin D has been shown to function in a tissue specific manner. Cyclin D3 is a member of D-type cyclins, and is critical in tumor progression and development [13,14]. It has been demonstrated that cyclin D3 is highly overexpressed and regulates cell cycle progression in human T cell acute lymphoblastic leukemia (TALL). And Notch signaling has been shown to regulate the cell cycle progression through the regulation on Cyclin D3 [15,16]. In CRC, Cyclin D3 expression is important for cell proliferation, and decreased expression of cyclins D1 and D3 resulted in G1 phase arrest and inhibition of cell proliferation in HT-29 CRC cells [17,18]. In several kinds of cancers, CREPT has been found to enhance the transcription of Cyclin D1 to promote cell proliferation [3]. In endometrial cancer, CREPT has been shown to promote cell proliferation and cell cycle progression through upregulating cyclin D1,
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Fig. 3. Overexpression of CREPT accelerates cell cycle and promotes cell proliferation in SW480 cells. A. The mRNA and protein level of CREPT in SW480 cells after the transfection of pcDNA3.1-CREPT for 48 h; B. CREPT overexpressing SW480 cells showed increased S phase; C. CREPT overexpressing SW480 cells showed increased cell proliferation; D. The expression of cyclin D3, CDK4 and CDK6 increased in CREPT overexpressing cells. *P < 0.05 versus control group; **P < 0.01 versus control group.
CDK4 and CDK6 [5]. In our study, we found that knockdown of CREPT in SW620 cells, which has high CREPT expression, led to obvious cell cycle arrest and significantly inhibited cell
proliferation. We also found that knockdown of CREPT can inhibit the expression of cyclin D3, CDK4 and CDK6. While overexpression of CREPT in SW480 cells, which has relatively low CREPT
Fig. 4. Overexpression of CREPT promotes tumor growth in vivo. A. Typical tumor tissues of CREPT overexpressing and control SW480 xenografts. B. Tumor volume of CREPT overexpressing and control SW480 xenografts; C. The CREPT overexpressing SW480 xenografts showed significantly increased tumor weight. **P < 0.01 versus control group.
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expression, can accelerate cell cycle and promote cell proliferation, and the overexpression of CREPT enhanced the expression of cyclin D3, CDK4 and CDK6. These results indicate that CREPT may control cell cycle progression and cell proliferation through the regulation on the expression of cyclin D3, CDK4 and CDK6 in CRC. In summary, we found that CREPT was highly expressed in CRC tissues, and the elevated CREPT expression is significantly correlated with the aggressive phenotype and poor prognosis of CRC. We also found that CREPT might regulate cell proliferation and cell cycle arrest through the regulation on the expression of cyclin D3, CDK4 and CDK6. Thus, we hypothesize that CREPT could be a promising target and potential prognostic indicator in CRC. However, further studies are still needed to determine how CREPT regulates CRC progression. Acknowledgments This work was supported by the National Natural Sciences Foundation of China (No. 81630069, 81372225). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.10.067. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA, A Cancer J. Clin. 65 (2015) 87e108. [2] J. Maurel, A. Postigo, Prognostic and predictive biomarkers in colorectal Cancer. From the preclinical setting to clinical practice, Curr. Cancer Drug Targets 15 (2015) 703e715. [3] D. Lu, Y. Wu, Y. Wang, F. Ren, D. Wang, F. Su, Y. Zhang, X. Yang, G. Jin, X. Hao, D. He, Y. Zhai, David M. Irwin, J. Hu, Joseph J.Y. Sung, J. Yu, B. Jia, Z. Chang, CREPT accelerates tumorigenesis by regulating the transcription of cell-cyclerelated genes, Cancer Cell 21 (2012) 92e104. [4] Y. She, J. Liang, L. Chen, Y. Qiu, N. Liu, X. Zhao, X. Huang, Y. Wang, F. Ren, Z. Chang, P. Li, CREPT expression correlates with poor prognosis in patients with retroperitoneal leiomyosarcoma, Int. J. Clin. Exp. Pathol. 7 (2014)
6596e6605. [5] Y. Wang, H. Qiu, W. Hu, S. Li, J. Yu, RPRD1B promotes tumor growth by accelerating the cell cycle in endometrial cancer, Oncol. Rep. 31 (3) (2014) 1389e1395. [6] J. Zheng, W. Qin, D. Jiao, J. Ren, M. Wei, S. Shi, W. Xi, H. Wang, A.G. Yang, Y. Huan, W. Wen, Knockdown of COUP-TFII inhibits cell proliferation and induces apoptosis through upregulating BRCA1 in renal cell carcinoma cells, Int. J. Cancer 139 (7) (2016) 1574e1585. [7] C. Liu, B. Yue, C. Yuan, S. Zhao, C. Fang, Y. Yu, D. Yan, Elevated expression of Thoc1 is associated with aggressive phenotype and poor prognosis in colorectal cancer, Biochem. Biophysical Res. Commun. 468 (2015) 53e58. [8] Y. Zhang, C. Liu, X. Duan, F. Ren, S. Li, Z. Jin, Y. Wang, Y. Feng, Z. Liu, Z. Chang, CREPT/RPRD1B, a recently identified novel protein highly expressed in tumors, enhances the -Catenin{middle dot}TCF4 transcriptional activity in response to Wnt signaling, J. Biol. Chem. 289 (2014) 22589e22599. [9] T. Liu, W.M. Li, W.P. Wang, Y. Sun, Y.F. Ni, H. Xing, J.H. Xia, X.J. Wang, Z.P. Zhang, X.F. Li, Inhibiting CREPT reduces the proliferation and migration of non-small cell lung cancer cells by down-regulating cell cycle related protein, Am. J. Transl. Res. 8 (2016) 2097e2113. [10] P. Hydbring, M. Malumbres, P. Sicinski, Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases, Nat. Rev. Mol. Cell Biol. 17 (2016) 280e292. [11] M.H. Lee, H.Y. Yang, Regulators of G1 cyclin-dependent kinases and cancers, Cancer Metastasis Rev. 22 (2003) 435e449. [12] C. Bertoli, J.M. Skotheim, R.A. de Bruin, Control of cell cycle transcription during G1 and S phases, Nat. Rev. Mol. Cell Biol. 14 (2013) 518e528. [13] K. Mastorci, E. Muraro, E. Pasini, C. Furlan, L. Sigalotti, M. Cinco, R. Dolcetti, E. Fratta, Toll-like receptor 1/2 and 5 ligands enhance the expression of cyclin D1 and D3 and induce proliferation in mantle cell lymphoma, PLoS One 11 (2016) e0153823. [14] Y. Chi, S. Huang, M. Liu, L. Guo, X. Shen, J. Wu, Cyclin D3 predicts disease-free survival in breast cancer, Cancer Cell Int. 15 (2015) 89. [15] C.M. Sawai, J. Freund, P. Oh, D. Ndiaye-Lobry, J.C. Bretz, A. Strikoudis, L. Genesca, T. Trimarchi, M.A. Kelliher, M. Clark, J. Soulier, S. Chen-Kiang, I. Aifantis, Therapeutic targeting of the cyclin D3:CDK4/6 complex in T cell leukemia, Cancer Cell 22 (2012) 452e465. [16] I. Joshi, L.M. Minter, J. Telfer, R.M. Demarest, A.J. Capobianco, J.C. Aster, P. Sicinski, A. Fauq, T.E. Golde, B.A. Osborne, Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases, Blood 113 (2009) 1689e1698. [17] P.B. Yaffe, M.R. Power Coombs, C.D. Doucette, M. Walsh, D.W. Hoskin, Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress, Mol. Carcinog. 54 (2015) 1070e1085. [18] Z. Liu, R. Wu, G. Li, P. Sun, Q. Xu, MiR-592 inhibited cell proliferation of human colorectal cancer cells by suppressing of CCND3 expression, Int. J. Clin. Exp. Med. 8 (2015) 3490e3497.
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High expression of CREPT promotes tumor growth and is correlated with poor prognosis in colorectal cancer Guoxu Zheng a, 1, Weimiao Li b, 1, Baile Zuo a, Zhangyan Guo a, Wenjin Xi a, Ming Wei c, Peng Chen d, Weihong Wen a, *, An-Gang Yang a, ** a
State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi'an, 710032, China Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, China Department of Urology, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, China d Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2016 Accepted 19 October 2016 Available online 20 October 2016
CREPT (cell cycle-related and expression elevated protein in tumor) is highly expressed in many kinds of cancer, and has been shown to be prognostic in certain cancers. However, the clinical significance of CREPT in colorectal cancer (CRC) has not been sufficiently investigated. In this study, we examined the CREPT expression in 225 clinical CRC tissues and paired adjacent normal tissues, and analyzed the correlation between CREPT expression and other clinicopathological features. We also evaluated the biological function of CREPT both in vitro and in vivo using knockdown or overexpressing CRC cells. Our results showed that CREPT expressed in 175 of 225 (77.8%) CRC patients and the CREPT expression was significantly associated with tumor differentiation (P ¼ 0.000), Dukes' stages (P ¼ 0.013) and metastasis (P ¼ 0.038). Patients with high CREPT expression tended to have shorter survival time. Multivariate analysis showed that positive CREPT expression can be used as an independent predictor for CRC prognosis. CREPT knockdown cells showed inhibited cell proliferation and arrested cell cycle, while CREPT overexpressing cells showed increased proliferation and promoted cell cycle. In addition, CREPT overexpression significantly promoted tumor growth in vivo. Mechanism study showed that CREPT may regulate cell proliferation and cell cycle through the regulation on cyclin D3, CDK4 and CDK6. © 2016 Elsevier Inc. All rights reserved.
Keywords: CREPT Cell proliferation Cell cycle Colorectal cancer Prognosis
1. Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with an estimated over 1 million new cases and more than half million deaths in 2012 [1]. Surgery is the most effective treatment for CRC, but most patients have already in the late stage at diagnosis because of the lack of effective diagnostic markers. In these cases, effectiveness of surgery is limited. Therefore, the identification of new accurate biological markers for diagnosis and prognosis are urgently required [2]. CREPT (cell cycle-related and expression elevated protein in
* Corresponding author. Department of Immunology, Fourth Military Medical University, 169 Changle West Road, Xi'an, 710032, China. ** Corresponding author. Department of Immunology, Fourth Military Medical University, 169 Changle West Road, Xi'an, 710032, China. E-mail addresses: [email protected] (W. Wen), [email protected] (A.-G. Yang). 1 Contributed equally. http://dx.doi.org/10.1016/j.bbrc.2016.10.067 0006-291X/© 2016 Elsevier Inc. All rights reserved.
tumor), also called RPRD1B (regulation of nuclear pre-mRNA domain containing protein 1B), is a novel protein that belongs to a new protein family with the RPR domain. It has been shown that CREPT is highly expressed in many kinds of cancers, such as lung cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer et al., and CERPT can promote tumor growth through binding with and recruiting RNA polymerase II (RNAPII) to the promoter of CYCLIN D1 [3]. CREPT was highly expressed in retroperitoneal leiomyosarcoma and its expression was significantly correlated with poor prognosis [4]. In endometrial cancer, the overexpression CREPT was correlated with tumor stage, histology type and depth of myometrial invasion. And CREPT can promote cell proliferation and accelerate cell cycle by upregulating cyclin D1, CDK4 and CDK6 in endometrial cancer cells, and can accelerate tumor growth in vivo [5]. However, the function of CREPT and the relationship between CREPT expression and prognosis in CRC remains unclear. In this study, we detected the CREPT expression in 225 CRC tissues and paired adjacent normal tissues and evaluated the
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predictive value of CREPT in the prognosis of CRC patients. We also examined the function of CREPT in CRC cells both in vitro and in vivo. 2. Materials and methods 2.1. Cell culture and tissue preparation Seven colorectal cancer cell lines (Caco-2, HCT115, HCT116, SW480, SW620, Lovo and HT29) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The SW480 and SW620 cells were cultured in L-15 medium (Macgen, China) supplemented with 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin-streptomycin, and cultured at 37 C and with 5% CO2 in a humidified incubator. Tumor tissues with paired adjacent normal tissues were obtained from 225 CRC patients at the Department of Colorectal Surgery, Tianjin Union Medical Center from 2004 to 2005. All the patients underwent surgery without chemotherapy or radiotherapy before operation and all the histology and clinical stages of the patients were classified according to the criteria of UICC (Union for International Cancer Control). The final patient follow-up date was January 1st, 2009, and the overall median survival time was 48 months (range: 4e61 months). This study was approved by the Ethics Committee of the Fourth Military Medical University (Xi'an, China), and all the patients gave their written informed consent.
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2.4. Cell cycle analysis Cell cycle distribution was analyzed by flow cytometry after PI staining. Cells were collected 48 h after transfection, and fixed overnight with 70% ice-cold ethanol, then stained with 50 mg/ml propidium iodide (MP Biomedicals, USA), and examined using a FACS scan flow cytometer (BD Biosciences, USA) at 488 nm. The results were analyzed by FlowJo 6.1 software. 2.5. Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The RNA was reverse transcribed using the Prime-Script RT Reagent Kit Perfect Real Time (TaKaRa, Japan). The qPCR was performed using a CFX96™ Real-Time PCR system (BioRad, USA) with SYBR Green reagents (TaKaRa, Japan). Gene expression was normalized to GAPDH. The primers used for real-time PCR were the following: CREPT, 50 -GCTGGAGAAGAAGCTCTCGG-3’ (forward) and 50 -ATTTGATTTGGCTTTGCGGAG-3’ (reverse); Cyclin D3, 50 -GACCATCGAAAAACTGTGCATCTA-3’ (forward) and 50 -CCCACTTGAGCTTCCCTAGGA-3’ (reverse); CDK4, 50 TGAGGGGGCCTCTCTAGCTT-30 (forward) and 50 -CAAGGGAGACCCTCACGCC-30 (reverse); CDK6, 50 -TGCACAGTGTCACGAACAGA3’ (forward) and 50 -ACCTCGGAGAAGCTGAAACA-30 (reverse); GAPDH, 50 -GGTGAAGGTCGGTGTGAACG-3’ (forward) and 50 CTCGCTCCTGGAAGATGGTG-3’ (reverse). 2.6. Western blot
2.2. Immunohistochemistry (IHC) Paraffin-embedded tissue sections were stained by immunohistochemistry (IHC) to detect the expression of CREPT in CRC tissues and adjacent normal tissues. IHC staining was performed as previously described with minor modifications [6]. Briefly, the slides were deparaffinized in xylene and rehydrated in a graded alcohol series and the endogenous peroxidase activity was blocked with 3% H2O2. Pre-immune rabbit serum was used to block nonspecific binding before the sections were incubated overnight with anti-CREPT primary antibody (GTX119969, GeneTex, USA) in a humidity chamber at 4 C. Slides were then washed three times with phosphate-buffered saline (PBS), and incubated with a biotinylated secondary antibody for 30 min at room temperature. The signal visualization was performed using 3, 30 -diaminobenzidine (DAB) chromogen for 2e3 min. The IHC evaluation was performed according to the intensity and extent of staining as previously described [7]. 2.3. siRNA and plasmids transfection The siRNAs targeting CREPT were synthesized by GenePharma (Shanghai, China). The CREPT expressing plasmid (pcDNA3.1CREPT) and the empty plasmid were obtained from the Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University. The siRNAs and plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. The siRNA duplexes (siCREPT-#1:50 GCAAGAACGAAGUGUUAUTT-30 , siCREPT-#2:50 -GUCUGUUACUAGCAGAAUATT-30 and siCREPT-#3:50 -ATGTCTGTTACTAGCAGAA30 ) target CREPT gene. The negative control duplex (50 -UUCUCCGAACGUGUCACGUTT-30 ) target a nonspecific sequence. For in vitro study, cells were harvested 48 h after transfection for analysis. To generate stable CREPT overexpressing cell lines, cells were screened by 800 mg/ml G418 after transfection and cells stably expressing CREPT were maintained in medium with 200 mg/ml G418.
Cells were harvested 48 h after transfection in lysis buffer (50 mM NaCl, 50 mM EDTA, 1% Triton X-100) containing protease inhibitor cocktail (Roche, USA). Cell lysates were separated by SDSPAGE and transferred onto nitrocellulose membranes [6]. The membranes were blocked with 5% nonfat milk diluted in PBS for 2 h at room temperature before being incubated with primary antibodies: anti-Cyclin D3, CDK4 and CDK6 (#2936, #12790, #3136, Cell Signaling Technology, USA); anti-CREPT (GTX119969, GeneTex, USA); and anti-b-actin (A4700; Sigma-Aldrich, USA). The membranes were then washed with PBS containing 0.05% Tween and incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The bands were visualized using a chemiluminescence reagent (New England Nuclear, USA). 2.7. MTT assay Cell proliferation was analyzed in vitro using tetrazolium salt 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent. Cells were transfected with specific siRNA or plasmid for 48 h before analysis. Briefly, 4000 cells from each group were plated in five 96-well plates in 200 mL of medium. For analysis, 20 mL of the MTT substrate (2.5 mg/mL) in PBS was added to each well. The plates were then returned to the incubater for an additional 4 h. The medium was then removed, and the cells were solubilized in 150 mL dimethylsulfoxide before colorimetric analysis (wavelength, 490 nm). Then, one plate was analyzed immediately after the cells adhered (approximately 4 h after plating), and the remaining plates were analyzed for the next 4 consecutive days. 2.8. Tumor growth analysis The protocol for animal study was approved by the Ethics Committee of the Fourth Military Medical University (Xi'an, China). Ten athymic Balb/c nude mice (5e6 weeks old, weighing 18 ± 0.39 g) were randomly assigned into two groups, with 5 mice per group (control and pcDNA3.1-CREPT group). CREPT
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overexpressing SW480 cells and control cells and were washed and resuspended to 5 106 cells/mL in PBS, and 0.2 mL was subcutaneously injected into the right back near the thigh of nude mice. Tumor diameter was measured with a Vernier caliper five days after injection and recorded every other five days until the 30th day. Tumor volume was calculated as the length width2/2, where the length and width are the longest and shortest axes in millimeters. At the end of the experiment (the 30th day), following euthanasia, tumors were excised for further analysis. 2.9. Statistical analysis Statistical analysis was performed using IBM SPSS statistical software (version 20.0). The Kaplan-Meier method was used to estimate survival curves, and the long-rank test was used for comparison. Cox regression was used for univariate and multivariate analysis. The differences in characteristics between the 2 groups were examined using Pearson's c2 test. All the P values were determined using 2-sided tests, and statistical significance was based on a P value of 0.05. 3. Results 3.1. CREPT is highly expressed in human colorectal cancer and is correlated with tumor stage and metastasis To evaluate the CREPT expression in clinical CRC tissues, we
performed IHC staining using 225 CRC tumor tissues and adjacent normal tissues. Results showed that 175 of 225 (77.8%) CRC tissues have positive CREPT expression, while CREPT expression was not detectable in all adjacent normal tissues (Fig. 1AeE). We also detected the expression of CREPT in seven colorectal cancer cell lines by qRT-PCR and Western blot. Results showed that the SW620 cells have high CREPT expression, while SW480 cells have pretty low CREPT expression in both mRNA and protein level (Fig. 1F), thus the SW620 and SW480 cells were used for later function study. We then analyzed the relationship between the CREPT expression and other clinicopathologic characteristics using Pearson chi-square test. We found that positive CREPT expression was significantly associated with tumor differentiation (P ¼ 0.000), Dukes' stages (P ¼ 0.013) and metastasis (P ¼ 0.038). However, CREPT expression was not correlated with gender, age and tumor site (Table 1). The results show that CREPT is highly expressed in CRC tumor tissues, and the CREPT level is correlated with tumor stage and metastasis. 3.2. High CREPT expression is associated with poor overall survival Kaplan-Meier analysis and log-rank test were used to analyze the relationship between CREPT expression and overall survival. Significant difference in overall survival was found between the CREPT-positive (þ, þþ, þþþ) and CREPT-negative groups (Fig. 1G, log-rank test: P ¼ 0.0024). Patients with positive CREPT expression tended to have a higher risk. In a multivariate analysis, we found
Fig. 1. CREPT is highly expressed in human colorectal cancer and high CREPT expression is correlated with poor overall survival. (AeE). Representative immunohistochemical staining of CREPT in clinical CRC tumor tissues and adjacent normal tissues. A. CREPT negative adjacent normal tissue. B. CREPT negative tumor tissue. C-E. CREPT positive tumor tissues: C, weak (þ); D, moderate (þþ); E, strong (þþþ). F. QRT-PCR and Western blot to show the CREPT expression in seven colorectal cancer cell lines. G. Patients with lower CREPT expression level showed better overall survival.
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Table 1 The correlation between clinicopathological features and CREPT expression of 225 CRC patients. Clinicopathological features
Age(years) ＜60 60 Gender Female Male Tumor site Colon Rectum Tumor differentiation Poorly/moderately Well Dukes' stages A-B C-D Metastasis No Yes
No. of cases 225
CREPT expression e
þ
þþ
þþ
104 121
17 33
36 40
33 28
18 20
106 119
22 28
35 41
29 32
20 18
73 152
18 32
25 51
18 43
12 26
191 34
34 16
64 12
58 3
35 3
125 100
34 16
45 31
28 33
18 20
130 95
34 16
46 30
31 30
19 19
c2
P value
2.671
0.012
0.601
0.438
0.180
0.671
14.504
0.000*
6.201
0.013*
4.293
0.038*
*P < 0.05 was considered statistically significant.
that positive CREPT expression is associated with decreased overall survival. The adjusted HR was 2.060 (95% CI: 1.081e3.926; p ¼ 0.028), indicating that CREPT could be used as an independent prognostic factor (Table 2). 3.3. Knockdown of CREPT induces cell cycle arrest and inhibits cell proliferation in SW620 cells To examine the function of CREPT in CRC cells, siRNAs targeting CREPT were transfected into SW620 cells to inhibit the CREPT expression. Compared with the control group, CREPT expression was significantly inhibited in cells transfected with three different kinds of CREPT-targeting siRNAs, both in mRNA and protein level (Fig. 2A). Cell cycle and MTT analysis were performed to examine the cell cycle progression and cell proliferation. Results showed that knockdown of CREPT led to obvious cell cycle arrest and significantly inhibited cell proliferation (Fig. 2B, C). We examined the expression level of several cell cycle related protein, including cyclin D3, CDK4 and CDK6, and found that the knockdown of CREPT can obviously inhibit their expression (Fig. 2D).
overexpression of CREPT can obviously accelerate cell cycle and significantly promote cell proliferation in SW480 cells (Fig. 3B, C). We also examined the expression level of cell cycle related protein, and found that the overexpression of CREPT obviously induced the expression of cyclin D3, CDK4 and CDK6 (Fig. 3D). These results indicate that CREPT may regulate regulated cell cycle progression and cell proliferation through the regulation of these cell cycle related protein in CRC cells. 3.5. Overexpression of CREPT promotes tumor growth in vivo To analysis whether overexpression of CREPT can promote tumor growth in vivo, we established CRC xenograft model by inoculating control SW480 cells and SW480 cells stably overexpressing CREPT into nude mice. Results showed that the CREPT overexpressing xenograft grew much faster than the control group (Fig. 4A, B). And the tumor weight in CREPT overexpressing group was significantly higher than the control group (Fig. 4C). These results indicate that CREPT has an oncogenic function in CRC. 4. Discussion
3.4. Overexpression of CREPT accelerates cell cycle and promotes cell proliferation in SW480 cells To obtain CREPT overexpressing cells, the pcDNA3.1-CREPT plasmid or control empty plasmid was transiently transfected into SW480 cells, which has relatively low CREPT expression. The overexpression of CREPT was confirmed in both mRNA and protein level (Fig. 3A). Cell cycle and MTT analysis were performed and results showed that, compared with the control group,
CREPT is a novel gene that was identified during homolog searching for new genes that are related to tumorigenesis. CREPT can promote cell proliferation by enhancing the transcription of Cyclin D1. CREPT prevents RNAPII from ‘‘reading through’’ and possibly promotes the recycling of RNAPII to the promoter of Cyclin D1 via the formation of a chromatin loop [3]. Recent study showed that Wnt/b-catenin signaling pathway was involved in CREPTmediated gene transcription activation. CREPT can bind with both
Table 2 Univariate and multivariate analysis of the overall survival in colorectal cancer. Univariate analysis
CREPT expression Age Gender Tumor site
Multivariate analysis
P value
HR (95% CI)
0.025* 0.411 0.654 0.122
2.074 0.779 1.091 0.682
HR: hazard ratio; CI: confidence interval. *P < 0.05 was considered statistically significant.
(1.096, (0.494, (0.692, (0.421,
3.924) 1.228) 1.719) 1.107)
P value
HR (95% CI)
0.028* 0.173 0.686 0.827
2.060 1.014 0.910 1.033
(1.081, (0.994, (0.578, (0.754,
3.926) 1.034) 1.434) 1.389)
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Fig. 2. Knockdown of CREPT induces cell cycle arrest and inhibits cell proliferation in SW620 cells. A. The mRNA and protein level of CREPT in SW620 cells after the transfection of siRNA-CREPT for 48 h; B. Downregulation of CREPT resulted in G1 phase arrest in SW620 cells. C. The proliferation of CREPT knockdown SW620 cells was inhibited; D. The expression of cyclin D3, CDK4 and CDK6 decreased in CREPT knockdown cells. *P < 0.05 versus NC group; **P < 0.01 versus NC group.
b-catenin and TCF4, and enhances the association of b-catenin with TCF4. Over-expression of CREPT enhances the activity of the bcatenin/TCF4 complex to initiate the transcription of Wnt target genes, thus promote cell proliferation and tumor invasion [8]. CREPT is highly expressed in many kinds of human cancers and is correlated with short survival time of cancer patients. CREPT has been found to be correlated with the histology type, tumor differentiation and tumor stage in endometrial cancer [5]. In non-small cell lung cancer, it has been shown that the CREPT expression was significantly increased in NSCLC compared with adjacent lung tissues. And the CREPT expression was correlated with histological type, differentiation, and pTNM stages of NSCLC [9]. In retroperitoneal leiomyosarcoma, it has also been shown that patients with high CREPT expression had significantly shorter overall postoperative survival [4]. Therefore, CREPT is an important factor in tumorigenesis and tumor progression in certain cancers. Previous study has shown that CRC tissues have high CREPT expression, but the sample number is limited, and the correlation between CREPT expression and clinicopathological features has not been analyzed. In this study, we evaluated the CREPT expression in 225 clinical CRC samples, and found that CREPT expressed in 175 of 225 (77.8%) CRC patients, while no CREPT expression was observed in paired adjacent normal tissues. These findings confirmed the oncogenic function of CREPT in CRC. We also studied the relationship between CREPT expression and clinicopathological features and found that positive CREPT expression was significantly
associated with tumor differentiation, Dukes' stages and metastasis. In addition, we found that the expression level of CREPT is significantly associated with overall survival of CRC patients. Our results indicated that CREPT expression is correlated with tumor progression, and positive CREPT expression can be used as an independent predictive marker for poor prognosis in CRC patients. Abnormal cell proliferation is an important factor for tumorigenesis, and cell proliferation is controlled by cell cycle machinery. The cell cycle progression is delicately controlled by the activity of different cyclin-dependent kinases (CDKs) and their regulatory subunits known as cyclins. Specific cyclin-CDK complexes are needed to ensure the cell cycle progresses in an ordered way [10e12]. Cyclin D has been shown to function in a tissue specific manner. Cyclin D3 is a member of D-type cyclins, and is critical in tumor progression and development [13,14]. It has been demonstrated that cyclin D3 is highly overexpressed and regulates cell cycle progression in human T cell acute lymphoblastic leukemia (TALL). And Notch signaling has been shown to regulate the cell cycle progression through the regulation on Cyclin D3 [15,16]. In CRC, Cyclin D3 expression is important for cell proliferation, and decreased expression of cyclins D1 and D3 resulted in G1 phase arrest and inhibition of cell proliferation in HT-29 CRC cells [17,18]. In several kinds of cancers, CREPT has been found to enhance the transcription of Cyclin D1 to promote cell proliferation [3]. In endometrial cancer, CREPT has been shown to promote cell proliferation and cell cycle progression through upregulating cyclin D1,
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Fig. 3. Overexpression of CREPT accelerates cell cycle and promotes cell proliferation in SW480 cells. A. The mRNA and protein level of CREPT in SW480 cells after the transfection of pcDNA3.1-CREPT for 48 h; B. CREPT overexpressing SW480 cells showed increased S phase; C. CREPT overexpressing SW480 cells showed increased cell proliferation; D. The expression of cyclin D3, CDK4 and CDK6 increased in CREPT overexpressing cells. *P < 0.05 versus control group; **P < 0.01 versus control group.
CDK4 and CDK6 [5]. In our study, we found that knockdown of CREPT in SW620 cells, which has high CREPT expression, led to obvious cell cycle arrest and significantly inhibited cell
proliferation. We also found that knockdown of CREPT can inhibit the expression of cyclin D3, CDK4 and CDK6. While overexpression of CREPT in SW480 cells, which has relatively low CREPT
Fig. 4. Overexpression of CREPT promotes tumor growth in vivo. A. Typical tumor tissues of CREPT overexpressing and control SW480 xenografts. B. Tumor volume of CREPT overexpressing and control SW480 xenografts; C. The CREPT overexpressing SW480 xenografts showed significantly increased tumor weight. **P < 0.01 versus control group.
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expression, can accelerate cell cycle and promote cell proliferation, and the overexpression of CREPT enhanced the expression of cyclin D3, CDK4 and CDK6. These results indicate that CREPT may control cell cycle progression and cell proliferation through the regulation on the expression of cyclin D3, CDK4 and CDK6 in CRC. In summary, we found that CREPT was highly expressed in CRC tissues, and the elevated CREPT expression is significantly correlated with the aggressive phenotype and poor prognosis of CRC. We also found that CREPT might regulate cell proliferation and cell cycle arrest through the regulation on the expression of cyclin D3, CDK4 and CDK6. Thus, we hypothesize that CREPT could be a promising target and potential prognostic indicator in CRC. However, further studies are still needed to determine how CREPT regulates CRC progression. Acknowledgments This work was supported by the National Natural Sciences Foundation of China (No. 81630069, 81372225). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.10.067. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA, A Cancer J. Clin. 65 (2015) 87e108. [2] J. Maurel, A. Postigo, Prognostic and predictive biomarkers in colorectal Cancer. From the preclinical setting to clinical practice, Curr. Cancer Drug Targets 15 (2015) 703e715. [3] D. Lu, Y. Wu, Y. Wang, F. Ren, D. Wang, F. Su, Y. Zhang, X. Yang, G. Jin, X. Hao, D. He, Y. Zhai, David M. Irwin, J. Hu, Joseph J.Y. Sung, J. Yu, B. Jia, Z. Chang, CREPT accelerates tumorigenesis by regulating the transcription of cell-cyclerelated genes, Cancer Cell 21 (2012) 92e104. [4] Y. She, J. Liang, L. Chen, Y. Qiu, N. Liu, X. Zhao, X. Huang, Y. Wang, F. Ren, Z. Chang, P. Li, CREPT expression correlates with poor prognosis in patients with retroperitoneal leiomyosarcoma, Int. J. Clin. Exp. Pathol. 7 (2014)
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