Characterization of a novel tumorigenic role of caspace 8

Caspase 8 is a well characterized protein that plays a pivotal role transducing the extrinsic apoptotic pathway. A hallmark of tumor cell is resistance to apoptosis; this feature is the result of several molecular aberrations in signalling pathways involved in the control and execution of cell death. In this context, Caspase 8 can represent a good target as its loss of function may severely impinge on apoptosis. Surprisingly, its expression is lost only in a small percentage of tumors, indicating that the retention of Caspase 8 expression in some tumors may be well tolerated and suggesting that it may even positively contribute to tumour progression. Consistent with this introduction, in the first part of this this project we show evidences supporting the idea that Caspase 8 expression may promote cell transformation in hepatocarcinoma and in glioblastoma cellular models. Interestingly, we could show that Src kinase, aberrantly activated in these cancer cellular models, may drive Caspase 8 phosphorylation on Tyr380. Using a cancer cellular model characterized by Src constitutive activation engineered to express either Caspase 8-wt or Caspase 8-Y380F we provide evidence that Caspase 8 expression and phosphorylation on Tyr380 but not its enzymatic activity promote in vitro cell transformation, Rac activation and resistance to anoikis. To further investigate the requirement for Caspase 8 expression in tumor progression we focused our studies on glioblastoma cellular models. Glioblastoma multiforme (GBM multiforme) is the most aggressive primary brain tumor in the adult nervous system and it is associated with a poor prognosis. Interestingly in this tumor a major role is played by the ability of cancer cells to strongly modulate tumor microenvironment by secreting interleukines and cytokines that overall sustain cancer cell survival and promote neo-angiogenesis. In the second part of the project we provide evidence that the inhibition of Caspase 8 expression, obtained by RNA interference, severely downregulates the mRNA levels and the production of IL-6, IL-8, IL-1β, MCP-1 and VEGF-A by glioblastoma cell lines. Consistently it impairs in vivo angiogenesis and in vitro proliferation and capillary tube-like network formation on matrigel triggered by conditioned media of cultured cells. How Caspase 8 is able to modulate the expression of these factors has not been elucidated yet. A possible molecular mechanism contemplates Caspase 8 interaction with NF-kB pathway. Several studies suggest an important role for nuclear factor kappa light chain enhancer of activated B cells (NF-kB) signalling in glioblastoma and implicate NF-kB activation as an important driver of the malignant phenotype that confers a negative prognosis in patients and resistance to therapeutic treatments, supporting the importance of a role of NF-kB to promote aggressiveness, invasion, neo-angiogenesis in this tumor. Importantly, previous studies have shown that NF-kB transcription factor may promote the expression of IL-6, IL-8, IL-1β, MCP-1 and VEGF-A. Moreover, it has been suggested that Caspase 8 may promote NF-kB activity. We demonstrate that Caspase 8 supports NF-kB nuclear translocation also in glioblastoma cell lines and provide evidence for a possible role of its phosphorylation on Tyr380.