SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway
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:
References (40)
Spontaneous evolution of intraepithelial lesions according to the grade and type of the implicated human papillomavirus (HPV)
Eur. J. Obstet. Gynecol. Reprod. Biol.
(1996)- et al.
DNA ploidy of cervical intraepithelial neoplasia (CIN)
Cancer Genet. Cytogenet.
(1997) - et al.
Caught up in a Wnt storm: Wnt signaling in cancer
Biochim. Biophys. Acta
(2003) - et al.
A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling
Dev. Cell.
(2003) - et al.
Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma
Gastroenterology
(2004) - et al.
Human secreted frizzled-related protein is down-regulated and induces apoptosis in human cervical cancer
Exp. Cell. Res.
(2002) - et al.
Cytoplasmic/nuclear expression without mutation of exon 3 of the beta-catenin gene is frequent in the development of the neoplasm of the uterine cervix
Gynecol. Oncol.
(2001) - et al.
Twist expression in patients with cervical cancer is associated with poor disease outcome
Ann. Oncol.
(2008) - et al.
Estimating the world cancer burden: Globocan 2000
Int. J. Cancer
(2001) - et al.
Human papillomavirus is a necessary cause of invasive cervical cancer worldwide
J. Pathol.
(1999)