TLR9 Agonists Oppositely Modulate DNA Repair Genes in Tumor versus Immune Cells and Enhance Chemotherapy Effects

Department of Human Morphology and Biomedical Sciences Città Studi, Università degli Studi di Milano, Italy.
Cancer Research (Impact Factor: 9.33). 08/2011; 71(20):6382-90. DOI: 10.1158/0008-5472.CAN-11-1285
Source: PubMed


Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.

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Available from: Michele Sommariva, May 15, 2014
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    • "Previous studies have showed that CpG-ODN can mediate anti-tumor effects on cancer cells due to its direct or indirect effects by inducing release of cytokines, and enhancing immune response [11]. Meanwhile, recent studies suggested that CpG-ODN may be considered as a potential chemosensitizer with weak side effects, such CpG-ODN 1826 [12,13] Clinical studies have also documented that CpG-ODN in combination with chemotherapy cannot only increase the treatment benefit of patients ,but also make patients with well tolerated [14,15]. Although a previous study had demonstrated the benefits of CpG-ODN for HCC treatment [16], the direct cytotoxicity of CpG-ODN against HCC cells and the potential mechanism are not clear. "
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    ABSTRACT: Recent studies indicated that a synthetic oligonucleotide containing un-methylated CpG oligodeoxynucleotides(CpG-ODN)has a potential function for cancer therapy. In this study, we evaluated the chemosensitizing effects of CpG-ODN in 5-fluorouracil (5-FU)-treated HepG2 human hepatoma cells. Cell viability assay were utilized to evaluate the direct cytotoxicity of CpG-ODN in the presence or absence of 5-FU in HepG2 cells, and apoptosis as well as cell-cycle was examined by flow cytometry analysis. The mRNA expression of Bcl-2, Livin and Survivin within HepG2 cells treated with CpG-ODN and/or 5-FU were analyzed by Real Time PCR assay in vitro. CpG-ODN in combination with 5-FU could decrease cell viability, increase apoptosis and further induce HepG2 cells cycle arrest at S phase when compared with CpG-ODN or 5-FU. CpG-ODN or 5-FU could down-regulate the mRNA expression of Bcl-2 within HepG2 cells. The mRNA expression of Livin and Survivin decreased in cells treated with CpG-ODN alone but increased in cells treated with 5-FU alone. However, CpG-ODN in combination with 5-FU could down-regulate the mRNA expression of Livin and Survivin within HepG2 cells. Our finding demonstrated that CpG-ODN enhanced the chemosentivity of 5-FU in HepG2 human hepatoma cells at least in part by down-regulating the expression of Livin and Survivin, leading to apoptosis and further inducing cell cycle arrest at S phase. Therefore, CpG-ODN may be a potential candidate as chemosensitizer for human hepatocellular carcinoma.
    Cancer Cell International 10/2013; 13(1):106. DOI:10.1186/1475-2867-13-106 · 2.77 Impact Factor
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    • "Many clinical studies have shown that the intra-tumoral presence of CD8+ cells, NK cells, CD4+ cells, and dendritic cells (DC) is positively correlated with survival, while the presence of macrophages and regulatory T cells predict poor responsiveness to therapy and survival [3], [4], [5]. There is increased interest in modulation of immune cells infiltrating the tumor microenvironment to enhance the therapeutic efficacy of radiation [6], [7].Patients received vaccine before the standard chemotherapy/radiotherapy to achieve a better result has successfully reported on prostate and head and neck cancer [8], [9], [10]. There is evidence that immune-mediated microenvironmental change has occurred during tumor progression and after therapy. "
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    ABSTRACT: The tumor microenvironment is a key determinant for radio-responsiveness. Immune cells play an important role in shaping tumor microenvironments; however, there is limited understanding of how natural killer (NK) cells can enhance radiation effects. This study aimed to assess the mechanism of reciprocal complementation of radiation and NK cells on tumor killing. Various tumor cell lines were co-cultured with human primary NK cells or NK cell line (NK-92) for short periods and then exposed to irradiation. Cell proliferation, apoptosis and transwell assays were performed to assess apoptotic efficacy and cell viability. Western blot analysis and immunoprecipitation methods were used to determine XIAP (X-linked inhibitor of apoptosis protein) and Smac (second mitochondria-derived activator of caspase) expression and interaction in tumor cells. Co-culture did not induce apoptosis in tumor cells, but a time- and dose-dependent enhancing effect was found when co-cultured cells were irradiated. A key role for caspase activation via perforin/granzyme B (Grz B) after cell-cell contact was determined, as the primary radiation enhancing effect. The efficacy of NK cell killing was attenuated by upregulation of XIAP to bind caspase-3 in tumor cells to escape apoptosis. Knockdown of XIAP effectively potentiated NK cell-mediated apoptosis. Radiation induced Smac released from mitochondria and neutralized XIAP and therefore increased the NK killing. Our findings suggest NK cells in tumor microenvironment have direct radiosensitization effect through Grz B injection while radiation enhances NK cytotoxicity through triggering Smac release.
    PLoS ONE 04/2013; 8(4):e61797. DOI:10.1371/journal.pone.0061797 · 3.23 Impact Factor
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    • "RNA extracted from omentum-adherent tumors of human IGROV-1 ovarian carcinoma-bearing mice treated i.p. with CpG-ODN or saline (control group) as described [3] was analyzed for miRNA expression using Illumina human miRNA_v2 array. Out of 1145 miRNAs represented on the Illumina chips, 567 mature miRNAs annotated on miRBase12.0, "
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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.
    PLoS ONE 03/2013; 8(3-3):e58849. DOI:10.1371/journal.pone.0058849 · 3.23 Impact Factor
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