Abstract 4358: Involvement of mTOR pathway in metastatic brain tumors.
Authors: Kwasnicki Amanda , Jeevan Dhruve , Goyal Anita , Braun Alex , Murali Raj , Jhanwar-Uniyal Meena 
Published: August 2014

Metastatic dissemination to the brain occurs in almost forty percent of all cancers, and is ten times more common than primary brain tumors. Mammalian target of Rapamycin (mTOR), an atypical PI3K related kinase, exists in two distinct multiprotein complexes (mTORC1 and mTORC2). mTORC1/2 regulate various cellular functions and are shown to be activated in primary brain tumors such as glioblastoma and medulloblastoma. In addition, metastatic tumor cells possess a unique reprogramming resulting in a stem cell like phenotype with invasive, migratory and proliferative potential similar to that seen in glioblastoma, where mTOR appears to play an essential role. However, the involvement of the mTOR pathway in metastatic brain tumors remains to be elucidated. This study aimed to test the hypothesis that the mTOR pathway plays a critical role in metastatic brain tumors. In order to achieve our goals, expression of mTOR and its components (Raptor and Rictor) were studied in metastatic brain tumors using immunohistochemical analysis. Further, the function of mTOR in relation to proliferation, migration, and cell spreading was studied in a metastatic breast cancer cell line (MBA-MD 231). The expression of epithelial marker E-Cadherin and meschenchymal marker Vimentin following the inhibition of mTOR using pharmacological inhibitors (Rapamycin and PP242) as well as short interfering RNA (mTOR, Raptor and Rictor) was examined by immunofluorescence analysis. Results demonstrated that a significant number of metastatic brain tumors expressed mTOR, Raptor, and Rictor; 50% expressing all three components. The metastatic potential showed that migration of cancer cells was inhibited significantly by mTORC1 inhibition (Rapamycin) and mTORC1/2 inhibition (PP242) by 29% and 64%, respectively. Utilizing the EdU incorporation technique, S-phase cell cycle entry analysis rendered a 52% decline in proliferation after Rapamycin treatment. Cell spreading/attachment analysis exhibited total attachment of control cells within 20 minutes of plating. This effect was prevented by pretreatment with Rapamycin or PP242. Immunoflourescence analysis showed that E-cadherin underwent forced nuclear localization following acute inhibition of mTOR, using Rapamycin or siRNA treatments. These results were substantiated by Western blotting, suggesting its role in cellular reprogramming, namely Epithelial-Mesenchymal Transition or its counterparts. However, Vimentin expression remained unaltered. In conclusion, these results provide evidence that mTOR is enhanced in metastatic brain tumors, suggesting its critical role in achieving a metastatic potential to the brain. Importantly, these findings underscore the therapeutic value of mTOR inhibition in the management of patients with metastatic brain tumors.

Citation Format: Amanda Kwasnicki, Dhruve Jeevan, Anita Goyal, Alex Braun, Raj Murali, Meena Jhanwar-Uniyal. Involvement of mTOR pathway in metastatic brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2013-4358