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Biomedicine & Pharmacotherapy 89 (2017) 1370–1377
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Original article
Sulfatase-2 promotes the growth and metastasis of colorectal cancer by activating Akt and Erk1/2 pathways Youmao Tao, Tao Han, Tao Zhang, Caixia Sun* Department of Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University, No. 126 Xiantai Street, Changchun 130033, China
A R T I C L E I N F O
Article history: Received 9 January 2017 Received in revised form 28 February 2017 Accepted 7 March 2017 Keywords: Sulfatase-2 Colorectal cancer Cancer growth Cancer metastasis Signaling pathway
A B S T R A C T
The molecular mechanisms underlying the growth and metastasis of colorectal cancer (CRC) remain largely unknown. Sulfatase-2 (SULF2) was found to play critical roles in human cancers. Recent study reported that SULF1/2 overexpression resulted in increased viability and proliferation, and augmented cell migration in CRC cells. However, the expression of SULF2 and its underlying molecular mechanisms in CRC remain unknown. In this study, we found that the expressions of SULF2 in CRC tissues and cell lines were significantly increased compared to control groups. Increased expression of SULF2 was associated with malignant clinical features and poor prognosis of CRC patients. Loss of SULF2 significantly prohibited the proliferation, cell cycle progression, migration and invasion of HT29 cells, while restoration of SULF2 significantly promoted these cellular functions of SW480 cells. In vivo tumorigenicity and liver metastasis assays confirmed that SULF2 knockdown significantly reduced the growth and metastatic abilities of HT29 cells in nude mice. Furthermore, SULF2 knockdown reduced the levels of p-Akt and p-Erk1/2 in HT29 cells, while SULF2 overexpression showed opposite effects on the expressions of these proteins in SW480 cells. In all, SULF2 promotes the growth and metastasis of CRC probably by activating Akt and Erk1/2 pathways. SULF2 potentially serves as a promising biomarker and therapeutic target in CRC. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Colorectal cancer (CRC), which ranks the fourth in cancerrelated deaths, is the third most frequent cancer worldwide [1,2]. During the last several decades, remarkable advances have been made in the early diagnosis and treatment of CRC [3]. However, the long term survival of CRC patients is still poor, especially for those in advanced stages [4]. Most patients in advanced stages cannot obtain effective treatments due to the occurrence of local and systemic metastasis of CRC cells. Unfortunately, the exact mechanisms responsible for the malignant growth and metastatic behaviors of CRC cells remain largely uncovered. Therefore, investigating the molecular mechanisms underlying the growth and metastasis of CRC cells will potentially contribute to
Abbreviations: CRC, colorectal cancer; SULF2, sulfatase2; IHC, immunohistochemistry; NR4A3, nuclear receptor subfamily 4 group A member 3; CD82, cluster of differentiation 82; PDGFC, platelet-derived growth factor C. * Corresponding author. E-mail address: [email protected] (C. Sun). http://dx.doi.org/10.1016/j.biopha.2017.03.017 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
identification of novel biomarkers and therapeutic targets of CRC patients. Sulfatase2 (SULF2), an extracellular heparan endosulfatase [5], has been found to play critical roles in the initiation and progression of human cancers including hepatocellular carcinoma (HCC) [6] gastric cancer [7], lung cancer [8] and breast cancer [9]. SULF2 regulates the sulfate patterns of HSPGs and lead to the release of cytokines on HSPGs [10]. These released cytokines, such as FGF1, VEFG and SDF-1, subsequently promote the malignant behaviors of cancer cells [5]. Recent study reported that SULF1/2 overexpression resulted in increased viability and proliferation, and augmented cell migration in CRC cells [11]. However, the clinical significance of SULF2 and mechanisms underlying its biological function in CRC have never been investigated. In this study, we found that the expressions of SULF2were increased in CRC tissues and cell lines. The positive expression of SULF2 was associated with unfavorable clinicopathological features and poor prognosis of CRC patients. Both gain- and loss-offunction experiments demonstrated that SULF2 promoted proliferation, cell cycle progression, migration and invasion of CRC cells. In vivo tumor formation and liver metastasis experiments showed
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that SULF2 knockdown inhibited the growth and metastasis of CRC cells in nude mice. Furthermore, SULF2 regulated the activation of Akt and Erk1/2 pathways, which were critical for the growth and metastasis of CRC cells. 2. Materials and methods 2.1. Clinical tissues Ninety pairs of CRC tissues and matched non-tumor tissues were collected from patients, who underwent enterectomy in the Department of Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University. All patients received and signed the informed consents for agreeing to donate the clinical samples for research. These clinical samples were stored in liquid nitrogen for RNA isolation and fixed in 10% formalin for immunohistochemistry, respectively. Patients who were categorized as having an event in the calculation of disease-free survival experienced local tumor recurrence in the form of either local or distant metastasis, or death due to CRC. Patients dying of all causes were considered as “death events” and were included as such in the overall survival analysis. The protocols for collecting and using these clinical samples were approved by the Research Ethics Committee of China-Japan Union Hospital of Jilin University. 2.2. Cell culture Human intestinal epithelial cells (HIEC) and human CRC cell lines including HCT116, HT29, Caco-2, SW620, and SW480 were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). All these cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific) along with penicillin (100 U/ml) and streptomycin (100 U/ml) (Sigma-Aldrich, St Louis, MO, USA) in an incubator in humidified atmosphere with 5% CO2 at 37 C. 2.3. Cell transfection A specific SULF2 shRNA and the non-targeting (NT) shRNA were obtained from Ambion/Life Technologies (Carlsbad, CA, USA). SULF2 overexpression vector and the empty control vector were purchased from ORIGENE (Rockville, MD, USA). These shRNAs or vectors were transfected into CRC cells using Lippofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The efficiency of SULF2 shRNA or SULF2 overexpression vector was evaluated by western blot. 2.4. RNA extraction and quantitative real-time PCR (qRT-PCR) RNA extraction from CRC tissues and cells were performed with ISOGEN (Nippon Gene, Tokyo, Japan) and TaqMan Fast Cells-to-CT Kit (Ambion/Life Technologies) based on manufacturer’s instructions. Then, the total RNA were subjected to cDNA construction following the protocols of SuperScript III First-strand Synthesis System (Invitrogen). Quantitative real-time PCR was performed using the StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers for SULF2 and GAPDH were synthesized and bought from GeneCopoeia (Guangzhou, China). GAPDH was used as the internal control. All experiments were conducted at least three times. 2.5. Western blot Total proteins were isolated using RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). A Bradford Protein Assay Kit (P0006) (Beyotime, Shanghai, China) was used to analyzed
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protein concentration. The isolated proteins were loaded on 10% SDS-PAGE for separation, and were then transferred to NC membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk/TBST, the membranes were incubated with primary antibodies overnight at 4 C. Primary antibodies used in this study included SULF2 (1:1000 dilution, Abcam, Cambridge, MA, USA), p-Akt (1:1000 dilution, Abcam), Akt (1:1000 dilution, Abcam), p-Erk1/2 (1:1000 dilution, Abcam), Erk1/2 (1:1000 dilution, Abcam) and GAPDH (1:1000 dilution, Abcam). Then, the membranes were washed with TBST and incubated with secondary antibodies (Cell Signaling Technology, Beverly, MA, USA). The protein bands on the membranes were detected with ECL Advance Western detection reagents (GE Healthcare, Buckinghamshire, UK) and visualized with ChemiDoc XRS plus system (BIORAD, Hercules, CA, USA). 2.6. Immunohistochemistry (IHC) staining CRC tissues and the adjacent non-tumor tissues were subjected to formalin fixation and paraffin-embedded. These tissues were then cut into 5 mm sections for IHC staining based on the standard protocols. The results of the IHC staining for SULF2 were evaluated by one pathologist in our hospital. Staining intensity was scored as no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. Staining quantity was graded as <25% = 1, 25%-75% = 2, and >75% = 3. The degree of staining was determined using a fourgrade evaluation system: IHC score = staining intensity staining quantity (negative, 0; weak positive, 1 and 2; moderate positive, 3 and 4; strong positive, 6 and 9). 2.7. Cell proliferation assay For cell proliferation, CRC cells that were treated with corresponding vectors were seeded into 96-well plates (1.5 103 cells per well). 24, 48, 72 and 96 h after transfection, the cell proliferation assay was performed by addition of 10 ml cell counting kit 8 (CCK8) solution (Beyotime) to each well, followed by incubation at 37 C for 2 h. Absorbance was measured at a wave length of 490 nm using a microplate reader (Flexstation III ROM V2.1.28, Molecular Devices, Sunnyvale, CA, USA). 2.8. Cell cycle analysis For cell cycle assay, CRC cells were collected 48 h after transfection. These cells were then fixed with 80% ethanol overnight at 4 C. These fixed cells were stained with Propidium Iodide (PI, 50 ug/mL, BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 20 min. The distribution of cell cycle was measured with FACS Calibur system (BD Biosciences). 2.9. Wound healing assay CRC cells that were transfected with SULF2 overexpression vector or SULF2 shRNA were seeded in 6-well plates to form single confluent cell layer. The wound were made with 10 ml pipette tip in the confluent cell layer. 24 h later, the width of wound was photographed and recorded with phase-contrast microscope. 2.10. Invasion assay The invasion assay was performed with Transwell chambers (BD Biosciences). 100 ml Matrigel (1:6 dilution) was added on the upper chamber before cell seeding. CRC cells that were transfected with SULF2 overexpression vector or SULF2 shRNA were suspended in 100 ml serum-free medium and were seeded into the upper chamber. DEME with 10% FBS was placed in the lower
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chamber as chemoattractant. 48 h later, cells invaded through the membranes were fixed with methanol and stained with crystal violet. The number of invaded cells was counted under microscope.
one way ANOVA were used to analyze continuous variable. Survival analysis was performed using Kaplan-Meier’s method and Logrank test. All data were presented as Mean SEM. P < 0.05 was considered to be statistically significant.
2.11. In vivo tumorigenicity and liver metastasis assay 3. Results For tumorigenicity assay, HT29 cells that were transfected with NT shRNA or SULF2 shRNA were subcutaneously injected into the right flank of 6-week-old nude mice (n = 5 mice/group). Tumors volumes were measured every five days from the first injection using the following formula: Volume = (Length Width2)/2. After 20 days, the mice were sacrificed, and the tumors were removed and weighted. For liver metastasis assay, HT29 cells that were transfected with NT shRNA or SULF2 shRNA were used for subcapsular splenic injection. 8 weeks after injection, all mice were euthanized and the livers were surgically removed to evaluate whether the hepatic metastatic foci were formed. All animal experiments were performed according to the animal experimental guidelines of China-Japan Union Hospital of Jilin University. 2.12. Statistical analysis GraphPad Prism 5 (GraphPad Software, Inc, San Diego, CA, USA) and SPSS 19 software (SPSS, Chicago, IL, USA) were used for statistical analysis in this study. Chi-squared test was employed to explore the association between two variables. Student’s t-test and
3.1. The expression of SULF2 is increased in CRC To investigate the expression status of SULF2 in CRC, IHC was performed in CRC and tumor-adjacent tissues for SULF2 staining. The results of IHC staining showed that the expressions of SULF2 in CRC tissues were higher (Fig. 1A–C: strong, moderate and weak staining of SULF2) than those in adjacent non-tumor tissues (Fig. 1D: negative staining of SULF2). Notably, 58 of 90 (64.44%) CRC tissues showed positive staining of SULF2, while SULF2 signal was detected in 25 of 90 (27.78%) adjacent non-tumor tissues. To further confirm the overexpression of SULF2 in CRC, we randomly selected 40 pairs of CRC tissues and matched non-tumor tissues, and measured the levels of SULF2 mRNA in these tissues using qRTPCR. The results showed that the levels of SULF2 mRNA were also significantly increased in CRC tissues compared with the tumoradjacent tissues (P < 0.05, Fig. 2A). Moreover, we measured the expression levels of SULF2 mRNA in CRC cell lines and HIEC cells. Compared with that in HIEC cells, the levels of SULF2 mRNA in CRC cell lines including HCT116, Caco2, HT29, SW620 and SW480 were significantly elevated (P < 0.05, Fig. 2B).
Fig. 1. Immunostaining of SULF2 in CRC and tumor-adjacent tissues. Representative IHC staining of SULF2 for CRC tissues and adjacent non-tumor tissues. (A) Strong, (B) moderate and (C) weak staining of SULF2 in CRC tissues. (D) Negative staining of SULF2 in adjacent non-tumor tissues. Scale bar: 50 mm.
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Fig. 2. Overexpression of SULF2 is associated with poor prognosis of CRC patients. (A) Expression differences of SULF2 mRNA between CRC tissues and matched tumoradjacent tissues. n = 40, *P < 0.05 by t-test. (B) Relative expression of SULF2 mRNA in five CRC cell lines (HCT116, Caco2, HT29, SW620 and SW480) and a normal human intestinal epithelial cell line (HIEC) detected by qRT-PCR. n = 3 repeats with similar results, *P < 0.05 by ANOVA. (C) and (D) CRC patients were divided into SULF2 positive expression group (n = 58) and SULF2 negative expression group (n = 32) based on the IHC scores. SULF2 positively expressing CRC patients showed a significant decreased overall survival and disease-free survival compared to SULF2 negatively expressing cases. P < 0.05 by Log-rank test.
3.2. Elevated expression of SULF2 is correlated with poor clinical features and prognosis of CRC patients To elucidate the clinical significance of SULF2 upregulation in CRC, CRC patients were divided into two groups (SULF2 positive expression group and SULF2 negative expression group). As shown in Table 1, compared with those in SULF2 negative expression
Table 1 The correlation between clinicopathological features and SULF2 expression in CRC. Features
n = 90
SULF2 expression Positive
Negative
Age (years)
<65 65
57 33
40 18
17 15
0.136
Sex
Male Female
50 40
29 29
21 11
0.153
Tumor grade
G1 + G2 G3 + G4
65 25
43 15
22 10
0.585
Size (cm)
<5 5
41 49
19 39
22 10
0.001*
Tumor invasion
T1 + T2 T3 + T4
23 67
11 47
12 20
0.054
Lymph node status
<1 1
47 43
24 34
23 9
0.006*
*
3.3. SULF2 promotes the proliferation and cell cycle progression of CRC cells To investigate the biological functions of SULF2 in CRC cells, HT29 cells were transfected a specific SULF2 shRNA. SULF2 shRNA significantly reduced SULF2 expression in HT29 cells (P < 0.05, Fig. 3A). Loss of SULF2 significantly inhibited the proliferative ability of HT29 cells as suggested by CCK8 assay (P < 0.05, Fig. 3B). Cell cycle analysis showed that SULF2 knockdown significantly increased the percentage of G1 phase cells (P < 0.05, Fig. 3C). On the other hand, SULF2 overexpression vector significantly increased SULF2 expression in SW480 cells (P < 0.05, Fig. 3D). Subsequently, overexpression of SULF2 significantly promoted the proliferation and cell cycle progression of SW480 cells (P < 0.05, respectively, Fig. 3E and 3F). 3.4. SULF2 knockdown inhibits the growth of HT29 cells in nude mice
Distant metastasis
Absent Present
75 15
45 13
30 2
0.049
TNM stage
I + II III + IV
42 48
21 37
21 11
0.007*
CRC: colorectal cancer; TNM: tumor-node-metastasis. Statistically significant.
*
P
group, patients in SULF2 positive expression group had large tumor size (P = 0.001), more lymph node metastasis (P = 0.006), more distant metastasis (P = 0.049), and advanced TNM stage (P = 0.007). Importantly, survival curve analysis showed that patients with SULF2 positive expression had significantly decreased disease-free survival (P = 0.013, Fig. 2C) and overall survival (P = 0.034, Fig. 2D).
To further confirm the effects of SULF2 on CRC cells in vitro, HT29 cells that were transfected with NT shRNA or SULF2 shRNA were subjected to tumorigenicity experiments. As shown in Fig. 4, SULF2 knockdown significantly inhibited the growth of HT29 cells in nude mice. Tumor growth curves and weight measurements confirmed that tumors arising from SULF2 knockdown group showed significantly reduced tumor weights and volumes (P < 0.05, Fig. 4).
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Fig. 3. SULF2 promotes the proliferation and cell cycle progression of CRC cells. (A) HT29 cells that were transfected with non-targeting (NT) shRNA or SULF2 shRNA were detected by immunoblotting. n = 3 repeats with similar results, *P < 0.05 by t-test. (B) CCK8 assays indicated that knockdown of SULF2 significantly reduced the proliferative ability of HT29 cells. n = 3 repeats with similar results, *P < 0.05 by ANOVA. (C) Cell cycle assays revealed that knockdown of SULF2 significantly increased the percentage of G1 phase cells. n = 3 repeats with similar results, *P < 0.05 by t-test. (D) SW480 cells that were transfected with empty vector (EV) or SULF2 overexpression vector were confirmed by western blotting. n = 3 repeats with similar results, *P < 0.05 by t-test. (E) Overexpression of SULF2 significantly enhanced the proliferative ability of SW480 cells. n = 3 repeats with similar results, *P < 0.05 by ANOVA. (F) Overexpression of SULF2 significantly decreased the percentage of G1 phase cells. n = 3 repeats with similar results, *P < 0.05 by t-test.
3.5. SULF2 potentiates the metastatic ability of CRC cells Next, we further investigated whether SULF2 could influence the metastatic ability of CRC cells. Wound healing assay and Transwell assay were performed to evaluate the influence of SULF2 on the metastatic ability of CRC cells. Would healing assay showed that knockdown of SULF2 significantly reduced the migration of HT29 cells (P < 0.05, Fig. 5A). Transwell assay demonstrated that SULF2 knockdown inhibited the invasion of HT29 cells (P < 0.05, Fig. 5B). On the other hand, overexpression of SULF2 increased the migratory and invasive abilities of SW480 cells (P < 0.05, respectively, Fig. 5C and D). Furthermore, subcapsular splenic
injection of HT29 cells were performed to evaluate whether SULF2 could affect metastatic ability of CRC cells in vivo. As shown in Fig. 5E, knockdown of SULF2 significantly reduced liver metastases of HT29 cells in nude mice (P < 0.05). 3.6. SULF2 activates Akt and Erk1/2 pathways in CRC cells Previous studies confirmed that activation of Akt and Erk1/2 pathways were critical for the proliferation and metastasis of CRC cells [12,13]. We performed western blot to confirm whether SULF2 promoted the growth and metastasis of CRC cells via activating Akt and Erk1/2 pathways. The results of western blot showed that
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Fig. 4. Knockdown of SULF2 inhibits the growth of HT29 cells in nude mice. HT29 cells that were transfected with SULF2 shRNA or non-targeting (NT) shRNA were subcutaneously injected into the dorsal flank of nude mice. Knockdown of SULF2 significantly inhibited the growth of HT29 cells in nude mice, as suggested by significant decreased tumor weights and tumor volumes. n = 5, *P < 0.05 byt test and ANOVA, respectively.
Fig. 5. SULF2 potentiates the metastatic ability of CRC cells. (A) HT29 cells were transfected with non-targeting (NT) shRNA or SULF2 shRNA. Wound healing assays indicated that knockdown of SULF2 significantly reduced the migration of HT29 cells. (B) Transwell assays revealed that knockdown of SULF2 significantly decreased the invasion of HT29 cells. n = 3 repeats with similar results, *P < 0.05 by t-test. (C) SW480 cells were transfected with empty vector (EV) or SULF2 overexpression vector. Overexpression of SULF2 significantly enhanced the migration of SW480 cells. (D) Overexpression of SULF2 significantly enhanced the invasion of SW480 cells. n = 3 repeats with similar results, *P < 0.05 by t-test. (E) Subscapular splenic injection were performed using SULF2 knockdown HT29 cells and control cells. Knockdown of SULF2 significantly inhibited the metastatic ability of HT29 cells in nude mice. n = 5, *P < 0.05 by t-test.
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Fig. 6. SULF2 activates Akt and Erk1/2 pathways in CRC cells. HT29 cells that were transfected with non-targeting (NT) shRNA or SULF2 shRNA were subjected to immunoblotting. Knockdown of SULF2 significantly reduced the levels of p-Akt and p- Erk1/2 in HT29 cells. SW480 cells that were transfected with empty vector (EV) or SULF2 overexpression vector were detected by western blotting. Overexpression of SULF2 significantly increased the levels of p-Akt and p- Erk1/2 in SW480 cells. n = 3 repeats with similar results, *P < 0.05 by t-test.
knockdown of SULF2 significantly reduced the levels of p-Akt and p- Erk1/2 in HT29 cells (P < 0.05, respectively, Fig. 6). On the other hand, overexpression of SULF2 significantly increased the levels of p-Akt and p-Erk1/2 in SW480 cells (P < 0.05, respectively, Fig. 6). These data indicate that SULF2 enhances the growth and metastasis of CRC cells probably by activating Akt and Erk1/2 pathways. 4. Discussion Malignant growth and systemic metastasis are the main reasons for the poor prognosis of cancer patients including CRC [14,15]. Thus, investigating the molecular mechanisms involved in the growth and metastasis of CRC is critical for improving the efficacy of CRC treatment and the prognosis of CRC patients. SULF2 has been demonstrated to be an important regulator in the initiation and progression of human cancers [16,17]. However, its expression and role are inconsistent in human cancers. Thus, it is important to clarify the expression and biological function of SULF2 in specific cancers. In this study, we confirmed that SULF2 expression was increased in CRC tissues and cells as suggested by IHC and qRT-PCR results. Importantly, clinical data analysis showed that elevated expression of SULF2 was associated with unfavorable clinicopathological features and poor prognosis of CRC patients. These data indicate that SULF2 can potentially serve as a novel biomarker for predicting the prognosis of CRC patients. Previous studies showed that SULF2 could enhance the growth and metastasis of HCC cells by activating Wnt [18,19] and FGF pathways [6]. SULF2 exerted an oncogenic role in breast cancer via up-regulating the expressions of c-fos induced growth factor (FIGF) and nuclear receptor subfamily 4 group A member 3 (NR4A3), and down-regulating the expressions of cluster of differentiation 82 (CD82) and platelet-derived growth factor C
(PDGFC) [20]. In this study, HT29 and SW480 cells that express high and low levels of SULF2, respectively, were used for loss- and gain-of-function experiments to determine the biological functions of SULF2 in CRC cells. Our results showed that overexpression of SULF2 promoted the proliferation, cell cycle progression, migration and invasion of SW480 cells while knockdown of SULF2 inhibited these cellular functions of HT29 cells. Moreover, in vivo tumorigenicity and liver metastasis experiments demonstrated that SULF2 knockdown significantly inhibited the growth and liver metastasis of CRC cells in nude mice. These data solidly demonstrate that SULF2 promotes the growth and metastasis of CRC cells both in vitro and in vivo. Previous studies regarding CRC showed that activation of Akt and Erk1/2 pathways were critical for the proliferation and metastatic behaviors of CRC cells [21,22]. In this study, SULF2 knockdown reduced the levels of phosphorylated Akt and Erk1/2 in HT29 cells, while SULF2 overexpression blocked the activation of Akt and Erk1/2 pathways in SW480 cells. These data indicate that SULF2 promotes the growth and metastasis of CRC cells possibly by activating Akt and Erk1/2 pathways. In conclusion, this study demonstrates that SULF2 is overexpressed in CRC tissues and cells lines. Elevated expression of SULF2 is correlated with unfavorable clinicopathological features and poor prognosis of CRC patients. Functionally, SULF2 enhances the growth and metastasis of CRC cells both in vitro and in vivo. Furthermore, we disclose that SULF2 exerts an oncogenic role probably by activating Akt and Erk1/2 pathways in CRC cells. 5. Conclusions To conclude, we recognize SULF2 overexpression as a biomarker for predicting poor prognosis of CRC patients. The overexpression of SULF2 creates a milieu of growth and metastasis facilitation that
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plays a promoting role in CRC progression. A mechanism by which overexpressed SULF2 promotes the growth and metastasis probably by targeting Akt and Erk1/2 pathways plays an important role in this process. This finding will improve understanding of mechanism involved in cancer progression and provide novel targets for the molecular treatment of CRC. Conflicts of interest All authors declare no conflicts of interest. Acknowledgement The authors thank all the patients who participated in this study. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA. Cancer J. Clin. 67 (1) (2017) 7–30. [2] A.R. Marley, H. Nan, Epidemiology of colorectal cancer, Int. J. Mol. Epidemiol. Genet. 7 (3) (2016) 105–114. [3] E. Van Cutsem, A. Cervantes, R. Adam, A. Sobrero, J.H. Van Krieken, D. Aderka, E. Aranda Aguilar, A. Bardelli, A. Benson, G. Bodoky, F. Ciardiello, A. D'Hoore, E. Diaz-Rubio, J.Y. Douillard, M. Ducreux, A. Falcone, A. Grothey, T. Gruenberger, K. Haustermans, V. Heinemann, P. Hoff, C.H. Kohne, R. Labianca, P. LaurentPuig, B. Ma, T. Maughan, K. Muro, N. Normanno, P. Osterlund, W.J. Oyen, D. Papamichael, G. Pentheroudakis, P. Pfeiffer, T.J. Price, C. Punt, J. Ricke, A. Roth, R. Salazar, W. Scheithauer, H.J. Schmoll, J. Tabernero, J. Taieb, S. Tejpar, H. Wasan, T. Yoshino, A. Zaanan, D. Arnold, ESMO consensus guidelines for the management of patients with metastatic colorectal cancer, Ann. Oncol. 27 (8) (2016) 1386–1422. [4] W. De Roock, H. Piessevaux, J. De Schutter, M. Janssens, G. De Hertogh, N. Personeni, B. Biesmans, J.-L. Van Laethem, M. Peeters, Y. Humblet, KRAS wildtype state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab, Ann. Oncol. 19 (3) (2008) 508–515. [5] S.D. Rosen, H. Lemjabbar-Alaoui, Sulf-2: an extracellular modulator of cell signaling and a cancer target candidate, Expert Opin. Ther. Targets 14 (9) (2010) 935–949. [6] J.P. Lai, D.S. Sandhu, C. Yu, T. Han, C.D. Moser, K.K. Jackson, R.B. Guerrero, I. Aderca, H. Isomoto, M.M. Garrity-Park, Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma, Hepatology 47 (4) (2008) 1211–1222.
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[7] K. Hur, T.S. Han, E.J. Jung, J. Yu, H.J. Lee, W.H. Kim, A. Goel, H.K. Yang, Upregulated expression of sulfatases (SULF1 and SULF2) as prognostic and metastasis predictive markers in human gastric cancer, J. Pathol. 228 (1) (2012) 88–98. [8] H. Lemjabbar-Alaoui, A. van Zante, M.S. Singer, Q. Xue, Y.-Q. Wang, D. Tsay, B. He, D.M. Jablons, S.D. Rosen, Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis, Oncogene 29 (5) (2010) 635–646. [9] M. Morimoto-Tomita, K. Uchimura, A. Bistrup, D.H. Lum, M. Egeblad, N. Boudreau, Z. Werb, S.D. Rosen, Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer, Neoplasia 7 (11) (2005) 1001–1010. [10] J.R. Bishop, M. Schuksz, J.D. Esko, Heparan sulphate proteoglycans fine-tune mammalian physiology, Nature 446 (7139) (2007) 1030–1037. [11] C.M. Vicente, M.A. Lima, E.A. Yates, H.B. Nader, L. Toma, Enhanced tumorigenic potential of colorectal cancer cells by extracellular sulfatases, Mol. Cancer Res. 13 (3) (2015) 510–523. [12] Q. Ye, W. Cai, Y. Zheng, B.M. Evers, Q.B. She, ERK and AKT signaling cooperate to translationally regulate survivin expression for metastatic progression of colorectal cancer, Oncogene 33 (14) (2014) 1828–1839. [13] B. Chen, X. Zeng, Y. He, X. Wang, Z. Liang, J. Liu, P. Zhang, H. Zhu, N. Xu, S. Liang, STC2 promotes the epithelial-mesenchymal transition of colorectal cancer cells through AKT-ERK signaling pathways, Oncotarget 7 (44) (2016) 71400– 71416. [14] A.F. Chambers, A.C. Groom, I.C. MacDonald, Metastasis: dissemination and growth of cancer cells in metastatic sites, Nat. Rev. Cancer 2 (8) (2002) 563– 572. [15] E.R. Fearon, Molecular genetics of colorectal cancer, Annu. Rev. Pathol.: Mech. Dis. 6 (2011) 479–507. [16] R. Nawroth, A. Van Zante, S. Cervantes, M. McManus, M. Hebrok, S.D. Rosen, Extracellular sulfatases, elements of the Wnt signaling pathway, positively regulate growth and tumorigenicity of human pancreatic cancer cells, PLoS One 2 (4) (2007) e392. [17] G.C. Leonardi, S. Candido, M. Cervello, D. Nicolosi, F. Raiti, S. Travali, D.A. Spandidos, M. Libra, The tumor microenvironment in hepatocellular carcinoma (review), Int. J. Oncol. 40 (6) (2012) 1733. [18] A.M. Lai, C.D. Oseini, C. Moser, S.F. Yu, C. Elsawa, I. Hu, T. Nakamura, I. Han, The oncogenic effect of sulfatase 2 in human hepatocellular carcinoma is mediated in part by glypican 3–dependent Wnt activation, Hepatology 52 (5) (2010) 1680–1689. [19] W. Gao, M. Ho, The role of glypican-3 in regulating Wnt in hepatocellular carcinomas, Cancer Rep. 1 (1) (2011) 14. [20] C. Zhu, L. He, X. Zhou, X. Nie, Y. Gu, Sulfatase 2 promotes breast cancer progression through regulating some tumor-related factors, Oncol. Rep. 35 (3) (2016) 1318–1328. [21] A. De Luca, M.R. Maiello, A. D'Alessio, M. Pergameno, N. Normanno, The RAS/ RAF/MEK/ERK and the PI3 K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches, Expert Opin. Ther. Targets 16 (suppl. 2) (2012) S17–S27. [22] Q. Ye, W. Cai, Y. Zheng, B.M. Evers, Q.-B. She, ERK and AKT signaling cooperate to translationally regulate survivin expression for metastatic progression of colorectal cancer, Oncogene 33 (14) (2014) 1828–1839.
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Original article
Sulfatase-2 promotes the growth and metastasis of colorectal cancer by activating Akt and Erk1/2 pathways Youmao Tao, Tao Han, Tao Zhang, Caixia Sun* Department of Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University, No. 126 Xiantai Street, Changchun 130033, China
A R T I C L E I N F O
Article history: Received 9 January 2017 Received in revised form 28 February 2017 Accepted 7 March 2017 Keywords: Sulfatase-2 Colorectal cancer Cancer growth Cancer metastasis Signaling pathway
A B S T R A C T
The molecular mechanisms underlying the growth and metastasis of colorectal cancer (CRC) remain largely unknown. Sulfatase-2 (SULF2) was found to play critical roles in human cancers. Recent study reported that SULF1/2 overexpression resulted in increased viability and proliferation, and augmented cell migration in CRC cells. However, the expression of SULF2 and its underlying molecular mechanisms in CRC remain unknown. In this study, we found that the expressions of SULF2 in CRC tissues and cell lines were significantly increased compared to control groups. Increased expression of SULF2 was associated with malignant clinical features and poor prognosis of CRC patients. Loss of SULF2 significantly prohibited the proliferation, cell cycle progression, migration and invasion of HT29 cells, while restoration of SULF2 significantly promoted these cellular functions of SW480 cells. In vivo tumorigenicity and liver metastasis assays confirmed that SULF2 knockdown significantly reduced the growth and metastatic abilities of HT29 cells in nude mice. Furthermore, SULF2 knockdown reduced the levels of p-Akt and p-Erk1/2 in HT29 cells, while SULF2 overexpression showed opposite effects on the expressions of these proteins in SW480 cells. In all, SULF2 promotes the growth and metastasis of CRC probably by activating Akt and Erk1/2 pathways. SULF2 potentially serves as a promising biomarker and therapeutic target in CRC. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Colorectal cancer (CRC), which ranks the fourth in cancerrelated deaths, is the third most frequent cancer worldwide [1,2]. During the last several decades, remarkable advances have been made in the early diagnosis and treatment of CRC [3]. However, the long term survival of CRC patients is still poor, especially for those in advanced stages [4]. Most patients in advanced stages cannot obtain effective treatments due to the occurrence of local and systemic metastasis of CRC cells. Unfortunately, the exact mechanisms responsible for the malignant growth and metastatic behaviors of CRC cells remain largely uncovered. Therefore, investigating the molecular mechanisms underlying the growth and metastasis of CRC cells will potentially contribute to
Abbreviations: CRC, colorectal cancer; SULF2, sulfatase2; IHC, immunohistochemistry; NR4A3, nuclear receptor subfamily 4 group A member 3; CD82, cluster of differentiation 82; PDGFC, platelet-derived growth factor C. * Corresponding author. E-mail address: [email protected] (C. Sun). http://dx.doi.org/10.1016/j.biopha.2017.03.017 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
identification of novel biomarkers and therapeutic targets of CRC patients. Sulfatase2 (SULF2), an extracellular heparan endosulfatase [5], has been found to play critical roles in the initiation and progression of human cancers including hepatocellular carcinoma (HCC) [6] gastric cancer [7], lung cancer [8] and breast cancer [9]. SULF2 regulates the sulfate patterns of HSPGs and lead to the release of cytokines on HSPGs [10]. These released cytokines, such as FGF1, VEFG and SDF-1, subsequently promote the malignant behaviors of cancer cells [5]. Recent study reported that SULF1/2 overexpression resulted in increased viability and proliferation, and augmented cell migration in CRC cells [11]. However, the clinical significance of SULF2 and mechanisms underlying its biological function in CRC have never been investigated. In this study, we found that the expressions of SULF2were increased in CRC tissues and cell lines. The positive expression of SULF2 was associated with unfavorable clinicopathological features and poor prognosis of CRC patients. Both gain- and loss-offunction experiments demonstrated that SULF2 promoted proliferation, cell cycle progression, migration and invasion of CRC cells. In vivo tumor formation and liver metastasis experiments showed
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that SULF2 knockdown inhibited the growth and metastasis of CRC cells in nude mice. Furthermore, SULF2 regulated the activation of Akt and Erk1/2 pathways, which were critical for the growth and metastasis of CRC cells. 2. Materials and methods 2.1. Clinical tissues Ninety pairs of CRC tissues and matched non-tumor tissues were collected from patients, who underwent enterectomy in the Department of Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University. All patients received and signed the informed consents for agreeing to donate the clinical samples for research. These clinical samples were stored in liquid nitrogen for RNA isolation and fixed in 10% formalin for immunohistochemistry, respectively. Patients who were categorized as having an event in the calculation of disease-free survival experienced local tumor recurrence in the form of either local or distant metastasis, or death due to CRC. Patients dying of all causes were considered as “death events” and were included as such in the overall survival analysis. The protocols for collecting and using these clinical samples were approved by the Research Ethics Committee of China-Japan Union Hospital of Jilin University. 2.2. Cell culture Human intestinal epithelial cells (HIEC) and human CRC cell lines including HCT116, HT29, Caco-2, SW620, and SW480 were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). All these cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific) along with penicillin (100 U/ml) and streptomycin (100 U/ml) (Sigma-Aldrich, St Louis, MO, USA) in an incubator in humidified atmosphere with 5% CO2 at 37 C. 2.3. Cell transfection A specific SULF2 shRNA and the non-targeting (NT) shRNA were obtained from Ambion/Life Technologies (Carlsbad, CA, USA). SULF2 overexpression vector and the empty control vector were purchased from ORIGENE (Rockville, MD, USA). These shRNAs or vectors were transfected into CRC cells using Lippofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The efficiency of SULF2 shRNA or SULF2 overexpression vector was evaluated by western blot. 2.4. RNA extraction and quantitative real-time PCR (qRT-PCR) RNA extraction from CRC tissues and cells were performed with ISOGEN (Nippon Gene, Tokyo, Japan) and TaqMan Fast Cells-to-CT Kit (Ambion/Life Technologies) based on manufacturer’s instructions. Then, the total RNA were subjected to cDNA construction following the protocols of SuperScript III First-strand Synthesis System (Invitrogen). Quantitative real-time PCR was performed using the StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers for SULF2 and GAPDH were synthesized and bought from GeneCopoeia (Guangzhou, China). GAPDH was used as the internal control. All experiments were conducted at least three times. 2.5. Western blot Total proteins were isolated using RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). A Bradford Protein Assay Kit (P0006) (Beyotime, Shanghai, China) was used to analyzed
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protein concentration. The isolated proteins were loaded on 10% SDS-PAGE for separation, and were then transferred to NC membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk/TBST, the membranes were incubated with primary antibodies overnight at 4 C. Primary antibodies used in this study included SULF2 (1:1000 dilution, Abcam, Cambridge, MA, USA), p-Akt (1:1000 dilution, Abcam), Akt (1:1000 dilution, Abcam), p-Erk1/2 (1:1000 dilution, Abcam), Erk1/2 (1:1000 dilution, Abcam) and GAPDH (1:1000 dilution, Abcam). Then, the membranes were washed with TBST and incubated with secondary antibodies (Cell Signaling Technology, Beverly, MA, USA). The protein bands on the membranes were detected with ECL Advance Western detection reagents (GE Healthcare, Buckinghamshire, UK) and visualized with ChemiDoc XRS plus system (BIORAD, Hercules, CA, USA). 2.6. Immunohistochemistry (IHC) staining CRC tissues and the adjacent non-tumor tissues were subjected to formalin fixation and paraffin-embedded. These tissues were then cut into 5 mm sections for IHC staining based on the standard protocols. The results of the IHC staining for SULF2 were evaluated by one pathologist in our hospital. Staining intensity was scored as no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. Staining quantity was graded as <25% = 1, 25%-75% = 2, and >75% = 3. The degree of staining was determined using a fourgrade evaluation system: IHC score = staining intensity staining quantity (negative, 0; weak positive, 1 and 2; moderate positive, 3 and 4; strong positive, 6 and 9). 2.7. Cell proliferation assay For cell proliferation, CRC cells that were treated with corresponding vectors were seeded into 96-well plates (1.5 103 cells per well). 24, 48, 72 and 96 h after transfection, the cell proliferation assay was performed by addition of 10 ml cell counting kit 8 (CCK8) solution (Beyotime) to each well, followed by incubation at 37 C for 2 h. Absorbance was measured at a wave length of 490 nm using a microplate reader (Flexstation III ROM V2.1.28, Molecular Devices, Sunnyvale, CA, USA). 2.8. Cell cycle analysis For cell cycle assay, CRC cells were collected 48 h after transfection. These cells were then fixed with 80% ethanol overnight at 4 C. These fixed cells were stained with Propidium Iodide (PI, 50 ug/mL, BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 20 min. The distribution of cell cycle was measured with FACS Calibur system (BD Biosciences). 2.9. Wound healing assay CRC cells that were transfected with SULF2 overexpression vector or SULF2 shRNA were seeded in 6-well plates to form single confluent cell layer. The wound were made with 10 ml pipette tip in the confluent cell layer. 24 h later, the width of wound was photographed and recorded with phase-contrast microscope. 2.10. Invasion assay The invasion assay was performed with Transwell chambers (BD Biosciences). 100 ml Matrigel (1:6 dilution) was added on the upper chamber before cell seeding. CRC cells that were transfected with SULF2 overexpression vector or SULF2 shRNA were suspended in 100 ml serum-free medium and were seeded into the upper chamber. DEME with 10% FBS was placed in the lower
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chamber as chemoattractant. 48 h later, cells invaded through the membranes were fixed with methanol and stained with crystal violet. The number of invaded cells was counted under microscope.
one way ANOVA were used to analyze continuous variable. Survival analysis was performed using Kaplan-Meier’s method and Logrank test. All data were presented as Mean SEM. P < 0.05 was considered to be statistically significant.
2.11. In vivo tumorigenicity and liver metastasis assay 3. Results For tumorigenicity assay, HT29 cells that were transfected with NT shRNA or SULF2 shRNA were subcutaneously injected into the right flank of 6-week-old nude mice (n = 5 mice/group). Tumors volumes were measured every five days from the first injection using the following formula: Volume = (Length Width2)/2. After 20 days, the mice were sacrificed, and the tumors were removed and weighted. For liver metastasis assay, HT29 cells that were transfected with NT shRNA or SULF2 shRNA were used for subcapsular splenic injection. 8 weeks after injection, all mice were euthanized and the livers were surgically removed to evaluate whether the hepatic metastatic foci were formed. All animal experiments were performed according to the animal experimental guidelines of China-Japan Union Hospital of Jilin University. 2.12. Statistical analysis GraphPad Prism 5 (GraphPad Software, Inc, San Diego, CA, USA) and SPSS 19 software (SPSS, Chicago, IL, USA) were used for statistical analysis in this study. Chi-squared test was employed to explore the association between two variables. Student’s t-test and
3.1. The expression of SULF2 is increased in CRC To investigate the expression status of SULF2 in CRC, IHC was performed in CRC and tumor-adjacent tissues for SULF2 staining. The results of IHC staining showed that the expressions of SULF2 in CRC tissues were higher (Fig. 1A–C: strong, moderate and weak staining of SULF2) than those in adjacent non-tumor tissues (Fig. 1D: negative staining of SULF2). Notably, 58 of 90 (64.44%) CRC tissues showed positive staining of SULF2, while SULF2 signal was detected in 25 of 90 (27.78%) adjacent non-tumor tissues. To further confirm the overexpression of SULF2 in CRC, we randomly selected 40 pairs of CRC tissues and matched non-tumor tissues, and measured the levels of SULF2 mRNA in these tissues using qRTPCR. The results showed that the levels of SULF2 mRNA were also significantly increased in CRC tissues compared with the tumoradjacent tissues (P < 0.05, Fig. 2A). Moreover, we measured the expression levels of SULF2 mRNA in CRC cell lines and HIEC cells. Compared with that in HIEC cells, the levels of SULF2 mRNA in CRC cell lines including HCT116, Caco2, HT29, SW620 and SW480 were significantly elevated (P < 0.05, Fig. 2B).
Fig. 1. Immunostaining of SULF2 in CRC and tumor-adjacent tissues. Representative IHC staining of SULF2 for CRC tissues and adjacent non-tumor tissues. (A) Strong, (B) moderate and (C) weak staining of SULF2 in CRC tissues. (D) Negative staining of SULF2 in adjacent non-tumor tissues. Scale bar: 50 mm.
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Fig. 2. Overexpression of SULF2 is associated with poor prognosis of CRC patients. (A) Expression differences of SULF2 mRNA between CRC tissues and matched tumoradjacent tissues. n = 40, *P < 0.05 by t-test. (B) Relative expression of SULF2 mRNA in five CRC cell lines (HCT116, Caco2, HT29, SW620 and SW480) and a normal human intestinal epithelial cell line (HIEC) detected by qRT-PCR. n = 3 repeats with similar results, *P < 0.05 by ANOVA. (C) and (D) CRC patients were divided into SULF2 positive expression group (n = 58) and SULF2 negative expression group (n = 32) based on the IHC scores. SULF2 positively expressing CRC patients showed a significant decreased overall survival and disease-free survival compared to SULF2 negatively expressing cases. P < 0.05 by Log-rank test.
3.2. Elevated expression of SULF2 is correlated with poor clinical features and prognosis of CRC patients To elucidate the clinical significance of SULF2 upregulation in CRC, CRC patients were divided into two groups (SULF2 positive expression group and SULF2 negative expression group). As shown in Table 1, compared with those in SULF2 negative expression
Table 1 The correlation between clinicopathological features and SULF2 expression in CRC. Features
n = 90
SULF2 expression Positive
Negative
Age (years)
<65 65
57 33
40 18
17 15
0.136
Sex
Male Female
50 40
29 29
21 11
0.153
Tumor grade
G1 + G2 G3 + G4
65 25
43 15
22 10
0.585
Size (cm)
<5 5
41 49
19 39
22 10
0.001*
Tumor invasion
T1 + T2 T3 + T4
23 67
11 47
12 20
0.054
Lymph node status
<1 1
47 43
24 34
23 9
0.006*
*
3.3. SULF2 promotes the proliferation and cell cycle progression of CRC cells To investigate the biological functions of SULF2 in CRC cells, HT29 cells were transfected a specific SULF2 shRNA. SULF2 shRNA significantly reduced SULF2 expression in HT29 cells (P < 0.05, Fig. 3A). Loss of SULF2 significantly inhibited the proliferative ability of HT29 cells as suggested by CCK8 assay (P < 0.05, Fig. 3B). Cell cycle analysis showed that SULF2 knockdown significantly increased the percentage of G1 phase cells (P < 0.05, Fig. 3C). On the other hand, SULF2 overexpression vector significantly increased SULF2 expression in SW480 cells (P < 0.05, Fig. 3D). Subsequently, overexpression of SULF2 significantly promoted the proliferation and cell cycle progression of SW480 cells (P < 0.05, respectively, Fig. 3E and 3F). 3.4. SULF2 knockdown inhibits the growth of HT29 cells in nude mice
Distant metastasis
Absent Present
75 15
45 13
30 2
0.049
TNM stage
I + II III + IV
42 48
21 37
21 11
0.007*
CRC: colorectal cancer; TNM: tumor-node-metastasis. Statistically significant.
*
P
group, patients in SULF2 positive expression group had large tumor size (P = 0.001), more lymph node metastasis (P = 0.006), more distant metastasis (P = 0.049), and advanced TNM stage (P = 0.007). Importantly, survival curve analysis showed that patients with SULF2 positive expression had significantly decreased disease-free survival (P = 0.013, Fig. 2C) and overall survival (P = 0.034, Fig. 2D).
To further confirm the effects of SULF2 on CRC cells in vitro, HT29 cells that were transfected with NT shRNA or SULF2 shRNA were subjected to tumorigenicity experiments. As shown in Fig. 4, SULF2 knockdown significantly inhibited the growth of HT29 cells in nude mice. Tumor growth curves and weight measurements confirmed that tumors arising from SULF2 knockdown group showed significantly reduced tumor weights and volumes (P < 0.05, Fig. 4).
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Fig. 3. SULF2 promotes the proliferation and cell cycle progression of CRC cells. (A) HT29 cells that were transfected with non-targeting (NT) shRNA or SULF2 shRNA were detected by immunoblotting. n = 3 repeats with similar results, *P < 0.05 by t-test. (B) CCK8 assays indicated that knockdown of SULF2 significantly reduced the proliferative ability of HT29 cells. n = 3 repeats with similar results, *P < 0.05 by ANOVA. (C) Cell cycle assays revealed that knockdown of SULF2 significantly increased the percentage of G1 phase cells. n = 3 repeats with similar results, *P < 0.05 by t-test. (D) SW480 cells that were transfected with empty vector (EV) or SULF2 overexpression vector were confirmed by western blotting. n = 3 repeats with similar results, *P < 0.05 by t-test. (E) Overexpression of SULF2 significantly enhanced the proliferative ability of SW480 cells. n = 3 repeats with similar results, *P < 0.05 by ANOVA. (F) Overexpression of SULF2 significantly decreased the percentage of G1 phase cells. n = 3 repeats with similar results, *P < 0.05 by t-test.
3.5. SULF2 potentiates the metastatic ability of CRC cells Next, we further investigated whether SULF2 could influence the metastatic ability of CRC cells. Wound healing assay and Transwell assay were performed to evaluate the influence of SULF2 on the metastatic ability of CRC cells. Would healing assay showed that knockdown of SULF2 significantly reduced the migration of HT29 cells (P < 0.05, Fig. 5A). Transwell assay demonstrated that SULF2 knockdown inhibited the invasion of HT29 cells (P < 0.05, Fig. 5B). On the other hand, overexpression of SULF2 increased the migratory and invasive abilities of SW480 cells (P < 0.05, respectively, Fig. 5C and D). Furthermore, subcapsular splenic
injection of HT29 cells were performed to evaluate whether SULF2 could affect metastatic ability of CRC cells in vivo. As shown in Fig. 5E, knockdown of SULF2 significantly reduced liver metastases of HT29 cells in nude mice (P < 0.05). 3.6. SULF2 activates Akt and Erk1/2 pathways in CRC cells Previous studies confirmed that activation of Akt and Erk1/2 pathways were critical for the proliferation and metastasis of CRC cells [12,13]. We performed western blot to confirm whether SULF2 promoted the growth and metastasis of CRC cells via activating Akt and Erk1/2 pathways. The results of western blot showed that
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Fig. 4. Knockdown of SULF2 inhibits the growth of HT29 cells in nude mice. HT29 cells that were transfected with SULF2 shRNA or non-targeting (NT) shRNA were subcutaneously injected into the dorsal flank of nude mice. Knockdown of SULF2 significantly inhibited the growth of HT29 cells in nude mice, as suggested by significant decreased tumor weights and tumor volumes. n = 5, *P < 0.05 byt test and ANOVA, respectively.
Fig. 5. SULF2 potentiates the metastatic ability of CRC cells. (A) HT29 cells were transfected with non-targeting (NT) shRNA or SULF2 shRNA. Wound healing assays indicated that knockdown of SULF2 significantly reduced the migration of HT29 cells. (B) Transwell assays revealed that knockdown of SULF2 significantly decreased the invasion of HT29 cells. n = 3 repeats with similar results, *P < 0.05 by t-test. (C) SW480 cells were transfected with empty vector (EV) or SULF2 overexpression vector. Overexpression of SULF2 significantly enhanced the migration of SW480 cells. (D) Overexpression of SULF2 significantly enhanced the invasion of SW480 cells. n = 3 repeats with similar results, *P < 0.05 by t-test. (E) Subscapular splenic injection were performed using SULF2 knockdown HT29 cells and control cells. Knockdown of SULF2 significantly inhibited the metastatic ability of HT29 cells in nude mice. n = 5, *P < 0.05 by t-test.
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Fig. 6. SULF2 activates Akt and Erk1/2 pathways in CRC cells. HT29 cells that were transfected with non-targeting (NT) shRNA or SULF2 shRNA were subjected to immunoblotting. Knockdown of SULF2 significantly reduced the levels of p-Akt and p- Erk1/2 in HT29 cells. SW480 cells that were transfected with empty vector (EV) or SULF2 overexpression vector were detected by western blotting. Overexpression of SULF2 significantly increased the levels of p-Akt and p- Erk1/2 in SW480 cells. n = 3 repeats with similar results, *P < 0.05 by t-test.
knockdown of SULF2 significantly reduced the levels of p-Akt and p- Erk1/2 in HT29 cells (P < 0.05, respectively, Fig. 6). On the other hand, overexpression of SULF2 significantly increased the levels of p-Akt and p-Erk1/2 in SW480 cells (P < 0.05, respectively, Fig. 6). These data indicate that SULF2 enhances the growth and metastasis of CRC cells probably by activating Akt and Erk1/2 pathways. 4. Discussion Malignant growth and systemic metastasis are the main reasons for the poor prognosis of cancer patients including CRC [14,15]. Thus, investigating the molecular mechanisms involved in the growth and metastasis of CRC is critical for improving the efficacy of CRC treatment and the prognosis of CRC patients. SULF2 has been demonstrated to be an important regulator in the initiation and progression of human cancers [16,17]. However, its expression and role are inconsistent in human cancers. Thus, it is important to clarify the expression and biological function of SULF2 in specific cancers. In this study, we confirmed that SULF2 expression was increased in CRC tissues and cells as suggested by IHC and qRT-PCR results. Importantly, clinical data analysis showed that elevated expression of SULF2 was associated with unfavorable clinicopathological features and poor prognosis of CRC patients. These data indicate that SULF2 can potentially serve as a novel biomarker for predicting the prognosis of CRC patients. Previous studies showed that SULF2 could enhance the growth and metastasis of HCC cells by activating Wnt [18,19] and FGF pathways [6]. SULF2 exerted an oncogenic role in breast cancer via up-regulating the expressions of c-fos induced growth factor (FIGF) and nuclear receptor subfamily 4 group A member 3 (NR4A3), and down-regulating the expressions of cluster of differentiation 82 (CD82) and platelet-derived growth factor C
(PDGFC) [20]. In this study, HT29 and SW480 cells that express high and low levels of SULF2, respectively, were used for loss- and gain-of-function experiments to determine the biological functions of SULF2 in CRC cells. Our results showed that overexpression of SULF2 promoted the proliferation, cell cycle progression, migration and invasion of SW480 cells while knockdown of SULF2 inhibited these cellular functions of HT29 cells. Moreover, in vivo tumorigenicity and liver metastasis experiments demonstrated that SULF2 knockdown significantly inhibited the growth and liver metastasis of CRC cells in nude mice. These data solidly demonstrate that SULF2 promotes the growth and metastasis of CRC cells both in vitro and in vivo. Previous studies regarding CRC showed that activation of Akt and Erk1/2 pathways were critical for the proliferation and metastatic behaviors of CRC cells [21,22]. In this study, SULF2 knockdown reduced the levels of phosphorylated Akt and Erk1/2 in HT29 cells, while SULF2 overexpression blocked the activation of Akt and Erk1/2 pathways in SW480 cells. These data indicate that SULF2 promotes the growth and metastasis of CRC cells possibly by activating Akt and Erk1/2 pathways. In conclusion, this study demonstrates that SULF2 is overexpressed in CRC tissues and cells lines. Elevated expression of SULF2 is correlated with unfavorable clinicopathological features and poor prognosis of CRC patients. Functionally, SULF2 enhances the growth and metastasis of CRC cells both in vitro and in vivo. Furthermore, we disclose that SULF2 exerts an oncogenic role probably by activating Akt and Erk1/2 pathways in CRC cells. 5. Conclusions To conclude, we recognize SULF2 overexpression as a biomarker for predicting poor prognosis of CRC patients. The overexpression of SULF2 creates a milieu of growth and metastasis facilitation that
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plays a promoting role in CRC progression. A mechanism by which overexpressed SULF2 promotes the growth and metastasis probably by targeting Akt and Erk1/2 pathways plays an important role in this process. This finding will improve understanding of mechanism involved in cancer progression and provide novel targets for the molecular treatment of CRC. Conflicts of interest All authors declare no conflicts of interest. Acknowledgement The authors thank all the patients who participated in this study. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA. Cancer J. Clin. 67 (1) (2017) 7–30. [2] A.R. Marley, H. Nan, Epidemiology of colorectal cancer, Int. J. Mol. Epidemiol. Genet. 7 (3) (2016) 105–114. [3] E. Van Cutsem, A. Cervantes, R. Adam, A. Sobrero, J.H. Van Krieken, D. Aderka, E. Aranda Aguilar, A. Bardelli, A. Benson, G. Bodoky, F. Ciardiello, A. D'Hoore, E. Diaz-Rubio, J.Y. Douillard, M. Ducreux, A. Falcone, A. Grothey, T. Gruenberger, K. Haustermans, V. Heinemann, P. Hoff, C.H. Kohne, R. Labianca, P. LaurentPuig, B. Ma, T. Maughan, K. Muro, N. Normanno, P. Osterlund, W.J. Oyen, D. Papamichael, G. Pentheroudakis, P. Pfeiffer, T.J. Price, C. Punt, J. Ricke, A. Roth, R. Salazar, W. Scheithauer, H.J. Schmoll, J. Tabernero, J. Taieb, S. Tejpar, H. Wasan, T. Yoshino, A. Zaanan, D. Arnold, ESMO consensus guidelines for the management of patients with metastatic colorectal cancer, Ann. Oncol. 27 (8) (2016) 1386–1422. [4] W. De Roock, H. Piessevaux, J. De Schutter, M. Janssens, G. De Hertogh, N. Personeni, B. Biesmans, J.-L. Van Laethem, M. Peeters, Y. Humblet, KRAS wildtype state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab, Ann. Oncol. 19 (3) (2008) 508–515. [5] S.D. Rosen, H. Lemjabbar-Alaoui, Sulf-2: an extracellular modulator of cell signaling and a cancer target candidate, Expert Opin. Ther. Targets 14 (9) (2010) 935–949. [6] J.P. Lai, D.S. Sandhu, C. Yu, T. Han, C.D. Moser, K.K. Jackson, R.B. Guerrero, I. Aderca, H. Isomoto, M.M. Garrity-Park, Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma, Hepatology 47 (4) (2008) 1211–1222.
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