|Year : 2016 | Volume
| Issue : 5 | Page : 19-22
Positive correlation of cysteine-rich 61 and target genes of Wnt/β-catenin pathway in esophageal squamous cell carcinoma
Peng Wang1, Li Li2, Ting Li3
1 Department of Clinical Laboratory, Lishui People's Hospital (6th Affiliated Hospital of Wenzhou Medical University), Lishui 323000, Zhejiang, P.R. China
2 Department of Pathology, Jiangsu Province Hospital of TMC, Jiangsu 210029, P.R. China
3 Department of Oncology, Lishui People's Hospital (6th Affiliated Hospital of Wenzhou Medical University), Lishui 323000, Zhejiang, P.R. China
|Date of Web Publication||7-Oct-2016|
Department of Oncology, Lishui People's Hospital (6th Affiliated Hospital of Wenzhou Medical University) Lishui 323000, Zhejiang
Source of Support: None, Conflict of Interest: None
Objectives: To analyze the relationship between cysteine-rich 61 (Cyr61) and target genes of Wnt/β-catenin pathway (CCND1 and MYC) in 40 esophageal squamous cell carcinoma (ESCC) tissue specimens. Another aim of this study was to verify whether Cyr61 could be regulated by Wnt/β-catenin signaling.
Materials and Methods: Forty ESCC tissue specimens with paired adjacent normal epithelial tissues were obtained. The expression of Cyr61, CCND1, and MYC was examined by quantitative real-time polymerase chain reaction. Regression analysis was further performed to evaluate the correlation between expression levels of Cyr61 and CCND1, MYC. Finally, the mRNA expression of Cyr61 was determined in ESCC cells after lithium chloride (LiCl) treatment, which activated intrinsic Wnt/β-catenin signaling.
Results: Cyr61, CCND1, and MYC were obviously increased in ESCC tissue specimens than the matched normal epithelial tissues. Positive correlation of Cyr61 expression with CCND1 and/or MYC was validated by regression analysis. In addition, the expression of Cyr61 could be modulated by LiCl, an activator of Wnt/β-catenin signaling.
Conclusion: Our data characterized that Cyr61 was aberrantly upregulated in ESCC tissues and was positively correlated to the targets of Wnt/β-catenin pathway. We also speculated that Cyr61 might be a direct target of this pathway and play a crucial role in ESCC progression.
Keywords: Correlation, cysteine-rich 61, esophageal squamous cell carcinoma, Wnt/β-catenin
|How to cite this article:|
Wang P, Li L, Li T. Positive correlation of cysteine-rich 61 and target genes of Wnt/β-catenin pathway in esophageal squamous cell carcinoma. J Can Res Ther 2016;12:19-22
|How to cite this URL:|
Wang P, Li L, Li T. Positive correlation of cysteine-rich 61 and target genes of Wnt/β-catenin pathway in esophageal squamous cell carcinoma. J Can Res Ther [serial online] 2016 [cited 2018 Aug 16];12:19-22. Available from: http://www.cancerjournal.net/text.asp?2016/12/5/19/191622
| > Introduction|| |
Cysteine-rich 61 (Cyr61), also known as CCN1, is encoded by a growth factor inducible immediate early gene, and shares a 40–50% amino-acid homology with other members of the CCN family. It is a secreted, and regulates multiple cellular events such as cell proliferation, apoptosis, adhesion, and migration.,, Abnormal expression of Cyr61 was observed in many types of tumors, but its role in these tumors varies. For instance, it was shown to promote tumor progression in glioblastoma,, breast cancer, and gastric cancer. However, it played the opposite roles such as inducing apoptosis, inhibiting cell proliferation in lung cancer, and endometrial cancer. In esophageal squamous cell carcinoma (ESCC) cells, a recent study showed that Cyr61 could contribute to the expression of CD204, a useful marker for tumor-associated macrophages (TAMs) contributing to the angiogenesis, progression, and prognosis of ESCC. They also showed that Cyr61 could promote cell migration in TAMs in the ESCC microenvironment via the MEK/ERK pathway. Previously, other groups have also found that Cyr61 was involved in growth, invasiveness, adhesion, transformation, and other processes of ESCC cells.,, In addition, Cyr61 is proved as a significant and independent prognostic factor for survival in ESCC. The activation and high level of Cyr61 expression can be induced by several agents, including serum, PDGF, HB-EGF, basic fibroblast growth factor, and cAMP.,,
Esophageal cancer ranks as the eighth most common malignancy and the sixth most frequent cause of cancer-related death in the worldwide. Among different subtypes of esophageal cancer, ESCC is the predominant type. The precise mechanisms that governing its progression remains largely unknown. We also know that Wnt/β-catenin signaling pathway plays an important role in esophageal cancer progression, metastasis, and invasion. And they found that nuclear translocation of β-catenin in ESCC cells required high level of Cyr61. In another study, it was found that Cyr61 could be regulated by Wnt/β-catenin signaling in hepatocellular carcinoma. However, the connection of Cyr61 expression with Wnt/β-catenin signaling molecules expression remains unclear. The aim of this study was to provide evidence to fulfill their correlations and answered whether Cyr61 could also be regulated by this pathway in ESCC cells.
| > Materials and Methods|| |
Patients and tissues
Forty pairs of primary ESCC and their corresponding adjacent normal tissues were collected from Lishui people's hospital, from 2013 to 2015, were included in this paper. These patients were selected based on the available complete clinical follow-up data and available tumor specimens before chemotherapy and/or radiation therapy. All patients gave their informed consent prior to their inclusion. The fresh tissues were snap-frozen in liquid nitrogen and stored at −80°C until analysis.
Two ESCC cell lines, Eca109 and Eca9706 were obtained from ATCC. Both cell lines were cultured in DMEM, supplemented with 10% FBS, and 1% Penicillin/Streptomycin, and incubated in 5% CO2 at 37°C.
Eca109 and Eca9706 cells were plated into 6-well plates at 3 × 105 cells per well. Twenty-four hour later, the cells were incubated with 0, 25 or 50 mM lithium chloride (LiCl) for 12 h before being lysed by TRIzol reagent.
Quantitative real-time real-time-polymerase chain reaction
RNA was extracted from tissue with TRIzol. CDNA was then synthesized by reserve transcription. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using SYBR Green PCR Master Mix. The sequences for all target gene primers were purchased from Applied Biosystems. GAPDH was used as an internal control.
Statistical analyses were performed by SAS 9.2 (http://www.sas.com/en_us/home.html). The significance of differences between normal tissues and tumors was estimated by paired Student's t-test. Correlation between expression levels of Cyr61 and Wnt/β-catenin target genes was analyzed using Spearman's linear correlation. Two-sided P values were calculated, and a P < 0.05 was chosen for statistical significance.
| > Results|| |
Overexpression of cysteine-rich 61 and Wnt/β-catenin signaling molecules in esophageal squamous cell carcinoma samples
We initiated our study by analyzing the expression of Cyr61 in 40 ESCC cancer tissues and paired adjacent normal epithelial tissues by quantitative qRT-PCR. As a result, its expression was remarkably overexpressed [Figure 1]a, which also confirmed the conclusion from the previous study. Since both cyclin D1 encoded by CCND1 and c-MYC encoded by MYC are crucial downstream effectors of the typical Wnt/β-catenin signaling pathway, we also detect their expression levels in the above ESCC samples. As shown in [Figure 1]b and [Figure 1]c, both CCND1 and MYC exhibited statistically significant overexpression in theses samples, and these results were inline with the former reports regarding the oncogenic activities of Wnt/β-catenin signaling in ESCC development.,
|Figure 1: Cysteine-rich 61 and Wnt/β-catenin signaling target genes were upregulated in EC samples. Relative expression of cysteine-rich 61 (a), CCND1 (b) and MYC (c) mRNA in paired human esophageal squamous cell carcinoma and normal esophageal tissues. Quantitative real-time polymerase chain reaction was performed on 40 pairs of esophageal squamous cell carcinoma tissues. Data were calculated from triplicates|
Click here to view
Cysteine-rich 61 levels were positively correlated with Wnt/β-catenin signaling molecules
To examine the correlation between Cyr61 and the well-known Wnt/β-catenin signaling molecules CCND1 and MYC, we performed Spearman's correlation analysis. The results shown in [Figure 2]a and [Figure 2]b verified a positive correlation between Cyr61 and CCND1/MYC expression at mRNA level (P < 0.0001). Our data thus indicated that Cyr61 might be positively regulated by Wnt/β-catenin pathway. Actually, it was shown in hepatocellular carcinoma that Cyr61 could be regulated by Wnt/β-catenin signaling. And we explored further details along our hypothesis in the follow-up.
|Figure 2: Spearman's correlation analysis between cysteine-rich 61 and Wnt/β-catenin target genes. The positive correlation between cysteine-rich 61 and CCND1 (a) or cysteine-rich 61 and MYC (b) expression in 40 esophageal squamous cell carcinoma samples was determined using Spearman's correlation analysis (P < 0.0001)|
Click here to view
Positive modulation of cysteine-rich 61 expression by the activator of Wnt/β-catenin signaling
To evaluate whether Cyr61 could be regulated by Wnt/β-catenin signaling in our ESCC cells, LiCl, an activator of Wnt/β-catenin signaling, was used to treat ESCC cell lines (Eca109 and Eca9706) at different concentrations (0, 25, 50 mM) for 12 h in vitro. [Figure 3] shows the increased mRNA expression of Cyr61 was observed along with the increasing LiCl doses in both cell lines. Therefore, our data supported that Cyr61 was also positively regulated by Wnt/β-catenin signaling in the ESCC cells. It makes sense to describe the critical roles of Cyr61 in these cells, and might also provide clues to understand the mechanisms that Cyr61 is involved in ESCC progression.
|Figure 3: Lithium chloride induced cysteine-rich 61 mRNA expression in Eca109 and Eca9706 cells. Eca109 and Eca9706 cells were treated with 0, 25 mM or 50 mM lithium chloride. After 12 h, cells were harvested for quantitative real-time polymerase chain reaction analysis. *P < 0.05, **P < 0.01|
Click here to view
| > Discussion|| |
Wnt/β-catenin signaling pathway plays an important part in not only embryonic development but also in cancer cell biology. Canonical Wnt signaling activation is a multistep and complex process involving in Wnt2, GSK3β, Axin, APC, β-catenin, TCF, c-MYC, and cyclin D1 molecules. Recently, the clinicopathological significance of this pathway was recognized in ESCC  and they suggested that the aberrant increased β-catenin expression could be an adverse underlying factor in carcinogenesis and progression of ESCC. Besides, many of the target genes of this pathway are involved in promoting cell proliferation.
In the present study, we showed that Cyr61, also known as CCN1, was overexpressed in the ESCC samples. Its expression in these samples positively and significantly correlated with the targets of the Wnt/β-catenin pathway, which were CCND1 and MYC specifically. Moreover, we found that Cyr61 could be regulated by the activator of the Wnt/β-catenin pathway, which further supported that Cyr61 was positively regulated by this pathway. Our results thus enrich the current knowledge about the Cyr61 in ESCC cells.
Cyr61 is a secreted, heparin-binding and extracellular matrix-associated angiogenic inducer. It participates in cell adhesion, migration, proliferation, and differentiation and characterized as both tumor suppressor and promoter of tumor progression and invasion, dependent on tumor types. For the ESCC cells, previous reports considered an oncogenic role of Cyr61, and it could be served as a significant and independent prognostic factor for survival.,,,, It was not surprising we found an increased expression of Cyr61 in ESCC samples, since it was proved by other groups. The meaning of our study was the finding that Cyr61 might be a direct target of the Wnt/β-catenin signaling pathway, confirmed by our regression analysis for Cyr61 expression with the Wnt/β-catenin signaling targets, as well as our LiCl treatment experiments. Therefore, we linked the phenomenon of abnormal expression of Cyr61 expression with the activated Wnt/β-catenin signaling pathway in ESCC cells.
| > Conclusion|| |
Our study indicates that Cyr61 is a target of the Wnt/β-catenin signaling pathway in ESCC cells, since its expression exhibits high correlations with that of the Wnt/β-catenin signaling targets, CCND1, and MYC, and could be positively regulated by LiCl. Further studies are underway to investigate the complex mechanisms of Cyr61 in ESCC tumorigenesis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Lau LF. CCN1/CYR61: The very model of a modern matricellular protein. Cell Mol Life Sci 2011;68:3149-63.
Chen Y, Du XY. Functional properties and intracellular signaling of CCN1/Cyr61. J Cell Biochem 2007;100:1337-45.
Chaqour B, Goppelt-Struebe M. Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins. FEBS J 2006;273:3639-49.
Xie D, Yin D, Tong X, O'Kelly J, Mori A, Miller C, et al.
Cyr61 is overexpressed in gliomas and involved in integrin-linked kinase-mediated Akt and beta-catenin-TCF/Lef signaling pathways. Cancer Res 2004;64:1987-96.
Cheng G, Zhang H, Zhang L, Zhang J. Cyr61 promotes growth of glioblastoma in vitro
and in vivo
. Tumour Biol 2015;36:2869-73.
Lin MT, Chang CC, Chen ST, Chang HL, Su JL, Chau YP, et al.
Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-kappaB-dependent XIAP up-regulation. J Biol Chem 2004;279:24015-23.
Lin MT, Zuon CY, Chang CC, Chen ST, Chen CP, Lin BR, et al.
Cyr61 induces gastric cancer cell motility/invasion via activation of the integrin/nuclear factor-kappaB/cyclooxygenase-2 signaling pathway. Clin Cancer Res 2005;11:5809-20.
Tong X, O'Kelly J, Xie D, Mori A, Lemp N, McKenna R, et al.
Cyr61 suppresses the growth of non-small-cell lung cancer cells via the beta-catenin-c-myc-p53 pathway. Oncogene 2004;23:4847-55.
Chien W, Kumagai T, Miller CW, Desmond JC, Frank JM, Said JW, et al.
Cyr61 suppresses growth of human endometrial cancer cells. J Biol Chem 2004;279:53087-96.
Shigeoka M, Urakawa N, Nishio M, Takase N, Utsunomiya S, Akiyama H, et al.
Cyr61 promotes CD204 expression and the migration of macrophages via MEK/ERK pathway in esophageal squamous cell carcinoma. Cancer Med 2015;4:437-46.
Xie JJ, Xu LY, Xie YM, Du ZP, Feng CH, Dong H, et al.
Involvement of Cyr61 in the growth, invasiveness and adhesion of esophageal squamous cell carcinoma cells. Int J Mol Med 2011;27:429-34.
Modak C, Mouazzen W, Narvaez R, Reavis KM, Chai J. CCN1 is critical for acid-induced esophageal epithelial cell transformation. Biochem Biophys Res Commun 2010;392:533-7.
Xie JJ, Xu LY, Wu JY, Shen ZY, Zhao Q, Du ZP, et al.
Involvement of CYR61 and CTGF in the fascin-mediated proliferation and invasiveness of esophageal squamous cell carcinomas cells. Am J Pathol 2010;176:939-51.
Huang X, Xiao DW, Xu LY, Zhong HJ, Liao LD, Xie ZF, et al.
Prognostic significance of altered expression of SDC2 and CYR61 in esophageal squamous cell carcinoma. Oncol Rep 2009;21:1123-9.
Lau LF, Nathans D. Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBO J 1985;4:3145-51.
Lau LF, Nathans D. Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: Coordinate regulation with c-fos or c-myc. Proc Natl Acad Sci U S A 1987;84:1182-6.
Nathans D, Lau LF, Christy B, Hartzell S, Nakabeppu Y, Ryder K. Genomic response to growth factors. Cold Spring Harb Symp Quant Biol 1988;53(Pt 2):893-900.
Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med 2003;349:2241-52.
Chai J, Modak C, Ouyang Y, Wu SY, Jamal MM. CCN1 induces ß-catenin translocation in esophageal squamous cell carcinoma through integrin a11. ISRN Gastroenterol 2012;2012:207235.
Li ZQ, Ding W, Sun SJ, Li J, Pan J, Zhao C, et al.
Cyr61/CCN1 is regulated by Wnt/ß-catenin signaling and plays an important role in the progression of hepatocellular carcinoma. PLoS One 2012;7:e35754.
Kolligs FT, Bommer G, Göke B. Wnt/beta-catenin/tcf signaling: A critical pathway in gastrointestinal tumorigenesis. Digestion 2002;66:131-44.
Deng F, Zhou K, Cui W, Liu D, Ma Y. Clinicopathological significance of wnt/ß-catenin signaling pathway in esophageal squamous cell carcinoma. Int J Clin Exp Pathol 2015;8:3045-53.
[Figure 1], [Figure 2], [Figure 3]