Abstract
Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2′-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2′-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.
Highlights
Insulin-like growth factor type I receptor (IGF-IR) signaling has a significant impact on development of many normal tissues, and on growth of malignant tumors [1]
KSG, DYQ, NNE, ADT and CMV short interfering RNAs (siRNAs) inhibited IGF-IR mRNA expression by approximately 70% compared to untreated cells and to cells transfected with control siRNAs (Figure 1A)
Dose-response experiments performed with ADT siRNA in EMT6 cells showed that 50 nM of ADT decreased IGF-IR mRNA by 83% compared to levels in untreated cells (Figure 1B)
Summary
Insulin-like growth factor type I receptor (IGF-IR) signaling has a significant impact on development of many normal tissues, and on growth of malignant tumors [1]. Epidemiological studies showed that increased serum concentration of insulin-like growth factor I (IGF-I) is associated with increased risk of developing tumors including those of the breast [2]. Other approaches using nucleic-acid based strategies have been used to investigate the IGF-IR/IGF-I pathway, including antisense oligonucleotides, antisense RNA expression plasmids, ribozymes, triplex-forming oligonucleotides and short interfering RNAs (siRNAs) [5,6,7,8,9,10]. Nucleic-acid based approaches are theoretically specific and selective, they may have the undesirable effect of silencing non-targeted mRNAs, more in the case of siRNAs and phosphorothioate antisense oligonucleotides [11]
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