Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.
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Carr TH, McEwen R, Dougherty B, Johnson JH, Dry JR, Lai Z, et al. Defining actionable mutations for oncology therapeutic development. Nat Rev Cancer. 2016;16:319–29.
Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev Cancer. 2012;12:237–51.
Friedman AA, Letai A, Fisher DE, Flaherty KT. Precision medicine for cancer with next-generation functional diagnostics. Nat Rev Cancer. 2015;15:747–56.
Melero I, Berman DM, Aznar MA, Korman AJ, Perez Gracia JL, Haanen J. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer. 2015;15:457–72.
Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: from discovery to therapy. Cancer Discov. 2016;6:353–67.
Strauss BE, Fontes RB, Lotfi CF, Skorupa A, Bartol I, Cipolla-Neto J, et al. Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats. Cancer Cell Int. 2002;2:2.
Costanzi-Strauss E, Strauss BE, Naviaux RK, Haas M. Restoration of growth arrest by p16INK4, p21WAF1, pRB, and p53 is dependent on the integrity of the endogenous cell-cycle control pathways in human glioblastoma cell lines. Exp Cell Res. 1998;238:51–62.
Chen J. The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harb Perspect Med. 2016;6:a026104.
Wang X, Simpson ER, Brown KA. p53: protection against tumor growth beyond effects on cell cycle and apoptosis. Cancer Res. 2015;75:5001–7.
Miciak J, Bunz F. Long story short: p53 mediates innate immunity. Biochim Biophys Acta. 2016;1865:220–7.
Beckerman R, Prives C. Transcriptional regulation byp53. Cold Spring Harb Perspect Biol. 2010;2:a000935.
Ablain J, Poirot B, Esnault C, Lehmann-Che J, De The H. p53 as an effector or inhibitor of therapy response. Cold Spring Harb Perspect Med. 2016;6:a026260.
Deben C, Deschoolmeester V, Lardon F, Rolfo C, Pauwels P. TP53 and MDM2 genetic alterations in non-small cell lung cancer: Evaluating their prognostic and predictive value. Crit Rev Oncol Hematol. 2016;99:63–73.
Lu X. Tied up in loops: positive and negative autoregulation of p53. Cold Spring Harb Perspect Biol. 2010;2:a000984.
Meek DW, Anderson CW. Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harb Perspect Biol. 2009;1:a000950.
Oren M, Rotter V. Mutant p53 gain-of-function in cancer. Cold Spring Harb Perspect Biol. 2010;2:a001107.
Haupt S, Raghu D, Haupt Y. Mutant p53 drives cancer by subverting multiple tumor suppression pathways. Front Oncol 2016;6:12.
Lane DP, Cheok CF, Lain S. p53-based cancer therapy. Cold Spring Harb Perspect Biol. 2010;2:a001222.
Khoo KH, Verma CS, Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov. 2014;13:217–36.
Nguyen DM, Spitz FR, Yen N, Cristiano RJ, Roth JA. Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 gene transfer. J Thorac Cardiovasc Surg. 1996;112:1372–6. discussion1376-7
Chen GX, Zhang S, He XH, Liu SY, Ma C, Zou XP. Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives. Onco Targets Ther. 2014;7:1901–9.
Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, et al. A census of human cancer genes. Nat Rev Cancer. 2004;4:177–83.
Helsten T, Kato S, Schwaederle M, Tomson BN, Buys TP, Elkin SK, et al. Cell-cycle gene alterations in 4,864 tumors analyzed by next-generation sequencing: implications for targeted therapeutics. Mol Cancer Ther. 2016;15:1682–90.
Jamal-Hanjani M, Wilson GA, McGranahan N, Birkbak NJ, Watkins TBK, Veeriah S, et al. Tracking the evolution of non-amall-cell Lung cancer. N Engl J Med. 2017;376:2109–21.
Sandig V, Brand K, Herwig S, Lukas J, Bartek J, Strauss M. Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death. Nat Med. 1997;3:313–9.
Ghaneh P, Greenhalf W, Humphreys M, Wilson D, Zumstein L, Lemoine NR, et al. Adenovirus-mediated transfer of p53 and p16(INK4a) results in pancreatic cancer regression in vitro and in vivo. Gene Ther. 2001;8:199–208.
Bajgelman MC, Costanzi-Strauss E, Strauss BE. Exploration of critical parameters for transient retrovirus production. J Biotechnol. 2003;103:97–106.
Ghattas IR, Sanes JR, Majors JE. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol. 1991;11:5848–59.
Strauss BE, Haas M. The region 3′ to the major transcriptional start site of the MDR1 downstream promoter mediates activation by a subset of mutant P53 proteins. Biochem Biophys Res Commun. 1995;217:333–40.
Naviaux RK, Costanzi E, Haas M, Verma IM. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol. 1996;70:5701–5.
Nyberg-Hoffman C, Shabram P, Li W, Giroux D, Aguilar-Cordova E. Sensitivity and reproducibility in adenoviral infectious titer determination. Nat Med. 1997;3:808–11.
Merkel CA, da Silva Soares RB, de Carvalho AC, Zanatta DB, Bajgelman MC, Fratini P, et al. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6. BMC Cancer 2010;10.
Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc. 2009;4:1798–806.
Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.
Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol. 1989;24:148–54.
Kaye FJ. RB and cyclin dependent kinase pathways: defining a distinction between RB and p16 loss in lung cancer. Oncogene. 2002;21:6908–14.
Robles AI, Harris CC. Clinical outcomes and correlates of TP53 mutations and cancer. Cold Spring Harb Perspect Biol. 2010;2:a001016.
Roth JA. Adenovirus p53 gene therapy. Expert Opin Biol Ther. 2006;6:55–61.
Muller PA, Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell. 2014;25:304–17.
Freed-Pastor WA, Prives C. Mutantp53: one name, many proteins. Genes Dev. 2012;26:1268–86.
Colombo F, Barzon L, Franchin E, Pacenti M, Pinna V, Danieli D, et al. Combined HSV-TK/IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results. Cancer Gene Ther. 2005;12:835–48.
Okada H, Pollack IF, Lotze MT, Lunsford LD, Kondziolka D, Lieberman F, et al. Gene therapy of malignant gliomas: a phase I study of IL-4-HSV-TK gene-modified autologous tumor to elicit an immune response. Hum Gene Ther. 2000;11:637–53.
Freytag SO, Stricker H, Pegg J, Paielli D, Pradhan DG, Peabody J, et al. Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate- to high-risk prostate cancer. Cancer Res. 2003;63:7497–506.
VanderVeen N, Raja N, Yi E, Appelman H, Ng P, Palmer D, et al. Preclinical efficacy and safety profile of allometrically scaled doses of doxycycline used to turn “on” therapeutic transgene expression from high-capacity adenoviral vectors in a glioma model. Hum Gene Ther Methods. 2016;27:98–111.
Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE. 2011;6:e18556.
Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc. 2012;2012:199–204.
We are grateful to Patrícia Léo and Juliana C. Gregório for the initial vector constructions. Financial support from the Sao Paulo Research Foundation (FAPESP): grant 98/15120-3 (ECS), grant 2015/26580-9 (BES), and fellowships 14/12322-5 (JGX), 11/21256-8 (RET).
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Xande, J.G., Dias, A.P., Tamura, R.E. et al. Bicistronic transfer of CDKN2A and p53 culminates in collaborative killing of human lung cancer cells in vitro and in vivo. Gene Ther 27, 51–61 (2020). https://doi.org/10.1038/s41434-019-0096-1