Mol Cell Biochem DOI 10.1007/s11010-015-2496-x
Derlin-1 is overexpressed in human colon cancer and promotes cancer cell proliferation Xueming Tan1 • Xiaolu He2 • Zhonghua Jiang3 • Xiaohong Wang3 • Limei Ma3 • Li Liu4 • Xiang Wang4 • Zhining Fan4 • Dongming Su5
Received: 30 January 2015 / Accepted: 18 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Derlin-1 is overexpressed in many types of solid tumors and plays an important role in cancer progression. However, the expression pattern and functions of Derlin-1 in human colon cancer are not fully understood. In the present study, we examined Derlin-1 expression in colon cancer cell lines and human tissues and investigated its role in colon cancer. We found that Derlin-1 expression was increased significantly in colon cancer tissues and its overexpression correlated with the tumor differentiation, Dukes stage, invasion, lymph node metastasis, distant metastasis, and poor overall survival. The silencing of Derlin-1 by shRNA led to the growth inhibition of colon cancer cells, which were associated with the promotion of apoptosis. Furthermore, Derlin-1 silencing significantly inhibited the activation of the PI3K/AKT signaling pathway. Taken together, our results showed that Derlin-1 is
overexpressed in colon cancer and promotes proliferation of colon cancer cells. Derlin-1 may be a potential therapeutic target for the treatment of colon cancer. Keywords Derlin-1 Human colon cancer Proliferation Apoptosis Abbreviations ER Endoplasmic reticulum NSCLC Non-small cell lung cancer UPR Unfolded protein response
Introduction Colon cancer is one of the most common types of cancer and leading cause of cancer death worldwide, and its incidence is increasing [1]. Colon cancer progression
Xueming Tan and Xiaolu He have contributed equally to this work. & Zhining Fan [email protected] & Dongming Su [email protected] Xueming Tan [email protected]
1
Department of Gastroenterology, Nanjing Dachang Hospital, 68 Taizishan Rd, Yanjiang Industrial Development Zone, Nanjing 210044, Jiangsu, China
2
Department of Apheresis, Nanjing Red Cross Blood Center, 3 Zizhulin, Nanjing 210003, Jiangsu, China
3
Department of Gastroenterology, The Second Affiliated Hospital of Nanjing Medical University, 121 Jiangjiayuan, Nanjing 210011, Jiangsu, China
4
Department of Endoscopy Center, The First Affiliated of Hospital of Nanjing Medical University, 300 Guangzhou St., Nanjing 210029, Jiangsu, China
5
The Metabolic Disease Research Centre, Nanjing Medical University, 140 Hanzhong St., Nanjing 210029, Jiangsu, China
Xiaolu He [email protected] Zhonghua Jiang [email protected] Xiaohong Wang [email protected] Li Liu [email protected] Xiang Wang [email protected]
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requires several genetic alterations that allow cells to escape from growth control and disable apoptotic signaling [1, 2]. Therefore, studies are needed to identify this type of cancer cell, and the signaling molecules that are specifically involved in cancer progression. Derlin-1, a partner of the p97 ATPase complex, was initially reported to mediate the elimination of misfolded proteins from the endoplasmic reticulum (ER) and retrotranslocation of proteins into the cytosol [3–5]. Accumulating evidence has strongly implicated that Derlin-1 plays an important and multifaceted role in cancer progression [6–12]. Derlin-1 was confirmed to promote hepatocellular carcinoma endothelial cell survival and growth [13]. It has also been shown to promote invasion of non-small cell lung cancer (NSCLC) by EGFR-ERK-mediated upregulation of MMP-2 and MMP-9 [7]. Additionally, Derlin-1 overexpression is associated with lymph node metastasis in human lung and breast cancer [6, 7]. These characteristics have made therapeutic targeting of Derlin-1 an attractive concept in cancer biology. However, the role of Derlin-1 in colon cancer has not yet been determined. In the present study, we examined the expression patterns of Derlin-1 in colon cancer tissues and its correlation with clinicopathological features. We found that Derlin-1 overexpression is associated with Dukes stages and lymph node metastasis in colon cancer. Furthermore, we showed that silencing of Derlin-1 by shRNA induced apoptosis and inhibited proliferation of colon cancer cells. The downregulation of Derlin-1 resulted in significant decreased activity of the PI3K/AKT signaling pathway. Taken together, our results showed that Derlin-1 played an important role in regulating the growth of colon cancer.
Materials and methods Patients and tissue samples This study was approved and supervised by the Research Ethics Committee of Nanjing Medical University (Nanjing, China). Written Informed Consents were obtained from all patients. Paired colon cancer and normal colon tissue were obtained between 2009 and 2011 from 20 patients who underwent primary surgical resection of colon cancer in the Department of Anorectal medical center Nanjing Hospital of traditional Chinese Medicine (Nanjing, China). Followup information was obtained by reviewing patients’ medical records. None of the patients received radiotherapy or chemotherapy before surgical resection. All these tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C until total RNA and protein were extracted.
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Tissue microarray and immunohistochemistry Colon cancer tissue microarrays (TMAs) were created by a contract service at the Outdo Biotechnology Company Ltd (Shanghai, China). The TMAs contained paraffinembedded paired colon cancer tissue and normal colon tissue samples from archival patient specimens. For immunohistochemistry, paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed in sodium citrate buffer for 2 h. The rabbit polyclonal antibody directed against Derlin-1 (1:100 dilution, Sigma, St. Louis, MO, USA) was added to sections and incubated at 4 °C overnight. HRP-conjugated secondary antibody was used according to the manufacturer’s instructions. The slides were then incubated with DAB to visualize Derlin-1 expression, followed by hematoxylin counterstaining. PBS was used rather than the primary antibody as a negative control. The images were captured using a RGB JVC solid-state camera connected to an Olympus BH2 microscope. Staining results were assessed independently and blindly by two pathologists. Immunostaining of Derlin-1 was scored following a semiquantitative scale by evaluating representative tumor areas in terms of intensity and percentage of cells that showed significantly higher immunostaining than control cells in normal colon tissues. Cytoplasmic immunostaining in tumor cells was considered positive staining. The level of staining intensity (SI) was subdivided into four groups: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of positive cells was regarded as 0 (none), 1 (1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). The scores of each tumor sample were multiplied to give a final score of 0–8, and the tumors were finally determined as having Derlin-1 positive expression when the tumor sample reached a score of 4; tumor samples with a score of \4 were considered as having negative expression. Cell lines and cell culture Human colon cancer cell lines HT-29, SW480, and RKO and human normal colon cell line HcoEpic were purchased from the institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) or Roswell Park Memorial Institute 1640 (Hyclone, Logan, UT, USA) supplemented with 10 % fetal bovine serum (FBS), 100U/ml penicillin and 100 ng/ml streptomycin (Invitrogen, California, USA) in humidified air at 37 °C with 5 % CO2.
Mol Cell Biochem
Western blotting Total protein from tissue and cells were extracted in lysis buffer (Vazyme Biotech, Nanjing, China) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-rad, California, USA). Protein samples were prepared with 5 9 SDS sample buffer and separated on 8–12 % SDS–polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes. Membranes were blocked in 5 % non-fat milk for 2 h at room temperature. Proteins were immunoblotted with appropriate antibodies overnight at 4 °C, followed by binding with peroxidaselabeled secondary antibodies for 1 h at room temperature. Chemoluminescence signals were quantified by ECL reagents (MultiSciences Biotech, Hangzhou, China), and analyzed using a Bio-Rad ChemiDocTM XRS ? Universal Hood II machine. RNA interference and transfection Derlin-1 knockdown colon cancer cells were produced by transient transfection methods as colon cancer cells are likely to have deregulated Derlin-1 levels. The shRNAs of Derlin-1 were kindly provided by Xiubin Liang’s Lab (The Metabolic Disease Research Center, Nanjing Medical University, Nanjing, China). For transfection, cells were seeded in culture plates, grown to 50–80 % confluency and transfected with plasmids using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s protocol. Following transfection, cells were incubated for another 48 h before being harvested for assays. RNA isolation and quantitative reversetranscriptase polymerase chain reaction (qRT-PCR) analyses Total RNA was isolated from frozen tissues using TRIZOL reagent (Invitrogen, California, USA) according to the manufacturer’s instructions. cDNA was synthesized from total RNA (1 lg) using PrimeScriptƻ R RT reagent Kit (Takara, Dalian, China) following the manufacturer’s instructions. qRT-PCR was performed using SYBRƻ RPremix Ex TaqTM (Takara, Dalian, China). Expression of Derlin-1 was determined using qRT-PCR analysis. GAPDH mRNA was used as an internal control for each sample, and the Ct value for each sample was normalized to GAPDH mRNA. Primers used were as follows: Derlin1, forward primer: 50 -TCGGACATCGGAGACTGGTT-30 , reverse primer: 50 -AGCCAGTAATCACGATGCAAA-30 ; GAPDH, forward primer 50 -AGGACTCATGTCCATGCC
AT-30 , Reverse primer: 50 -ACCCTGTTGCTGTAGCCA AA-30 . Cell proliferation assay The shRNA–Derlin-1 or empty vector-transfected (3000/ well) cells were allowed to grow in 96-well plates and cultured for 96 h. At each time point, cells were stained with Cell Proliferation Reagent Kit I (MTT; Roche Applied Science, Basel, Switzerland) for 4 h at 37 °C. DMSO was used to dissolve the formazan, and the absorbance at 490 nm was measured using a microplate reader. Cell apoptosis assay The shRNA–Derlin-1 or empty vector-transfected SW480 cells were cultured in six-well plates for 48 h. The cells were harvested by trypsinization. Following double staining with FITC-annexin V and propidium iodide (PI), the cells were analyzed using flow cytometry (FACScan; BD Biosciences, San Jose, CA) and equipped with a CellQuest software (BD Biosciences). The cells were categorized into early apoptotic cells, late apoptotic cells, dead cells, and viable cells. The percentage of apoptotic cells were compared to the control group from each experiment. All of the samples assayed were from triplicate experiments. Statistical analysis Statistical analysis was performed using SPSS 17.0 software (IBM, IL, USA). Statistically significant differences were performed using Two-tailed Student’s t test or Chi square test. The results are expressed as the mean ± SD. The threshold for statistical significance was P \ 0.05.
Results Derlin-1 is overexpressed in the majority of human colon cancer To understand the clinical significance of Derlin-1 expression in colon cancer, we performed immunohistochemistry on 20 cases and tissue microarray on 90 cases of colon cancer tissues and the normal colon tissues. Representative staining of colon cancer sections exhibited negative, low, moderate, and high immunostaining for Derlin1 (Fig. 1a). Representative images of immunohistochemical staining of tissues with Derlin-1 antibody between colon tumor and normal tissues showed that the expression of Derlin-1 was significantly higher in colon cancer tissues than in the normal colon tissues (Fig. 1b). It was also confirmed with TMAs of patients with colon cancer which
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showed the positive expression of Derlin-1 was more frequent in 63 of 90 (70 %) cases of colon cancer compared with normal colon tissues (Table 1; Fig. 1c). In addition, of the 90 cases with normal colon samples, the positive expression of Derlin-1 was observed in 45.56 % (41 of 90 cases) (Table 1). These results indicated that Derlin-1 positive expression in colon cancer tissues was more frequent than expression in normal tissues. The expression of Derlin-1 was also analyzed using Western blot analysis and real-time PCR in cancer tissues and normal tissues, and the results showed that Derlin-1 expression in colon cancer tissues was significantly higher compared with the normal colon tissues at both the mRNA and protein levels (Fig. 2a, b), which were consistent with the data obtained in the immunohistochemical study.
Table 1 Derlin-1 expression level between colon cancer tissues and normal colon tissues (n = 90) Position
Derlin-1 expression level Positive
P value
Negative
Colon cancer tissues
63
27
Normal colon tissues
41
49
0.001**
** P \ 0.01 was considered statistically significant
and Dukes stage (P = 0.006). Furthermore, Kaplan–Meier survival analysis showed a significantly lower overall survival in patients with Derlin-1 positive expression than those with negative Derlin-1 expression (n = 90, *P \ 0.0001; Fig. 3).
Derlin-1 expression correlates with tumor grade, metastasis, and poor overall survival
Downregulation of Derlin-1 inhibits the proliferation of SW480 cells
The relationship between Derlin-1 expression and various clinicopathological features was investigated. As shown in Table 2, no statistical difference was found between Derlin-1 positive expression and clinical parameters such as age (P = 0.352), sex (P = 0.174). However, it showed the significant statistical associations between Derlin-1 positive expression and tumor differentiation (P = 0.002), tumor invasive depth (P = 0.014), tumor lymph node metastasis (P = 0.005), distant metastasis (P = 0.002),
To investigate the role of Derlin-1 in colon cancer cells, endogenous Derlin-1 expression was inhibited by shRNA in SW480 cells, which have high endogenous Derlin-1 levels (Fig. 4a, b). The Western blot result showed that Derlin-1-specific shRNA could significantly inhibit Derlin1 expression (Fig. 4c). We then investigated the effect of Derlin-1 shRNA on the growth of SW480 cells. Cells transfected with empty vector pcDNA 3.1(?) (pcDNA) or Derlin-1 shRNA (shRNA) were cultured for the times as
Fig. 1 Expression of Derlin-1 is increased in human colon cancer. Representative immunohistochemical staining for Derlin-1 protein in colon cancer tissues and normal human colon tissues. a representative staining of colon cancer sections exhibiting negative, low, moderate, and high immunostaining for Derlin-1. Negative of signal was scored as ‘0’; Low-intensity signal was scored as ‘1’; Moderate-intensity
signal was scored as ‘2’; High-intensity signal was scored as ‘3’. Original magnification, 9200. b Representative images of immunohistochemical staining of normal tissues and tumor tissues with Derlin-1 antibody; original magnification, 9400. c Immunohistochemical staining of tissue microarray in normal tissues and tumor tissues with Derlin-1 antibody; original magnification, 9200
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depletion also reduced the level of phospho-PI3K, suggesting that Derlin-1 plays a role in the activation of the PI3K/AKT pathway.
Discussion
Fig. 2 Derlin-1 expression in colon cancer tissues and paired normal colon tissues detected by Western blot and real-time PCR analyzed. a A representative Western blot analysis of derlin-1 protein in colon cancer (T) and paired normal colon tissues (N) from four patients is shown. In each line is the expression of b-actin used as a loading control to normalize the Derlin-1 protein levels in each sample. b Determination of Derlin-1 mRNA level in colon cancer tissues and paired non-cancerous normal tissues of colon cancer patients by realtime PCR. *P \ 0.05
indicated in Fig. 4d and the cell proliferation was determined by MTT assay. The results demonstrated that Derlin-1 shRNA inhibited cell growth in a time-dependent manner, which began 72 h (P \ 0.05) after transfection (Fig. 4d). To determine whether the growth-inhibitory effects of Derlin-1 shRNA could result from cell death, we measured cell apoptosis by flow cytometry. As shown in Fig. 4e and f, the rate of apoptotic cells in empty vector pcDNA 3.1(?) (pcDNA) or Derlin-1 shRNA (shRNA) transfected SW480 cells were 14.97 and 21.55 %, respectively, suggesting that the observed decrease in the number of cells upon Derlin-1 shRNA transfection was caused by cell death. Downregulation of Derlin-1 inhibits activation of the PI3K/AKT pathway Previous studies have shown that the PI3K/AKT pathway is critical for the proliferation of colon cancer cell [14–16], thus we determined whether Derlin-1 plays a role in regulating the PI3K/AKT pathway. We first examined the level of phosphorylated (active) forms of AKT in SW480 cells transfected with Derlin-1 shRNA. As shown in Fig. 5, Derlin-1 depletion significantly decreased AKT activation, but not the expression of AKT. Further analysis of upstream components of AKT showed that Derlin-1
Derlin-1 has been proven to be upregulated in various types of cancer and to be involved in cancer progression, such as lung cancer [6, 7, 9, 11]. However, little is known about its expression pattern in colon cancer and its role in colon cancer progression. The present study was aimed to address this issue by examining the expression patterns of Derlin-1 in colon cancer tissues, its correlation to clinicopathological features, and its effect on cancer cell proliferation. In the present study, we found that of colon cancer tissues, 63 of 90 (70 %) expressed moderate to high levels of Derlin-1, whereas percentage of Derlin-1 expression in normal colon tissues is 45.56 % (41 of 90 cases). These data demonstrate that the levels of Derlin-1 protein were elevated in the majority of colon cancer tissues compared with normal colon tissues. Our data are consistent with previous observations that Derlin-1 is overexpressed in different types of human cancers such as breast, lung, and liver tumors [6, 7, 9]. Derlin-1 expression has been shown to be more strongly present in higher-grade breast carcinomas than in lower-grade tumors [6], suggesting that Derlin-1 expression may correlate with a more malignant phenotype. Further studies are needed to decipher whether the expression of Derlin-1 is indicative of the malignant phenotype of colon cancer. Recent studies have demonstrated that Derlin-1 expression correlates with tumor grade and lymph node metastasis in several different types of human cancers, such as breast and lung cancer [6, 7]. Although our previous studies have demonstrated that Derlin-1 is overexpressed in colon cancer, its correlation to clinicopathological features of colon cancer has not yet been reported. In the present study, we showed that overexpression of Derlin-1 significantly correlated with lymph node metastasis and Duke stage, which was in accord with previous reports that Derlin-1 overexpression is associated with lymph node metastasis in human breast and lung cancers [6, 7]. More important, Derlin-1 overexpression correlated with poor overall survival in patients with colon cancer. The levels of Derlin-1 in colon cancer cell lines were much higher than in the normal cell line, especially in the SW480 cell line, which have strong invasive abilities. Collectively, these observations suggest that Derlin-1 may play a potential role in colon cancer invasion and metastasis. A recent study demonstrated that Derlin-1 is overexpressed in NSCLC and promotes invasion by EGFR-ERK-mediated upregulation of MMP-2 and MMP-9 [7]. The role and mechanism
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Mol Cell Biochem Table 2 Distribution of Derlin1 status in colon cancer according to clinicopathological characteristics
Number of patients
Positive
Negative
P value
Age B65
36
23
13
[65
54
40
14 0.352
Sex Male
47
36
11
Female
43
27
16 0.174
Differentiation Well
18
7
11
Moderate
56
41
15
Poor
16
15
1 0.002**
Invasive depth Submucosal/muscularis Serosal layer/serous outside
29 61
15 48
14 13 0.014*
Lymph node metastasis Absent
36
19
17
Present
54
44
10 0.005**
Distant metastasis Absent
73
46
27
Present
17
17
0 0.002**
Dukes stage A
29
15
B
7
4
14 3
C
37
27
10
D
17
17
0 0.006**
* P \ 0.05 was considered statistically significant ** P \ 0.01 was considered statistically significant
Fig. 3 Overall survival rate was analyzed by Kaplan–Meier survival analysis. Kaplan–Meier survival analysis showed that the overall survival rate was significantly lower in patients with Derlin-1-positive expression than in patients with negative Derlin-1 expression (N = 90, P \ 0.0001)
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remain to be determined of Derlin-1 in controlling invasion and metastasis of colon cancer. Our findings also concern the growth of colon cancer cells affected by Derlin-1, which are associated with colon cancer progression. Recent studies have demonstrated a correlation between Derlin-1 and proliferation in several cancer cell lines and have implicated this association in cancer progression [6, 11]. In the present study, we examined whether Derlin-1 downregulation affects the proliferation of SW480 cells, which have high endogenous Derlin-1 levels. Our MTT assay showed that Derlin-1 knockdown SW480 cells displayed decreased cell proliferation. In addition, flow cytometry analysis clearly showed that transfection of Derlin-1 shRNA induces apoptosis of SW480 cells. These results indicated that Derlin-1 plays an important role in regulating the
Mol Cell Biochem
Fig. 4 Derlin-1 knockdown inhibits cancer cell proliferation. a, b Derlin-1 expression is determined in a normal colon cell line HcoEpic and three colon cancer cell lines by Western blot analyses. Derlin-1 expression level in four cell lines after normalization with bactin was shown in (b). *P \ 0.05. c Derlin-1 expression in SW480 cells after transfection with Derlin-1 shRNA (shRNA), pcDNA 3.1(?) empty vector (pcDNA) or not (Control) was measured by Western blot analysis. b-actin served as a loading control. d The
effect of Derlin-1 shRNA on cell growth. SW480 cells transiently transfected with Derlin-1 shRNA (shRNA) or pcDNA 3.1(?) empty vector (pcDNA) were cultured for 96 h. Cell proliferation was measured by MTT assay. *P \ 0.05, **P \ 0.01. e, f Flow cytometry analysis of SW480 cells transfected with Derlin-1 shRNA (shRNA) or pcDNA 3.1(?) empty vector (pcDNA), the percentages of cells obtained are indicated below (e). The percentage of apoptosis was shown in (f). *P \ 0.05
proliferation of colon cancer cells. Previous research has shown that Derlin-1 is overexpressed in human breast carcinoma and protects the cancer cells from ER stressinduced apoptosis [6]. Almost all solid tumors including colon cancer encounter ER stress, such as hypoxia, and tumor growth depends on an intact unfolded protein response (UPR) [17–21]. Furthermore, Derlin-1 expression has been shown to be upregulated by inducers of ER stress and Derlin-1 reportedly mediates retrotranslocation of
misfolded proteins from ER lumen into the cytosol [3, 22– 24]. Therefore, based on our study, we suggest that Derlin1 expression may protect cancer cells from stresses encountered during tumor growth of colon cancer. Future experiments are needed to explore this issue. It has been demonstrated recently that Derlin-1 is involved in the activation of many signaling pathway, such as MAPK–ERK signaling pathway [7]. However, its involvement in the activation of PI3K/AKT signaling
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Fig. 5 Effect of Derlin-1 shRNA on the activation of PI3 K/AKT pathway. SW480 cells were transiently transfected with Derlin-1 shRNA (shRNA) or pcDNA 3.1(?) empty vector (pcDNA). Total and phosphorylated PI3K and AKT expression levels in cells were detected by Western blot analysis
pathway has not yet been reported. In this study, we found that Derlin-1 silencing triggered the inactivation of AKT in SW480 cells. In addition, Derlin-1 silencing significantly inhibited the phosphorylation of PI3K, suggesting that the activation of PI3K/AKT is regulated by Derlin-1 in colon cancer cells. It is known that the activated PI3K/AKT pathway directly modulates cell growth of many types of cancer cells, including colon cancer cell [14–16]. Therefore, it is reasonable to speculate that the decreased cell proliferation observed in Derlin-1 shRNA-transfected SW480 cells is due to the decreased PI3K/AKT activity. In summary, our results showed that Derlin-1 is overexpressed in colon cancer and high expression level of Derlin1 correlates with tumor grade, metastasis, and poor overall survival. Knockdown of Derlin-1 by shRNA inhibits the proliferation of colon cancer cells and the PI3K/AKT signaling pathway. However, further studies are necessary for exploring the role of Derlin-1 in tumor invasion, metastasis, and other aspects of colon cancer progression, which may widen our knowledge about Derlin-1 as an oncogene. Acknowledgments The work was supported by the National Natural Science Foundation of China (81172266), the Life Health Technology Foundation of Jiangsu province (BL2012031), and the Natural Science Foundation of Jiangsu province (BK2011859) to ZNF.
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Derlin-1 is overexpressed in human colon cancer and promotes cancer cell proliferation Xueming Tan1 • Xiaolu He2 • Zhonghua Jiang3 • Xiaohong Wang3 • Limei Ma3 • Li Liu4 • Xiang Wang4 • Zhining Fan4 • Dongming Su5
Received: 30 January 2015 / Accepted: 18 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Derlin-1 is overexpressed in many types of solid tumors and plays an important role in cancer progression. However, the expression pattern and functions of Derlin-1 in human colon cancer are not fully understood. In the present study, we examined Derlin-1 expression in colon cancer cell lines and human tissues and investigated its role in colon cancer. We found that Derlin-1 expression was increased significantly in colon cancer tissues and its overexpression correlated with the tumor differentiation, Dukes stage, invasion, lymph node metastasis, distant metastasis, and poor overall survival. The silencing of Derlin-1 by shRNA led to the growth inhibition of colon cancer cells, which were associated with the promotion of apoptosis. Furthermore, Derlin-1 silencing significantly inhibited the activation of the PI3K/AKT signaling pathway. Taken together, our results showed that Derlin-1 is
overexpressed in colon cancer and promotes proliferation of colon cancer cells. Derlin-1 may be a potential therapeutic target for the treatment of colon cancer. Keywords Derlin-1 Human colon cancer Proliferation Apoptosis Abbreviations ER Endoplasmic reticulum NSCLC Non-small cell lung cancer UPR Unfolded protein response
Introduction Colon cancer is one of the most common types of cancer and leading cause of cancer death worldwide, and its incidence is increasing [1]. Colon cancer progression
Xueming Tan and Xiaolu He have contributed equally to this work. & Zhining Fan [email protected] & Dongming Su [email protected] Xueming Tan [email protected]
1
Department of Gastroenterology, Nanjing Dachang Hospital, 68 Taizishan Rd, Yanjiang Industrial Development Zone, Nanjing 210044, Jiangsu, China
2
Department of Apheresis, Nanjing Red Cross Blood Center, 3 Zizhulin, Nanjing 210003, Jiangsu, China
3
Department of Gastroenterology, The Second Affiliated Hospital of Nanjing Medical University, 121 Jiangjiayuan, Nanjing 210011, Jiangsu, China
4
Department of Endoscopy Center, The First Affiliated of Hospital of Nanjing Medical University, 300 Guangzhou St., Nanjing 210029, Jiangsu, China
5
The Metabolic Disease Research Centre, Nanjing Medical University, 140 Hanzhong St., Nanjing 210029, Jiangsu, China
Xiaolu He [email protected] Zhonghua Jiang [email protected] Xiaohong Wang [email protected] Li Liu [email protected] Xiang Wang [email protected]
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requires several genetic alterations that allow cells to escape from growth control and disable apoptotic signaling [1, 2]. Therefore, studies are needed to identify this type of cancer cell, and the signaling molecules that are specifically involved in cancer progression. Derlin-1, a partner of the p97 ATPase complex, was initially reported to mediate the elimination of misfolded proteins from the endoplasmic reticulum (ER) and retrotranslocation of proteins into the cytosol [3–5]. Accumulating evidence has strongly implicated that Derlin-1 plays an important and multifaceted role in cancer progression [6–12]. Derlin-1 was confirmed to promote hepatocellular carcinoma endothelial cell survival and growth [13]. It has also been shown to promote invasion of non-small cell lung cancer (NSCLC) by EGFR-ERK-mediated upregulation of MMP-2 and MMP-9 [7]. Additionally, Derlin-1 overexpression is associated with lymph node metastasis in human lung and breast cancer [6, 7]. These characteristics have made therapeutic targeting of Derlin-1 an attractive concept in cancer biology. However, the role of Derlin-1 in colon cancer has not yet been determined. In the present study, we examined the expression patterns of Derlin-1 in colon cancer tissues and its correlation with clinicopathological features. We found that Derlin-1 overexpression is associated with Dukes stages and lymph node metastasis in colon cancer. Furthermore, we showed that silencing of Derlin-1 by shRNA induced apoptosis and inhibited proliferation of colon cancer cells. The downregulation of Derlin-1 resulted in significant decreased activity of the PI3K/AKT signaling pathway. Taken together, our results showed that Derlin-1 played an important role in regulating the growth of colon cancer.
Materials and methods Patients and tissue samples This study was approved and supervised by the Research Ethics Committee of Nanjing Medical University (Nanjing, China). Written Informed Consents were obtained from all patients. Paired colon cancer and normal colon tissue were obtained between 2009 and 2011 from 20 patients who underwent primary surgical resection of colon cancer in the Department of Anorectal medical center Nanjing Hospital of traditional Chinese Medicine (Nanjing, China). Followup information was obtained by reviewing patients’ medical records. None of the patients received radiotherapy or chemotherapy before surgical resection. All these tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C until total RNA and protein were extracted.
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Tissue microarray and immunohistochemistry Colon cancer tissue microarrays (TMAs) were created by a contract service at the Outdo Biotechnology Company Ltd (Shanghai, China). The TMAs contained paraffinembedded paired colon cancer tissue and normal colon tissue samples from archival patient specimens. For immunohistochemistry, paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed in sodium citrate buffer for 2 h. The rabbit polyclonal antibody directed against Derlin-1 (1:100 dilution, Sigma, St. Louis, MO, USA) was added to sections and incubated at 4 °C overnight. HRP-conjugated secondary antibody was used according to the manufacturer’s instructions. The slides were then incubated with DAB to visualize Derlin-1 expression, followed by hematoxylin counterstaining. PBS was used rather than the primary antibody as a negative control. The images were captured using a RGB JVC solid-state camera connected to an Olympus BH2 microscope. Staining results were assessed independently and blindly by two pathologists. Immunostaining of Derlin-1 was scored following a semiquantitative scale by evaluating representative tumor areas in terms of intensity and percentage of cells that showed significantly higher immunostaining than control cells in normal colon tissues. Cytoplasmic immunostaining in tumor cells was considered positive staining. The level of staining intensity (SI) was subdivided into four groups: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of positive cells was regarded as 0 (none), 1 (1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). The scores of each tumor sample were multiplied to give a final score of 0–8, and the tumors were finally determined as having Derlin-1 positive expression when the tumor sample reached a score of 4; tumor samples with a score of \4 were considered as having negative expression. Cell lines and cell culture Human colon cancer cell lines HT-29, SW480, and RKO and human normal colon cell line HcoEpic were purchased from the institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) or Roswell Park Memorial Institute 1640 (Hyclone, Logan, UT, USA) supplemented with 10 % fetal bovine serum (FBS), 100U/ml penicillin and 100 ng/ml streptomycin (Invitrogen, California, USA) in humidified air at 37 °C with 5 % CO2.
Mol Cell Biochem
Western blotting Total protein from tissue and cells were extracted in lysis buffer (Vazyme Biotech, Nanjing, China) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-rad, California, USA). Protein samples were prepared with 5 9 SDS sample buffer and separated on 8–12 % SDS–polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes. Membranes were blocked in 5 % non-fat milk for 2 h at room temperature. Proteins were immunoblotted with appropriate antibodies overnight at 4 °C, followed by binding with peroxidaselabeled secondary antibodies for 1 h at room temperature. Chemoluminescence signals were quantified by ECL reagents (MultiSciences Biotech, Hangzhou, China), and analyzed using a Bio-Rad ChemiDocTM XRS ? Universal Hood II machine. RNA interference and transfection Derlin-1 knockdown colon cancer cells were produced by transient transfection methods as colon cancer cells are likely to have deregulated Derlin-1 levels. The shRNAs of Derlin-1 were kindly provided by Xiubin Liang’s Lab (The Metabolic Disease Research Center, Nanjing Medical University, Nanjing, China). For transfection, cells were seeded in culture plates, grown to 50–80 % confluency and transfected with plasmids using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s protocol. Following transfection, cells were incubated for another 48 h before being harvested for assays. RNA isolation and quantitative reversetranscriptase polymerase chain reaction (qRT-PCR) analyses Total RNA was isolated from frozen tissues using TRIZOL reagent (Invitrogen, California, USA) according to the manufacturer’s instructions. cDNA was synthesized from total RNA (1 lg) using PrimeScriptƻ R RT reagent Kit (Takara, Dalian, China) following the manufacturer’s instructions. qRT-PCR was performed using SYBRƻ RPremix Ex TaqTM (Takara, Dalian, China). Expression of Derlin-1 was determined using qRT-PCR analysis. GAPDH mRNA was used as an internal control for each sample, and the Ct value for each sample was normalized to GAPDH mRNA. Primers used were as follows: Derlin1, forward primer: 50 -TCGGACATCGGAGACTGGTT-30 , reverse primer: 50 -AGCCAGTAATCACGATGCAAA-30 ; GAPDH, forward primer 50 -AGGACTCATGTCCATGCC
AT-30 , Reverse primer: 50 -ACCCTGTTGCTGTAGCCA AA-30 . Cell proliferation assay The shRNA–Derlin-1 or empty vector-transfected (3000/ well) cells were allowed to grow in 96-well plates and cultured for 96 h. At each time point, cells were stained with Cell Proliferation Reagent Kit I (MTT; Roche Applied Science, Basel, Switzerland) for 4 h at 37 °C. DMSO was used to dissolve the formazan, and the absorbance at 490 nm was measured using a microplate reader. Cell apoptosis assay The shRNA–Derlin-1 or empty vector-transfected SW480 cells were cultured in six-well plates for 48 h. The cells were harvested by trypsinization. Following double staining with FITC-annexin V and propidium iodide (PI), the cells were analyzed using flow cytometry (FACScan; BD Biosciences, San Jose, CA) and equipped with a CellQuest software (BD Biosciences). The cells were categorized into early apoptotic cells, late apoptotic cells, dead cells, and viable cells. The percentage of apoptotic cells were compared to the control group from each experiment. All of the samples assayed were from triplicate experiments. Statistical analysis Statistical analysis was performed using SPSS 17.0 software (IBM, IL, USA). Statistically significant differences were performed using Two-tailed Student’s t test or Chi square test. The results are expressed as the mean ± SD. The threshold for statistical significance was P \ 0.05.
Results Derlin-1 is overexpressed in the majority of human colon cancer To understand the clinical significance of Derlin-1 expression in colon cancer, we performed immunohistochemistry on 20 cases and tissue microarray on 90 cases of colon cancer tissues and the normal colon tissues. Representative staining of colon cancer sections exhibited negative, low, moderate, and high immunostaining for Derlin1 (Fig. 1a). Representative images of immunohistochemical staining of tissues with Derlin-1 antibody between colon tumor and normal tissues showed that the expression of Derlin-1 was significantly higher in colon cancer tissues than in the normal colon tissues (Fig. 1b). It was also confirmed with TMAs of patients with colon cancer which
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showed the positive expression of Derlin-1 was more frequent in 63 of 90 (70 %) cases of colon cancer compared with normal colon tissues (Table 1; Fig. 1c). In addition, of the 90 cases with normal colon samples, the positive expression of Derlin-1 was observed in 45.56 % (41 of 90 cases) (Table 1). These results indicated that Derlin-1 positive expression in colon cancer tissues was more frequent than expression in normal tissues. The expression of Derlin-1 was also analyzed using Western blot analysis and real-time PCR in cancer tissues and normal tissues, and the results showed that Derlin-1 expression in colon cancer tissues was significantly higher compared with the normal colon tissues at both the mRNA and protein levels (Fig. 2a, b), which were consistent with the data obtained in the immunohistochemical study.
Table 1 Derlin-1 expression level between colon cancer tissues and normal colon tissues (n = 90) Position
Derlin-1 expression level Positive
P value
Negative
Colon cancer tissues
63
27
Normal colon tissues
41
49
0.001**
** P \ 0.01 was considered statistically significant
and Dukes stage (P = 0.006). Furthermore, Kaplan–Meier survival analysis showed a significantly lower overall survival in patients with Derlin-1 positive expression than those with negative Derlin-1 expression (n = 90, *P \ 0.0001; Fig. 3).
Derlin-1 expression correlates with tumor grade, metastasis, and poor overall survival
Downregulation of Derlin-1 inhibits the proliferation of SW480 cells
The relationship between Derlin-1 expression and various clinicopathological features was investigated. As shown in Table 2, no statistical difference was found between Derlin-1 positive expression and clinical parameters such as age (P = 0.352), sex (P = 0.174). However, it showed the significant statistical associations between Derlin-1 positive expression and tumor differentiation (P = 0.002), tumor invasive depth (P = 0.014), tumor lymph node metastasis (P = 0.005), distant metastasis (P = 0.002),
To investigate the role of Derlin-1 in colon cancer cells, endogenous Derlin-1 expression was inhibited by shRNA in SW480 cells, which have high endogenous Derlin-1 levels (Fig. 4a, b). The Western blot result showed that Derlin-1-specific shRNA could significantly inhibit Derlin1 expression (Fig. 4c). We then investigated the effect of Derlin-1 shRNA on the growth of SW480 cells. Cells transfected with empty vector pcDNA 3.1(?) (pcDNA) or Derlin-1 shRNA (shRNA) were cultured for the times as
Fig. 1 Expression of Derlin-1 is increased in human colon cancer. Representative immunohistochemical staining for Derlin-1 protein in colon cancer tissues and normal human colon tissues. a representative staining of colon cancer sections exhibiting negative, low, moderate, and high immunostaining for Derlin-1. Negative of signal was scored as ‘0’; Low-intensity signal was scored as ‘1’; Moderate-intensity
signal was scored as ‘2’; High-intensity signal was scored as ‘3’. Original magnification, 9200. b Representative images of immunohistochemical staining of normal tissues and tumor tissues with Derlin-1 antibody; original magnification, 9400. c Immunohistochemical staining of tissue microarray in normal tissues and tumor tissues with Derlin-1 antibody; original magnification, 9200
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depletion also reduced the level of phospho-PI3K, suggesting that Derlin-1 plays a role in the activation of the PI3K/AKT pathway.
Discussion
Fig. 2 Derlin-1 expression in colon cancer tissues and paired normal colon tissues detected by Western blot and real-time PCR analyzed. a A representative Western blot analysis of derlin-1 protein in colon cancer (T) and paired normal colon tissues (N) from four patients is shown. In each line is the expression of b-actin used as a loading control to normalize the Derlin-1 protein levels in each sample. b Determination of Derlin-1 mRNA level in colon cancer tissues and paired non-cancerous normal tissues of colon cancer patients by realtime PCR. *P \ 0.05
indicated in Fig. 4d and the cell proliferation was determined by MTT assay. The results demonstrated that Derlin-1 shRNA inhibited cell growth in a time-dependent manner, which began 72 h (P \ 0.05) after transfection (Fig. 4d). To determine whether the growth-inhibitory effects of Derlin-1 shRNA could result from cell death, we measured cell apoptosis by flow cytometry. As shown in Fig. 4e and f, the rate of apoptotic cells in empty vector pcDNA 3.1(?) (pcDNA) or Derlin-1 shRNA (shRNA) transfected SW480 cells were 14.97 and 21.55 %, respectively, suggesting that the observed decrease in the number of cells upon Derlin-1 shRNA transfection was caused by cell death. Downregulation of Derlin-1 inhibits activation of the PI3K/AKT pathway Previous studies have shown that the PI3K/AKT pathway is critical for the proliferation of colon cancer cell [14–16], thus we determined whether Derlin-1 plays a role in regulating the PI3K/AKT pathway. We first examined the level of phosphorylated (active) forms of AKT in SW480 cells transfected with Derlin-1 shRNA. As shown in Fig. 5, Derlin-1 depletion significantly decreased AKT activation, but not the expression of AKT. Further analysis of upstream components of AKT showed that Derlin-1
Derlin-1 has been proven to be upregulated in various types of cancer and to be involved in cancer progression, such as lung cancer [6, 7, 9, 11]. However, little is known about its expression pattern in colon cancer and its role in colon cancer progression. The present study was aimed to address this issue by examining the expression patterns of Derlin-1 in colon cancer tissues, its correlation to clinicopathological features, and its effect on cancer cell proliferation. In the present study, we found that of colon cancer tissues, 63 of 90 (70 %) expressed moderate to high levels of Derlin-1, whereas percentage of Derlin-1 expression in normal colon tissues is 45.56 % (41 of 90 cases). These data demonstrate that the levels of Derlin-1 protein were elevated in the majority of colon cancer tissues compared with normal colon tissues. Our data are consistent with previous observations that Derlin-1 is overexpressed in different types of human cancers such as breast, lung, and liver tumors [6, 7, 9]. Derlin-1 expression has been shown to be more strongly present in higher-grade breast carcinomas than in lower-grade tumors [6], suggesting that Derlin-1 expression may correlate with a more malignant phenotype. Further studies are needed to decipher whether the expression of Derlin-1 is indicative of the malignant phenotype of colon cancer. Recent studies have demonstrated that Derlin-1 expression correlates with tumor grade and lymph node metastasis in several different types of human cancers, such as breast and lung cancer [6, 7]. Although our previous studies have demonstrated that Derlin-1 is overexpressed in colon cancer, its correlation to clinicopathological features of colon cancer has not yet been reported. In the present study, we showed that overexpression of Derlin-1 significantly correlated with lymph node metastasis and Duke stage, which was in accord with previous reports that Derlin-1 overexpression is associated with lymph node metastasis in human breast and lung cancers [6, 7]. More important, Derlin-1 overexpression correlated with poor overall survival in patients with colon cancer. The levels of Derlin-1 in colon cancer cell lines were much higher than in the normal cell line, especially in the SW480 cell line, which have strong invasive abilities. Collectively, these observations suggest that Derlin-1 may play a potential role in colon cancer invasion and metastasis. A recent study demonstrated that Derlin-1 is overexpressed in NSCLC and promotes invasion by EGFR-ERK-mediated upregulation of MMP-2 and MMP-9 [7]. The role and mechanism
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Mol Cell Biochem Table 2 Distribution of Derlin1 status in colon cancer according to clinicopathological characteristics
Number of patients
Positive
Negative
P value
Age B65
36
23
13
[65
54
40
14 0.352
Sex Male
47
36
11
Female
43
27
16 0.174
Differentiation Well
18
7
11
Moderate
56
41
15
Poor
16
15
1 0.002**
Invasive depth Submucosal/muscularis Serosal layer/serous outside
29 61
15 48
14 13 0.014*
Lymph node metastasis Absent
36
19
17
Present
54
44
10 0.005**
Distant metastasis Absent
73
46
27
Present
17
17
0 0.002**
Dukes stage A
29
15
B
7
4
14 3
C
37
27
10
D
17
17
0 0.006**
* P \ 0.05 was considered statistically significant ** P \ 0.01 was considered statistically significant
Fig. 3 Overall survival rate was analyzed by Kaplan–Meier survival analysis. Kaplan–Meier survival analysis showed that the overall survival rate was significantly lower in patients with Derlin-1-positive expression than in patients with negative Derlin-1 expression (N = 90, P \ 0.0001)
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remain to be determined of Derlin-1 in controlling invasion and metastasis of colon cancer. Our findings also concern the growth of colon cancer cells affected by Derlin-1, which are associated with colon cancer progression. Recent studies have demonstrated a correlation between Derlin-1 and proliferation in several cancer cell lines and have implicated this association in cancer progression [6, 11]. In the present study, we examined whether Derlin-1 downregulation affects the proliferation of SW480 cells, which have high endogenous Derlin-1 levels. Our MTT assay showed that Derlin-1 knockdown SW480 cells displayed decreased cell proliferation. In addition, flow cytometry analysis clearly showed that transfection of Derlin-1 shRNA induces apoptosis of SW480 cells. These results indicated that Derlin-1 plays an important role in regulating the
Mol Cell Biochem
Fig. 4 Derlin-1 knockdown inhibits cancer cell proliferation. a, b Derlin-1 expression is determined in a normal colon cell line HcoEpic and three colon cancer cell lines by Western blot analyses. Derlin-1 expression level in four cell lines after normalization with bactin was shown in (b). *P \ 0.05. c Derlin-1 expression in SW480 cells after transfection with Derlin-1 shRNA (shRNA), pcDNA 3.1(?) empty vector (pcDNA) or not (Control) was measured by Western blot analysis. b-actin served as a loading control. d The
effect of Derlin-1 shRNA on cell growth. SW480 cells transiently transfected with Derlin-1 shRNA (shRNA) or pcDNA 3.1(?) empty vector (pcDNA) were cultured for 96 h. Cell proliferation was measured by MTT assay. *P \ 0.05, **P \ 0.01. e, f Flow cytometry analysis of SW480 cells transfected with Derlin-1 shRNA (shRNA) or pcDNA 3.1(?) empty vector (pcDNA), the percentages of cells obtained are indicated below (e). The percentage of apoptosis was shown in (f). *P \ 0.05
proliferation of colon cancer cells. Previous research has shown that Derlin-1 is overexpressed in human breast carcinoma and protects the cancer cells from ER stressinduced apoptosis [6]. Almost all solid tumors including colon cancer encounter ER stress, such as hypoxia, and tumor growth depends on an intact unfolded protein response (UPR) [17–21]. Furthermore, Derlin-1 expression has been shown to be upregulated by inducers of ER stress and Derlin-1 reportedly mediates retrotranslocation of
misfolded proteins from ER lumen into the cytosol [3, 22– 24]. Therefore, based on our study, we suggest that Derlin1 expression may protect cancer cells from stresses encountered during tumor growth of colon cancer. Future experiments are needed to explore this issue. It has been demonstrated recently that Derlin-1 is involved in the activation of many signaling pathway, such as MAPK–ERK signaling pathway [7]. However, its involvement in the activation of PI3K/AKT signaling
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Fig. 5 Effect of Derlin-1 shRNA on the activation of PI3 K/AKT pathway. SW480 cells were transiently transfected with Derlin-1 shRNA (shRNA) or pcDNA 3.1(?) empty vector (pcDNA). Total and phosphorylated PI3K and AKT expression levels in cells were detected by Western blot analysis
pathway has not yet been reported. In this study, we found that Derlin-1 silencing triggered the inactivation of AKT in SW480 cells. In addition, Derlin-1 silencing significantly inhibited the phosphorylation of PI3K, suggesting that the activation of PI3K/AKT is regulated by Derlin-1 in colon cancer cells. It is known that the activated PI3K/AKT pathway directly modulates cell growth of many types of cancer cells, including colon cancer cell [14–16]. Therefore, it is reasonable to speculate that the decreased cell proliferation observed in Derlin-1 shRNA-transfected SW480 cells is due to the decreased PI3K/AKT activity. In summary, our results showed that Derlin-1 is overexpressed in colon cancer and high expression level of Derlin1 correlates with tumor grade, metastasis, and poor overall survival. Knockdown of Derlin-1 by shRNA inhibits the proliferation of colon cancer cells and the PI3K/AKT signaling pathway. However, further studies are necessary for exploring the role of Derlin-1 in tumor invasion, metastasis, and other aspects of colon cancer progression, which may widen our knowledge about Derlin-1 as an oncogene. Acknowledgments The work was supported by the National Natural Science Foundation of China (81172266), the Life Health Technology Foundation of Jiangsu province (BL2012031), and the Natural Science Foundation of Jiangsu province (BK2011859) to ZNF.
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