La survivina : un nuovo target terapeutico contro il cancro

1. La survivina:un nuovo target terapeutico contro il cancro

2. Il tumore si sviluppa da un’unica cellula dell’organismo che “impazzisce”, cioè rompe le regole basilari ed essenziali del comportamento cellulare fisiologico. Il singolo clone mutante inizia a proliferare sino a prendere il sopravvento sulla popolazione cellulare sana.

3. Le cellule tumorali hanno due caratteristiche essenziali: - si riproducono senza rispettare le normali limitazioni della proliferazione cellulare;- sono capaci di invadere il tessuto circostante e di formare metastasi

4. Cancerogenesi - Il tumore deriva da una singola cellula aberrante;- Il cancro è causato dal lento accumulo di numerose mutazioni in una singola linea cellulare (incubazione). Progressione tumorale: un lieve disordine iniziale del comportamento cellulare evolve gradualmente in un cancro conclamato.

5. Perché sono necessarie numerose mutazioni? Una cellula per poter diventare tumorale deve acquisire un certo livello di instabilità genetica, cioè deve mostrare un’aumentata tendenza ad accumulare mutazioni livello “ottimale” di instabilità genetica

6. Le mutazioni che vanno via via accumulandosi in una cellula tumorale interessano principalmente quei geni coinvolti nella regolazione del ciclo cellulare e dell’apoptosi. Il ciclo cellulare è la serie di eventi che avvengono in una cellula eucariotica tra una divisione cellulare e quella successiva.

7. PROTOONCOGENI e ONCOSOPPRESSORI In condizioni normali la regolazione del ciclo cellulare e della proliferazione dipende dall’equilibrio tra i prodotti dei protooncogeni e degli oncosoppressori

8. PROTOONCOGENI ONCOSOPPRESSORI ONCOGENI cellula normale ONCOSOPPRESSORI Mutazioni Delezioni Amplificazione genica cellula tumorale

9. L’apoptosi Aspetti morfologici: La cellula apoptotica perde i contatti con le cellule adiacenti Il nucleo si condensa e poi si frammenta La cellula si frammenta in corpuscoli chiamati corpi apoptotici I corpi apoptotici vengono riconosciuti e fagocitati dai macrofagi

10. IAP Family • c-IAP1 • c-IAP2, • XIAP • NAIP • Apollon • ML-IAP/livin • ILP-2 • survivina

11. Proteine IAP • Numerose copie di domini BIR • RING finger motif al COOH-terminale • Sito di ubiquitinazione Survivina • Unico dominio BIR • Ring finger motif assente • α-elica al COOH-terminale • Struttura dimerica • Espressione regolata dal ciclo cellulare

12. localizzazione citoplasmatica La survivina è presente in tre isoforme diverse derivanti da splicing alternativo: • survivina full-lenght (142 aa) • survivina-2B (165 aa) • survivina-ΔEx-3 (137 aa) localizzazione nucleare

13. Survivin-Δx3 and Survivin-2B: Two Novel Splice Variants of the Apoptosis Inhibitor Survivin with Different Antiapoptotic Properties CsabaMahotka, Michael Wenzel, Erik Springer, Helmut E. Gabbert, and Claus D. Gerharz (1999, CancerResearch) 500 bp 431 bp Electrophoresis of quantitative RT-PCR products. (RCC cell line) 329 bp Differential antiapoptotic potential of the novel survivin splice variants. Coding sequences of survivin and its two splice variants were ligated into the expression vector pEGFP-N3 and transfected into the human hepatoma cell line HepG2. Cell death was induced in transient transfectants by exposure to methotrexate (100 mg/ml). Survivin-DEx3 transfectants exhibited cell survival frequencies closely corresponding to that of survivin transfectants. In contrast, survivin-2B transfectants showed a marked reduction of cell survival.

14. L’espressione della survivina è legata al ciclo cellulare Control of apoptosis and mitotic spindle checkpoint by survivin Fengzhi Li, Grazia Ambrosini, Emily Y. Chu,Janet Plescia, Simona Tognin, Pier Carlo Marchisio & Dario C. Altieri (1998, Nature) Cell-cycle synchronization. HeLa cells (ATCC) were treated with 400mM mimosine (to arrest cells in G1), 2mM thymidine (to arrest cells in S) or 0.4 mgml-1 nocodazole (to arrest cells in G2/M) (Sigma), for 16 h at 37 °C , or were arrested at the G1/S boundary by sequential culture with 2mM thymidine and 400mM mimosine. Figure 1 Cell-cycle-dependent expression of survivin in G2/M. a, Northern blot of survivin (left) or GAPDH (right) RNA in exponentially growing (no treatment) or drug-synchronized HeLa cells (mimosine, G1; thymidine, S; nocodazole, G2/M).

15. Survivin promoter activity. Survivin±luciferase promoter constructs pLuc-230 (-39 to -268), pLuc-cyc1B (-36 to -268) and pLuc-cyc1.2 (+1 to -268)(numbering from the initiating methionine) were generated by PCR and confirmed by DNA sequencing. HeLa cells were transiently transfected by LipofectAMINE, synchronized and analysed for luciferase activity in a Lumat LB 9510 luminometer. Values were normalized for b-galactosidase activity at A405.

16. Ruolo nella mitosi Human Survivin Is a Kinetochore-associated Passenger Protein Dimitrios A. Skoufias, Cristiana Mollinari, Françoise B. Lacroix, and Robert L. Margolis (2000, JCB) Immunofluorescence microscopy of transfected cells stained with anti–HA (green) and propidium iodide (red) for chromatin. In different interphase cells, survivin localizes predominantly either in the cytoplasm (a) or the nucleus (b). In prophase through metaphase (c–e), survivin localizes in distinct spots on the chromatin. In anaphase, leaves the chromatin, associates with the spindle midzone (f), extending to the cortex (g), and remains localized in telophase (h) and the midbody (i).

17. Ruolo nella mitosi Se la survivina viene silenziata? • centrosomi sovrannumerari • fusi multipolari • interruzione citochinesi Esperimenti su topi knockout 100% morte embrioni Cellule poliploidi e multinucleate

18. Cell division and cell survival in the absence of survivin Dun Yang*, Alana Welm, and J. Michael Bishop (2004, PNAS) Fig. 2. Depletion of survivin elicits nuclear abnormalities. (A) The effect of survivin RNAi on cell-cycle distribution. RPE cells were either mock-treated or treated with shRNAs (suv1 and suv1m). Cell-cycle distribution was analyzed by flow cytometry 3 days after shRNA transfection. (B) Nuclear abnormalities in cells treated with survivin RNAi. Immunostaining of cells was performed 3 days after transfection with suv1 (a–e) or suv1m ( f). Tubulin was detected with fluorescein isothiocyanate-conjugated mouse anti--tubulin antibody (green), and nuclei were stained with DAPI (red). White arrowheads and arrows identify mininuclei and DNA bridges, respectively. (C) Percentage of cells with nuclear abnormalities illustrated in B. Each column represents the average of three independent experiments, and each experiment was done in triplicate. More than 500 cells were counted in each measurement. Error bars represent SD.

19. late telophaseearlytelophaseanaphasemetaphase Fig. 3. Chromosome behavior and microtubule assembly during mitosis are abnormal in survivin-depleted cells. RPE cells were fixed 2 days after treatment with either suv1m or suv1. Immunofluorescence was used to detect tubulin and survivin (green and blue, respectively, in the merged images). DNA was stained with DAPI (red in the merged images). Cells in metaphase (a, e, a, and e), anaphase (b, f, b, and f), early telophase (c, g, c, and g), and late telophase (d, h, d, and h) are shown. Arrowheads point to misaligned chromosomes, red arrows denote lagging chromosomes or chromatin bridges, and white arrows identify visible cortex contraction.

20. Ruolo nell’apoptosi Ipotesi sul meccanismo mediante il quale la survivina inibisce l’apoptosi: • inibizione diretta della caspasi-3 • interazione con la caspasi-9 • inibizione del complesso pro-apoptoticoSmac/DIABLO

21. Control of apoptosis and mitotic spindle checkpoint by survivin Fengzhi Li, Grazia Ambrosini, Emily Y. Chu, Janet Plescia, Simona Tognin, Pier Carlo Marchisio & Dario C. Altieri (1998, Nature) C84A: survivin dominant negative mutant (BIR mutated) Taxol: microtubule-stabilizing agent Displacement of endogenous survivin from microtubules by forced expression of the C84A BIR mutant resulted in a progressive increase in caspase-3 activity in synchronized HeLa cells. Taxol-induced apoptosis in NIH3T3 transfectants is inhibited by wild-type survivin, Bcl-2 and XIAP, but not by the survivin coil-less mutant M1±G99. pcDNA3 is a control vector. L’α-elica al COOH-terminale e il dominio BIR sono essenziali per l’attività anti-apoptotica

22. Regolazione della survivina La survivina è attivata mediante fosforilazione della Thr34 ad opera della chinasi ciclina-dipendente cdc2-ciclinaB1.

23. La survivina e’ uno dei geni antiapoptoticitrascrizionalmente repressi da p53. p53 è un fattore di trascrizione che regola il ciclo cellulare e svolge la funzione di oncosoppressore. E’ infatti in grado di bloccare la progressione cellulare o di indurre apoptosi. Nel 50% delle neoplasie p53 è mutata o deleta. C’è una connessione tra la perdita di p53 wild-type e l’over-espressione della survivina nel tumore?

24. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway Asra Mirza, Marnie McGuirk, Tish N Hockenberry, QunWu1, Hena Ashar, Stuart Black, Shu Fen Wen2, Luquan Wang1, Paul Kirschmeier, W Robert Bishop, Loretta L Nielsen, Cecil B Pickett1 and Suxing Liu (2002, Oncogene) • La survivina è repressa sia trascrizionalmente che a livello proteico da p53 2774qw1: human ovarian cancer cell line expressing mutant p53 Infected with replication-deficient adenovirus expressing wild-type p53

25. L’abbassamento dei livelli di mRNA è dovuto alla repressione dell’attività del promotore della survivina The human survivin gene has two copies of a p53-binding sequence in its promoter region. These two putative p53-binding sequences are located at nucleotide position 77 (p53S1) and 71294 (p53S2) relative to the proximal transcriptional start site (+1). To explore a possible role of these sequences in the repression of survivin gene expression by p53, a series of 5' deletions of the survivin promoter sequence (nucleotides 71895 to +366) were prepared using PCR.

26. The indicated survivin promoter constructs were transiently transfected into 2774 cells in the presence of wild-type p53 or empty expression vector alone. The empty luciferase reporter vector (BASIC) was used as a control for p53 induced non-specific change in transcription. Il sito p53S1 è indispensabile per la repressione da parte di p53?! La repressione dell’espressione della survivina operata da p53 non è mediata dal binding al DNA The sequence of the p53S1 in the construct Del-2 was mutated by base substitutions. Transient transfection experiments indicated that these mutants remained sensitive to p53-dependent repression of luciferase activity.

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28. Espressione della survivina nei tessuti normali La survivina è espressa ad elevati livelli durante lo sviluppo embrionale ma il gene che la codifica rimane quiescente nella maggior parte dei tessuti differenziati. I tessuti differenziati in cui la survivina è espressa (a bassi livelli) sono: • il timo • le cellule staminali CD34+ del midollo osseo • la mucosa gastrica

29. Il timo I timocitidouble-positive (CD4+/CD8+) mostrano overespressione della survivina mentre questa è espressa a livelli moderati nei single-positive e completamente assente nei linfociti T maturi.

30. Le cellule staminali CD34+ del midollo osseo CD34 è una glicoproteina di membrana che media l’adesione cellula-cellula. E’ espressa soprattutto negli stadi iniziali del processo di differenziazione dei tessuti ematopoietici. La produzione di survivina e’ mantenuta costante durante tutto il ciclo cellulare

31. Espressione della survivina nel tumore I tumori nei quali è stata dimostrata l’upregolazione della survivina sono i seguenti: • polmone • seno • colon • stomaco • esofago • pancreas • utero • ovaio • fegato

32. Prognosi più infausta L’overespressione della survivina nei tumori correla con: • un fenotipo più aggressivo e invasivo • una minore responsività agli agenti chemioterapici Il livello di espressione della survivina può essere considerato un fattore predittivo per la risposta al protocollo terapeutico

33. La survivina come target terapeutico contro il cancro La survivina è ritenuta un buon target terapeutico perché: • è richiesta dalla cellula per la proliferazione cellulare • ha attività anti-apoptotica • è espressa ad elevati livelli nei tessuti neoplastici e non in quelli adulti differenziati • è coinvolta nell’angiogenesi

34. RNA interference (siRNA o shRNA) • Taglio dell’mRNA della survivina tramite un ribozimamRNA-specifico • Espressione di un cDNA antisenso per la survivina • Espressione di survivinadominant negative mutant (T34A) • Utilizzo di piccole molecole antagoniste (CDK Inhibitors): • flavopiridolo: impedisce la fosforilazione della Thr34 • purvalanolo A: induce la dissociazione del complesso survivina-caspasi-9

35. Meccanismo RNA interference La RNA interference è un meccanismo mediante il quale alcuni frammenti di RNA a doppio filamento sono in grado di interferire con l'espressione genica.

36. Survivin Stable Knockdown by siRNA Inhibits Tumor Cell Growth and Angiogenesis in Breast and Cervical Cancers Qing-Xia Li1, Jing Zhao, Jia-Yun Liu2, Lin-Tao Jia, Hong-Yan Huang, Yan-Ming Xu, Yong Zhang., Rui Zhang, Cheng-Ji Wang, Li-Bo Yao, Si-Yi Chen, An-Gang Yang (2006, Cancer Biology and Therapy) Figure 1. shRNA-mediated inhibition of survivin expression. (A) survivin mRNA levels were determined by RT-PCR in parental SKBr-3 and HeLa cells. (B) Western blot analysis of survivin protein in parental SKBr-3 and HeLa cells. SKBr-3: human breast cancer cell line; HeLa: cervical carcinoma cell line The inhibition of survivin gene expression by RNAi in both SKBr-3 and HeLa cells can promote spontaneous apoptosis, increase apoptotic rates in response to several proapoptotic stimuli or resensitize these cancer cells to chemical drugs.

37. Che cos’è un ribozima? Un ribozima è una molecola di RNA in grado di catalizzare una reazione chimica.

38. ribozima Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo Marzia Pennati, Mara Binda, Michelandrea De Cesare, Graziella Pratesi, Marco Folini, Lorenzo Citti, Maria Grazia Daidone, Franco Zunino and Nadia Zaffaroni (2004, Carcinogenesis) The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv or the catalytically inactive ribozyme mutRZsurv. In vitro cleavage of the 362-nt synthetic 32P-RNA substrate by RZsurv into the cleavage products (310 and 52-nt). The reaction products were resolved on 5% polyacrylamide/7 M urea gel and visualized by autoradiography.

39. JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan compared with JR8/mutRZsurv cells. Fig. 3. Clonogenic cell survival curves obtained after exposure of JR8/ RZsurv (filled circle) and JR8/mutRZsurv (filled square) cells to topotecan for 24 h. Survivin inhibition enhances the antitumor activity of topotecan in vivo.

40. cDNA antisenso

41. cDNA antisenso The survivingene was identified by hybridization with the EPR-1 cDNA, and its coding sequence was found to be extensively complementary to that of EPR-1 Pulsed field gel electrophoresis and FISH studies suggested the existenceof a single EPR-1/Survivin locus spanning 75–130 kb on chromosome 17q25. The use of single strand-specific probes further demonstrated that EPR-1 and Survivin originated from two structurally different messages of 1.3 and 1.9 kb Induction of Apoptosis and Inhibition of Cell Proliferation by survivin Gene Targeting Grazia Ambrosini, Colette Adida, Giorgio Sirugo and Dario C. Altieri (1998, The Journal ofBiologicalChemestry) HeLa cells were stably transfected with an EPR-1 cDNA under the control of an metallothionein-inducible promoter. In these experiments, ZnSO4 induction of EPR-1 mRNA suppressed the expression of endogenous survivin, which resulted in massive apoptosis in growth factor-deprived HeLa cells. 9.6% 18.2%

42. dominant-negativemutant Inhibition of melanoma tumor growth in vivo by survivintargeting Douglas Grossman, Paul J. Kim, Jeffrey S. Schechner, and Dario C. Altieri (2001, PNAS) Expression of a phosphorylation-defective survivin mutant (Thr34Ala) triggered apoptosis in several human melanoma cell lines and enhanced cell death induced by the chemotherapeutic drugcisplatinin vitro. The melanoma cell lines YUSAC2, LOX, and YUGEN8 were transfected separately with the indicated GFP-containing plasmids. At 48 h after transfection, cell nuclei were scored morphologically as normal or apoptotic by fluorescence microscopy. Data are expressed as percent apoptosis based on counting approximately 100 cells per condition Thr34Ala expressed Enhancement of cisplatin-induced apoptosis by expression of survivin Thr34Ala. YUSAC2yT34A-C4 cells were cultured for 3 days in the presence or absence of tet and 3 mM cisplatin.

43. Conditional expression of survivin Thr34Ala in YUSAC2 melanoma cells prevented tumor formation upon s.c. injection into CB.17 severe combined immunodeficient-beige mice. When induced in established melanoma tumors, survivin Thr34Ala inhibited tumor growth by 60–70%. YUSAC2yT34A-C4 tumors were established in 30 animals maintained on tet. When tumors became apparent (day 0), tet was maintained in the drinking water of 10 animals (tet +) and withheld from 20 animals (tet -), and tumors were monitored for 3 weeks. Withdrawal of Tet in established tumors was associated with a significant reduction (70-80%) in growth rate.

44. Riepilogando…

45. Grazie a tutti per l’attenzione! Irene Bagaloni