Increased Sensitivity to Chemotherapy Induced by CpG-ODN Treatment Is Mediated by microRNA Modulation

Loris De Cecco,Martina Berardi,Alessandra Cataldo,Michele Sommariva,Delia Mezzanzanica,Silvana Canevari,Andrea Balsari,Marilena V Iorio,Elda Tagliabue,Ramiro Garzon

doi:10.1371/journal.pone.0058849

Loris De Cecco, Martina Berardi + Show 8 more

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https://doi.org/10.1371/journal.pone.0058849

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Abstract

We recently reported that peritumoral CpG-ODN treatment, activating TLR-9 expressing cells in tumor microenvironment, induces modulation of genes involved in DNA repair and sensitizes cancer cells to DNA-damaging cisplatin treatment. Here, we investigated whether this treatment induces modulation of miRNAs in tumor cells and their relevance to chemotherapy response. Array analysis identified 20 differentially expressed miRNAs in human IGROV-1 ovarian tumor cells from CpG-ODN-treated mice versus controls (16 down- and 4 up-regulated). Evaluation of the role of the 3 most differentially expressed miRNAs on sensitivity to cisplatin of IGROV-1 cells revealed significantly increased cisplatin cytotoxicity upon ectopic expression of hsa-miR-302b (up-modulated in our array), but no increased effect upon reduced expression of hsa-miR-424 or hsa-miR-340 (down-modulated in our array). Accordingly, hsa-miR-302b expression was significantly associated with time to relapse or overall survival in two data sets of platinum-treated ovarian cancer patients. Use of bio-informatics tools identified 19 mRNAs potentially targeted by hsa-miR-302b, including HDAC4 gene, which has been reported to mediate cisplatin sensitivity in ovarian cancer. Both HDAC4 mRNA and protein levels were significantly reduced in IGROV-1 cells overexpressing hsa-miR-302b. Altogether, these findings indicate that hsa-miR-302b acts as a “chemosensitizer” in human ovarian carcinoma cells and may represent a biomarker able to predict response to cisplatin treatment. Moreover, the identification of miRNAs that improve sensitivity to chemotherapy provides the experimental underpinning for their possible future clinical use.

Highlights

Oligodeoxynucleotides (ODN) containing dinucleotides with unmethylated CpG motifs (CpG-ODN) are potent activators of both the innate and adaptive immune systems [1;2]
In a xenograft model of human IGROV-1 ovarian cancer, we recently showed that treatment with CpG-ODN induced down-modulation of DNA repair genes in tumor cells and that peritumoral injection of CpGODN in the peritoneal cavity was for inducing this downmodulation [3] and for the antitumor activity of CpG-ODN [4]
RNA extracted from omentum-adherent tumors of human IGROV-1 ovarian carcinoma-bearing mice treated i.p. with CpGODN or saline as described [3] was analyzed for miRNA expression using Illumina human miRNA_v2 array

Summary

Introduction

Oligodeoxynucleotides (ODN) containing dinucleotides with unmethylated CpG motifs (CpG-ODN) are potent activators of both the innate and adaptive immune systems [1;2]. In a xenograft model of human IGROV-1 ovarian cancer, we recently showed that treatment with CpG-ODN induced down-modulation of DNA repair genes in tumor cells and that peritumoral injection of CpGODN in the peritoneal cavity was for inducing this downmodulation [3] and for the antitumor activity of CpG-ODN [4]. Considering the CpG-ODN species specificity and to the lack of TLR9 expression on IGROV1 cells, the effect cannot be mediated by a direct interaction between the oligonucleotide and tumor cells, instead it is likely that peritumoral TLR9-expressing cells, such as innate immune cells and/or endothelial cells, fibroblasts and epithelial cells, directly respond to CpG-ODN and downregulate DNA repair in tumor cells through a direct cell-cell interaction and/or by secreting soluble factors. Several experimental and clinical findings have implicated miRNAs in the response to chemotherapy [13], demonstrating a role for miRNAs in the modulation of genes involved in DNA repair [14;15]

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