NUOVI BERSAGLI TERAPEUTICI NEL CANCRO PROSTATICO: TELOMERASI E SURVIVINA

Transcription

1 NUOVI BERSAGLI TERAPEUTICI NEL CANCRO PROSTATICO: TELOMERASI E SURVIVINA Nadia Zaffaroni U.O. 10 Dipartimento di Oncologia Sperimentale e Laboratori 23 novembre 2005

2 Identification of critical determinants of the transition from androgendependent to androgen-independent prostate cancer - alterations in the androgen receptor - induction of anti-apoptotic genes - role of HER kinases - telomerase reactivation - Identification of therapeutic targets for the androgenindependent disease - Target validation in well-characterized preclinical models - Development of mechanism-based therapeutic approaches

3 The anti-apoptosis protein SURVIVIN belongs to the family of IAPs (inhibitors of apoptosis proteins) and inhibits terminal caspases SURVIVIN

4 The anti-apoptosis protein SURVIVIN interacts with microtubules through its 42-aminoacid coiled-coil domain and controls the progression of cancer cells throughout mitosis

5 Cell cycle-dependent transcription Post-translational stabilization through: - phosphorylation by p34 cdc2 - interaction with HSP90

6 The anti-apoptosis protein SURVIVIN a is overexpressed in most human tumors but not in normal tissues a is associated with clinical progression in a large number of human tumor types

7 Main reasons for considering survivin an attractive therapeutic target in cancer differential expression in tumours versus normal tissues its potential requirement for maintaining tumour cell viability

8 STRATEGIES TO INHIBIT SURVIVIN

9 SURVIVIN MOLECULAR ANTAGONISTS antisense oligonucleotides ribozymes sirnas targeting survivin mrna Loss of survivin was able to trigger caspase-dependent apoptosis to enhance chemotherapy-induced cell death to dysregulate mitotic progression to promote tumor eradication when combined with immunotherapy and chemotherapy

10 Effects of survivin inhibition on prostate cancer cells DU145/CTR DU145/RZ Counts FL-2H Tumor Volume (mm 3 ) Days after cell injection

11 Silencing of survivin by sirnas inhibits prostate cancer cell growth and induces apoptosis sirna Lipo sirnacontrol sirna Lipo sirnacontrol sirna Lipo sirnacontrol sirna Lipo sirnacontrol sirna Lipo sirnacontrol sirna PCNA Survivin 100 sirna cell growth (% of control) 20 40% 0 control sirna

12 The interaction with Hsp90 appears necessary for survivin activation Rational design of a peptido-mimetic molecule able to interfere with the Hsp90/survivin binding site

13 The molecule destabilizes survivin and other Hsp90 client proteins. Moreover, it shows a cytotoxic effect greater than that induced by the Hsp90 inhibitor 17-AAG

14 Survivin inhibitors in clinical trials Antisense phosphorothioate oligonucleotide (Eli Lilly)

15 TELOMERES Telomeres are DNA-protein structures at the ends of eukariotic chromosomes and are essential for maintaining the stability and integrity of chromosomes by preventing degradation and end-to to-end fusion. Telomeres of human somatic cells shorten with each round of cell division because of the incomplete replication of linear DNA. The sequential loss of telomeric DNA limits the proliferative lifespan of cells to a definite number of cell divisions before they enter a state of replicative senescence.

16 HUMAN TELOMERASE Telomerase is a ribonucleoprotein complex that maintains and elongates telomeres by the de novo synthesis of telomeric repeats (TTAGGG). Telomerase components: htert the catalytic reverse transcriptase subunit htr the template-containing RNA subunit

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18 Telomerase activity is controlled by htert transcription and alternative splicing phosphorylation/dephosphorylation of htert and htep1 proteins interaction of telomerase holoenzyme with telomere-associated proteins (TRF1 and tankyrase) ) and molecular chaperons (p23 and Hsp90).

19 Telomerase activity is generally absent in normal somatic cells. Telomerase is reactivated in 80-90% of human cancers of different histotypes. The ectopic expression of htert in cooperation with the oncogenes SV40 large-t and H-ras results in direct tumorigenic conversion of human epithelial and fibroblast cells Telomerase is a potential selective target for anticancer therapy

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21 Approaches for the inhibition of telomerase activity RNA component (htr) - antisense oligonucleotides - peptide nucleic acids - ribozymes - sirnas Catalytic subunit (htert) - nucleotide analogues - antisense oligonucleotides - dominant negative mutants Telomeric DNA - DNA quadruplex-interacting agents

22 Small-molecule molecule ligands for the human telomeric DNA quadruplex IC50 (µm)( Quadruplex:duplex selectivity Anthraquinones Porphyrins Acridines >50 n.d. Fluorenones > Tetracyclic quinone

23 Cellular effects of htr antagonists Telomerase activity Always inhibited (to a variable extent with the different approaches) Telomere length Telomere shortening was observed after several passages in culture (months!) Cell growth Inhibition of cell proliferation was observed after telomere shortening

24 htert antagonists

25 2 -O-Methyl-RNA-phosphorothioate oligomers splicing intron 5 site exon 6 alternative splicing site uccucgccuccacucacacaggtggaugugacgggcgcguacgacaccaucccccaggac 36 nt delected in the α splicing variant 3 agcggaggugaguguguc caccuacacugcccgcgc 3 5 guccaccuacacugcccg uguguccaccuacacugc gaguguguccaccuacac 5 ggugaguguguccaccua 5 ggaggugaguguguccac aggagcggaggugagugu 5 8

26 TELOMERASE ACTIVITY IN DU145 CELLS EXPOSED TO 2 -O- METHYL-RNA-PTOs OLIGOMERS Control OLIGOMERS Blank +85 C TSR8 Quantification of telomerase activity 100 Telomerase activity (% of control) 50 ITAS 0 R1 R2 R3 R4 R5 R6 R7 R8 Oligomers Brambilla et al., Cell Mol Life Science, 2004

27 ANALYSIS OF APOPTOTIC CELLS (TUNEL assay) Scramble 250 Number of cells % R7 oligomer 40% 10 4 % of apoptotic cells Scramble R7 oligomer Recovery (h) FL Folini et al., Eur J Cancer, 2005

28 Telomerase inhibition by a hammerhead ribozyme targeting htert mrna induces immediate apoptosis in four ovarian cancer cell lines. The kinetics of induction of cell death is not dependent on telomere length, and no telomere shortening is observed (Saretzki et al., Cancer Gene Ther, 2002)

29 sirnas targeting htert mrna inhibit human prostate cancer cell growth TSR8 mock sirnas blank +85 C Cell number (x10 6 ) transfection recovery (h)

30 A photochemically-internalized PNA targeting htert mrna inhibits human prostate cancer cell growth Photochemically internalised-pna 100 Telomerase activity ( % of control) Relative cell survival (% of control) Light exposure (seconds) 0 6h 24h 48h 48h htert-pna Scramble

31 L inibizione di htert, ma non di htr, mediata da oligomeri antisenso appare in grado di inibire la proliferazione cellulare e indurre apoptosi in cellule tumorali umane in assenza di accorciamento dei telomeri htert ha funzioni citoprotettive/anti-apoptotiche indipendenti dalla sua attività catalitica di allungamento dei telomeri

32 Telomerase inhibitors in clinical trials Antisense phosphoramidate oligonucleotide targeting htr (Geron)