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MicroRNAs control the expression of various genes, and several miRNAs are aberrantly expressed in tumors. MiRNA-145 is markedly downregulated in various cancers including colon carcinoma, and in vitro studies have established its pro-apoptotic and anti-proliferative role. MiR-33a has only recently been connected to cancer through its capacity to downregulate the oncogenic kinase Pim-1. Despite the functional relevance of miRNAs suppressed in tumors, only a few studies have explored their therapeutic application. This miRNA replacement therapy is largely hampered by the lack of powerful non-viral delivery tools for their in vivo administration. In this paper, we establish, for the first time, miRNA replacement therapy by polyethylenimine (PEI)-mediated delivery of unmodified miRNAs. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules are delivered into tumors, resulting in profound anti-tumor effects in s.c. colon carcinoma xenograft mouse models. Tumor inhibition is based on miR-145-mediated reduced proliferation, increased apoptosis and the concomitant repression of c-Myc and ERK5, which we identify for the first time as being regulated by miR-145. Furthermore, we introduce the systemic injection of PEI-complexed miR-33a as a novel therapeutic principle aiming at the in vivo downregulation of the oncogenic kinase Pim-1. Anti-tumor effects of the treatment with PEI/miRNA complexes are comparable to PEI/siRNA-mediated Pim-1 knockdown in vivo. Taken together, this is the first study to establish chemically unmodified miRNAs, complexed with PEI, as an efficient and biocompatible strategy for miRNA replacement therapy, and to introduce PEI/miR-145 and PEI/miR-33a complexes as anti-tumor compounds in colon carcinoma.

Various miRNAs are aberrantly expressed in cancer. In colon carcinoma, decreased levels of the pro-apoptotic and anti-proliferative miRNA-145 are observed, while the role of miR-33a has not been analysed yet. In this study, we demonstrate the tumor-inhibitory role of miR-33a and newly identify the proto-oncogenic kinase Pim-1 as its direct target. Comparably to siRNA-mediated knockdown of Pim-1, miR-33a reduces Pim-1 expression and thus inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression. Most recent in vitro and in vivo data from our group further establish the so far unknown functional relevance of Pim-1 in colon carcinoma and identify Pim-1 as attractive target gene. Among others, RNAi-mediated Pim-1 knockdown reduces tumor growth, induces apoptosis and leads to major changes in oncogenic signal transduction, including the inhibition of STAT3. The therapeutic application of miRNAs strongly relies on the development of suitable delivery tools, and we introduce polyethylenimine (PEI)-based nanoparticles for systemic or local miRNA administration in vivo. PEIs mediate miRNA protection, delivery to target organs, cellular uptake and intracellular release. MiRNA replacement therapy through systemic or local injection of PEI/miR-145 complexes results in efficient miRNA delivery and in antitumor effects in s.c. colon carcinoma xenograft mouse models. Likewise, tumor growth inhibition is observed upon treatment with PEI-complexed miR-33a. This is due to the miR-33a-mediated downregulation of Pim-1 expression, comparable with the Pim-1 knockdown through PEI/siRNA complexes. Conclusions: (i) The PEI-complexation of miRNAs represents a novel strategy in miRNA replacement therapy, (ii) miR-145 / miR-33a may be promising candidate miRNAs, and (iii) Pim-1 is a newly identified, attractive target gene in colon carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-482. doi:1538-7445.AM2012-LB-482