FOXO transcription factors control cellular formation of reactive oxygen species (ROS), which critically contribute to cell survival and cell death in neuroblastoma. In this thesis we report that C10orf10, also named “decidual protein induced by progesterone” (DEPP), is a direct transcriptional target of FOXO3 and is regulated by three distinctive FOXO3 binding sites located on the DEPP promoter. As there is little known about the physiological function of DEPP we investigated whether DEPP expression affects essential cellular functions like proliferation, migration, apoptosis and autophagy, which are frequently deregulated in cancer cells.
In neuroblastoma FOXO3-triggered apoptosis involves a biphasic ROS accumulation. The effect of DEPP expression on cellular ROS was measured by live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. We found that DEPP knockdown in SH-EP/FOXO3-shDEPP and NB15/FOXO3-shDEPP cells inhibits ROS accumulation during FOXO3 activation and attenuates FOXO3-induced apoptosis, whereas its overexpression in SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells raises cellular ROS levels and sensitizes to cell death. Furthermore, the elevated cellular ROS levels due to ectopic DEPP expression triggered the induction of autophagy. In neuronal cells cellular steady state ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that enables protein import into peroxisomes, we analyzed the effect of DEPP on peroxisomal function by measuring the catalase enzyme activity. Catalase activity was reduced by conditional DEPP overexpression and significantly increased in SH-EP/FOXO3-shDEPP knockdown cells. Consistent with the changes in catalase activity, ectopic DEPP expression increased H2O2-induced apoptosis, whereas SH-EP/FOXO3-shDEPP and NB15/FOXO3-shDEPP cells were much more resistant to H2O2 treatment. Furthermore we provide evidence that DEPP stimulates the Wnt-signaling pathway via its PPPSP motif and thereby affects the expression of the PPARG transcription factor, which is critical for the cellular ROS balance as it directly regulates the catalase enzyme. We found PPARG protein levels strongly upregulated in DEPP knockdown cells.
Using live cell imaging and fluorescent peroxisomal and mitochondrial probes as well as subcellular fractionation and immunoblot analysis we could demonstrate that DEPP localizes to both, peroxisomes and mitochondria in neuroblastoma cells.
In addition co-immunoprecipitation analyses revealed that DEPP is targeted to peroxisomes in a PTS2-dependent manner as it interacts with the peroxisomal PEX7 receptor.
The combined data indicate that DEPP expression reduces peroxisomal activity, thereby impairs the cellular ROS detoxification capacity and contributes to death sensitization to all forms of apoptotic cell death that involve accumulation of ROS as a second messenger. Hence, the chemotherapeutic agent etoposide elevated the mRNA level of endogenous DEPP and ectopic DEPP expression significantly increased the sensitivity to etoposide- induced apoptosis, which is an important finding of therapeutic value.
In addition we found that DEPP expression triggers the activation of ERK1/2, resulting in p21 upregulation and reduced cell proliferation. However, DEPP knockdown significantly impaired the formation of actin filopodia and cellular migration abilities. The reduced cellular migration was also reflected by changed integrin expression patterns of ITGA2, ITGA7, ITGA11 and ITGB3, which suggests that low DEPP expression might impair the formation of cancer metastasis in vivo.
In future the better understanding how DEPP controls cellular ROS levels, autophagy, apoptosis as well as migration capacities in neuroblastoma cells may help to develop new therapeutic strategies.