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Sezione III: Attività per progetti
Milestone B5: Generation of colon cancer cell lines that stably express either
Caspase-8-wt or Caspase-8-Y380F – All the experiments described before will
provide a correlative connection between Src activity, Caspase-8 phosphorylation
on Tyr380 and cancer development. However, we are aware that the
impairment of Src kinase activity has a broad effect on the oncogenic potential
of these cells, which may be only partially linked to Caspase-8 phosphorylation.
To unambiguously address the functional contribution of Tyr380
phosphorylation on colon cancer progression, based on the results of the
screening performed in Milestone B2, we will develop colon cancer cell lines
where endogenous Caspase-8 expression has been genetically ablated by
siRNA. Caspase-8 deficiency often correlates with Fas-resistance. Therefore, an
alternative approach to generate colon cancer cells that express low or no levels
of Caspase-8, will be the selection in the presence of anti-Fas antibodies of
clones resistant to Fas-induced apoptosis. Those clones resistant to Fas that
indeed express low levels of Caspase-8 will be then selected. Cells with low levels
of Caspase-8 will be then reconstituted by stable constitutive or inducible
expression with Caspase-8-wt (phosphorylatable) or caspase-8-Y380F
(unphosphorylatable).
Overall these approaches will identify cell lines that will be used to perform
all the experiments described in Milestone B3-4 in the presence or in the
absence of phosphorylated Caspase-8 and to unambiguously identify the contribution
of Tyr380 phosphorylation in tumor progression.
Milestone B6: Caspase-8 tyrosine phosphorylation in anoikis – Apoptosis
can be induced by numerous triggers, one of them being the loss of cell
anchorage of normally adherent cells.It has been reported that the expression
of the oncogenic form of Src, v-Src confers resistance to anoikis (10) .
Moreover, Src activation in human metastatic colon tumor cells impairs
anoikis of these cells (28) . The possibility that Src activation prevents anoikis
also through Caspase-8 tyrosine phosphorylation will be investigated. Caspase-8
tyrosine phosphorylation will be assayed by immunoblots on
immunoprecipitated Caspase-8 from cell extract from human colon tumor
cell lines, using antiphosphotyrosines and the phospho-Tyr380-Caspase-8
antibodies. Moreover, colon cancer cell lines generated in B5, that express
either Caspase-8-wt or Caspase-8Y380F, will be used in anoikis experiment in
the presence or in the absence of a panel of Src kinases inhibitors. These
studies might allow the identification of a new mechanism involved in tumor
progression and in the metastasis process.
Task C: Role of caspase-mediated cleavage of Abl family
non receptor tyrosine kinases in anoikis
Milestone C1: Role of caspase-mediated cleavage of c-Abl in anoikis –We
have previously reported that c-Abl is cleaved by caspases in the cytoplasm
upon stimulation of death receptors. Cleavage causes the loss of a C-terminal
fragment of about 20kDa, containing the Nuclear Export Signal (NES) and the
actin binding region. The loss of the NES upon caspase cleavage, causes the
636 2006