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Supplementary Figure Legends 1-5 from MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

Supplementary Methods and Materials from MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

Supplementary Figures 1-5 from MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

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

The therapeutic application of RNA interference (RNAi) for the knockdown of tumor-relevant genes critically relies on the efficient delivery of small interfering RNAs (siRNAs) or shRNA-encoding DNA plasmids in vivo. Previously, we have established polyethylenimine (PEI)/siRNA complexes as a highly efficient and ease-to-use system for gene targeting and a universally applicable platform for therapeutic RNAi. Here, we analyse the anti-tumor effects of the therapeutic knockdown of various members of the HER family, i.e. HER1 (c-erbB1, EGFR), HER2 (c-erbB2/neu) and HER3 (c-erbB3) in s.c. ovarian carcinoma xenografts in mice. While little effects are seen upon HER1 knockdown, the treatment of the mice with PEI/complexed siRNAs targeting HER2 or HER3 lead to a significant reduction in tumor growth. Notably, additive anti-tumor effects are observed upon double knockdown of HER2 and HER3. The biocompatibility of this PEI-based siRNA delivery platform for in vivo RNAi can be further increased by modifications of the polyethylenimines. To this end, we systematically analyse physicochemical and biological properties of DNA and siRNA complexes prepared from a set of maltose-, maltotriose- or maltoheptaose-modified hyperbranched PEIs (termed (oligo-)maltose-modified PEIs; OM-PEIs). While (oligo-)maltose grafting generally leads to reduced in vitro DNA transfection efficacies, this effect is less profound in OM-PEI/siRNA complexes for the induction of RNAi. This is particularly true for NH2-monosubstituted OM-PEIs. Independent of the maltose architecture, DNA and siRNA complexes based on maltose-grafted PEI show considerably lower toxicity as compared to PEI complexes. More importantly, pharmacokinetics in mice are markedly altered in OM-PEI/siRNA complexes. While their halflives in the blood circulation upon i.v. injection are generally increased as compared to PEI/siRNA complexes, the tissue distribution of radioactively labelled (OM-)PEI complexed siRNA molecules critically relies on the degree and pattern of (oligo-)maltose grafting. Marked differences in siRNA tissue distribution profiles are observed between different OM-PEIs. Enhanced tissue siRNA levels upon treatment of mice with OM-PEI/siRNA complexes as compared to PEI-based complexes are observed. Taken together, we (i) demonstrate the therapeutic anti-tumor effects of PEI/siRNA-mediated (double) knockdown of members of the HER family and (ii) establish OM-PEI complexes as novel promising tools for the in vivo application of siRNAs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 594.