Abstract
Background
The MDM2 oncogene is amplified or overexpressed in many human cancers and MDM2 levels are associated with poor prognosis. MDM2 not only serves as a negative regulator of p53 but also has p53-independent activities. This study investigates the functions of the MDM2 oncogene in colon cancer growth and the potential value of MDM2 as a drug target for cancer therapy, by inhibiting MDM2 expression with an antisense antihuman-MDM2 oligonucleotide.
Materials and Methods
The selected antisense mixed-backbone oligonucleotide was evaluated for its in vitro and in vivo antitumor activity in human colon cancer models: LS174T cell line containing wild-type p53 and DLD-1 cell line containing mutant p53. The levels of MDM2, p53 and p21 proteins were quantified by Western blot analysis.
Results
In vitro antitumor activity was found in both cell lines, resulting from specific inhibition of MDM2 expression. In vivo antitumor activity of the oligonucleotide occurred in a dose-dependent manner in both models and synergistically or additive therapeutic effects of MDM2 inhibition and the cancer chemotherapeutic agents 10-hydroxycamptothecin and 5-fluorouracil were also observed.
Conclusions
These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53-independent mechanisms. We speculate that MDM2 inhibitors have a broad spectrum of antitumor activities in human cancers regardless of p53 status. This study should provide a basis for future development of anti-MDM2 antisense oligonucleotides as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.
Similar content being viewed by others
References
Prives C, Hall PA. (1999) The p53 pathway. J. Pathol. 187: 112–126.
Fakharzadeh SS, Trusko SP, George DL. (1991) Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. EMBO J. 10: 1565–1569.
Piette J, Neel H, Marechal V. (1997) Mdm2: keeping p53 under control. Oncogene 15: 1001–1010.
Momand J, Zambetti GP. (1997) Mdm-2: “big brother” of p53. J. Cell. Biochem. 64: 343–352.
Lozano G, Montes de Oca Luna R. (1998) MDM2 function. Biochim. Biophys. Acta 1377: M55–M59.
Juven-Gershon T, Oren M. (1999) Mdm2: the ups and downs. Mol. Med. 5: 71–83.
Freedman DA, Wu L, Levine AJ. (1999) Functions of the MDM2 oncoprotein. Cell Mol. Life Sci. 55: 96–107.
Freedman DA, Levine AJ. (1999) Regulation of p53 protein by MDM2 oncoprotein-Thirty eighth G.H.A. Clowes memorial award lecture. Cancer Res. 59: 1–7.
Momand J, Jung D, Wilczynski S, Niland J. (1998) The MDM2 gene amplification database. Nucleic Acids Res. 26: 3453–3459.
Bueso-Ramos CE, Yang Y, deLeon E, McCown P, Stass, SA, Albitar M. (1993) The human MDM-2 oncogene is overexpressed in leukemias. Blood 82: 2617–2623.
Watanabe T, Hotta T, Ichikawa A, et al. (1994) The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin. Blood 84: 3158–3165.
Landers JE, Haines DS, Strauss JF, George DL. (1994) Enhanced translation: a novel mechanism of mdm2 oncogene overexpression identified in human tumor cells. Oncogene 9: 2745–2750.
Landers JE, Cassel SL, George DL. (1997) Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer Res. 57: 3562–3568.
Lonardo F, Ueda T, Huvos AG, Healey J, Ladanyi M. (1997) P53 and MDM2 alterations in osteosarcomas. Cancer 79: 1541–1547.
Wurl P, Meye A, Berger D, et al. (1997) Prognostic relevance of C-terminal mdm2 detection is enhanced by p53 positivity in soft tissue sarcomas. Diagnostic Mol. Pathol. 6: 249–254.
Stefanou DG, Nonni AV, Agnantis NJ, Athanassiadou SE, Briassoulis E, Pavlidis N. (1998) P53/MDM-2 immunohistochemical expression correlated with proliferative activity in different subtypes of human sarcomas: a ten-year follow-up study. Anticancer Res. 18: 4673–4682.
Yokoyama R, Schneider-Stock R, Radig K, Wex T, Roessner A. (1998) Clinicopathologic implications of MDM2, p53 and K-ras gene alterations in osteosarcomas: MDM2 amplification and p53 mutations found in progressive tumors. Pathol. Res. Pract. 194: 615–621.
Jiang M, Shao Z-M, Wu J, et al. (1997) P21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis. Int. J. Cancer (Pred. Oncol.) 74: 529–534.
Tanner B, Hengstler JG, Laubscher S, et al. (1997) Mdm2 mRNA expression is associated with survival in ovarian cancer. Int. J. Cancer (Pred. Oncol.) 74: 438–442.
Dellas A, Schultheiss E, Almendral AC, et al. (1997) Altered expression of mdm2 and its association with p53 protein status, tumor-cell-proliferation rate and prognosis in cervical neoplasia. Int. J. Cancer (Pred. Oncol.) 74: 421–425.
Girod SC, Pfeiffer P, Ries J, Pape H-D. (1998) Proliferative activity and loss of function of tumor suppressor genes as biomarkers in diagnosis and prognosis of benign and preneoplastic oral lesions and oral squamous cell carcinoma. Br. J. Oral Maxillofacial Surg. 36: 252–260.
Agrawal S, Mathur M, Srivastava A, Ealhan R. (1999) MDM2/p53 co-expression in oral premalignant and malignant lesions: potential prognostic implication. Oral Oncol. 35: 209–216.
Ehrmann J, Kolar Z, Vojtesek B, Kala M, Komenda S, Oulton A. (1997) Prognostic factors in astrocytomas: relationship of p53, MDM-2, Bcl-2 and PCNA immunohistochemical expression to tumor grade and overall patient survival. Neoplasma 44: 299–304.
Shimada Y, Imamura M, Shibagaki I, et al. (1997) Genetic alterations in patients with esophageal cancer with short- and long-term survival rates after curative esophagectomy. Ann. Surg. 226: 162–168.
Valassiadou K, Stenfanaki K, Tzardi M, et al. (1997) Immunohistochemical expression of p53, bcl2, mdm2 and waf1/p21 proteins in colorectal adenocarcinomas. Anticancer Res. 17: 2571–2576.
Korkolopoulou P, Christodoulou P, Kapralos P, et al. (1997) The role of p53, MDM2 and c-erb B-2 oncoproteins, epidermal growth factor receptor and proliferation markers in the prognosis of urinary bladder cancer. Pathol. Res. Pract. 193: 767–775.
Osman I, Scher H, Zhang Z-F, et al. (1997) Alterations affecting the p53 control pathway in Bilharzial-related bladder cancer. Clin. Cancer Res. 3: 531–536.
Shiina H, Igawa M, Shigeno K, et al. (1999) Clinical significance of mdm2 and p53 expression in bladder cancer. A comparison with cell proliferation and apoptosis. Oncology 56: 239–247.
Ozdemir E, Kakehi Y, Okuno H, Habuchi T, Okada Y, Yoshida O. (1997) Strong correlation of basement membrane degradation with p53 inactivation and/or MDM2 overexpression in superficial urithelial carcinomas. J. Urol. 158: 206–211.
Marks DI, Kurz BW, Link MP, et al. (1997) Altered expression of p53 and mdm-2 proteins at diagnosis is associated with early treatment failure in childhood acute lymphoblastic leukemia. J. Clin. Oncol. 15: 1158–1162.
Sanchez E, Chacon I, Plaza MM, et al. (1998) Clinical outcome in diffuse large B-cell lymphoma is dependent on the relationship between different cell-cycle regulator proteins. J. Clin. Oncol. 16: 1931–1939.
Prives C. (1998) Signaling to p53: breaking the MDM2-p53 circuit. Cell 95: 5–8.
Bottger A, Bottger V, Sparks A, Liu WL, Howard SF, Lane DP. (1997) Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo. Curr. Biol. 7: 860–869.
Midgley CA, Lane DP. (1997) P53 protein stability in tumor cells is not determined by mutation but is dependent on Mdm2 binding. Oncogene 15: 1179–1189.
Bottger A, Bottger A, Garcia EC, et al. (1997) Molecular characterization of the hdm2-p53 interaction. J. Mol. Biol. 269: 744–756.
Blattner C, Sparks A, Lane D. (1999) Transcription factor E2F-1 is upregulated in response to DNA damage in a manner analogous to that of p53. Mol. Cell. Biol. 19: 3704–3713.
Chen L, Agrawal S, Zhou W, Zhang R, Chen J. (1998) Synergistic activation of p53 by inhibition of MDM2 expression and DNA damage. Proc. Natl. Acad. Sci. U.S.A. 95: 195–200.
Chen L, Agrawal S, Zhou W, Zhang R, Chen J. (1999) Ubiquitous induction of p53 in tumor cells by antisense inhibition of MDM2 expression. Mol. Med. 5: 21–34.
Wang H, Oliver P, Zeng X, et al. (1999) MDM2 oncogene as a target for cancer therapy: an antisense approach. Int. J. Oncol. 15: 653–660.
Mandel JS. (1996) Screening for colon and rectal cancer. Cancer Cont. 3: 170–177.
Agrawal S, Jiang Z, Zhao Q, et al. (1997) Mixed-backbone oligonucleotides as second generation antisense oligonucleotides: In vitro and in vivo studies. Proc. Natl. Acad. Sci. U.S.A. 94: 2620–2625.
Zhang R, Li Y, Cai Q, Liu T, Sun H, Chambless B. (1998) Preclinical pharmacology of the natural product anticancer agent 10-hydroxycamptothecin, an inhibitor of topoisomerase I. Cancer Chemother. Pharm. 41: 257–267.
Cai Q, Lindsey JR, Zhang R. (1997) Regression of human colon cancer xenografts in SCID mice following oral administration of water-insoluble camptothecins, natural product topoisomerase I inhibitors. Int. J. Oncol. 10: 953–960.
Wang H, Cai Q, Zeng X, Yu D, Agrawal S, Zhang R. (1999) Anti-tumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to RIαsubunit of protein kinase A after oral administration. Proc. Natl. Acad. Sci. U.S.A. 96: 13989–13994.
Liu W, Zhang R. (1998) Upregulation of p21/WAF1/CIP1 in human breast cancer cell lines MCF-7 and MDA-MB-468 undergoing apoptosis induced by natural product anticancer, agents 10-hydroxycamptothecin and camptothecin through p53-dependent and independent pathways. Int. J. Oncol. 12: 793–804.
Freedman DA, Levine AJ. (1998) Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Mol. Cell. Biol. 18: 7288–7293.
Zhang R, Wang H. (2000) MDM2 oncogene as a novel target for human cancer therapy. Curr. Pharm. Design 6: 393–416.
Wang H, Nan L, Yu D, Agrawal S, Zhang R. (2001) Antisense anti-MDM2 oligonucleotides as a novel therapeutic approach to human breast cancer: in vitro and in vivo activities and mechanisms. Clin. Cancer Res. 7: 3613–3624.
Wurl P, Meye A, Schmidt H, et al. (1998) High prognostic significance of mdm2/p53 co-overexpression in soft tissue sarcomas of the extremities. Oncogene 16: 1183–1185.
Burton EC, Lamborn KR, Forsyth P, et al. (2002) Aberrant p53, MDM2, and proliferation differ in glioblastomas from long-term compared with typical survivors. Clin. Cancer Res. 8: 180–187.
Lu M-L, Wikman F, Orntoft TF, et al. (2002) Impact of alterations affecting the p53 pathway in bladder cancer on clinical outcome, assessed by conventional and array-based methods. Clin. Cancer Res. 8: 171–179.
Dorigo O, Turla ST, Lebedeva S, Gjerset RA. (1998) Sensitization of rat glioblastoma multiforme to cisplatin in vivo following restoration of wild-type p53 function. J. Neurosurg. 88: 535–540.
Seth P, Katayose D, Li Z, et al. (1997). A recombinant adenovirus expressing wild type p53 induces apotosis in drug resistant human breast cancers: a gene therapy approach for drug-resistant cancers. Cancer Gene Ther. 4: 383–390.
Xiao Z, Chen J, Levine AJ, et al. (1995) Interaction between the retinoblastoma protein and the oncoprotein MDM2. Nature 375: 694–698.
Martin K, Trouche D, Hagemeier C, Sorensen TS, La Thangue NB, Kouzarides T. (1995) Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein. Nature 375: 691–694.
Thomas A, White E. (1998) Suppression of the p300-dependent mdm2 negative-feedback loop induces the p53 apoptotic function. Genes Dev. 12: 1975–1985.
Pomerantz J, Schreiber-Agus N, Liegeois NJ, et al. (1998) The Ink4a tumor suppressor gene product, p19ARF, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell 92: 713–723.
Honda R, Yasuda H. (1999) Association of p19 ARF with mdm2 inhibits ubiquitin ligase activity of mdm2 for tumor suppressor p53. EMBO J. 18: 22–27.
Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ. (1998) Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Natl. Acad. Sci. U.S.A. 95: 8292–8297.
Zeng X, Chen L, Jost CA, et al. (1999) Mdm2 suppresses p73 function without promoting p73 degradation. Mol. Cell. Biol. 19: 3257–3266.
Juven-Gershon T, Shifman O, Unger T, Elkeles A, Haupt Y, Oren M. (1998) The Mdm2 oncoprotein interacts with the cell fate regulator numb. Mol. Cell. Biol. 18: 3974–3982.
Marechal V, Elenbass B, Piette J, Nicolas JC, Levine AJ. (1994) The ribosomal L5 protein is associated with mdm2 and mdm-2-p53 complexes. Mol. Cell. Biol. 14: 7414–7420.
Elenbaas B, Dobbelstein M, Roth J, Shenk T, Levine AJ. (1996) The MDM2 oncoprotein binds specifically to RNA through its RING finger domain. Mol. Med. 2: 439–451.
Fiddler TA, Smith L, Tapscott SJ, Thayer MJ. (1996) Amplification of MDM2 inhibits MyoD-mediated myogenesis. Mol. Cell Biol. 16: 5048–5057.
Olson DC, Marechal V, Momand J, Chen J, Romocki C, Levine A. (1993) Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes. Oncogene 8: 2353–2360.
Sigalas I, Calvert AH, Anderson JJ, Neal DE, Lunec J. (1996) Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences: transforming ability and frequent detection in human cancer. Nat. Med. 2: 912–917.
Matsumoto R, Tada M, Nozaki M, Zhang C-L, Sawamura Y, Abe H. (1998) Short alternative splice transcripts of the mdm2 oncogene correlate to malignancy in human astrocytic neoplasms. Cancer Res. 58: 609–613.
Pinkas J, Naber SP, Butel JS, Medina D, Jerry DJ. (1999) Expression of MDM2 during mammary tumorigenesis. Int. J. Cancer 81: 292–298.
Lundgren K, Montes de Oca Luna R, Mcneill YB, et al. (1997) Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53. Genes Dev. 11: 714–725.
Sun P, Dong P, Dai K, Hannon GJ, Beach D. (1998) p53-independent role of MDM2 in TGFβ1 resistance. Science 282: 2270–2272.
Blaydes JP, Wynford-Thomas, D. (1998) The proliferation of normal human fibroblasts is dependent upon negative regulation of p53 function by mdm2. Oncogene 16: 3317–3322.
Komarov PG, Komarova EA, Kondratov RV, et al. (1999) A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285: 1733–1736.
Wang H, Prasad G, Buolamwini JK, Zhang R. (2001) Antisense anticancer oligonucleotide therapeutics. Curr. Cancer Drug Targets 1: 177–196.
Acknowledgments
This study was supported by National Institute of Health grant R01 CA 80698 (to R.Z.). We thank Dr. J. Chen for providing anti-MDM2 antibodies, L. P. Le, J. Sutton, M. Shackelford, and J. Hosmer for their excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wang, H., Nan, L., Yu, D. et al. Anti-Tumor Efficacy of a Novel Antisense Anti-MDM2 Mixed-Backbone Oligonucleotide in Human Colon Cancer Models: p53-Dependent and p53-Independent Mechanisms. Mol Med 8, 185–199 (2002). https://doi.org/10.1007/BF03402011
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03402011