NOTCH COOPERATES WITH PATHWAYS INVOLVED IN TUMOR PROGRESSION

Notch signaling mediates central cellular functions through direct cell-to-cell contact. Deregulation of Notch activity can alter cell proliferation and it is linked to many human cancers. Similarly to other oncogenes, Notch cooperates with many pathways during tumor progression. My study aimed to evaluate the effect of interactions of Notch pathway with AKT signaling and with CXCR4/SDF1a axis. Notch signaling is known to play role as the main trigger of T cell acute lymphoblastic leukaemia (T-ALL), aggressive hematologic cancer that accounts for 10-15% of pediatric and 25% of adult ALL cases. Recent evidences indicate the reciprocal regulation between the constitutively active Notch and AKT pathways in T-ALL cells. However, the mechanism of this interaction is still unclear. This prompted me to investigate the molecular mechanisms of AKT-dependent Notch1 regulation in T-ALL cells. Previous results in T-ALL cells shown that Akt signaling withdrawal induced by LY294002, a specific PI3K inhibitor, triggers Notch1 degradation via lysosomal machinery. Further, I showed that LY294002 treatment increased monoubiquitin-dependent accumulation of transmembrane Notch1 protein. Co-immunoprecipitation and densitometry analysis of confocal images revealed that Notch1 specifically co-precipitated with the ubiquitin E3 ligase, c-Cbl. This is involved a covalent attachment of ubiquitin to Notch1 and directing it to the lysosome. Finally, a confocal microscopy analysis showed Notch1 localization in the lysosome. These data suggest that AKT pathway can control the steady-state level of Notch1 rescuing it from lysosomal degradation by interfering with c-Cbl triggered mono-ubiquitination and lysosomal degradation. Notch crosstalk with the chemokine system CXCR4/SDF1awas studied in cell models of ovarian cancer (OC). OC ranks fifth in tumor mortality among women. Recent evidences support a role of Notch pathway in OC progression where its activation is related to tumor progression. Similarly, CXCR4/SDF1a correlates with OC cell survival, tumor growth and metastasis. To investigate the consequences of Notch-CXCR4 crosstalk in OC biology, a panel of OC cell lines was characterized by high expression of Notch pathway as well as by elevated levels of CXCR4 and its ligand SDF1α comparing to a normal ovarian cell line. Treatment with an inhibitor of Notch activation, DAPT, was associated with a reduced time-dependent OC cell proliferation and cell cycle arrest in G0/G1 phase. By contrast DAPT did not affect apoptosis, neither sensitize OC cell lines to other pro-apoptotic and anti-proliferative thiazolinedione’s drugs. In addition, Notch withdrawal determined a significant CXCR4 and SDF1α mRNA and protein inhibition. Further studies also displayed Notch ability to control the biological function of CXCR4 signaling, i.e. growth and migration in response to SDF1a. A confirmation of the specific role of Notch1 in the regulation of CXCR4 signaling was obtained by transient ectopic expression of Notch1 in OC cells that resulted in increased expression levels of CXCR4 and SDF1a. The evidence that Notch deregulation might affect CXCR4/SDF1a axis expression and function is relevant in the light of CXCR4 role in promoting OC cell proliferation and SDF1a-mediated dissemination of OC cells in the abdominal cavity. Altogether, these results suggest that Notch pathway might be a promising therapeutic target to hamper tumor progression and improve the efficacy of present anti-cancer therapy.