Investigating drug targets and improving drug delivery for anti-cancer treatment of osteosarcoma

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Osteosarcoma is cancer of bone, most common in children and young adults. Metastasis is either present at the time of diagnosis or develops later during the course of treatment in most patients. The survival rate with metastatic osteosarcoma is very low, therefore new therapeutic interventions are needed. Our lab has demonstrated that Riluzole, a glutamate release inhibitor, is effective in inhibiting proliferation and inducing apoptosis both in human and mouse osteosarcoma. Furthermore, we have demonstrated that Riluzole blocks the activity of the mGluR5 receptor signaling to inhibit growth in osteosarcoma cells. Yes associated protein (YAP) is a transcription co-activator involved in cell proliferation. The evidence in literature shows that YAP phosphorylated at serine 127 facilitates cytoplasmic sequestration and degradation of YAP. Furthermore, YAP is phosphorylated at Y357 by C-Abl kinase under DNA damage-induced stress. Interestingly, phosphorylation of YAP at Y357 promotes strong interaction with p73, a transcription co-activator, to induce transcription of pro- apoptotic genes. Our data has shown that Riluzole decreased phosphorylation of YAP at serine 127 and increased nuclear localization of YAP. Furthermore, Riluzole also changed the localization of a YAP mutant, YAPS5A (five serine residues at 61, 109, 127, 164, and 397 changed to alanine) from cytoplasm to nucleus suggesting that change is localization is independent of phosphorylation at these sites. We hypothesize that the increase in nuclear localization of YAP facilitates transcription of pro-apoptotic genes. We want to determine if YAP is directly involved in Riluzole-induced apoptosis and if YAP is regulated by C-Abl to activate pro-apoptotic genes in osteosarcoma cells. We will use human metastatic osteosarcoma cells, LM7, and mouse cells lines, OS482, to study the effect of: a) Riluzole on phosphorylation of YAP at Y357 b) C-Abl inhibitors on phosphorylation of YAP at Y357 and Riluzole-induced apoptosis c) Riluzole on osteosarcoma cells with YAP knockdown or C-Abl knockdown to confirm the role of YAP and C-Abl in apoptosis d) Riluzole on YAP and P73 binding and on transcription of Bax promoter in a luciferase reporter assay. Our recent in vitro data has demonstrated that Riluzole released from the iron oxide nanocage is more effective in inducing apoptosis in LM7 cells compared to free Riluzole or Riluzole released from the nanosphere. We hypothesize that Riluzole released from Riluzole-loaded nanocage will be more effective, compared to free Riluzole, in reducing metastasis in a nude mouse model. We will implant osteosarcoma cells, LM7.eGFP.ffLuc, in the tail vein of nude mouse. We will randomly sort the animals in 4 groups and carry out the following treatments. 1) no treatment, 2) free Riluzole, 3) nanocage alone, 4) Riluzole-loaded nanocage. We will treat the animals and monitor metastasis using bioluminescence imaging in all groups. We will perform whole animal magnetic resonance imaging (MRI) for biodistribution of iron oxide nanocage carriers and then use quantitative susceptibility mapping (QSM) to quantify the concentration of iron oxide nanocages at metastasis sites. Our data has demonstrated that glutamate signaling via mGluR5 is important in colony forming ability of LM7 cells. In Aim 3, we will assess the efficiency of mGluR5 siRNA delivery to decrease tumor size in a xenograft mouse model. We will use mGluR5 siRNA-loaded iron oxide nanocage and induce siRNA release using magnetic hyperthermia in mice. We will perform whole animal MRI/QSM and ex vivo MRI/QSM to determine the nanocage concentration in each organ and tumor site. We will perform and analyze the experiments in Aim 2 and Aim 3 in collaboration with my colleague, Dr. Hiroshi Matsui, who is an expert in bionanotechnology. In brief, we expect to: a) demonstrate a direct role of YAP in Riluzole-induced apoptosis of osteosarcoma cells b) assess the efficacy of the delivery method of Riluzole in preventing metastasis in xenograft nude mouse model c) deliver mGluR5 siRNA via nanoparticle to prevent tumor establishment in a xenograft mouse model. Riluzole is used as a FDA approved therapeutic drug for Amyotrophic Lateral Sclerosis (ALS). This study is a drug- repurposing study in which Riluzole is used as an anti-cancer agent for osteosarcoma. !