Research ArticleCancer

Synthetic mRNA nanoparticle-mediated restoration of p53 tumor suppressor sensitizes p53-deficient cancers to mTOR inhibition

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Science Translational Medicine  18 Dec 2019:
Vol. 11, Issue 523, eaaw1565
DOI: 10.1126/scitranslmed.aaw1565
  • Fig. 1 In vitro transfection efficiency of the redox-responsive mRNA NPs in p53-null Hep3B cells.

    (A) Transmission electron microscopy (TEM) images of the hybrid mRNA NPs before incubation (in PBS) or after incubation in 10 mM DTT for 2 or 4 hours at 37°C. (B) Confocal laser scanning microscopy (CLSM) images of p53-null Hep3B cells after incubation with naked Cy5-labeled mRNA (red) for 6 hours and with engineered Cy5-labeled mRNA NPs for 1, 3, or 6 hours. Endosomes were stained by LysoTracker Green (green), and nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. (C) In vitro transfection efficiency (percentage of EGFP-positive cells) was determined by flow cytometry. Data shown as means ± SEM (n = 3), and statistical significance was determined using two-tailed t test (**P < 0.01). (D) Histogram analysis of the in vitro transfection efficiency by FlowJo software.

  • Fig. 2 Restoration of p53 functions in p53-null Hep3B cells by the mRNA NPs and in vitro mechanisms for p53 restoration–mediated antitumor effect.

    (A) Immunofluorescence (IF) staining of p53 in the p53-null Hep3B cells treated by empty NP or p53-mRNA NPs. Scale bars, 50 μm. (B) Viability of the p53-null Hep3B liver cancer cells after treatment with PBS, empty NPs, naked p53-mRNA (0.830 μg/ml), or p53-mRNA NPs (mRNA concentrations: 0.103, 0.207, 0.415, or 0.830 μg/ml) by alarmBlue assay. Statistical significance was determined using two-tailed t test (*P < 0.05, **P < 0.01). (C) Colony formation assays of Hep3B cells after treatment with empty NPs versus p53-mRNA NPs in six-well plates. (D) Apoptosis of Hep3B cells as determined by flow cytometry after treatment with empty NPs, naked p53-mRNA, or p53-mRNA NPs. (E) Histogram analysis of the cell apoptosis (%) by FlowJo software. Data shown as means ± SEM (n = 3), and statistical significance was determined using two-tailed t test (***P < 0.001). (F) Cell cycle distributions of Hep3B cells after treatment with PBS, empty NPs, naked p53-mRNA, or p53-mRNA NPs (mRNA concentration: 0.830 μg/ml). (G) Western blot (WB) analysis of cell cycle–related protein expression (p21 and CyclinE1) after treatment with p53-mRNA NPs (mRNA concentration: 0.830 μg/ml). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. (H) WB analysis of the mitochondrial apoptotic signaling pathway in p53-null Hep3B cells treated with PBS, empty NPs, naked p53-mRNA, or p53-mRNA NPs (mRNA concentration: 0.830 μg/ml). BCL-2, BAX, PUMA, C-CAS9, and C-CAS3 proteins were detected. Actin was used as the loading control. (I) TEM images of the mitochondrial morphology in Hep3B cells from control, empty NPs, and p53-mRNA NP groups (mRNA concentration: 0.830 μg/ml; blue arrow, normal mitochondria; red arrow, swelling mitochondria). Scale bars, 2 μm for the top images and 1 μm for the enlarged images (bottom).

  • Fig. 3 Mechanisms of the p53-mRNA NP–mediated sensitization to everolimus in p53-null Hep3B cells.

    (A) Viability of Hep3B cells after treatment with everolimus, as measured by AlamarBlue assay. Data shown as means ± SEM (n = 3). (B) WB analysis of total mTOR, p-mTOR, and p-p70S6K after treatment with everolimus at different concentrations. Actin was measured as the loading control. (C) WB analysis of p-mTOR, LC3B-1, and LC3B-2. Actin was measured as the loading control. (D) TEM images of Hep3B cells before and after 24 hours of treatment with everolimus (32 nM). Autophagosomes were labeled by yellow arrows. Scale bars (left to right), 2, 5, and 1 μm. (E) CLSM images of GFP-LC3–transfected Hep3B cells from different treatment groups. Scale bars, 50 μm. (F) WB analysis of p53, p-mTOR, total m-TOR, p-4EBP1, BECN1, LC3B-1, LC3B-2, BCL-2, BAX, C-CAS9, and C-CAS3 in Hep3B cells after different treatments. Actin was used as the loading control. (G) Left: TEM images of Hep3B cells in control, p53-mRNA NPs, everolimus, and p53-mRNA NPs + everolimus groups (mRNA concentration: 0.415 μg/ml; everolimus concentration: 32 nM). Scale bars, 2 μm for the raw images and 1 μm for the enlarged images. Yellow arrows, autophagosomes; red arrows, mitochondria. Right: Statistical analysis of the numbers of autophagosomes (Auto; yellow) and swollen mitochondria (Mito; red) after different treatments. (H) Viability of Hep3B cells in different groups (control, EGFP-mRNA NPs, p53-mRNA NPs, everolimus, or p53-mRNA NPs + everolimus) as measured by AlamarBlue assay (mRNA concentration: 0.415 μg/ml; everolimus concentration: 32 nM). Data shown as means ± SEM (n = 3), and statistical significance was determined using two-tailed t test (**P < 0.01, ***P < 0.001). (I) Colony formation of Hep3B cells in different treatment groups in six-well plates. (J) Flow cytometry analysis of the cell apoptosis (AnnV+PI and AnnV+PI+). The percentage of apoptotic Hep3B cells was shown in the histogram. Statistical significance was determined using two-tailed t test (***P < 0.001).

  • Fig. 4 Antitumor effects of p53-mRNA NPs are synergistic with everolimus in p53-null HCC xenograft model.

    (A) Blood circulation profiles of naked Cy5-labeled mRNA and Cy5-labeled mRNA NPs (at an mRNA dose of 750 μg/kg of animal weight). NP25, NP50, and NP75 represent three different ratios of DSPE-PEG/DMPE-PEG (25:75, 50:50, and 75:25) hybrid in the lipid-PEG layer of hybrid NPs. Data shown as means ± SEM (n = 3). (B) Time-lapse near-infrared fluorescence imaging of nude mice bearing p53-null HCC xenograft tumors after intravenous injection of free Cy5-mRNA, Cy5-mRNA NP25, Cy5-mRNA NP50, or Cy5-mRNA NP75. The tumors were annotated with white arrows. (C) Scheme of tumor inoculation [subcutaneous (s.c.)] and treatment schedule in Hep3B tumor–bearing athymic nude mice. Twelve days after tumor inoculation, mice were treated with PBS [intravenous (i.v.)], EGFP-mRNA NPs (intravenous), p53-mRNA NPs (intravenous), everolimus (oral), or p53-mRNA NPs (intravenous) + everolimus (oral) every 3 days for six rounds (mRNA dose, 750 μg/kg; everolimus dose, 5 mg/kg). Tumors from different groups were harvested 18 days after the last treatment. (D) Photos of excised tumors from mice bearing Hep3B xenografts in different treatment groups on day 33 (n = 5). (E to I) Individual tumor growth kinetics in (E) control, (F) EGFP-mRNA NPs, (G) everolimus, (H) p53-mRNA NPs, and (I) p53-mRNA NPs + everolimus group (n = 5). (J) Average tumor growth kinetics for all treatment groups. Data shown as means ± SEM (n = 5), and significance was determined using two-tailed t test (***P < 0.001). (K) Average tumor volumes at experimental endpoint (day 33) in all groups. Data shown as means ± SEM (n = 5), and statistical significance was determined using two-tailed t test (***P < 0.001). (L) IF images of p53 (red) and C-CAS3 (green) costained Hep3B tumor sections at 12, 24, 48, and 60 hours after intravenous injection of p53-mRNA NPs. PBS (60 hours after intravenous injection) was used as control group. Scale bars, 100 μm.

  • Fig. 5 In vivo mechanisms underlying the p53-mRNA NP–mediated sensitization of p53-null HCC xenograft model to everolimus.

    (A) Immunohistochemistry (IHC) images from tumor sections of Hep3B tumor–bearing xenograft mice before and after treatment with p53-mRNA NPs (mRNA dose, 750 μg/kg). The protein expressions of p53, apoptotic markers (BAX and C-CAS3), and proliferation markers (Ki67 and PCNA) were evaluated by IHC staining (blue: nucleus; brown: p53, BAX, C-CAS3, Ki67, or PCNA). Scale bars, 100 μm. (B) CLSM images of fixed tumor tissues with the TUNEL staining (blue: nucleus; red: apoptosis) from PBS, EGFP-mRNA NPs, p53-mRNA NPs, everolimus, and p53-mRNA NPs + everolimus groups. Scale bars, 100 μm. (C) WB analysis of p53, LC3B-1, LC3B-2, BECN1, p62, BCL-2, BAX, C-CAS9, C-CAS3, and p-4EBP1 in the Hep3B xenograft tumors after different treatments. Actin was used as the loading control.

  • Fig. 6 Therapeutic efficacy in the p53-null orthotopic HCC tumors and the liver metastases of p53-null NSCLC.

    (A) Scheme of tumor inoculation and different treatments in luciferase-expressing Hep3B (Hep3B-Luc) orthotopic tumor–bearing nude mice. Twenty-one days after tumor inoculation, mice were treated with PBS (intravenous), EGFP-mRNA NPs (intravenous), p53-mRNA NPs (intravenous), everolimus (oral), or p53-mRNA NPs (intravenous) + everolimus (oral) every 3 days for four rounds (mRNA dose, 750 μg/kg; everolimus dose, 5 mg/kg). (B) Bioluminescence images of the Hep3B-Luc orthotopic tumor–bearing nude mice at days 0, 6, and 12. (C) Average radiance [×106 photons per second (s) per cm2 per steradian (sr)] of tumor burden determined by bioluminescence imaging at different time points. (D) Average radiance of tumor burden at the endpoint (day 12). Data shown as means ± SEM (n = 3), and statistical significance was determined using two-tailed t test (*P < 0.05, **P < 0.01). (E) Scheme of tumor inoculation and different treatments in p53-null H1299 metastatic tumor–bearing nude mice. Twenty-eight days after tumor inoculation, mice were treated with PBS (intravenous), EGFP-mRNA NPs (intravenous), p53-mRNA NPs (intravenous), everolimus (oral), or p53-mRNA NPs (intravenous) + everolimus (oral) every 3 days for five rounds (mRNA dose, 750 μg/kg; everolimus dose, 5 mg/kg). Organs from different groups were harvested three days after the final treatment. (F) Histological examination of liver tissues from each group by H&E staining. The metastatic lesions (red dotted ovals) were identified as cell clusters with darkly stained nuclei. Scale bars, 100 μm. (G) The number of metastatic nodules in the liver from each group. One liver was randomly selected from each group with a blind method, and the liver section from each group was divided into four regions for counting of the metastasis nodules. Data shown as means ± SEM (n = 4 regions), and statistical significance was determined using two-tailed t test (*P < 0.05, **P < 0.01).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/523/eaaw1565/DC1

    Materials and Methods

    Fig. S1. Study summary.

    Fig. S2. The structure schematic of synthetic mRNA.

    Fig. S3. The chemical structure of 3′-O-Me-m7G(5′)ppp(5′)G ARCA cap.

    Fig. S4. Chemicals for NP synthesis.

    Fig. S5. Characterization of the engineered hybrid mRNA NPs.

    Fig. S6. Size of EGFP-mRNA NPs and Luc-mRNA NPs with various formulations.

    Fig. S7. Encapsulation efficiency of EGFP-mRNA NPs and Luc-mRNA NPs with various formulations.

    Fig. S8. Normalized luminescence intensity of Hep3B cells after treatment with various Luc-mRNA NP formulations at the mRNA dose of 0.830 μg/ml.

    Fig. S9. Endosomal escape of mRNA NPs.

    Fig. S10. Transfection efficacy verified by CLSM imaging.

    Fig. S11. Transfection efficacy verified by flow cytometry.

    Fig. S12. Transfection efficacy after quenching intracellular GSH.

    Fig. S13. In vitro toxicity of the synthetic EGFP-mRNA NPs.

    Fig. S14. IF staining of p53 in p53-null H1299 cells.

    Fig. S15. WB analysis of p53 protein expression.

    Fig. S16. In vitro therapeutic efficacy of the synthetic p53-mRNA NPs in p53-null H1299 cells.

    Fig. S17. Apoptosis of p53-null H1299 cells as determined by flow cytometry after different treatments.

    Fig. S18. G1-phase cell cycle arrest induced by p53-mRNA NPs.

    Fig. S19. WB analysis of apoptotic signaling pathway in p53-null H1299 cells after different treatments.

    Fig. S20. TEM images of mitochondrial morphology in p53-null H1299 cells after different treatments.

    Fig. S21. In vitro toxicity of the mutant p53-R175H-mRNA NPs.

    Fig. S22. Cytotoxicity of everolimus in p53-null H1299 cells.

    Fig. S23. Effect of everolimus on autophagy activation in p53-null H1299 cells.

    Fig. S24. WB analysis of autophagy and apoptotic signaling pathways in p53-null H1299 cells.

    Fig. S25. Analysis of the autophagosomes and swollen mitochondria in p53-null H1299 cells after different treatments.

    Fig. S26. In vitro therapeutic efficacy of the combination of p53-mRNA NPs with everolimus in p53-null H1299 cells.

    Fig. S27. In vitro apoptosis of p53-null H1299 cells after different treatments.

    Fig. S28. In vitro toxicity of the combination of everolimus with venetoclax.

    Fig. S29. In vitro toxicity of the combination of everolimus with siBcl-2.

    Fig. S30. The relative mRNA expression of p53.

    Fig. S31. The relative mRNA expression of ULK1, ATG7, BECN1, and ATG12.

    Fig. S32. The relative mRNA expression of DRAM1, ISG20L1, and SESN1.

    Fig. S33. The relative mRNA expression of TIGAR.

    Fig. S34. WB analysis of AMPK and TIGAR pathways.

    Fig. S35. Schematic representation of the possible mechanism by which p53 tumor suppressor inhibits protective autophagy and sensitizes tumor cells to everolimus.

    Fig. S36. BioD of different mRNA NPs in HCC xenograft tumor model.

    Fig. S37. BioD of different mRNA NPs in NSCLC xenograft tumor model.

    Fig. S38. Blood vessel staining in tumor sections.

    Fig. S39. Efficacy and safety of different treatments in HCC xenograft model.

    Fig. S40. Antitumor effects of p53-mRNA NPs are synergistic with everolimus in NSCLC xenograft model.

    Fig. S41. Murine p53 restoration in p53-null murine liver cancer RIL-175 cells.

    Fig. S42. Therapeutic efficacy of murine p53-mRNA NPs in immunocompetent mice bearing p53-null RIL-175 tumors.

    Fig. S43. Expression of p53 protein in HCC xenograft model after treatment with p53-mRNA NPs.

    Fig. S44. Expression of p53 protein in NSCLC xenograft model after treatment with p53-mRNA NPs.

    Fig. S45. IHC images from tumor sections of H1299 tumor–bearing mice before and after treatment with p53-mRNA NPs.

    Fig. S46. In vivo toxicity of the p53-mRNA NP–mediated strategy for everolimus rescue assessed by histopathological and hematological analysis.

    Fig. S47. IHC images from major organs and tumor sections of the HCC xenograft model.

    Fig. S48. Evaluation of immune responses after the treatment with mRNA NPs.

    Fig. S49. Scans of the liver metastases from different treatment groups in Fig. 6.

    Table S1. Compositions of different NP formulations.

    Table S2. Different p53-mRNA sequences used in this study.

    Table S3. Primer sequences for qRT-PCR.

  • This PDF file includes:

    • Materials and Methods
    • Fig. S1. Study summary.
    • Fig. S2. The structure schematic of synthetic mRNA.
    • Fig. S3. The chemical structure of 3′-O-Me-m7G(5′)ppp(5′)G ARCA cap.
    • Fig. S4. Chemicals for NP synthesis.
    • Fig. S5. Characterization of the engineered hybrid mRNA NPs.
    • Fig. S6. Size of EGFP-mRNA NPs and Luc-mRNA NPs with various formulations.
    • Fig. S7. Encapsulation efficiency of EGFP-mRNA NPs and Luc-mRNA NPs with various formulations.
    • Fig. S8. Normalized luminescence intensity of Hep3B cells after treatment with various Luc-mRNA NP formulations at the mRNA dose of 0.830 μg/ml.
    • Fig. S9. Endosomal escape of mRNA NPs.
    • Fig. S10. Transfection efficacy verified by CLSM imaging.
    • Fig. S11. Transfection efficacy verified by flow cytometry.
    • Fig. S12. Transfection efficacy after quenching intracellular GSH.
    • Fig. S13. In vitro toxicity of the synthetic EGFP-mRNA NPs.
    • Fig. S14. IF staining of p53 in p53-null H1299 cells.
    • Fig. S15. WB analysis of p53 protein expression.
    • Fig. S16. In vitro therapeutic efficacy of the synthetic p53-mRNA NPs in p53-null H1299 cells.
    • Fig. S17. Apoptosis of p53-null H1299 cells as determined by flow cytometry after different treatments.
    • Fig. S18. G1-phase cell cycle arrest induced by p53-mRNA NPs.
    • Fig. S19. WB analysis of apoptotic signaling pathway in p53-null H1299 cells after different treatments.
    • Fig. S20. TEM images of mitochondrial morphology in p53-null H1299 cells after different treatments.
    • Fig. S21. In vitro toxicity of the mutant p53-R175H-mRNA NPs.
    • Fig. S22. Cytotoxicity of everolimus in p53-null H1299 cells.
    • Fig. S23. Effect of everolimus on autophagy activation in p53-null H1299 cells.
    • Fig. S24. WB analysis of autophagy and apoptotic signaling pathways in p53-null H1299 cells.
    • Fig. S25. Analysis of the autophagosomes and swollen mitochondria in p53-null H1299 cells after different treatments.
    • Fig. S26. In vitro therapeutic efficacy of the combination of p53-mRNA NPs with everolimus in p53-null H1299 cells.
    • Fig. S27. In vitro apoptosis of p53-null H1299 cells after different treatments.
    • Fig. S28. In vitro toxicity of the combination of everolimus with venetoclax.
    • Fig. S29. In vitro toxicity of the combination of everolimus with siBcl-2.
    • Fig. S30. The relative mRNA expression of p53.
    • Fig. S31. The relative mRNA expression of ULK1, ATG7, BECN1, and ATG12.
    • Fig. S32. The relative mRNA expression of DRAM1, ISG20L1, and SESN1.
    • Fig. S33. The relative mRNA expression of TIGAR.
    • Fig. S34. WB analysis of AMPK and TIGAR pathways.
    • Fig. S35. Schematic representation of the possible mechanism by which p53 tumor suppressor inhibits protective autophagy and sensitizes tumor cells to everolimus.
    • Fig. S36. BioD of different mRNA NPs in HCC xenograft tumor model.
    • Fig. S37. BioD of different mRNA NPs in NSCLC xenograft tumor model.
    • Fig. S38. Blood vessel staining in tumor sections.
    • Fig. S39. Efficacy and safety of different treatments in HCC xenograft model.
    • Fig. S40. Antitumor effects of p53-mRNA NPs are synergistic with everolimus in NSCLC xenograft model.
    • Fig. S41. Murine p53 restoration in p53-null murine liver cancer RIL-175 cells.
    • Fig. S42. Therapeutic efficacy of murine p53-mRNA NPs in immunocompetent mice bearing p53-null RIL-175 tumors.
    • Fig. S43. Expression of p53 protein in HCC xenograft model after treatment with p53-mRNA NPs.
    • Fig. S44. Expression of p53 protein in NSCLC xenograft model after treatment with p53-mRNA NPs.
    • Fig. S45. IHC images from tumor sections of H1299 tumor–bearing mice before and after treatment with p53-mRNA NPs.
    • Fig. S46. In vivo toxicity of the p53-mRNA NP–mediated strategy for everolimus rescue assessed by histopathological and hematological analysis.
    • Fig. S47. IHC images from major organs and tumor sections of the HCC xenograft model.
    • Fig. S48. Evaluation of immune responses after the treatment with mRNA NPs.
    • Fig. S49. Scans of the liver metastases from different treatment groups in Fig. 6.
    • Table S1. Compositions of different NP formulations.
    • Table S2. Different p53-mRNA sequences used in this study.
    • Table S3. Primer sequences for qRT-PCR.

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