Two ways of epigenetic silencing of TFPI2 in cervical cancer.

Alexandra Fullár,Katalin Karászi,Péter Hollósi,Lászlóné Oláh,Gábor Lendvai,Gábor Sobel,József Dudás,Andrea Reszegi,Ilona Kovalszky,Zoltán Papp,Arun Rishi

doi:10.1371/journal.pone.0234873

Abstract

ObjectiveComparison of human mRNA microarray results from tumor-associated and normal cervical fibroblasts revealed significant TFPI2 downregulation in tumor-associated fibroblasts isolated from cervical cancer, indicating that TFPI2 downregulation may play an important role in the pathogenesis of the disease. In the present work, we investigated the mechanism of TFPI2 downregulation in tumor-associated fibroblasts and tumor cells.MethodsIn vitro models of monocultures and co-cultures were established with tumor cells and fibroblasts to explore the changes of TFPI-2 expression and epigenetic modifications of the TFPI2 gene.ResultsThe TFPI2 gene was hypermethylated only in tumor cells. Reduction of TFPI-2 protein levels in tumor-associated fibroblasts, although the gene was not methylated, suggested alternative regulatory mechanisms of gene expression, such as inhibition by microRNAs. The expression pattern of miR-23a, a gene thought to inhibit TFPI2 translation, showed changes strongly correlated to detected TFPI-2 protein alterations. Transfections with miR-23a mimics resulted in a decrease of TFPI-2 protein expression whereas miR-23a inhibitors increased the TFPI-2 amount. Due to downregulation of miR-23a expression by HPV in cancer cells, TFPI2 was silenced by promoter methylation. In contrary, miR-23a was active in HPV-free fibroblasts and inactivated TFPI2.ConclusionThese results indicate dual epigenetic inhibition of TFPI2 on the transcription level by promoter methylation in cancer cells and on the translation level by miR-23a in tumor-associated fibroblasts. As a consequence, inactivation of the TFPI2 gene plays a strategic role in the progression of cervical cancer.

Highlights

High-risk human papilloma virus (HPV) infection is responsible for the development of cervical cancer in 99.7% of cases studied [1]
Due to downregulation of miR-23a expression by HPV in cancer cells, tissue factor pathway inhibitor-2 (TFPI2) was silenced by promoter methylation
The remaining authors have no competing interests to disclose. These results indicate dual epigenetic inhibition of TFPI2 on the transcription level by promoter methylation in cancer cells and on the translation level by miR-23a in tumor-associated fibroblasts

Summary

Introduction

High-risk human papilloma virus (HPV) infection is responsible for the development of cervical cancer in 99.7% of cases studied [1]. The majority of women infected with HPV do not develop cervical cancer, suggesting that additional environmental factors are required for tumor development. The question addressed in this work is, whether the microenvironment could be involved in this process. Earlier we reported that the cervical connective tissue in HPV-mediated carcinogenesis plays a major role in the transformation process [2]. In the present work we demonstrate that during tumorigenesis normal stromal fibroblasts can transform into a tumorassociated phenotype. The transformed cells support the formation and subsequent invasion of cancer [3]

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Results

Conclusion