Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo

Abstract

Intracellular delivery and endosomal escape of functional small interfering RNAs (siRNAs) remain major barriers limiting the clinical translation of RNA interference (RNAi)-based therapeutics. Recently, we demonstrated that a cell-penetrating endosome-disruptive peptide we synthesized, termed 599, enhanced the intracellular delivery and bioavailability of siRNAs designed to target the CIP2A oncoprotein (siCIP2A) into oral cancer cells and consequently inhibited oral cancer cell invasiveness and anchorage-independent growth in vitro. Thus, to further assess the therapeutic potential of the 599 peptide in mediating RNAi-based therapeutics for oral cancer and its prospective applicability in clinical settings, the objective of the current study was to determine whether intratumoral dosing of the 599 peptide-siCIP2A complex could induce silencing of CIP2A and consequently impair tumor growth using a xenograft oral cancer mouse model. Our results demonstrate that the 599 peptide is able to protect siRNAs from degradation by serum and ribonucleases in vitro and upon intratumoral injection in vivo, confirming the stability of the 599 peptide-siRNA complex and its potential for therapeutic utility. Moreover, 599 peptide-mediated delivery of siCIP2A to tumor tissue induces CIP2A silencing without any associated toxicity, consequently resulting in reduction of the mitotic index and significant inhibition of tumor growth. Together, these data suggest that the 599 peptide carrier is a clinically effective mediator of RNAi-based cancer therapeutics.

Keywords: CIP2A; Cell-penetrating peptide; Fusogenic peptide; Oral cancer; RNAi; siRNA delivery.

Figure 1

The 599 peptide protects siRNAs from RNase A and serum ribonuclease-mediated degradation in vitro. Naked and 599 peptide-complexed siRNAs targeting the CIP2A oncogene (siCIP2A) at a 50:1 (599:siCIP2A) molar ratio were incubated in the absence or presence of either (A) RNase A or (B) sera for 1 hour at 37°C. The position of the intact siRNA is indicated. Fetal bovine serum (FBS); human serum (HS).

Figure 2

The 599 peptide protects siRNAs from degradation in vivo. (A) Representative in vivo images of mice 1 to 72 hours post-treatment with Cy5.5-fluorescent-labeled siCIP2A (Cy5.5-siCIP2A) alone or in complex with the 599 peptide (599+Cy5.5-siCIP2A). Ex vivo images of the tumors and internal organs dissected from the respective mice 72 hours post-treatment are also shown. (B) Quantitative analysis of siRNA fluorescence intensity over time in tumor tissues treated with either naked Cy5.5-siCIP2A or 599+Cy5.5-siCIP2A. Data are mean ± SEM of three independent samples, where **P<0.01 and ***P<0.001 compared to 599+Cy5.5-siCIP2A treated tumors (Student’s t test). Black and white image (B/W); site of tumor (arrow); heart (H); brain (B); lung (Lu); kidney (K); liver (Li), spleen (S); tumor (T); arbitrary units (A.U.).

Figure 3

The 599 peptide mediates delivery of bioactive siRNAs into xenograft mouse tumors following a single-dose treatment. (A) Fluorescence microscopy analysis of DY547-conjugated siCIP2A (D-siCIP2A; red) uptake into tumor tissues 48 hours post-treatment with or without complexation to the 599 peptide. Nuclei (blue) were counterstained with DAPI. Scale bar: 50 μm. (B) Western blot analysis of CIP2A protein expression levels in excised xenograft tumor tissues 48 hours post-treatment with 599 peptide complexed to D-siCIP2A (599+D-siCIP2A), compared to tissues treated with D-siCIP2A alone or 599 peptide complexed to a control non-targeting siRNA (599+siNT). The CIP2A protein levels in untreated tumor tissues were also analyzed for comparison. Tissue samples from two mice (M1 and M2) per treatment group were analyzed. GAPDH protein levels were monitored to ensure equal loading of samples. (C) Indirect immunofluorescence analysis of CIP2A protein expression levels (green) and D-siCIP2A (red) uptake in excised xenograft tumor tissues 48 hours post-treatment with 599+D-siCIP2A compared to untreated tissues or tissues treated with D-siCIP2A alone or 599+siNT. Nuclei (blue) were counterstained with DAPI. Representative images of xenograft tumors from mice in the untreated group and treatment groups are shown. Scale bar: 100 μm.

Figure 4

The 599+siCIP2A complex significantly impairs tumor growth in mice following a three-dose schedule. The (A) weights and (B) volumes of excised xenograft tumors 8 days post-initial treatment with 599+siCIP2A, compared to tissues treated with siCIP2A alone or 599+siNT. The weights and volumes of untreated tumor tissues were also analyzed for comparison. Data are mean ± SEM of four independent samples (3 samples for untreated group), where *P<0.05, **P<0.01, and ***P<0.001 compared to 599+siCIP2A treated tumors (one-way ANOVA).

Figure 5

The 599+siCIP2A complex mediates silencing of CIP2A in tumor tissues following a three-dose schedule. (A) Real-time PCR analysis of CIP2A mRNA levels in excised xenograft tumor tissues 8 days post-initial treatment with 599+siCIP2A, compared to untreated tissues or tissues treated with siCIP2A alone or 599+siNT. The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of four independent samples (3 samples for untreated group) performed in duplicate, where **P<0.01 compared to 599+siCIP2A treated tumors (one-way ANOVA). (B) Western blot analysis of CIP2A protein expression levels in excised xenograft tumor tissues 8 days post-initial treatment with 599+siCIP2A, compared to untreated (Unt) tissues or tissues treated with siCIP2A alone or 599+siNT. Tissue samples from four mice (M1-4) per treatment group were analyzed, except for the untreated group which included samples from only two mice. β-actin protein levels were monitored to ensure equal loading of samples. (C) Densitometric quantification of CIP2A protein expression levels (from Western blot shown in panel B) in treated tissues normalized to untreated tissues. CIP2A protein levels were normalized to β-actin. Data are mean ± SEM of four independent samples, where **P<0.01 and ****P<0.0001 compared to 599+siCIP2A treated tumors (one-way ANOVA).

Figure 6

The 599+siCIP2A complex does not trigger an interferon response or induce acute toxicities in xenograft mouse tumor tissues. (A) Real-time PCR analysis of mRNA levels of the interferon-responsive genes, OAS1 and STAT1, in excised xenograft tumor tissues 2 days post-final treatment with 599+siCIP2A, compared to untreated tissues or tissues treated with siCIP2A alone or 599+siNT. The OAS1 and STAT1 mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of four independent samples (3 samples for untreated group) performed in duplicate, where P>0.05, not significant (ns), compared to 599+siCIP2A treated tumors (one-way ANOVA). (B) Histopathological examinations of hematoxylin and eosin-stained tumor tissues treated with 599+siCIP2A, compared to untreated tissues or tissues treated with siCIP2A alone or 599+siNT. Representative images of xenograft tumors from mice in the untreated group and treatment groups are shown. Scale bar: 50 μm.

Figure 7

Treatment of tumors with 599 peptide complexed to siCIP2A causes a significant decrease in the number of mitotic figures. (A) Representative high-power fields of hematoxylin and eosin-stained tumor tissues treated with 599+siCIP2A, compared to untreated tissues or tissues treated with siCIP2A alone or 599+siNT. Arrows indicate mitotic figures. Scale bar: 20 μm. (B) Graphic representation of mitotic index in twenty high-power fields (HPF) from excised xenograft tumor tissues 8 days post-initial treatment with 599 peptide+siCIP2A, compared to untreated tissues or tissues treated with siCIP2A alone or 599+siNT. Data are mean ± SEM of four independent samples (3 samples for untreated and siRNA alone-treated groups), where **P<0.01 and ***P<0.001 compared to 599+siCIP2A treated tumors (one-way ANOVA).