0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
U.O.6 – Laboratorio di Cell Signaling
Daniela Barilà
Specific contribution of the unit to the project
The aberrant downregulation of the apoptotic program significantly contributes
to tumour development. Caspase-8 (C8), a central player of apoptosis.
Interestingly, several cancers show genetic alterations that lead to lack of Caspase-8
activity, supporting the idea that defective C8 function promotes cancer.
Significantly C8 activity is compromised in tumours of the nervous system
such as gliomas and neuroblastomas [Stupack (2006) Nature; Ashley
(2005) Cancer; Teitz (2000) Nat Med]. Importantly, we have recently identified
C8, as new substrate of Src tyrosine kinase. Src triggers C8 phosphorylation
on Y380 and this phosphorylation represses C8 proapoptotic activity [Cursi
(2006) EMBO Journal]. Src kinases, are often aberrantly activated in a variety
of cancers [Summy (2003)] and the extent of increased Src family activity correlates
with malignancy. Using a phospho-Y380-C8 antibody, made in house
(p-Y380), we could show that C8 is phosphorylated on Y380 in human colon
cancer.
Importantly, Y380 plays a role in the modulation of cell adhesion and
migration [Helfer (2006) Cancer Research; Senft (2007) Cancer Research; Finlay
(2007) Cancer Research], and along with Stupack lab, UCSD, USA we
could show that its phosphorylation plays a role in the metastatic process
[Berbero, submitted]. Using an immunohystochemistry approach we will perform
a large scale study on different neuroblastomas and gliomas samples
derived from human patients upon surgical resection. The presence of C8,
pY380C8 and active Srcfamily kinases will be evaluated and correlated with
several clinical parameters. Furthermore in collaboration with Stupack we
plan to use the neuroblastoma C8 -/- cell lines, reconstituted with C8-wt or
with C8-Y380F as model system to study the physiological relevance of this
event in vivo in tumour formation and metastases development. All together
this study will identify of pY380C8 as a novel molecular mechanism to promote
cancer and as a putative diagnostic and prognostic marker.
Methods
Recently we set up conditions to perform immunohystochemistry analysis
using our anti-phospho-Ty380-Caspase-8.
Immunohystochemistry will be carried out using our anti-phospho-
Ty380-Caspase-8 antibody on sections derived from glioma and neuroblastoma
tumours.
Using this approach we will score at least one hundred cases for each
tumour. All specimens will be formalin-fixed and paraffin embedded and
processed for immunohystochemistry as previously described [Wilson (2006)
British Journal of Cancer]. Adjacent sections will be stained with the following
antibodies: mAb Clone28, that specifically recognizes activated Src, and anti-
758 2009