A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer

Abstract

Targeted cancer therapies, although often effective, have limited utility owing to preexisting primary or acquired secondary resistance. Consequently, agents are sometimes used in combination to simultaneously affect multiple targets. MicroRNA mimics are excellent therapeutic candidates because of their ability to repress multiple oncogenic pathways at once. Here we treated the aggressive Kras;p53 non-small cell lung cancer mouse model and demonstrated efficacy with a combination of two tumor-suppressive microRNAs (miRNAs). Systemic nanodelivery of miR-34 and let-7 suppressed tumor growth leading to survival advantage. This combinatorial miRNA therapeutic approach engages numerous components of tumor cell-addictive pathways and highlights the ability to deliver multiple miRNAs in a safe and effective manner to target lung tissue.

Figure 1

miR-34a and let-7b reduce tumor cell proliferation and invasiveness in a synergistic manner. (A–C) Cells were transfected with miRNA mimics: control, let-7b, miR-34a, or half the dose of each. (A) Sulphorhodamine B (SRB) assays were performed 7 days post transfection. Data represent 5 transfections for each treatment. Error bars depict standard deviation. (B) Change in SRB stained cells from day four to five post transfection is shown as a proxy for change in proliferation, n=3 for each treatment. Error = standard deviation (C) Dose-response curves were created over a wide range of miRNA concentrations (3 – 100nM). Effect represents decreased proliferation. ▲ let-7b and miR-34a, ■ miR-34a,● let-7b (D) Invading H23 cells were stained 24 hours after seeding. Three fields of view were photographed and the average number of invading cells per field is graphically represented. (E) Human lung cancer cell lines, A549, H441, and H23 were transfected with 25 nM of miRNA mimics: control (c), let-7b (l7), miR-34a (34), or half the dose of both let-7b and miR-34a (d) or left untransfected (u). Forty-eight hours following transfection, protein lysates were prepared, resolved, transferred to PVDF, and evaluated for relevant let-7b and mir-34a target genes. Actin serves as a loading control. ND – not detected. Densitometry measurements relative to untransfected cells are shown following normalization to actin. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, unless otherwise indicated, based on one-way analysis of variance (ANOVA) with Tukey’s post hoc correction.

Figure 2

Systemic delivery of miRNAs encapsulated in neutral lipid emulsion (NLE) slows tumor growth. (A) Schematic of dosing schedule. (B) Animals were sacrificed 24 or 48 hours after the final injection; lungs were perfused, harvested, and processed for hematoxylin and eosin staining (H & E). Bars = 1mm (C) Tumor nodules were counted from three sections obtained from the left lobe of each animal and are shown as average nodules/section/left lobe. (D) Overall tumor burden represents total tumor area averaged from three sections obtained from each treated animal relative to the total area of the lung. (baseline: n=3, NLE: n=3, let-7b: n=5*, miR-34a: n=4, let-7b and miR-34a: n=5) (* Note: two of the let-7b sections were not included in the nodule count since the sections obtained were predominantly one large nodule.) * p-value < 0.05, unless otherwise indicated, based on one-way analysis of variance (ANOVA) with Tukey’s post hoc correction. ns = not significant.

Figure 3

Systemically delivered miRNAs accumulate in whole blood and lung tissue, and do not elevate serum cytokines. Serum, whole blood, and lung tissue was harvested 24 or 48 hours following the final injection. (A–C) Serum was evaluated for relevant cytokines: TNF-α (A), IL-6 (B) and IL-1β (C). Serum from animals treated with LPS was included as a positive control (n=3). (D–G) RNA was extracted from lung tissue (D and F) or whole blood (E and G) and evaluated for accumulation of miR-34a (D and E) or and let-7b (F and G) by RT-qPCR. Data are shown as copies per 1 ng of total RNA. (baseline: n=7, control: n=9, let-7b: n=9, miR-34a: n=8, let-7b and miR-34a: n=8) † Statistically significant from miR-NC treated based on one-way ANOVA of log-transformed data followed by a Dunnett’s post hoc test (multiple comparison test).

Figure 4

Reduction in tumor size following systemic delivery of combinatorial miRNAs encapsulated in a clinically relevant NOV340 delivery agent. (A) Schematic of dosing schedule. (B) Representative H & E stain of the left lobe of animals from each treatment group (histology from every animal is shown in Supplementary Fig. 3). Bars = 1 mm (C) Quantification of tumor area relative to total lung area. (D) The volumes of individual tumors were recorded and averaged for each treatment group. Each bar represents over 280 tumors from each treatment group. Standard error of the mean is depicted. (E) Images of KI-67 staining from representative sections for each treatment group. (F) Proliferation index calculated as KI-67 positive cells/mm². (C, D, F) * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 based on one-way analysis of variance (ANOVA) with Tukey’s post hoc correction. ns = not significant. (G) Survival data of Kras^G12D/+; p53^flx/flx treated mice (arrows indicate injection days). Survival p-values = 0.07 for control vs. let-7b and miR-34 combination, and 0.05 for control vs. miR-34a following log-rank (Mantel-Cox) test.