ROLE OF SERPINB3 IN GLIOBLASTOMA CANCER STEM CELLS Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of 18-20 months. Despite therapeutic interventions including surgery, radiation, and chemotherapy, multiple clones of chemo- and radiotherapy-resistant cells repopulate the tumor, resulting in recurrence and a high rate of patient mortality. These cells are referred to as cancer stem cells (CSCs) due to their ability to self-renew and generate the cellular heterogeneity present in the tumor. Our lab identified junctional adhesion molecule-A (JAM-A) on CSCs and, through functional studies, demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. We determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the intermediate signaling network is yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from GBM CSCs and performed mass spectrometry to determine the proteins to which JAM-A directly binds. This analysis led to the identification of the serine/cysteine protease inhibitor SerpinB3 as a binding partner. Interestingly, SerpinB3 does not contain the conserved PDZ domain that is present on nearly every other known JAM-A binding partner. Although multiple pro-tumorigenic mechanisms, including regulation of TGF-?1 and inhibition of apoptosis, have been proposed for SerpinB3 in the context of other cancers, very little is known about the function of the protein in GBM CSCs, and its relationship to JAM-A is yet to be elucidated. Using in vitro CSC functional assays, I have accumulated evidence that SerpinB3 is necessary for the maintenance of CSCs and that reduction of SerpinB3 attenuates TGF-?1 expression. Based on these observations, I hypothesize that SerpinB3 interaction with JAM-A is essential for the maintenance of GBM CSCs through regulation of TGF-?1 and inhibition of apoptosis.
Aim 1 will test the hypothesis that SerpinB3 maintains the CSC state through inhibition of apoptosis and upregulation of TGF-?1. I will disrupt the lysosomal membrane with siramesine to elucidate the role of SerpinB3 in the inhibition of apoptosis. Additionally, I will investigate the role of SerpinB3 in the regulation of TGF-?1 signaling in CSCs.
Aim 2 will test the hypothesis that targeting the JAM-A/SerpinB3 interaction will compromise self-renewal and GBM growth. I will utilize DSSO crosslinking to determine the region of interaction between the two proteins. Finally, I will determine the consequence of disrupting the JAM-A/SerpinB3 interaction on the CSC state with small interfering peptides. Successful completion of this project will advance our understanding of how the CSCs state is maintained in GBM via specific JAM-A intracellular binding domains, bridging cellular communication and cell signaling. The studies outlined in this fellowship will provide me an opportunity to gain experience in brain tumor research and allow me to continue my training though scientific meetings and mentorship opportunities, preparing for a career as a physician scientist.
ROLE OF SERPINB3 IN GLIOBLASTOMA CANCER STEM CELLS Glioblastoma (GBM) is the most common primary malignant brain tumor, and a population of cancer stem cells (CSCs) allows the tumor the tumor to quickly regrow after aggressive standard-of-care treatment. The objective of my project is to determine the mechanism through which GBM CSCs signal through junctional adhesion molecule-A (JAM-A) via Serpin B3, a serine/cysteine protease inhibitor that interacts with JAM-A and has been shown to signal through TGF-? in other cancers. The successful completion of this proposal will further our understanding of the signaling networks used by GBM CSCs for their self-renewal, identify new therapeutic targets and provide essential training toward my development as a physician scientist.
