Knockdown of β-catenin with dicer-substrate siRNAs reduces liver tumor burden in vivo

Abstract

Despite progress in identifying molecular drivers of cancer, it has been difficult to translate this knowledge into new therapies, because many of the causal proteins cannot be inhibited by conventional small molecule therapeutics. RNA interference (RNAi), which uses small RNAs to inhibit gene expression, provides a promising alternative to reach traditionally undruggable protein targets by shutting off their expression at the messenger RNA (mRNA) level. Challenges for realizing the potential of RNAi have included identifying the appropriate genes to target and achieving sufficient knockdown in tumors. We have developed high-potency Dicer-substrate short-interfering RNAs (DsiRNAs) targeting β-catenin and delivered these in vivo using lipid nanoparticles, resulting in significant reduction of β-catenin expression in liver cancer models. Reduction of β-catenin strongly reduced tumor burden, alone or in combination with sorafenib and as effectively as DsiRNAs that target mitotic genes such as PLK1 and KIF11. β-catenin knockdown also strongly reduced the expression of β-catenin-regulated genes, including MYC, providing a potential mechanism for tumor inhibition. These results validate β-catenin as a target for liver cancer therapy and demonstrate the promise of RNAi in general and DsiRNAs in particular for reaching traditionally undruggable cancer targets.

Figure 1

Results of β-catenin Dicer-substrate short-interfering RNAs (DsiRNA) 1° screen. (a) Knockdown results for 368 Common DsiRNAs; (b) results for 72 human-Unique DsiRNAs. Red and blue symbols indicate results for two quantitative polymerase chain reaction assays. The 1° screen successfully identified active DsiRNAs, many of which knocked down β-catenin >90% at 1 nmol/l. mRNA, messenger RNA.

Figure 2

Optimization of β-catenin Dicer-substrate short-interfering RNAs (DsiRNAs). (a) Examples of 3° and 4° screening results. Each DsiRNA was tested both at 1 nmol/l (red bars) and at 0.1 nmol/l (blue bars) (mean + SEM). In the 3° screen, each DsiRNA sequence was tested with six different 2′-OMe patterns on the antisense strand. In the 4° screen, each DsiRNA sequence was then tested with one or two different antisense strand 2′-OMe patterns (identified from the 3° screen as optimal) combined with four different sense strand 2′-OMe patterns. Potent, 2′-OMe–modified DsiRNAs were identified. (b) Messenger RNA (mRNA) knockdown activity of β-catenin 25/27mer DsiRNAs, compared with activity of corresponding 21mer siRNAs (data for 21mers without 5′ phosphates is shown). Each duplex was tested at 0.1 nmol/l (left) and 1 nmol/l (right) (means ± SEM). Each 25/27mer DsiRNA (blue diamonds) was compared in activity with its corresponding 21mer (red squares); the siRNA pairs are ordered by rank activity of the DsiRNAs at 0.1 nmol/l. Many of the 25/27mer DsiRNAs showed higher knockdown activity than the corresponding 21mers.

Figure 3

Lack of immunostimulatory activity of 2′-OMe–modified Dicer-substrate short-interfering RNAs (DsiRNAs). Mice were dosed once intravenously, at 10 mg/kg, with β-catenin DsiRNAs in lipid nanoparticle (LNP) F30.1, a formulation designed to facilitate immunostimulation. Seven days postdose, serum was analyzed for an immune response, indicated by antibody to PEG. The heavily 2′-OMe–modified DsiRNA β-cat-253-M14/M35 did not show significant in vivo immunostimulation activity, compared with mice treated with phosphate-buffered saline (PBS) or with LNP F30.1 without DsiRNA (Empty Particle (EP), dashed line), in contrast with DsiRNA β-cat-3393-M0/M11 with lower 2′-OMe modification (**P < 0.01, relative to EP; mean ± SEM).

Figure 4

In vivo gene targeting efficacy of β-catenin Dicer-substrate short-interfering RNAs (DsiRNAs). (a) Inhibition of β-catenin and Axin2 messenger RNA (mRNA) expression in liver by β-catenin DsiRNAs. Mice were treated with β-cat-253-M14/M35, β-cat-900-M14/M12, or β-cat-3393-M14/M12, or the control DsiRNA HPRT1-716-M0/M26 to HPRT1 (5 mg/kg per dose, six doses over 2 weeks). Forty-eight hours after the last dose, mice were killed and liver mRNA levels were quantitated by quantitative polymerase chain reaction (qPCR). β-catenin was significantly knocked down by β-cat-900-M14/M12 and β-cat-3393-M14/M12 but not by human-specific β-cat-253-M14/M35. Corresponding with β-catenin knockdown, expression of Axin2, a β-catenin transcriptional target, was reduced (****P < 0.0001; *P < 0.05; means + SEM are shown). (b) Knockdown of β-catenin mRNA in Hep 3B orthotopic tumors, quantitated by qPCR. β-cat-253-M14/M35 was dosed at 5 mg/kg, in five doses over 15 days, starting ~2 weeks after Hep 3B cell implantation. Sorafenib was dosed at 10 mg/kg orally once a day × 14 days. Forty-eight hours after the last DsiRNA dose, mice were killed and tumors were isolated. β-catenin was knocked down by β-cat-253-M14/M35 (means ±SEM are shown; ****P < 0.0001). (c) Knockdown of β-catenin protein in tumors. Tumor samples from the experiment in Figure 5b were analyzed by immunblotting for β-catenin and for β-actin for normalization; 1 through 10 represent individual mice. (d) Knockdown of β-catenin mRNA in Hep 3B tumors, visualized by ViewRNA with hybridization probes specific for β-catenin (same experiment as Figure 4b,c; original magnification × 200; the phosphate-buffered saline (PBS) or β-catenin DsiRNA-treated mice from which these sections were analyzed also received sorafenib). ViewRNA analysis of the control housekeeping gene PPIB did not show reduced expression after β-catenin DsiRNA-LNP treatment. LNP, lipid nanoparticle.

Figure 5

In vivo tumor inhibition by β-catenin Dicer-substrate short-interfering RNAs (DsiRNAs). (a) Reduced orthotopic Hep 3B tumor weight in mice treated with β-cat-253-M14/M35 (5 mg/kg intravenously, six doses over 2 weeks) in combination with sorafenib (10 mg/kg orally (po), once a day (qd) × 14 days). Mice were killed 48 hours after the last DsiRNA dose, and tumors were dissected from the liver and weighed. Means ± SEM are shown; statistics (one-way ANOVA with Bonferroni posttest) were calculated relative to phosphate-buffered saline (PBS) + sorafenib. β-catenin DsiRNA administration reduced Hep 3B tumors (**P < 0.01), as did the DsiRNAs KSP-2455-M14/M3 and PLK1/H-p1497-M108/M85 to the genes KSP1 and PLK1. (b) Reduction of Hep 3B tumor weight by β-catenin DsiRNA treatment. DsiRNAs in lipid nanoparticle (LNP) 2072 were dosed at 5 mg/kg, in five doses over 15 days, starting ~2 weeks after implantation. Sorafenib was dosed at 10 mg/kg po qd × 14 days. Tumors were significantly reduced by β-cat-253-M14/M35 treatment, both alone and in combination with sorafenib (means ± SEM).

Figure 6

Reduction of Hep 3B tumor burden and target gene expression by multiple β-catenin Dicer-substrate short-interfering RNAs (DsiRNAs). (a) Reduction of Hep 3B tumor weight. Intravenous administration of β-cat-253-M14/M35 or β-cat-900-M14/M12 DsiRNAs in lipid nanoparticle (LNP) 2072 (2 or 5 mg/kg three times a week × 2 weeks) began ~2 weeks after Hep 3B cells were orthotopically implanted. Forty-eight hours after the last dose, mice were killed, and tumors were weighed. Means ±SEM are shown; statistics were calculated using one-way ANOVA and Bonferroni posttest analysis. P < 0.0001 for all groups compared with phosphate-buffered saline (PBS). (b) Knockdown of β-catenin messenger RNA (mRNA) and reduced expression of downstream genes Axin2 and MYC in tumors by β-catenin DsiRNAs (means + SEM).

Figure 7

Reduction of Hep G2 tumor burden by β-catenin Dicer-substrate short-interfering RNA (DsiRNA) treatment. Intravenous administration of β-cat-253-M14/M35 or the control DsiRNA HPRT1-716-M21/M36 to HPRT1 in LNP2072 (5 mg/kg three times a week × 2 weeks, seven doses total) began ~2 weeks after Hep G2 cells were implanted. Forty-eight hours after the last dose, animals were killed, and tumors were weighed. Means ± SEM are shown; statistics were calculated using one-way ANOVA and Bonferroni posttest analysis. β-cat-253-M14/M35, but not an HPRT1 DsiRNA, significantly reduced Hep G2 tumor weight.