ANTI-TUMOR ACTIVITY OF CPG-ODN IN OVARIAN XENOGRAFT TUMORS

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN), Toll-like receptor 9 (TLR9) agonists, are able to induce innate/adaptive immune responses and can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. It was recently reported that peritumoral CpG-ODN treatment in preclinical models of ovarian cancer, activating TLR-9 expressing cells in tumor microenvironment, induces modulation of DNA repair genes and sensitizes cancer cells to DNA-damaging Cisplatin treatment. In this thesis 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 (16 down- and 4 up-regulated) in human IGROV-1 ovarian tumor cells from CpG-ODN-treated mice versus controls. Evaluation of the role of the 3 most differentially expressed miRNAs on sensitivity to Cisplatin of IGROV-1 cells revealed significant 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). The impact of expression levels of all 20 differentially expressed miRNAs were associated with time to replase and overall survival probability in two data sets of ovarian cancer patients treated with platinum. It was found that hsa-miR-302b expression was significantly associated with time to relapse or overall survival in these 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. In the second part of this thesis we tested the efficacy of CpG-ODN in combination with other possible therapeutic agents in ovarian carcinoma ascites-bearing athymic mice, to mimic clinical treatment situations in advanced human ovarian disease. Mice injected i.p. with IGROV-1 ovarian cancer cells were treated at different stages of ascites progression for 4 weeks with CpG-ODN alone or in combination with Bevacizumab, Polyinosinic:Polycytidylic acid (Poly(I):Poly(C)), Gefitinib, Cetuximab and Cisplatin. In mice treated when ascitic fluid began to accumulate, CpG-ODN combined with Bevacizumab, Poly(I): Poly(C) or Gefitinib did not significantly increase Median Survival Times (MST), as compared with that using CpG-ODN alone, whereas MST in mice treated with CpG-ODN plus Cetuximab was significantly increased (>103 days for combination vs 62 days for CpG alone; P = 0.0008), with 4/8 mice alive at the end of the experiment. In mice showing evident and established ascites, evaluated with increase of abdominal volume and body weight (27.9 ± 0.8 g after vs 23 ± 1.1 g before tumor cell injection), treatment with Cisplatin in addition to CpG-ODN/Cetuximab led to significantly increased MST (105.5 days; P = 0.001), with all mice still alive at 85 days, over that using CpG ODN/Cetuximab (66 days), Cetuximab/Cisplatin (18.5 days), Cisplatin (23 days) or saline (16 days). At a very advanced stage of disease (body weight: 31.4 ± 0.9 g), when more than half of control mice had to be sacrificed 6 days after starting treatments, the triple-combination therapy still increased MST (45 days; P = 0.0089) vs controls. These data indicated that CpG-ODN combination therapies that enhance the immune response in the tumor microenvironment and concomitantly target tumor cells are highly efficacious even in experimental advanced malignancies. Although differences in the distribution of TLR9 in mice and humans and the enrichment of this receptor on innate immune cells of athymic mice must be considered, our results indicate a promising strategy to treat ovarian cancer patients with bulky ascites.