Transkriptionales Targeting zur zielgerichteten Krebsgentherapie des Mammakarzinoms

Zusammenfassung

Fragestellung: Die Gentherapie ist ein Therapiekonzept der molekularen Medizin, welches durch ein Targeting der Vektoren die gerichtete Expression therapeutischer Gene für eine systemische und tumorspezifische Krebstherapie ermöglicht. Tumorspezifische Promotoren (TSPs) sind geeignet, um eine selektive transgene Expression in der Krebszelle zu erreichen. Heparanase (HPR) wird in Mammakarzinomzellen überexprimiert und spielt eine wichtige Rolle im Rahmen der Metastasenentwicklung. Ziel dieser Studie war es zu untersuchen, ob HPR ein geeigneter Promotor zur Krebsgentherapie des Mammakarzinoms ist.

Material und Methodik: Untersuchungen zur HPR-Expression erfolgten mittels quantitativer RT-PCR in etablierten Mammakarzinomzelllinien, primären aufgereinigten Mammakarzinomzellen von Patientinnen und gesunden Kontrollzellen. Zur Untersuchung der Promotoraktivität wurde ein Adenoviraler (Ad) Vektor (AdHPRluc) konstruiert. Dieser exprimiert Luziferase als Reportergen unter der transkriptionalen Kontrolle des HPR-Promotors. Zur Kontrolle diente ein Ad-Vector, welcher den ubiquitär exprimierten CMV-Promotor (AdCMVluc) enthält.

Ergebnisse: Die quantitative RT-PCR zeigte eine 4,5 - 44,6fach, (p < 0,05) erhöhte Expression des HPR-Gens in verschiedenen Mammakarzinomzelllinien im Vergleich zu gesunden Mammazelllinien. AdHPRluc zeigte eine hohe Aktivität in verschiedenen Mammakarzinomzelllinien (5,5 - 12,7 % im Vergleich zu CMV) und primären Mammakarzinomzellen (8,8 - 14,4 %).

Schlussfolgerung: Der Promotor des HPR-Gens ist geeignet für transkriptionale Targetingstrategien im Rahmen einer adenoviralen Krebsgentherapie des Mammakarzinoms.

Abstract

Purpose: Gene therapy with adenoviral (Ad) vectors is a promising new approach for different tumor types. Strategies to restrict adenoviral-mediated transgene expression are important to avoid side effects due to gene transfer into healthy cells. Heparanase (HPR) is highly overexpressed in human cancers including breast cancer but low or undetectable in differentiated, healthy tissue.

Material and Methods: To evaluate the utility of HPR as a TSP for breast cancer gene therapy, RT-PCR was performed to evaluate the expression of the HPR gene in various established breast cancer cell lines, primary human breast cancer tissue samples and normal breast cell lines. We constructed an Ad vector, AdHPR.luc, encoding luciferase under the control of the HPR promoter to determine relative activity in a variety of breast cancers, normal human breast cell lines and purified breast cancer tissue samples. An Ad vector containing the ubiquitously expressed CMV promoter (AdCMV.luc) was used as control.

Results: Quantitative RT-PCR revealed a 4.5 - 44.6fold, (p < 0.05) increased expression of the HPR gene in several breast cancer cell lines compared to a normal breast control cell line. When compared to the ubiquitous CMV promoter, the HPR promoter showed a high level of activity in four breast cancer cell lines (5.5 - 12.7 % compared to CMV) and primary breast cancer patient samples (8.8 - 14.4 %), whereas activity in normal breast cells was low.

Conclusion: These findings show that the HPR pathway is a target for the development of breast cancer directed gene therapy strategies.

Schlüsselwörter

Krebsgentherapie - Mammakarzinom - Heparanase - transkriptionales Targeting

Key words

Breast cancer - tissue-specific promoters - transcriptional targeting - cancer gene therapy

Literatur

• 1 Hemminki A, Alvarez R D. Adenoviruses in oncology: a viable option?.  BioDrugs. 2002;  16 77-87
• 2 Hitt M M, Addison C L, Graham F L. Human adenovirus vectors for gene transfer into mammalian cells.  Adv Pharmacol. 1997;  40 137-206
• 3 Mulvihill S, Warren R, Venook A. et al . Safety and feasibility of injection with an E1 B-55 kDa gene-deleted, replication-selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase I trial.  Gene Ther. 2001;  8 308-315
• 4 Bauerschmitz G J, Hemminki A, Curiel D T, Dall P. Tumour-dependent replicating adenoviruses in the treatment of carcinomas.  Zentralbl Gynakol. 2004;  126 280-281
• 5 Bilbao G, Gomez-Navarro J, Curiel D T. Targeted adenoviral vectors for cancer gene therapy.  Adv Exp Med Biol. 1998;  451 365-374
• 6 Bilbao R, Gerolami R, Bralet M P. et al . Transduction efficacy, antitumoral effect, and toxicity of adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir therapy of hepatocellular carcinoma: the woodchuck animal model.  Cancer Gene Ther. 2000;  7 657-662
• 7 Huard J, Lochmuller H, Acsadi G, Jani A, Massie B, Karpati G. The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants.  Gene Ther. 1995;  2 107-115
• 8 Tomko R P, Xu R, Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses.  Proc Natl Acad Sci USA. 1997;  94 3352-3356
• 9 Nettelbeck D M, Jerome V, Muller R. Gene therapy: designer promoters for tumour targeting.  Trends Genet. 2000;  16 174-181
• 10 Hulett M D, Freeman C, Hamdorf B J, Baker R T, Harris M J, Parish C R. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis.  Nat Med. 1999;  5 803-809
• 11 Nakajima M. Heparanases and tumor metastasis.  Tanpakushitsu Kakusan Koso. 1992;  37 1753-1758
• 12 Parish C R, Freeman C, Hulett M D. Heparanase: a key enzyme involved in cell invasion.  Biochim Biophys Acta. 2001;  1471 M99-108
• 13 Vlodavsky I, Goldshmidt O, Zcharia E. et al . Molecular properties and involvement of heparanase in cancer progression and normal development.  Biochimie. 2001;  83 831-839
• 14 Xiao Y, Kleeff J, Shi X, Buchler M W, Friess H. Heparanase expression in hepatocellular carcinoma and the cirrhotic liver.  Hepatol Res. 2003;  26 192-198
• 15 Takaoka M, Naomoto Y, Ohkawa T. et al . Heparanase expression correlates with invasion and poor prognosis in gastric cancers.  Lab Invest. 2003;  83 613-622
• 16 Staquicini F I, Moreira C R, Nascimento F D. et al . Enzyme and integrin expression by high and low metastatic melanoma cell lines.  Melanoma Res. 2003;  13 11-18
• 17 Maxhimer J B, Quiros R M, Stewart R. et al . Heparanase-1 expression is associated with the metastatic potential of breast cancer.  Surgery. 2002;  132 326-333
• 18 Speirs V, Green A R, Walton D S. et al . Short-term primary culture of epithelial cells derived from human breast tumours.  Br J Cancer. 1998;  78 1421-1429
• 19 Elkin M, Cohen I, Zcharia E. et al . Regulation of heparanase gene expression by estrogen in breast cancer.  Cancer Res. 2003;  63 8821-8826
• 20 Vlodavsky I, Friedmann Y, Elkin M. et al . Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.  Nat Med. 1999;  5 793-802
• 21 He T C, Zhou S, da Costa L T, Yu J, Kinzler K W, Vogelstein B. A simplified system for generating recombinant adenoviruses.  Proc Natl Acad Sci USA. 1998;  95 2509-2514
• 22 Koshikawa N, Takenaga K, Tagawa M, Sakiyama S. Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells.  Cancer Res. 2000;  60 2936-2941
• 23 Yamamoto M, Alemany R, Adachi Y, Grizzle W E, Curiel D T. Characterization of the cyclooxygenase-2 promoter in an adenoviral vector and its application for the mitigation of toxicity in suicide gene therapy of gastrointestinal cancers.  Mol Ther. 2001;  3 385-394
• 24 Barker S D, Coolidge C J, Kanerva A. et al . The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy.  J Gene Med. 2003;  5 300-310
• 25 Garver Jr R I, Goldsmith K T, Rodu B, Hu P C, Sorscher E J, Curiel D T. Strategy for achieving selective killing of carcinomas.  Gene Ther. 1994;  1 46-50
• 26 Zhang D H, Salto-Tellez M, Chiu L L, Shen L, Koay E S. Tissue microarray study for classification of breast tumors.  Life Sci. 2003;  73 3189-3199