Funding

Grant Data

Pluripotent cells have the potential to differentiate into all cell types of an organism and understanding the mechanisms that govern pluripotency status is crucial for regenerative medicine. Evidence indicates the involvement of the proteostasis machinery, regulated by the heat shock organizing protein/stress inducible protein 1 (STIP1) and its ligands, Hsp70 and Hsp90, in embryogenesis and maintenance of embryonic stem cells (ESCs) pluripotency. Exciting results from a research project, initiated in a sabbatical period of Dr. Lopes at the University of Western Ontario, demonstrate the ability of STIP1 in the regulation of key pluripotency factors in stem cells. Based on these preliminary results, a critical component for these collaborative efforts is the generation of several mouse models targeting the STIP1 gene. Dr. Prado’s group pioneered the study of the function of STIP1 in embryogenesis of mammals in vivo. Moreover, his group recently demonstrated that the deletion of STIPI in mice is embryonic lethal which suggests that STIP1 is an essential factor in the early embryonic development stage. Several STIP1 mouse lines, expressing different levels of STIP1, are available and researchers at Western are the only group in the world to maintain the newly generated STIP1”TPR1 lineage (expressing STIP1 truncated isoform). These transgenic animal strains are fundamental for our proposal to understand how STIP1 and its partners regulate proteostasis in pluripotent stem cells. We hypothesized that STIP1 is a key regulator of Hsp90 and Hsp70 chaperone machinery during development to support self-renewal and differentiation of ESCs. Our overarching aims are: i) to determine whether STIP1 regulates the levels of clients and co-chaperones required for embryonic development; ii) To determine if changes in STIP1 levels regulate self-renewal and pluripotency of ESCs; iii) To determine mechanisms by which increased and/or decreased STIP1 levels regulate embryonic stem cell resilience. To address these issues ESCs, derived from transgenic mice blastocysts expressing different STIP1 levels, will be used for cell proliferation, differentiation, survival, self-renewal, and embryoid bodies formation assays. This collaboration will allow Dr. Lopes to continue to benefit from participating in a research environment with a consolidated research group that will contribute to establishing new collaborations with potential impact on Graduate Programs for both Institutions, with short training visits of young students and fellows to both research centers to develop advanced research.

Grant data

Glioblastoma (GBM), the most aggressive and frequent form of brain malignant tumors in adults, contains a subpopulation of tumor cells called GBM stem cells (GSCs), essential for tumor maintenance, invasion and resistance to therapy. Functionally relevant GSCs plasma membrane markers present potential as a therapeutic target for the treatment of this aggressive disease. Our group has shown that the GPI-anchored membrane glycoprotein, prion protein (PrPC) is enriched in GSCs and is co-expressed with conventional GSC markers. The loss-of-function of PrPC in GSC results in the inhibition of self-renewal, proliferation and capacity for tumor formation. We have proposed that PrPC acts as a scaffold protein on the cell surface, recruiting and organizing molecules on signaling platforms with different biological consequences. In light of these findings, this study aims to investigate the role of PrPC as a key molecule in GSC biology, modulating pathway signaling platforms associated with stemness and differentiation processes and as a target for GBM therapy. We proposed to address this question by using four different paradigms: 1) in vitro assays using patient-derived GSCs cultures to assess the function of PrPC and partners in the biology of these cells; 2) Transcriptome profiling using Exon Microarray and RNA-seq to define the PrPC and partners expression pattern, to quantify PrPC targets and downstream signaling pathways up/downregulated according to PrPC expression; 3) in vivo assays using GSCs to target PrPC, its partners and/or intracellular signaling-related proteins in combination with temozolomide; 4) in vitro assays to test the effect of extracellular vesicles into directed specification of GSCs toward the neuronal lineage as alternative for anti-GBM therapy. Thus, this proposal will employ the scaffold concept for PrPC as an alternative for the development of new strategies for the treatment of GBM and to demonstrate that PrPC might act as a key regulator interconnecting functions related to GSCs stemness maintenance.

Grant Data

Our group has characterized STI1 (stress inducible protein one), a co-chaperone, as a specific cellular prion protein ligand (PrPC), a normal isoform from that protein neurodegenerative disease-related. PrPC-STI1 interaction is able to modulate neuronal survival and differentiation. Futhermore, PrPC plays an important role in the neural stem cell (NSCs) biology and preliminary data from our group show that its interaction with STI1 participates in the auto-renewal of NSCs. NSCs have been involved not only in adult SNC neurogenesis but also in the genesis and control of brain neoplasias particularly gliomas, meduloblastomas and ependimonas. Recent data show that STI1 is secreted either by normal or tumoral astrocytes (glioblastoma cell line) and its interaction with PrPC modulates cellular proliferation of human glioblastoma. Interestingly, recent studies in vivo show that NSCs can be recruited to glioblastoma xenograft being this event associate to decreasing of tumor size and increasing of animal survival. Thus, those data suggest a putative role for STI1 in the brain tumor biology, since the blockage of its interaction with PrPc and its participation in NSCs auto-renewal could modulates the tumor progression. Regarding NSCs as a relevant model to explore gliomas biology and to screening new therapeutic targets, this project has as main goal investigate the PrPc-STI1 participation in the NSCs biology of adult animals and study a possible association of NSCs in brain neoplasias. Finally, the implementation of this project would establish new researcher fields in our laboratory: neurogenesis and study of brain tumor and its association to neurogenesis.

Grant Data

Stress inducible protein 1 was originally identified as a co-chaperone able to modulate heat shock protein (Hsp) activities. In fact, several evidence suggest that STI1 is not only an adapter protein to link Hsps. The interaction of STI1 with various partners such as Hsp family members, transcription factors, prion protein (PrPC) among others, demonstrates its functional versatility. Several cellular localization of STI1 indicate that this protein could participate in the formation of multiprotein complexes in the nucleus, cytoplasm and/or extracellular milieu, with distinct biological activities. Interestingly, when at the extracellular space, the soluble form of STI1 is able to interact with PrPC, a cell surface glycoprotein, and orchestrates relevant biological phenomena related to nervous system development and neural plasticity, such as self-renewal of neural progenitors, control of neural differentiation and survival, and memory consolidation. Recent findings show STI1 involved in the regulation of pluripotency status of embryonic stem cells, suggesting a role for STI1 during embryogenesis. Given those data that demonstrate several biological functions associated to STI1, and in attempt to explore the role of STI1 in mammalian development, a constitutive knockout mouse, deficient for STI1 gene, was generated. Preliminary data show that in homozygous knockout animals, the STI1 deletion is lethal and leads to embryo degeneration which seems to occur between E6.5 and E10.5, indicating that STI1 could play an essential role during mammalian development. Hence, the culture of pluripotent embryonic stem cells, become an approach more adequate to study the mechanism involved in the lethality observed in STI1 knockout embryos during early development. Regarding that little is known about the contribution of STI1 to maintenance of embryonic stem cells and embryo development, and based on literature data that show the key role for STI1 in events that govern the brain plasticity during the development, the main goal of this proposal is to investigate the role of STI1 in the embryo development using embryonic stem cell as a model and evaluate the role of its main partner, PrPC, in these processes.

Grant Data

Glioblastoma (GBM), the most aggressive and frequent form of brain malignant tumors in adults, contains a subpopulation of tumor cells, called GBM stem cells (GSC), essential for tumor maintenance, metastasis and resistance to therapy. GSC plasma membrane markers functionally relevant present potential as a therapeutic target for the treatment of this aggressive disease. Our group has shown that the GPI-anchored membrane glycoprotein, prion protein (PrPC) is enriched in GSC and is co-expressed with conventional GSC markers, such as CD133. The loss-of-function of PrPC in GSC results in inhibition of self-renewal, proliferation, and capacity for tumor formation. Several ligands have been described for PrPC, including some extracellular and transmembrane matrix functionally relevant to the biology of GSC. CD133, CD44 and integrins ±6 and ±7, major GSC markers, are able to interact and/or share with PrPC the same membrane microdomain, being preferentially located in lipid rafts. It has been proposed that PrPC acts as a scaffold protein on the cell surface, recruiting and organizing molecules on signaling platforms with different biological consequences. Giving these data, this study aims to investigate the role of PrPC as a key molecule in the maintenance of the undifferentiated state of GSC by modulating signaling platforms associated with stemness and as a target for GBM therapy. For this purpose, PrPC loss-of-function studies will be conducted using the CRISPR/Cas9 technique in GSC. The expression, cell surface distribution and intracellular trafficking of potential molecules components of the signaling module will be addressed by immunophenotyping, biochemical assays, confocal microscopy, and functional assays of self-renewal and proliferation. The participation of PrPC and its partners, members of the signaling platform, will also be evaluated in chemoresistance assays using temozolomide. Thus, we propose to use the scaffold concept for PrPC as an alternative for the development of new strategies for the treatment of glioblastoma, besides to demonstrate that PrPC can act as a key regulator of GSC stemness maintenance.