Cellular proteome alterations in response to hypoxia inducible factor HIF-2α in normoxic neuroblastoma cells

Il neuroblastoma (NB) è un tumore embrionale del sistema nervoso simpatico, che deriva da cellule della cresta neurale. È il tumore extracranico più diffuso tra i bambini di età inferiore a un anno e rappresenta circa il 7% di tutti i tumori infantili. L'ipossia si sviluppa comunemente durante la crescita tumorale ed è associata ad una prognosi infausta con resistenza ai trattamenti terapeutici. Molte evidenze suggeriscono una correlazione tra i fattori ipossia-inducibile (HIF), HIF-1α e HIF-2α, con il grado di differenziamento e quindi l’aggressività tumorale. In particolare, nel Neuroblastoma HIF-2α è stabile anche in condizioni di normossiche e continua ad essere attivo anche dopo 48-72 ore dall’ipossia. In NB HIF-2α, localizzato principalmente nelle nicchie peri-vascolare del tumore ed è correlato ad una prognosi infausta. Studi recenti hanno dimostrato il coinvolgimento di HIF-2α nella proliferazione e nell’aumento della aggressività tumorale. Lo scopo del progetto è quello di acquisire ulteriori informazioni sui meccanismi molecolari indotti dall’over-espressione di HIF-2α che sono alla base della resistenza ai convenzionali trattamenti terapeutici. Il nostro progetto, quindi, propone un approccio proteomico, basato sull’analisi DIGE e sull’identificazione di antigeni di membrana (FACS), per identificare nuovi bersagli prognostici e terapeutici per il trattamento clinico di forme di NB più aggressive e resistenti ai protocolli terapeutici convenzionali. Attraverso l’indagine DIGE e studi funzionali sono stati identificati nel nostro sistema sperimentale diverse proteine coinvolte nel metabolismo cellulare e nei processi di regolazione dell’mRNA. Inoltre sono stati identificati nuovi antigeni di membrana differenzialmente espressi soffermandoci sulle proprietà antiadesive e proinvasive di CD55, marker specifico di HIF-2α.Quindi, CD55 potrebbe essere usato nella diagnosi e per la stratificazione di pazienti affetti da forme più aggressive di Neuroblastoma.

Neuroblastoma (NB) is an embryonal tumor of neuroectodermal cells derived from precursor or immature cells of the sympathetic nervous system (SNS). This disease rappresents the most common extracranial tumor in infants, accounting for 8% to 10% of all childhood cancer and for approximately 15% of cancer deaths in children. The deep knowledge of NB biology is imperative toward the development of novel therapy. Hypoxia is a typical feature of several solid tumors microenvironment and is associated with a poor prognosis and resistance to therapy. The relationship among hypoxia, tumor phenotypes and clinical parameters in NB is not well characterized. Tumor adaptation to hypoxia is mainly mediated by two transcription factors: the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α. HIF-2α is stable also in normoxia condition and continues to be active even after 48–72 h of hypoxia in some neuroblastoma cell lines thus indicate that HIF-2α plays a critical role in driving the hypoxic response. Interesting, HIF-2α is correlated with poor patient prognosis in NB and is localizated in tumor peri-vascular niches. These findings indicate that HIF-2α protein expression in NB samples at normoxic levels might affect the aggressive tumor phenotype. The main aim of my phD program has been to get new insights into the molecular mechanism of tumor aggressiveness mediated by HIF-2α protein overexpression in NB cells. Interesting, HIF-2α overexpressing cells acquire an undifferentiated phenotype and the ability to grow as neurospheres in soft agar. Then I applied two different proteomic approaches, DIGE analysis and FACS detection of membrane antigens to identify new putative prognostic and therapeutic hypoxia-related targets to be used in clinical treatment of aggressive NB forms. The identified proteins have important roles in a variety of pathways such as “citrate cycle”, “glycolysis” and “splicesoma” thus indicating that HIF-2α over-expression affects the cellular metabolic balance and increases the processes of mRNA regulation. These findings might provide an innovative therapeutic strategy by combining anti-metabolic drugs and pathways inhibitors.Among the cell surface antigens which were differentialy HIF-2α regulated CD55 was the most significantly expressed marker in our cellular system. I assessed CD55 has anti-adhesive and pro-invading functions that might provide the basis for NB solid tumors to survive as microscopic residual disease. Furthermore, the use of CD55 antibody-based visualization as in PET (Positron Emission Tomography) imaging will have implications for the development of more accurate diagnosis and prognosis in challenging cases and for driving personalized treatment. In conclusion, the HIF-2α novel markers identified in this study might improve patients risk stratification and could be also used as putative drug targets being immunotherapy is one of the most promising anticancer treatment.