NUOVI BERSAGLI TERAPEUTICI NEL CANCRO PROSTATICO: TELOMERASI E SURVIVINA

Nadia ZaffaroniU.O. 10

Dipartimento di Oncologia Sperimentale e Laboratori

23 novembre 2005

Identification of critical determinants of the transition from androgen-dependent 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 androgen-independent disease

- Target validation in well-characterized preclinical models- Development of mechanism-based therapeutic approaches

The anti-apoptosis protein SURVIVINbelongs to the family of IAPs (inhibitors of apoptosisproteins) and inhibits terminal caspases

SURVIVIN

The anti-apoptosis protein SURVIVINinteracts with microtubules through its 42-aminoacidcoiled-coil domain and controls the progression of cancer cells throughout mitosis

Cell cycle-dependent transcription

Post-translational stabilization through:

- phosphorylation by p34cdc2

- interaction with HSP90

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 largenumber of human tumor types

Main reasons for considering survivin anattractive therapeutic target in cancer

• differential expression in tumours versusnormal tissues

• its potential requirement for maintainingtumour cell viability

STRATEGIES TO INHIBIT SURVIVIN

SURVIVIN MOLECULAR ANTAGONISTS

• antisense oligonucleotides• ribozymes• siRNAstargeting 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

FL-2H

DU145/CTR DU145/RZ

0 200 400 600 800 1000 0 200 400 600 800 1000

Cou

nts

Days after cell injection

Tum

or V

olum

e (m

m3 )

0 10 20 30 40 50 601

10

100

1000

Pennati et al, Oncogene, 2004

Effects of survivin inhibition on prostate cancer cells

Survivin

PCNAsiR

Nacon

trol

Lipo

siRNA

siRNac

ontro

l

Lipo

siRNA

siRNac

ontro

l

Lipo

siRNA

siRNac

ontro

l

Lipo

siRNA

siRNac

ontro

l

Lipo

siRNA

2 3 7 10 45 siRNA

Silencing of survivin by siRNAs inhibits prostate cancer cell growthand induces apoptosis

10 2 0

20

40

60

80

100

cell

grow

th(%

of c

ontr

ol)

control 3

40%

siRNA

siRNA2

Paduano et al., Mol Cancer Ther, 2006

The interaction with Hsp90 appears necessary forsurvivin activation

Rational design of a peptido-mimetic molecule able tointerfere with the Hsp90/survivin binding site

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

Plescia et al., Cancer Cell, 2005

Survivin inhibitors in clinical trials

• Antisense phosphorothioateoligonucleotide (Eli Lilly)

•• TelomeresTelomeres are are DNADNA--proteinprotein structuresstructures at the at the endsends of of eukarioticeukarioticchromosomeschromosomes and are and are essentialessential forfor maintainingmaintaining the the stabilitystability and and integrityintegrity of of chromosomeschromosomes byby preventingpreventing degradationdegradation and and endend--toto--endendfusionfusion..

•• TelomeresTelomeres of of humanhuman somaticsomatic cellscells shortenshorten withwith eacheach round of round of cellcelldivisiondivision becausebecause of the incomplete of the incomplete replicationreplication of of linearlinear DNA. DNA.

•• The The sequentialsequential lossloss of of telomerictelomeric DNA limits the DNA limits the proliferativeproliferative lifespanlifespanof of cellscells toto a definite a definite numbernumber of of cellcell divisionsdivisions beforebefore theythey enterenter a statea stateof of replicativereplicative senescencesenescence..

TELOMERES

HUMAN TELOMERASEHUMAN TELOMERASE

TelomeraseTelomerase isis a a ribonucleoproteinribonucleoprotein complexcomplex thatthat maintainsmaintains andandelongates telomeres by the elongates telomeres by the de novode novo synthesis of telomeric synthesis of telomeric repeatsrepeats(TTAGGG).(TTAGGG).

TelomeraseTelomerase componentscomponents::hTERT hTERT the catalytic reverse transcriptase the catalytic reverse transcriptase

subunitsubunithTR hTR the the templatetemplate--containingcontaining RNA RNA

subunitsubunit

TelomeraseTelomerase activityactivity isis controlledcontrolled byby

•• hTERT transcription and alternative hTERT transcription and alternative splicingsplicing

•• phosphorylationphosphorylation//dephosphorylationdephosphorylation of of hTERThTERT and hTEP1 proteinsand hTEP1 proteins

•• interaction of telomerase holoenzyme interaction of telomerase holoenzyme withwith telomeretelomere--associatedassociatedproteinsproteins (TRF1 and (TRF1 and tankyrasetankyrase) and ) and molecularmolecular chaperonschaperons (p23 and(p23 andHsp90).Hsp90).

TelomeraseTelomerase activityactivity isis generallygenerally absentabsent in in normalnormal somaticsomaticcellscells..

TelomeraseTelomerase isis reactivatedreactivated in 80 in 80 -- 90% of 90% of humanhuman cancerscancers of of differentdifferent histotypeshistotypes..

The The ectopicectopic expressionexpression of of hTERThTERT in in cooperationcooperation withwith the the oncogenesoncogenes SV40 SV40 largelarge--TT and and HH--rasras resultsresults in direct in direct tumorigenictumorigenic conversionconversion of of humanhuman epithelialepithelial and and fibroblastfibroblastcellscells

TelomeraseTelomerase isis a a potentialpotential selectiveselective target target forfor anticancer anticancer therapytherapy

Approaches for the inhibition of telomerase activity

RNA RNA componentcomponent ((hTRhTR) ) -- antisenseantisense oligonucleotidesoligonucleotides-- peptide peptide nucleicnucleic acidsacids-- ribozymesribozymes-- siRNAssiRNAs

CatalyticCatalytic subunitsubunit ((hTERThTERT) ) -- nucleotidenucleotide analoguesanalogues-- antisenseantisense oligonucleotidesoligonucleotides-- dominantdominant negative negative mutantsmutants

TelomericTelomeric DNA DNA -- DNADNA quadruplexquadruplex--interactinginteractingagentsagents

SmallSmall--moleculemolecule ligandsligands forfor the the humanhuman telomerictelomeric DNA DNA quadruplexquadruplex

IC50 (IC50 (µµM)M) QuadruplexQuadruplex:duplex:duplexselectivityselectivity

Anthraquinones Anthraquinones 1.0 1.0 -- 20 20 0.8 0.8 -- 3.33.3Porphyrins Porphyrins 6.5 6.5 -- 6565 1.41.4AcridinesAcridines 1.0 1.0 -- >50 >50 n.d.n.d.FluorenonesFluorenones 8.0 8.0 -- >50>50 3.73.7TetracyclicTetracyclic quinonequinone 5.4 5.4 2.4 2.4

CellularCellular effectseffects of of hTRhTR antagonistsantagonists

TelomeraseTelomerase activityactivity AlwaysAlways inhibitedinhibited ((toto a a variablevariable extentextentwithwith the the differentdifferent approachesapproaches))

TelomereTelomere lengthlength TelomereTelomere shortening was observed shortening was observed after after severalseveral passagespassages in culture in culture ((monthsmonths!)!)

CellCell growthgrowth Inhibition of Inhibition of cellcell proliferationproliferation waswasobservedobserved after after telomeretelomere shorteningshortening

hTERT antagonists

5’ 3’uccucgccuccacucacacaggtggaugugacgggcgcguacgacaccaucccccaggacintron 5 exon 6

alternativesplicing site

splicingsite

caccuacacugcccgcgcagcggaggugaguguguc

uguguccaccuacacugc

3’ 5’

3’ 5’

guccaccuacacugcccg3’

3’

5’

5’

gaguguguccaccuacac 5’3’

ggugaguguguccaccua 5’3’

ggaggugaguguguccac 5’3’

aggagcggaggugagugu 5’3’

36 nt delected in the α splicing variant

1

2

3

4

5

6

7

8

2’-O-Methyl-RNA-phosphorothioate oligomers

TELOMERASE ACTIVITY IN DU145 CELLS EXPOSED TO 2’-O-METHYL-RNA-PTOs OLIGOMERS

Cont

rol

1 2 3 4 5 6 7 8 Blank

+85°

CTS

R8

ITAS

OLIGOMERS

0

50

100

Quantification of telomerase activity

Oligomers

Telo

mer

ase

activ

ity(%

of c

ontr

ol)

R1 R2 R3 R4 R5 R6 R7 R8

Brambilla et al., Cell Mol Life Science, 2004

104101100 102 1030

50

200

300

150

250

350

3%

Scramble

104101100 102 1030

50

200

300

150

250

350

40%

R7 oligomer

Num

bero

f cel

ls

FL-1

0

25

50

% o

f apo

ptot

icce

lls

Scramble R7 oligomer

0 24 48 72 0 24 48 72

Recovery (h)

ANALYSIS OF APOPTOTIC CELLS(TUNEL assay)

Folini et al., Eur J Cancer, 2005

Telomerase inhibition by a hammerhead ribozyme targeting hTERTmRNA induces immediate apoptosis in four ovarian cancer cell lines.

The kinetics of induction of cell death is not dependent on telomerelength, and no telomere shortening is observed (Saretzki et al., Cancer Gene Ther, 2002)

TSR8

moc

k

16 22 27 41 56 63 8 10 13 blan

k+8

5°C

siRN

As

24 48 24 96transfection recovery (h)

Cel

lnum

ber(

x106 )

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

siRNAs targeting hTERT mRNA inhibit human prostate cancer

cell growth

Telo

mer

ase

activ

ity( %

of c

ontr

ol)

Photochemicallyinternalised-PNA

6h 24h 48h 48h

hTERT-PNA

Scramble

0

50

100

0

50

100

0 40 60 80Light exposure (seconds)

Rel

ativ

e ce

llsu

rviv

al(%

of c

ontr

ol)

A photochemically-internalized PNA targeting hTERT mRNA inhibits human prostate

cancer cell growth

L’inibizione di hTERT, ma non di hTR, mediata da oligomeriantisenso 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

Telomerase inhibitors in clinical trials

• Antisense phosphoramidateoligonucleotide targeting hTR (Geron)