MicroRNAs (miRNA) aberrantly expressed in tumors may offer novel therapeutic approaches to treatment. miR-145 is downregulated in various cancers including colon carcinoma in which in vitro studies have established proapoptotic and antiproliferative roles. miR-33a was connected recently to cancer through its capacity to downregulate the oncogenic kinase Pim-1. To date, miRNA replacement therapy has been hampered by the lack of robust nonviral delivery methods for in vivo administration. Here we report a method of miRNA delivery by using polyethylenimine (PEI)-mediated delivery of unmodified miRNAs, using miR-145 and miR-33a to preclinically validate the method in a mouse model of colon carcinoma. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules were delivered into mouse xenograft tumors, where they caused profound antitumor effects. miR-145 delivery reduced tumor proliferation and increased apoptosis, with concomitant repression of c-Myc and ERK5 as novel regulatory target of miR-145. Similarly, systemic injection of PEI-complexed miR-33a was validated as a novel therapeutic targeting method for Pim-1, with antitumor effects comparable with PEI/siRNA-mediated direct in vivo knockdown of Pim-1 in the model. Our findings show that chemically unmodified miRNAs complexed with PEI can be used in an efficient and biocompatible strategy of miRNA replacement therapy, as illustrated by efficacious delivery of PEI/miR-145 and PEI/miR-33a complexes in colon carcinoma. Cancer Res; 71(15); 5214-24. Ó2011 AACR.

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3 0 -untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.

We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signaling pathways and establish the functional relevance of Pim-1 in colon carcinoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric polyethylenimine/small interfering RNA nanoparticles as a promising therapeutic approach.

Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12–15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability.

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Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17-5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3'-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications.

Skull base chordomas are challenging tumors due to their deep surgical location and resistance to conventional radiotherapy. Chemotherapy plays a marginal role in the treatment of chordoma resulting from lack of preclinical models due to the difficulty in establishing tumor cell lines and valuable in vivo models. Here, we established a cell line from a recurrent clival chordoma. Cells were cultured for more than 30 passages and the expression of the chordoma cell marker brachyury was monitored using both immunohistochemistry and Western blot. Sensitivity of chordoma cells to the inhibition of specific signaling pathways was assessed through testing of a commercially available small molecule kinase inhibitor library. In vivo tumorigenicity was evaluated by grafting chordoma cells onto immunocompromised mice and established tumor xenografts were treated with rapamycin. Rapamycin was administered to the donor patient and its efficacy was assessed on follow-up neuroimaging. Chordoma cells maintained brachyury expression at late passages and generated xenografts closely mimicking the histology and phenotype of the parental tumor. Rapamycin was identified as an inhibitor of chordoma cell proliferation. Molecular analyses on tumor cells showed activation of the mammalian target of rapamycin signaling pathway and mutation of KRAS gene. Rapamycin was also effective in reducing the growth of chordoma xenografts. On the basis of these results, our patient received rapamycin therapy with about six-fold reduction of the tumor growth rate upon 10-month follow-up neuroimaging. This is the first case of chordoma in whom chemotherapy was tailored on the basis of the sensitivity of patient-derived tumor cells.

Liver cancer is the fourth leading cause of cancer-related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2-specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST-1 assays and spheroid assays), 3D-colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT-qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh-7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)-complexed siRNA. The knockdown of PIM2 resulted in potent anti-proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh-7 cells. In vivo, the therapeutic nanoparticle-mediated delivery of PIM2 siRNA led to profound anti-tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.