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. 2018 Jan;17(1):200-208.
doi: 10.3892/mmr.2017.7853. Epub 2017 Oct 20.

Wnt/β‑catenin pathway activation and silencing of the APC gene in HPV‑positive human cervical cancer‑derived cells

Erick Ayala-Calvillo  1 , Luis Humberto Mojica-Vázquez  2 , Alejandro García-Carrancá  3 , Leticia González-Maya  1
Affiliations

Wnt/β‑catenin pathway activation and silencing of the APC gene in HPV‑positive human cervical cancer‑derived cells

Erick Ayala-Calvillo et al. Mol Med Rep. 2018 Jan.

Abstract

Although persistent infections with high‑risk human papilloma virus (HPV) constitute the most significant cofactor for the development of cervical cancer, they are insufficient on their own. Mutations or epigenetic inactivation of the tumor suppressor adenomatous polyposis coli (APC), the two acting as prominent oncogenic mechanisms in a number of types of cancer, are frequently associated with aberrant activation of the Wnt/β‑catenin pathway. According to these observations, it was hypothesized that APC alteration may lead to β‑catenin deregulation and the abnormal expression of direct targets of the Wnt pathway in HPV‑infected cervical cancer cells. The present study confirmed that the stabilization of β‑catenin correlates with enhanced transcriptional activity of the β‑catenin/T‑cell factor complex in cervical cancer cell lines. Sequence analysis of the 'hot‑spot' in the mutation cluster region did not exhibit genetic alterations that may be associated with APC gene inactivation. In addition, it was identified that there was a good correlation with the hypermethylation status of the APC promoter 1A and the abnormal accumulation of endogenous β‑catenin in cell lines and biopsies infected with HPV16, although not HPV18. Removal of the epigenetic markers led to an increase in APC levels and a reduction of β‑catenin expression in two transcriptional targets of the Wnt pathway: Matrix metalloproteinase‑7 and vascular endothelial growth factor. The present study suggested that the increase in Wnt activity in certain cervical cancer‑derived cells may be associated with an alteration in the methylation status of the APC gene promoter 1A.

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Figures

Figure 1.
Figure 1.
β-catenin expression and transcriptional activity in cervical cancer cell lines. (A) β-catenin protein was determinate by western blot analysis. An anti-β-actin antibody was used as the control for total protein loading. (B) Cell lines were transfected with TOPFLASH or FOPFLASH luciferase reporter constructs. Luciferase activity was determined in triplicate and the results are expressed as TOP/FOP correlation mean ± standard deviation for independent triplicate cultures. Significant induction of β-catenin activity (*P<0.05; **P<0.001) was calculated using an one-way analysis of variance and Tukey's multiple comparisons test. Top, TOPFLASH; Fop, FOPFLASH.
Figure 2.
Figure 2.
Sequence analysis of the APC gene. The APC mutation cluster regions from HeLa, CasKi, SiHa, C33A and SW480 cells were amplified by polymerase chain reaction and sequenced. The histogram from the codons 1335 to 1339 are shown as representative results of the sequence analysis with a mutation at codon 1338 (CAG/TAG) in the control SW480 cells. APC, adenomatous polyposis coli.
Figure 3.
Figure 3.
Hypermethylation analysis of APC gene promoter 1A. Methylation-specific PCR analysis were performed to determine APC promoter 1A methylation patterns in (A) cervical cancer cell lines and (B) biopsies. The DNA was treated with sodium bisulphite and PCR-amplified with primer pairs specific for unmethylated (108 bp) and methylated (98 bp) APC gene promoter 1A alleles. The PCR products were separated on an agarose gel. The HPV status of specimens is depicted as the specific HPV type present (16/18) or negative (−). The status of β-catenin is depicted as altered (+) or normal (−). KatoIII cells were included as positive control. APC, adenomatous polyposis coli; PCR, polymerase chain reaction; bp, base pair; HPV, human papilloma virus; U, unmethylated; M, methylated.
Figure 4.
Figure 4.
Analysis of APC gene expression in cervical cancer cell lines. (A) The expression of the APC gene in cancer cell lines was analyzed by RT-PCR. (B) The effect of the demethylating agents DAC (3 µM) and TSA (0.5 µM) on APC gene expression was measured by RT-PCR in cancer cell lines. Expression of β-globin mRNA was assessed in all samples as the control. The PCR products were separated on an agarose gel. APC, adenomatous polyposis coli; RT-PCR, reverse transcription-polymerase chain reaction; DAC, decitabine; TSA, trichostatin-A; bp, base pair.
Figure 5.
Figure 5.
β-catenin repression and Wnt pathway activity. (A) β-catenin protein levels were determined by western blot analysis in CaSki, KatoIII and HFF cells prior to and following treatment with DAC (3 µM) and TSA (0.5 Μm). An anti β-actin antibody was used as the control for total protein loading. (B) VEGF and MMP-7 mRNA levels were analyzed via RT-PCR in CaSki, KatoIII and HFF cells prior to and following treatment with DAC (3 µM) and TSA (0.5 µM). The expression of β-globin mRNA was assessed in all samples as the control. The PCR products were separated on an agarose gel. HFF, human foreskin fibroblasts; DAC, decitabine; TSA, trichostatin-A; VEGF, vascular endothelial growth factor; MMP-7, matrix metalloproteinase-7; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair.
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