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Cross-talk between non receptor tyrosine kinases and caspases in cancer and in apoptosis
One of the major concerns of cancer research is the resistance of many
tumors to chemotherapic drugs, irradiation and death receptors stimulation.
Therefore an important target is the study of mechanisms that can selectively
sensitize tumor cells, and not normal ones, to the induction of apoptosis. The
aim of this part of the project is to test the hypothesis that the impairment of
phosphorylation of Caspase-8 could sensitize tumor cells to apoptosis.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)
exhibits potent antitumor activity upon systemic administration in mice without
showing the harmful side effects observed with other apoptosis-inducing
members of the death receptor family such as TNF and Fas. Therefore, TRAIL
might have great potential in the treatment of human cancer (47) . However,
about 60% of tumor cell lines are resistant to TRAIL and many labs are setting
up different approaches to sensitize these cell lines to TRAIL (48) .
Many tumors are also resistant to more classical treatments, such as
chemotherapic drugs (doxorubicin, bleomycin, 5-FU, etoposide, cisplatin) and
radiation.
Different tumor cell lines, selected as described in Milestone B2, will be
induced to undergo apoptosis upon Fas or TRAIL stimulation as well as by
drug or radiation treatments, in the presence or in the absence of a panel of
Src kinase inhibitors (49) .
We are aware that these experiments will provide evidence for a general
role of Src kinase activity in the resistance of these cells to apoptosis. To
investigate whether tyrosine phosphorylation of Caspase-8 might contribute
to this event the levels of tyrosine phosphorylated Caspase-8 will be analyzed
by immunoblotting with the specific phospho-Tyr380-Caspase-8 antibody.
These experiments, although mainly correlative, will provide preliminary evidences
for a role of Caspase-8 tyrosine phosphorylation in tumor resistance
to apoptosis and will be useful in designing more specific approaches (Milestone
B5).
Milestone B4: Generation of a cell culture/xenograft model that allows to
study the contribute of Caspase-8 phosphorylation on Tyr380 in colon cancer
development – To further investigate the role of Caspase-8 tyrosine phosphorylation
in cancer, a cell culture/xenograft approach will be developed (50) . This
approach is aimed at the elucidation of the correlation between Src activity,
caspase-Tyr380 phosphorylation and tumor development. Therefore, colon
cancer cell lines where Caspase-8 phosphorylation on Tyr380 has been previously
identified and correlated with Src kinase activity (Milestone B2), will be
injected in the flank of SCID mice and allowed to form palpable tumors for few
days. The role of Caspase-8 Tyr phosphorylation will be investigated comparing
the tumor development triggered by cells that show Caspase-8 phosphorylation
with the same cells where Caspase-8 phosphorylation has been impaired
by Src kinase defective expression. Moreover, the effect on tumor growth of
several Src kinase inhibitors as well as chemoterapeutic agents, previously
shown to downregulate Caspase-8 Tyr phosphorylation in vitro (Milestone B3),
will be investigated by treatment of the xenografted animals with these compounds
followed by tumor volume calculation.
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