Nanoparticle-complexed antimiRs for inhibiting tumor growth and metastasis in prostate carcinoma and melanoma

Abstract

Background: MiRNAs act as negative regulators of gene expression through target mRNA degradation or inhibition of its translation. In cancer, several miRNAs are upregulated and play crucial roles in tumorigenesis, making the inhibition of these oncomiRs an interesting therapeutic approach. This can be achieved by directly complementary single-stranded anti-miRNA oligonucleotides (antimiRs). A major bottleneck in antimiR therapy, however, is their efficient delivery. The nanoparticle formation with polyethylenimine (PEI) may be particularly promising, based on the PEI's ability to electrostatically interact with oligonucleotides. This leads to their protection and supports delivery. In the present study, we explore for the first time PEI for antimiR formulation and delivery. We use the branched low molecular weight PEI F25-LMW for the complexation of different antimiRs, and analyse tumor- and metastasis-inhibitory effects of PEI/antimiR complexes in different tumor models.

Results: In prostate carcinoma, transfection of antimiRs against miR-375 and miR-141 leads to tumor cell inhibition in 2D- and 3D-models. More importantly, an in vivo tumor therapy study in prostate carcinoma xenografts reveals anti-tumor effects of the PEI/antimiR complexes. In advanced melanoma and metastasis, we identify by a microRNA screen miR-150 as a particularly relevant oncomiR candidate, and validate this result in vitro and in vivo. Again, the systemic application of PEI/antimiR complexes inhibiting this miRNA, or the previously described antimiR-638, leads to profound tumor growth inhibition. These effects are associated with the upregulation of direct miRNA target genes. In a melanoma metastasis mouse model, anti-metastatic effects of PEI/antimiR treatment are observed as well.

Conclusions: We thus describe PEI-based complexes as efficient platform for antimiR therapy, as determined in two different tumor entities using in vivo models of tumor growth or metastasis. Our study also highlights the therapeutic relevance of miR-375, miR-141, miR-150 and miR-638 as target miRNAs for antimiR-mediated inhibition.

Keywords: Antimir; PEI; PEI/antimiR nanoparticles; Polyethylenimine; Therapeutic miRNA inhibition.

Fig. 1

AntimiR-375 and antimiR-141 reduce tumor growth of prostate cancer in vitro and in vivo. a LNCaP prostate cancer cells were cultured under standard culture conditions and exposed to antimiR-375 or antimiR-141. Cell proliferation was measured using the colorimetric WST-1 assay. b PC3 and DU145 prostate cancer cells were transfected with antimiR-375 or antimiR-141 and grown at low density and stained with Hematoxylin. c, d DU145 prostate cancer cells were transfected with antimiRs and analyzed for anchorage-dependent (c, on plastic) and anchorage-independent (d, in soft agar) colony formation capacity. e LNCaP, PC3 and DU145 prostate cancer cells were transfected with antimiRs as above and caspase activity was measured as a marker for apoptosis induction

Fig. 2

Nanoparticle complex stability and transfection rates. a For the determination of complexation efficacy by agarose gel electrophoresis, 0.5 µg antimiR were complexed with PEI at the different weight ratios. The complexes were mixed and separated on a 2% agarose gel pre-stained with Sybr™ Gold. b Complex stabilities were measured by heparin displacement assay. c PEI/antimiR complex sizes were determined by Dynamic Light Scattering (DLS). d Transmission electron microscopy (TEM) of PEI/antimiR complexes. e,f Uptake of PEI complexes containing Cy3-labeled antimiR was analyzed by flow cytometry and confocal microscopy

Fig. 3

Treatment of mouse prostate cancer xenografts with PEI/antimiR complexes. a For the generation of tumor xenografts in athymic nude mice (Foxn1nu) 5 × 10⁶ PC3 cells in 150 µL PBS were injected subcutaneously into both flanks of mice. The PEI/antimiR complexes injected intraperitoneally every 2–3 days at the time points indicated in the figure. Right half of the picture shows representative pictures of mice injected with scrambled RNA (control) or antimiRs. b Analysis of tumor lysates by Western blotting, for alterations in the expression levels of miR-375 target genes SEC23A and PHLPP1. Left: representative samples; right: quantitation of all Western blot samples

Fig. 4

miR-150 drives melanoma growth in vivo. a TaqMan^® low-density arrays were used to measure the expression of 667 human miRNAs in a set of melanocytes, primary melanomas and cutaneous melanoma metastases. Relative expression of indicated miRNAs is shown. The heatmap shows high expression in green and low expression in red. b Fold changes (FC) of miRNA expression is indicated when comparing melanocytes (ME), primary melanomas (PM), lymph node metastases (LNM) and distant cutaneous metastases (DCM), respectively. c Melanoma cells were transfected with miR-150 and seeded at low density. Colony formation was counted by light microscopy. d miR-150 was overexpressed in B16V mouse melanoma cells after transfection of the microRNA mimic, and tumor cells were injected into flanks of C57BL6N mice. Tumors were surgically removed after 14 days and weighted. e B16V melanoma cells were transfected using antimiRs against miR-150 or antimiR-control oligos. Mice were sacrificed after 14 days and surgically removed tumors were weighted. f LOX cells were transfected with antimiRs and analyzed for growth rates (here: in the interval 96 h–120 h after transfection)

Fig. 5

Treatment of local melanoma xenografts with antimiR/PEI complexes in mice. a For the generation of melanoma xenografts, 5 × 10⁶ LOX cells were injected subcutaneously into both flanks of athymic nude mice. The PEI/antimiR complexes were injected intraperitoneally every 2–3 days at the time points indicated in the figure. Tumor sizes were determined by measuring all three dimensions. b Representative pictures of mice. c Representative examples of immunofluorescence staining of melanoma xenografts with antibodies directed against the miR-150 target MDM4. The right panel shows the quantitation of immunofluorescence staining

Fig. 6

Treatment of distant melanoma metastases with antimiR/PEI complexes in mice. a,b For the analysis of anti-metastatic effects of PEI/antimiR-mediated inhibition, 5 × 10⁶ LOX melanoma cells were injected intravenously into the tail veins of NOD scid gamma mice. After 7 days, mice were randomized into specific treatment groups (antimiR-150, antimiR-638 and scrambled RNA). The PEI/antimiR complexes were injected intraperitoneally every 2 days. Lungs will be surgically removed after 4 weeks and lung metastasis was evaluated by counting of number of melanoma metastatic lesions in representative sections of each lung and the number of affected lungs. The total number of metastases per group and number of affected lung samples is given as bar graphs. c Representative examples of metastatic lesions in mouse lungs (arrows)