Cell death signaling in cancer cells treated by Photodynamic Therapy

Glioblastoma multiformes (GBMs) are an extremely aggressive and infiltrating type of brain
cancer. Despite heavy therapies and extensive fundamental and applied research, the
median survival of patients remains about 15 months after diagnosis for over a decade.
Therefore, there is an emergency to find new approaches and therapeutic targets for
treating this cancer. Photodynamic therapy (PDT) recently demonstrated a high potential in
the treatment of GBM. Moreover, GBMs induced cell death by PDT is dependent of an
atypical RIP3-dependent programmed necrosis. In parallel, GBMs activate a pro-survival
autophagic pathway in order to recycle PDT-damaged structures and organelles.
In our PhD thesis, we investigated the regulation of this autophagic process and found that
TSC2 protein had an important role in autophagy activation by PDT. Indeed, PDT treatment
quickly activates the kinase MK2, which phosphorylates TSC2 on serine 1254. We then
showed that phosphorylation of this serine was crucial for autophagy activation, which
makes TSC2 a crucial pro-survival factor in GBMs treated with PDT. Finally, we demonstrated
that protein 14-3-3 ζ (YWHAZ) interacts with TSC2 and protects TSC2 serine 1254
phosphorylation from phosphatase actions after PDT. In the same time we conducted a
proteomic analysis on RIP3 immunoprecipitate. The major implication of this analysis is the
demonstration that RIP3 interacts with TSC2 and YWHAZ. Finally, we showed that RIP3
interacts better with the non-phosphorylated form of TSC2 than with the phosphorylated
form, suggesting a RIP3 interference in the TSC2-dependent autophagy activation process.
These data were submitted for publication in "Scientific Reports".
In the second part of this thesis, we also investigated the influence of RIP3 expression on
osteosarcoma (U2OS) cell death. U2OS expressing or not RIP3 were treated with PDT and
cell death mechanisms have been investigated. We first demonstrated that in both cell lines,
apoptosis was the major cell death mechanism. Secondly, we noticed an over-activation of
various caspases in cells expressing RIP3 despite a stronger resistance to PDT. This could be
explained by a lower activation of autophagy in cells not expressing RIP3. Thirdly, we showed
that in RIP3 expressing cells, residual necrosis was RIP1-dependent. We therefore suggest
that RIP3 is able to influence the cell death process. These data were published in "Laser in
surgery and medicine."