SapC-DOPS has antitumor efficacy in glioblastoma through the induction autophagy-associated cell death (2013)

Purpose: Glioblastoma multiforme (GBM) is the most common and most aggressive form of primary brain tumor. Despite decades of improvements in radiation and chemotherapy, the median survival remains less than 15 months. The aggressive and drug resistant phenotype of these tumors is compounded by their protection behind the blood-brain tumor barrier (BBTB). Thus, there is an unmet and urgent need to develop new treatment modalities. Saposin C (SapC) is a sphingolipid activating protein found ubiquitously throughout the body which functions to catabolize glycosphingolipids. When SapC is coupled with dioleoylphosphatidylserine (DOPS), stable nanovesicles are formed which can fuse with phosphatidylserine on the surface of cancer cells causing cell death. The main objective of this study was to evaluate the efficacy of SapC-DOPS against GBM. This was done by evaluating the ability of SapC-DOPS to cross the BBTB to target GBMs in vivo and elucidating the mechanism of SapC-DOPS-induced killing. \nResearch Mehtods: Live animal imaging using lipophilic far-red fluorophore (CVM) labeled SapC-DOPS was done using an IVIS 200 Series imaging system. Spontaneous glioma mouse models (GFAP-cre;Nf1loxP/+;p53-/loxP;PtenloxP/+) or (GFAP-creER;PtenloxP/loxP;p53loxP/loxP;Rb1loxP/loxP;p107-/-) were used for targeting experiments. Synergy analysis was completed using CompuSyn software. Western blot, fluorescent microscopy, FACS analysis, MTT viability assays, and transmission electron microscopy were used to check for molecular markers.\nFindings: Using live animal imaging intravenously delivered SapC-DOPS was found to specifically target intracranial tumors in mice bearing spontaneous brain tumors, as well in nude mice intracranially implanted with human GBM cells. Targeting of SapC-DOPS within the tumor was confirmed by immunofluorescence in frozen brain sections. Lack of any significant fluorescence signal in normal non-neoplastic brain parenchyma also attested to the specificity of the targeting. Treatment of tumor bearing mice with SapC-DOPS significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P<.0001). Western blot analysis of primary GBM neurospheres treated with SapC-DOPS did not show activation of apoptosis as measured by cleaved caspase 9 and cleaved PARP, and lacked activation of DNA damage markers phospho-ATM and γ-H2AX. Consistent with this, treatment with a pan-caspase inhibitor Z-VAD-FMK did not rescue SapC-DOPS-induced killing (P>0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed a significant increase in punctuated GFP expression following treatment, indicative of autophagosome formation (P<.001). Inducers of autophagy can be diverse, but many times share similar signaling through MAPK activation and/or inhibition of mTOR. Evaluation of MAPK signaling, showed increased phosphorylation of ERK and JNK, but not p38, following SapC-DOPS treatment. To clarify the involvement of these pathways we treated glioma neurospheres with SapC-DOPS in the presence or absence of an ERK or JNK inhibitor. Inhibition of JNK, but not ERK was able to prevent LC3-I lipidation by SapC-DOPS treatment and resulted in a partial rescue in cell viability (P<.01). Interestingly, we did not observe a decrease in the activation of mTOR, a known regulator of autophagy, as determined by the phosphorylation of 4EBP1 and p70S6K. We hypothesized that inducing autophagy through mTOR inhibition in addition to SapC-DOPS treatment would lead to enhanced autophagic signaling and synergistic cell death. Using Rapamycin, a well characterized mTOR inhibitor and inducer of autophagy, we examined possible synergistic interactions using the Chou Talalay. Combination of SapC-DOPS and Rapamycin yielded strong synergistic interactions against primary GBM neurospheres using the Chou Talalay Analysis (combination index<.4). \nImplications: The data presented here highlight the ability of systemic SapC-DOPS to effectively cross the BBTB to target GBM in vivo. SapC-DOPS was found to induce potent autophagy in these cells through activation of JNK and showed strong synergistic interactions in combination with alternative autophagy inducing therapeutic Rapamycin. These findings suggest therapeutic implications for treating GBM using SapC-DOPS alone and in combination with an AKT/mTOR inhibitor.