Abstract
BackgroundKlotho was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome. Recently, KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies. Abnormal activation of the canonical Wnt pathway due to epigenetic deregulation of Wnt antagonists is thought to play a crucial role in cervical tumorigenesis. In this study, we examined epigenetic silencing of KLOTHO in human cervical carcinoma.ResultsLoss of KLOTHO mRNA was observed in several cervical cancer cell lines and in invasive carcinoma samples, but not during the early, preinvasive phase of primary cervical tumorigenesis. KLOTHO mRNA was restored after treatment with either the DNA demethylating agent 2'-deoxy-5-azacytidine or histone deacetylase inhibitor trichostatin A. Methylation-specific PCR and bisulfite genomic sequencing analysis of the promoter region of KLOTHO revealed CpG hypermethylation in non-KLOTHO-expressing cervical cancer cell lines and in 41% (9/22) of invasive carcinoma cases. Histone deacetylation was also found to be the major epigenetic silencing mechanism for KLOTHO in the SiHa cell line. Ectopic expression of the secreted form of KLOTHO restored anti-Wnt signaling and anti-clonogenic activity in the CaSki cell line including decreased active β-catenin levels, suppression of T-cell factor/β-catenin target genes, such as c-MYC and CCND1, and inhibition of colony growth.ConclusionsEpigenetic silencing of KLOTHO may occur during the late phase of cervical tumorigenesis, and consequent functional loss of KLOTHO as the secreted Wnt antagonist may contribute to aberrant activation of the canonical Wnt pathway in cervical carcinoma.
Highlights
Klotho was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome
Transcriptional repression of KLOTHO in human cervical cancer To investigate epigenetic gene silencing of the secreted Frizzledrelated proteins (SFRPs) class of secreted Wnt antagonists in human cervical carcinoma, we first examined the mRNA levels of SFRP1, SFRP2, SFRP4, SFRP5, and KLOTHO in eight cervical cancer cell lines (Figure 1A)
To examine whether the transcriptional repression of KLOTHO occurs in primary cervical tumors, we performed RT-PCR for KLOTHO in 4 samples of lowgrade squamous intraepithelial lesion (LSIL), 6 samples of high-grade squamous intraepithelial lesion (HSIL), and 10 samples of invasive carcinoma (Figure 1B)
Summary
Klotho was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome. KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies. Abnormal activation of the canonical Wnt pathway due to epigenetic deregulation of Wnt antagonists is thought to play a crucial role in cervical tumorigenesis. We examined epigenetic silencing of KLOTHO in human cervical carcinoma. Previous studies have revealed that epigenetic gene silencing of soluble Wnt antagonists, such as secreted Frizzledrelated proteins (SFRPs), constitutes one of the major alterations resulting in constitutive activation of the canonical Wnt pathway in many tumors, colorectal [8], bladder [9], gastric [10], and breast cancer [11].
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