Direct targeting of amplified gene loci for proapoptotic anticancer therapy

Direct targeting of amplified gene loci for proapoptotic anticancer therapy

Abstract

Gene amplification drives oncogenesis in a broad spectrum of cancers. A variety of drugs have actually been established to prevent the protein items of magnified motorist genes, however their medical effectiveness is frequently obstructed by drug resistance. Here, we present a healing method for targeting cancer-associated gene amplifications by triggering the DNA damage reaction with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in growths, whereas cells without amplifications procedure lower levels of DNA damage. Concentrating on cancers driven by HER2 amplification, we discover that TFOs targeting HER2 cause copy number-dependent DNA double-strand breaks (DSBs) and trigger p53- independent apoptosis in HER2-positive cancer cells and human growth xenografts by means of a system that is independent of HER2 cellular function. This method has actually shown in vivo effectiveness similar to that of existing accuracy medications and supplied a practical option to fight drug resistance in HER2-positive breast and ovarian cancer designs. These findings use a basic method for targeting growths with enhanced genomic loci.

Access alternatives

Subscribe to Journal

Get complete journal gain access to for 1 year

99,00 EUR

just 8,25 EUR per problem

Tax computation will be settled throughout checkout.

Rent or Buy short article

Get time minimal or complete post gain access to on ReadCube.

from$ 8.99

All rates are NET rates.

Data schedule

The authors state that the information supporting the findings of this research study are offered within the paper and its Supplementary Information Source information are offered with this paper.

References

  1. 1.

    Chen, Y. et al. Recognition of druggable cancer motorist genes magnified throughout TCGA datasets. PLoS ONE 9, e98293(2014).

    Article

    Google Scholar

  2. 2.

    Matsui, A., Ihara, T., Suda, H., Mikami, H. & Semba, K. Gene amplification: systems and participation in cancer. Biomol. Ideas 4, 567–582(2013).

    CAS
    Article

    Google Scholar

  3. 3.

    Santarius, T., Shipley, J., Brewer, D., Stratton, M. R. & Cooper, C. S. A census of enhanced and overexpressed human cancer genes. Nat. Rev. Cancer10, 59–64(2010).

    CAS
    Article

    Google Scholar

  4. 4.

    Albertson, D. G. Gene amplification in cancer. Trends Genet.22, 447–455(2006).

    CAS
    Article

    Google Scholar

  5. 5.

    Ohshima, K. et al. Integrated analysis of gene expression and copy number determined possible cancer chauffeur genes with amplification-dependent overexpression in 1,454 strong growths. Sci. Rep. 7, 641 (2017).

    Article

    Google Scholar

  6. 6.

    Moasser, M. M. & Krop, I. E. The developing landscape of HER2 targeting in breast cancer. JAMA Oncol. 1, 1154–1161(2015).

    Article

    Google Scholar

  7. 7.

    Slamon, D. J. et al. Research studies of the HER-2/ neu proto-oncogene in human breast and ovarian cancer. Science244, 707–712(1989).

    CAS
    Article

    Google Scholar

  8. 8.

    Baselga, J., Albanell, J., Molina, M. A. & Arribas, J. Mechanism of action of trastuzumab and clinical upgrade. Semin. Oncol.28, 4–11(2001).

    CAS
    Article

    Google Scholar

  9. 9.

    Swain, S. M. et al. Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N. Engl. J. Med.372, 724–734(2015).

    CAS
    Article

    Google Scholar

  10. 10

    Wilks, S. T. Potential of conquering resistance to HER2-targeted treatments through the PI3K/Akt/mTOR path. Breast24, 548–555(2015).

    Article

    Google Scholar

  11. 11

    Petty, R. D. et al. Gefitinib and EGFR gene copy number aberrations in esophageal cancer. J. Clin. Oncol.35, 2279–2287(2017).

    CAS
    Article

    Google Scholar

  12. 12

    Pao, W. et al. Obtained resistance of lung adenocarcinomas to gefitinib or erlotinib is related to a 2nd anomaly in the EGFR kinase domain. PLoS Med. 2, e73(2005).

    Article

    Google Scholar

  13. 13

    Ricciardi, A. S., McNeer, N. A., Anandalingam, K. K., Saltzman, W. M. & Glazer, P. M. Targeted genome adjustment through triple helix development. Methods Mol. Biol.1176, 89–106(2014).

    CAS
    Article

    Google Scholar

  14. 14

    Gaddis, S. S. et al. A web-based online search engine for triplex-forming oligonucleotide target series. Oligonucleotides16, 196–201(2006).

    CAS
    Article

    Google Scholar

  15. 15

    Ebbinghaus, S. W. et al. Triplex development hinders HER-2/ neu transcription in vitro. J. Clin. Invest.92, 2433–2439(1993).

    CAS
    Article

    Google Scholar

  16. 16

    Kaushik Tiwari, M. & Rogers, F. A. XPD-dependent activation of apoptosis in action to triplex-induced DNA damage. Nucleic Acids Res.41, 8979–8994(2013).

    CAS
    Article

    Google Scholar

  17. 17

    Kaushik Tiwari, M., Adaku, N., Peart, N. & Rogers, F. A. Triplex structures cause DNA double hair breaks by means of duplication fork collapse in NER lacking cells. Nucleic Acids Res.44, 7742–7754(2016).

    Article

    Google Scholar

  18. 18

    Rogers, F. A., Vasquez, K. M., Egholm, M. & Glazer, P. M. Site-directed recombination through bifunctional PNA– DNA conjugates. Proc. Natl Acad. Sci. U.S.A.99, 16695–16700(2002).

    CAS
    Article

    Google Scholar

  19. 19

    Wang, G., Seidman, M. M. & Glazer, P. M. Mutagenesis in mammalian cells caused by triple helix development and transcription-coupled repair work. Science271, 802–805(1996).

    CAS
    Article

    Google Scholar

  20. 20

    Szollosi, J., Balazs, M., Feuerstein, B. G., Benz, C. C. & Waldman, F. M. ERBB-2( HER2/ neu) gene copy number, p185 HER-2 overexpression, and intratumor heterogeneity in human breast cancer. Cancer Res.55, 5400–5407(1995).

    CAS
    PubMed

    Google Scholar

  21. 21

    Vergote, I. et al. Neoadjuvant chemotherapy or main surgical treatment in phase IIIC or IV ovarian cancer. N. Engl. J. Med.363, 943–953(2010).

    CAS
    Article

    Google Scholar

  22. 22

    Jenjaroenpun, P. & Kuznetsov, V. A. TTS mapping: integrative WEB tool for analysis of triplex development target DNA series, G-quadruplets and non-protein coding regulative DNA aspects in the human genome. BMC Genomics10, S9 (2009).

    Article

    Google Scholar

  23. 23

    Cook, P. J. et al. Tyrosine dephosphorylation of H2AX regulates apoptosis and survival choices. Nature458, 591–596(2009).

    CAS
    Article

    Google Scholar

  24. 24

    zum Buschenfelde, C. M., Hermann, C., Schmidt, B., Peschel, C. & Bernhard, H. Antihuman skin development aspect receptor 2 (HER2) monoclonal antibody trastuzumab improves cytolytic activity of class I-restricted HER2-specific T lymphocytes versus HER2-overexpressing growth cells. Cancer Res.62, 2244–2247(2002).


    Google Scholar

  25. 25

    Cuello, M. et al. Down-regulation of the erbB-2 receptor by trastuzumab (Herceptin) improves growth necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2. Cancer Res.61, 4892–4900(2001).

    CAS
    PubMed

    Google Scholar

  26. 26

    Deng, Y. et al. The result of hyperbranched polyglycerol finishings on drug shipment utilizing degradable polymer nanoparticles. Biomaterials35, 6595–6602(2014).

    CAS
    Article

    Google Scholar

  27. 27

    Bindra, R. S. & Glazer, P. M. Repression of RAD51 gene expression by E2F4/p130 complexes in hypoxia. Oncogene26, 2048–2057(2007).

    CAS
    Article

    Google Scholar

  28. 28

    Balashanmugam, M. V. et al. Preparation and characterization of unique PBAE/PLGA polymer mix microparticles for DNA vaccine shipment. ScientificWorldJournal2014, 385135 (2014).

    Article

    Google Scholar

  29. 29

    Seo, Y. E. et al. Nanoparticle-mediated intratumoral inhibition of miR-21 for enhanced survival in glioblastoma. Biomaterials 201, 87–98(2019).

    CAS
    Article

    Google Scholar

  30. 30

    Oeck, S. et al. The Focinator v2-0– visual user interface, 4 channels, colocalization analysis and cell stage recognition. Radiat. Res.188, 114–120(2017).

    CAS
    Article

    Google Scholar

  31. 31

    Oeck, S., Malewicz, N. M., Hurst, S., Rudner, J. & Jendrossek, V. The Focinator– a brand-new open-source tool for high-throughput foci examination of DNA damage. Radiat. Oncol.10, 163 (2015).

    Article

    Google Scholar

  32. 32

    Mandl, H. K. et al. Enhancing naturally degradable nanoparticle size for tissue-specific shipment. J. Control. Release314, 92–101(2019).

    CAS
    Article

    Google Scholar

Download referrals

Acknowledgements

This work was supported by grants from the National Cancer Institute (NCI) of the National Institutes of Health (NIH) R21 CA185192 to F.A.R., the Breast Cancer Alliance Exceptional Project Grant to F.A.R., National Institute of General Medical Sciences R01 GM126211 to F.A.R. and NIH R01 CA149128 to W.M.S. E.Q. was supported by training grants T32 GM07205 and 5T32 GM007223-43

Author info

Author notes

  1. These authors contributed similarly: Daniel A. Colon-Rios, Hemanta C. Rao Tumu.

Affiliations

  1. Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA

    Meetu Kaushik Tiwari, Daniel A. Colon-Rios, Hemanta C. Rao Tumu, Yanfeng Liu, Adam Krysztofiak, Cynthia Chan & Faye A. Rogers

  2. Department of Genetics, Yale School of Medicine, New Haven, CT, USA

    Elias Quijano

  3. Department of Biomedical Engineering, Yale School of Medicine, New Haven, CT, USA

    Elias Quijano, Eric Song, Hee-Won Suh & W. Mark Saltzman

  4. Department of Pathology, Yale School of Medicine, New Haven, CT, USA

    Demetrios T. Braddock

  5. Department of Chemical & Environmental Engineering, Yale University, New Haven, CT, USA

    W. Mark Saltzman

  6. Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA

    W. Mark Saltzman

  7. Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA

    Faye A. Rogers

Contributions

F.A.R. developed and developed the research study, added to conclusion of experiments and composed the manuscript. M.K.T. added to study style and performed most of the research study. D.A.C.-R. carried out research studies to assess TFOs targeting introns of the HER2 gene and research studies to assess system of action. H.C.R.T. carried out tumor development hold-up research studies in the orthotopic mouse design for breast cancer, immunofluorescence of growth tissue and transcription inhibition research studies. Y.L. carried out the tumor development hold-up research studies in mouse designs for breast and ovarian cancers. E.Q. produced and identified NPs. A.K. added to the analysis of confocal microscopy images and metrology of immunofluorescence images. C.C. added to DNA damage and apoptosis experiments in ovarian cancer cell lines. E.S. added to TFO growth uptake research studies. D.T.B. performed pathology analysis of growth xenograft samples. H.W.S. and W.M.S. helped with NP innovation.

Corresponding author

Correspondence to.
Faye A. Rogers

Ethics statements

Competing interests

Yale University has actually submitted patent applications associated to this work (innovator F.A.R.).

Additional info

Peer evaluation info Nature Biotechnology thanks Carlo V. Catapano and the other, confidential, customer( s) for their contribution to the peer evaluation of this work.

Publisher’s note Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations.

Extended information

Extended Data Fig. 1 Experiments Supporting Main Fig. 2

( a) Representative pictures of neutral comet assays carried out 24 h after HER2-205 treatment in MCF7 and BT474 cells (scale bars, 200 μm). ( b) Quantification of cells with higher than 5 γH2AX and/or 53 BP1 foci per nuclei in BT474 cells treated with HER2-205 or MIX24(suggest ± SD; two-way ANOVA with Tukey test post-hoc; P< 0.0001, P< 0.01; 50 cells per sample, n= 2 independent experiments). ( c) Triplex development causes apoptosis in HER2-positive breast cancer cell lines as determined by Western blot analysis of cleaved PARP (n= 3 independent experiments). ( d) Detection of HER2 copies in interphase nuclei by double color FISH with HER2 probe (red) and chromosome 17 probe (green), scale bars, 2.5 μm. ( e) Immunofluorescence of γH2AX in PE01 ovarian cancer cells 24 h post-treatment with HER2-205 or MIX24(scale bars, 5 μm). ( f) Representative immunofluorescence pictures of γH2AX foci in SKOV3 ovarian cancer cells 24 h following treatment with HER2-205 or MIX24(scale bars, 2.5 μm). ( g) Frequency of PE01 and SKOV3 cells favorable for γH2AX following 24 h treatment (mean ± SD; two-way ANOVA with Tukey test post-hoc; P< 0.001, P< 0.01; 50 cells per sample, n= 2 independent experiments). ( h) Quantification of triplex-induced DNA double hair breaks utilizing the neutral comet assay as determined by tail minute (mean ± SEM; two-way ANOVA with Tukey test post-hoc, P< 0.0001; n=150 comets). ( i) Monolayer development assay shows a decline in cell survival in PE01 and SKOV3 cells treated with HER2-20572 h after treatment. ( j) Western blot analysis of activation of apoptosis as determined by cleaved PARP in ovarian cancer cells following TFO treatment (n= 3 independent experiments).

Source information

Extended Data Fig. 2 Experiments Supporting Main Fig. 5

( a) ChIP analysis of γH2AX in BT474 cells spotted increased DNA damage at the targeted HER2 gene following HER2-5922 treatment. Information exist as mean ± SEM and evaluated by two-way ANOVA with Tukey test post-hoc, P< 0.001, n= 3 independent experiments. ( b) Quantification of phosphorylated ATM by circulation cytometry following treatment with HER2-205 Information exist as mean ± SEM and evaluated by one-way ANOVA with Tukey test post-hoc, P< 0.05, n= 3 independent experiments. ( c) Analysis of HER2 gene expression by RT-PCR 12 h post-treatment with HER2-targeted TFOs (mean ± SD; two-ANOVA with Tukey test post-hoc; ns, not considerable; n= 3 independent experiments). ( d) Quantification of triplex-induced DNA double hair breaks utilizing the neutral comet assay as determined by tail minute 12 h post TFO treatment (mean ± SEM; one-ANOVA with Tukey test post-hoc; P< 0.0001; n= 3 independent experiments). ( e) Western blot analysis of activation of apoptosis as determined by cleaved PARP and pH2AX Y14212 h following TFO treatment (representative immunoblots, n= 2 independent experiments). Western blot analysis of the phosphorylation status of HER household receptors ( f) HER3, ( g) HER4, and ( h) EGFR (HER1) in numerous breast cancer cell lines following HER2-205 treatment (representative immunoblots, n= 2). ( i) Analysis of HER2 gene expression by RT-PCR 12 h post-treatment with HER2-targeted TFOs (suggest ± SEM; one-way ANOVA with Tukey test post-hoc; ns, not substantial; n= 3 independent experiments). ( j) Analysis of HER2 gene expression by RT-PCR 20 h following pretreatment with the transcription inhibitor, α-amanitin (mean ± SD; one-way ANOVA with Tukey test post-hoc; P< 0.0001; n= 4 experiments).

Source information

Extended Data Fig. 3 Biodistribution of nanoparticle formulas.

Comparison of PLGA and PLA-HPG NPs in vivo. ( a) Uptake of DiD-loaded NPs, PLGA/DCM, PLGA/EtOAc and PLA-HPG, 12 h after systemic administration through retro-orbital injection. Growth cryosections imagine DAPI (blue) and DiD (red) (scale bars, 50 μm; n= 2 growths). ( b) Biodistribution of DiD-loaded PLA-HPG NPs 12 h after systemic administration. DiD fluorescence in separated organs after retro-orbital injection with DiD encapsulated NPs (2 mg). Cryosections envision DAPI (blue) and DiD (red) (scale bars, 50 μm; n= 2 animals).

Extended Data Fig. 4 Experiments Supporting Main Fig. 6

Biodistribution of TAMRA-HER2-205 encapsulated PLA-HPG nanoparticles (NPs). ( a) Representative confocal pictures of tissue areas 12 hours post intravenous administration by means of retro-orbital injection of a 2 mg dosage of NPs (scale bars, 50 μm). ( b) Representative confocal pictures of TAMRA-HER2-205 biodistribution in tissues 24 hours post treatment (scale bars, 50 μm). ( c) TAMRA fluorescence was measured at both 12 and 24 hours after dosing (2 mg of NPs) and TFO uptake in each tissue is reported as mean fluorescence strength (MFI) (indicate ± SEM, n= 2 mice). Analytical significance was computed by one-way ANOVA and Kruskal-Wallis test P< 0.0001, P< 0.01). ( d) Analysis of TAMRA-HER2-205 biodistribution 12 h post treatment. Fluorescence strength observed in each tissue is reported as a portion of the combined overall fluorescence strength found in spleen, kidney, liver and growth (growth information is revealed and measured in Fig. 6a, b). Overall location of the pie chart signifies the amount of the outright fluorescence within the 4 organs, representing the overall TFO uptake by these organs, and each piece offers the relative HER2-205 uptake for each organ. ( e) Analysis of TAMRA-HER2-205 biodistribution 24 h post systemic administration. Fluorescence strength observed in each tissue is reported as a portion of the combined overall fluorescence strength spotted in spleen, kidney, liver and growth (growth information is revealed and measured in Fig. 6a, b). Overall location of the pie chart signifies the amount of the outright fluorescence within the 4 organs, representing the overall TFO uptake by these organs, and each piece provides the relative HER2-205 uptake for each organ.

Extended Data Fig. 5 Experiments Supporting Main Fig. 6.

( a) Nanoparticle Characterization. Nanoparticle size as determined by vibrant light scattering. Nanoparticle surface area charge determined by zeta capacity. Nanoparticle loading of TFOs determined by extraction and analysis. All information is outlined as mean ± SEM, n= 3 experiments. ( b) Representative pictures of confocal microscopy of γH2AX immunofluorescence in growths 24 h post-treatment with HER2-205 PLA-HPG NPs and metrology of γH2AX foci is reported as mean fluorescence strength (MFI) (indicate ± SEM; Kolmogorov-Smirnov test; P< 0.001, P< 0.01; n= 4 tumors/timepoint; scale bars, 10 μm). ( c) Representative pictures of confocal microscopy of cleaved caspase 3 immunofluorescence in growths 12 h post-treatment with HER2-205 PLA-HPG NPs and metrology of triggered caspase 3 is reported as mean fluorescence strength (mean ± SEM; Kolmogorov-Smirnov test; P< 0.0001; n= 4 tumors/timepoint; scale bars, 10 μm). ( d) HER2 immunofluorescence analysis of BT474 growth areas from mice 12 h and 24 h after treatment with a single dosage of HER2-205 PLA-HPG NPs (2 mg). Information represented as mean ± SEM and evaluated by one-way ANOVA Kruskal-Wallis test (n= 4 tumors/time point; ns, not considerable). Scale bar, 10 μm. ( e) Confocal microscopy pictures of growth areas examined by immunofluorescence 12 h and 24 h following a single dosage of TAMRA-HER2-205 PLA-HPG NPs (scale bars, 10 μm).

Extended Data Fig. 6 ESI-MS and HPLC characterization of TFOs.

( a) Analytical ESI-MS spectrum of HER2-205 ( b) Analytical reverse-phased HPLC of HER2-205 ( c) Analytical ESI-MS spectrum of HER2-5922 ( d) Analytical reverse-phased HPLC of HER2-5922

Extended Data Fig. 7 Flow cytometry profiles.

A single cell uniform population was made use of for FCS/SSC gating of the beginning cell population. ( a) Flow cytometry profiles of BT474 cells stained for Annexin V-FITC/PI to determine apoptotic cells. Cells were collected 24 h after treatment. Lower best quadrant represents the combined portion of early and late apoptotic cells. ( b) Flow cytometry profiles of BT474 cells stained for pATM. Cells were gathered 24 h after treatment. Package suggests eviction for high levels of pATM and numbers represent portion of cells with high levels of pATM.

Supplementary info

About this post

Cite this short article

Kaushik Tiwari, M., Colon-Rios, D.A., Tumu, H.C.R. et al. Direct targeting of magnified gene loci for proapoptotic anticancer treatment.
Nat Biotechnol(2021). https://doi.org/101038/ s41587-021-01057 -5

Download citation

Read More

Author: admin

Leave a Reply

Your email address will not be published. Required fields are marked *