Elsevier

Gynecologic Oncology

Volume 112, Issue 3, March 2009, Pages 646-653
Gynecologic Oncology

SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway

https://doi.org/10.1016/j.ygyno.2008.10.026Get rights and content

Abstract

Objectives

Aberrant activation of the Wnt/β-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as Wnt antagonists and play important implications in carcinogenesis. Recently, we have shown that SFRP1 and SFRP2 are frequently downregulated through promoter hypermethylation. However, the function of SFRP1 and SFRP2 in cervical cancer remains unclear.

Methods

To improve our understanding of the role of SFRP1 and SFRP2 in cervical cancer cells, we use overexpression or shRNA approach in cervical cancer cell lines.

Results

Restoration of the expression of SFRP1 and SFRP2 attenuated Wnt signaling in CaSki cells, decreased abnormal accumulation of free β-catenin in the nucleus, and suppressed cancer cell growth. In addition, different statuses of β-catenin accumulation in the cytoplasm of CaSki or HeLa3rd cells were observed, suggesting that different Wnt pathways are executed. Furthermore, we demonstrated that SFRP1 and SFRP2 enhance the expression of the epithelial marker E-cadherin, through inhibition of the expression of SLUG, TWIST and SNAIL, three transcription factors involved in the epithelial mesenchymal transition (EMT) program. Finally, in a xenograft animal model, we showed that SFRP1 suppresses tumorigenicity of cancer cells in vivo.

Conclusions

Taken together, these data strongly suggest that epigenetic silencing of SFRP genes leads to oncogenic activation of the Wnt pathway and contributes to cervical cancer progression through the EMT program.

Introduction

Cervical cancer is one of the major causes of death in women worldwide [1]. Infection with oncogenic human papillomavirus (HPV), which can be detected in virtually all cases of cervical cancers, is the most significant risk factor in the etiology of this type of cancer [2]. The interaction of the E6/E7 oncoprotein (encoded by high-risk HPV types) with the tumor-suppressor gene p53/Rb causes abnormal cell-cycle regulation, which constitutes the major mechanistic theme of malignant transformation [3]; however, HPV infection is necessary but not sufficient to cause cervical cancer. About 60% of low-grade squamous intraepithelial lesion (LSIL) regress, 30% persist, 5–10% progress to high-grade SIL (HSIL), and only less than 1% become squamous cervical cancer (SCC) [4]. The molecular mechanisms underlying such an inefficient HPV-initiated cervical carcinogenesis remain elusive. Genetic changes with resultant genomic instability have long been recognized as an important mechanism for cervical carcinogenesis [5], [6]. Increasing reports of DNA methylation findings in cervical cancer and precancerous lesions [7], [8], [9], [10] support a role for this phenomenon in cervical cancer development.

The Wnt family of proteins comprises a large variety of secreted growth factors that regulate cell differentiation, proliferation, migration, and organogenesis during embryonic development [11]. Recent reports provide evidence that activation of the Wnt pathway leads to inhibition of tumor cell apoptosis in several human cancers, including colon cancer, breast cancer, melanomas, and hepatocellular carcinoma [12], [13], [14]. Wnt proteins bind to the frizzled receptor and subsequently activate the canonical and noncanonical Wnt pathways [15], [16], [17]. In the canonical pathway, signal transduction activates disheveled (Dsh), which releases β-catenin from the axin-adenomatous polyposis coli (APC)-glycogen synthase kinase 3 (GSK3) complex, causing stabilization and accumulation of β-catenin in the cytoplasm [15]. After translocation into the nucleus, β-catenin interacts with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and activates its downstream target oncogenes; e.g., c-Myc and Cyclin D1[11], [18], [19], [20]. Nuclear β-catenin is thought to be the hallmark of activation of the canonical Wnt pathway [21], [22]. On the other hand, through the activation of Dsh, Wnt also activates a β-catenin-independent noncanonical pathway through calcium flux, G-proteins and inactivates c-Jun NH2-terminal kinase (c-JNK) induced apoptosis [23], [24].

Secreted frizzled-related proteins (SFRPs), a family of five secreted glycoproteins, are extracellular signaling molecules that antagonize the Wnt signaling pathway. In a previous study, we have demonstrated that SFRP1 is a candidate TSG that is silenced in hepatocarcinogenesis through promoter hypermethylation [25], [26]. Recently, we also demonstrated that SFRP genes are inactivated by promoter methylation in cervical cancers [27]. Uren et al. detected increased cytoplasmic and nuclear staining of β-catenin in invasive cervical carcinomas and proposed a model for cervical cancer progression. They suggest that the transformation of HPV-immortalized human keratinocytes requires a second hit, namely the activation of the canonical Wnt pathway [28]. Therefore, the interaction of the Wnt pathway with its antagonist in the tumorigenesis of cervical cancer warrants a more detailed investigation.

Epithelial–mesenchymal transitions are known to be key steps during embryonic morphogenesis, and they have now been implicated in the progression of primary tumors towards metastases. Many signaling pathways are involved in the EMT process, including the Wnt, Notch, NF-κB, TGFβ, and RTK/Ras signaling pathways. Repression of E-cadherin by transcription factors such as SNAIL, SLUG, or TWIST emerges as an important EMT-driving step.

Based on these observations, we proposed that epigenetic silencing of SFRP genes leads to oncogenic activation of the Wnt pathway and contribute to cervical cancer progression through the EMT program. In this study, to improve our understanding of the role of SFRP1 and SFRP2 in cervical cancer development, we used reexpression of these genes to test whether they could attenuate Wnt signaling in β-catenin-dependent cervical cancer cells. Moreover, to assess the effect of SFRP1 on cell growth, we knocked down SFRP1 expression in SiHa cell lines. Finally, we also used a xenograft model to demonstrate the tumor suppressor effect of SFRP1.

Section snippets

Cell lines

CaSki and SiHa cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Subpopulations of HeLa cells were selected according to their differential invasiveness using Transwell plates [29]. Briefly, the polycarbonate membranes (8 μm pore size) of the Transwell inserts were coated with a reconstituted basemembrane gel (Matrigel, Becton Dickinson Labware, Bedford, MA). Cells were then seeded into the wells. Following incubation for 72 h at 37 °C, the inserts were

β-catenin accumulation in cervical cancer cell line

Oncogenic activation of the Wnt signaling pathway is common in human cancers. To the best of our knowledge, β-catenin accumulation is critical in the canonical Wnt signaling pathway. Uren et al. have showed strong β-catenin staining in cervical dysplasia and cancer tissues by immunohistochemistry [28]. Through Western blot analysis, we found abundant β-catenin accumulated within the cytoplasm and nuclei of CaSki cells (Fig. 1A). When we subsequently analyzed TCF/LEF-regulated transcriptional

Discussion

The Wnt pathway is known to be involved in tumorigenesis in many human cancers, including colon cancer, breast cancer, lung cancer, melanomas, and hepatocellular carcinoma [12], [13], [14]. Dysregulation of this pathway can be caused by mutations in any molecular components (e.g., CTNNB1, AXIN, or FZD7) in colon cancers, hepatocellular carcinomas (HCC) and other cancers [32], [33], [34]. Suzuki et al. demonstrated recently that the epigenetic loss of SFRP gene function might contribute to the

Conflict of interest statement

The authors have no conflicts of interest in relation to this article.

Acknowledgments

We thank Dr. Hiromu Suzuki (First Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan) for kindly providing the construct plasmid. This work was supported in part by National Science Council, Taiwan, Republic of China (ROC); grant numbers: the NSC95-2320-B-016-019-MY2; NSC96-3112-B-016-003; NSC97-3112-B-016-002; the Department of Health, Taiwan, Republic of China; grant number; DOH97-TD-I-111-TM005; Tri-Service General Hospital, Taiwan, ROC; grant numbers:

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