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Mammano, Enzo (2008) Applicazione del Reverse Phase Protein Array per l'analisi del profilo fosfoprotemico nelle metastasi epatiche da carcinoma del colon-retto e identificazione di nuovi bersagli terapeutici. [Ph.D. thesis]

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Abstract (english)

Background
Hepatic metastases from colorectal cancer are still characterized by a poor prognosis as only 20% of patients can undergo surgical resection and in the other 80% chemotherapy achieves short results with a 5-years survival lower than 5%. Protein-kinases play a crucial role in determinating alterations of intracellular signal transmission that are basis for neoplastic and metastatic process. Reverse phase protein array is a technique that allow simultaneous analysis of the phosphorilation state from different specimens and consequently the activation of protein-kinases.
Aim of the study
Our purpose is to compare the phosphoproteomic profile expression of a kinases group involved in intracellular signal transmission of sybchronous liver metastases from colorectal cancer with the matched primary tumors and with metachronous lung metastases, to evaluate if there are a common phenotype for every type of metastases from colorectal cancer and a specific phenotype for liver metastases.
Materials and methods
Using reverse phase protein array we analyzed the activity of 87 protein-kinases involved in multiple intracellular signalling pathways of 34 specimens from patients with colorectal cancer and synchronous liver metastases and of 16 specimens from patients with metachronous colorectal cancer lung metastases.
Results
The levels of 38 out of 87 endpoints analyzed were significantly altered between the primary tumors and the liver metastases. The majority of these are components of only two major signaling pathways: these include the PI3 Kinase/AKT/mTOR prosurvival pathway and the growth factor receptor signaling pathways (which also feed into PI3K signaling). We also observed an increase in phosphorylation of PDGFRb (Y751) (p value = 0.0181), and cKit Y703 (p value = 0.005) in the liver metastases as compared to the primary tumors: both these growth factor receptors also signal through the PI3K/AKT pathway. To confirm this finding, Western blot analysis of AKT (S473) and c-Kit (Y703) was performed on 5 of the cases using a separately obtained cell population obtained by LCM: the results show that the elevations observed by RPPA are accurate.
Comparing the liver metastases to the matched primary colorectal cancver tumor and the unmatched pulmonary metastasis phosphorylation of Pyk2 (Y402) and VEGFR (Y996) were found to be higher and phosphorylation of PKCzeta/lambda (T403/410) and BAD (S136) lower in the liver metastases (p <0.05).
Conclusions
Our results highlight that:
1- There is a common metastatic phenotype in neoplastic cells which is not related to target organ, the PI3 Kinase/AKT/mTOR prosurvival pathway.
2- There is also a phenotype specific for the liver metastases that include the overexpression of VEGFR2 (Y996) e Pyk2 (Y402) in the liver metastases.
Moreover these results suggest some new targets for the therapeutic strategy of colorectal metastases: an inhibitor of PI3K-AKT, in association with drugs as Gleevec (Imatinib mesilate) that inhibit PDGFR receptors and c-KIT, might be used as for colorectal metastases. More selectively, for the treatment of liver metastases, an association of Bevacizumab (VEGFR inhibitor) with a Pyk2 inhibitor could be useful and effective.

Abstract (italian)

Introduzione
La presenza di metastasi epatiche da carcinoma del colon-retto è contraddistinta da una prognosi infausta, poiché solo il 20% dei pazienti può essere sottoposto a resezione chirurgica e nella restante parte dei casi i risultati della chemioterapia sono scarsi, con sopravvivenza a 5 anni inferiore al 5%. Le protein-chinasi hanno un ruolo decisivo nel determinare le alterazioni della trasmissione del segnale intracellulare che caratterizzano sia il processo neoplastico che quello metastatico. Il reverse phase protein array è una metodica che permette di analizzare contemporaneamente in più campioni lo stato di fosforilazione e quindi di attivazione di numerose protein-chinasi.
Scopo dello studio
Scopo del progetto di ricerca è il confronto del profilo d’espressione fosfoproteomico di un gruppo di protein-chinasi implicate nella trasmissione del segnale intracellulare nel tumore del colon-retto primitivo e nelle rispettive metastasi epatiche e polmonari, per verificare se esiste un profilo d’espressione fosfoproteomico comune per le metastasi, inattivo nel tumore primitivo e parallelamente se esiste un profilo d’espressione specifico per le metastasi epatiche.
Materiali e metodi
Usando la tecnica reverse phase protein array, abbiamo analizzato l’attività di 87 protein-chinasi coinvolte in diverse vie di trasduzione del segnale intracellulare di 34 campioni provenienti da pazienti con adenocarcinoma del colon-retto con metastasi epatiche sincrone e di 16 campioni di metastasi polmonari metacrone da adenocarcinoma del colon-retto.

Risultati
I livelli di 38 protein chinasi sono risutati significativamente alterati nelle metastasi epatiche risptto ai rispettivi tumori primitivi. La maggior parte di queste protein-chinasi appartengono a due pathways di trasmissione del segnale intracellulare: il pathway PI3 kinase/AKT/mTOR e il pathway che regola l’apoptosi legata ai recettori per i growth factors, che prevede comunque l'attivazione di PI3 kinase/AKT/mTOR. Un’ulteriore conferma dell’importanza di tale via di segnale è data dall’iperattivazione nelle metastasi epatiche di 2 recettori tirosin-chinasici connessi con il pathway PI3 kinase/AKT/mTOR: PDGFRb Y751 (p = 0.0181) e cKit Y703 (p = 0.005), recettori invece ipofosforilati nel tumore primitivo. Questi risultati sono stati confermati mediante analisi con Western Blot dell’espressione di pAKT e di p-cKit su 5 casi. Confrontando le metastasi epatiche con le metastasi polmonari il livello di fosforilazione di 4 protein-chinasi è risultato alterato solo nelle metastasi epatiche: più precisamente la fosforilazione di Pyk2 (Y402) e VEGFR (Y996) è aumentata, mentre quella di PKCzeta/lambda (T403/410) e BAD (S136) è diminuita nelle metastasi epatiche rispetto alle metastasi polmonari ed al tumore primitivo (p < 0.05).
Conclusioni:
Questi risultati ci hanno portato alle seguenti considerazioni:
1- Esiste un pathway intracellulare iperattivato sia nelle metastasi epatiche che polmonari, rispetto al tumore primitivo, rappresentato dalla via mediata da PI3K-AKT
2- La sovraespressione di VEGFR2 (Y996) e Pyk2 (Y402) nelle metastasi epatiche ha permesso di delineare un secondo profilo fosfoproteomico specifico delle metastasi epatiche.
Questi risultati hanno inoltre evidenziato nuovi possibili bersagli terapeutici per una terapia personalizzata con inibitori delle protein chinasi: un inibitore di PI3K-AKT assieme ad un inibitore di PDGFRb e c-Kit (Imatinib mesilato) per inibire il profilo fosfoproteomico comune alle metastasi epatiche e polmonari; un inibitore della via Pyk2-FAK (PF-562,271-01 Pfizer, Groton, Conn) per inibire il profilo fosfoproteomico tipico delle metastasi epatiche.

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EPrint type:Ph.D. thesis
Tutor:Nitti, Donato
Ph.D. course:Ciclo 21 > Corsi per il 21simo ciclo > ONCOLOGIA E ONCOLOGIA CHIRURGICA
Data di deposito della tesi:29 January 2009
Anno di Pubblicazione:2008
Key Words:liver metastases, target tharapy, protein kinase, phosphoproteomic
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/18 Chirurgia generale
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Oncologiche e Chirurgiche
Codice ID:1148
Depositato il:29 Jan 2009
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