Supervisor's Name: Dr Janusz Rak
Supervisor's Email: janusz.rak [at] mcgill.ca
Supervisor's Phone: 514-412-4400 ext. 22342
Supervisor's Website: http://www.thechildren.com/en/research/mch.aspx?myID=89
Supervisor's department: Biochemistry; Pediatrics (primary appointment)
Course number: BIOC396 (Biochemistry)
Term: Winter 2012-2013
Project start date: Monday, January 7, 2013
Project end date: Tuesday, April 16, 2013
Project title: The role of p53 target TSAP6 in emission of oncosomes by brain tumour cells
Rationale: The MCH group (Rak laboratory) has previously described a brain tumour (glioblastoma) vesiculation process whereby activation of oncogenic pathways (EGFRvIII, Ras) provokes increased biogenesis and emission of membrane-derived organelle-like structures known as extracellular vesicles (EVs) 1. Notably, glioblastoma (GBM) derived EVs harbour the very oncogenic gene products that trigger their emission, for which reason they were termed oncosomes (1). The latter mediate were found to mediate the entry of oncogenes into the vascular system, their intercellular trafficking and horizontal transformation of indolent cells. However, the mechanisms, rate limiting steps and the exact pathogenetic role of oncosome formation remain unknown. In this regard a recent study reported formation of one particular type of EV (exosomes) being linked to the signalling pathway involving p53 and its target TSAP6 (2). Moreover in TSAP6-/- mice, the exosome formation was found to be diminished (3). Following these leads the laboratiry has recently generated 5-8 different GBM cell lines from TSAP+/+ and TSAP-/- transgenic GBM tumours that have emerged in mice injected with plasmids containing mutant Ras expressed under control of the sleeping beauty transposase (4).
Experimental design: In light of these findings the main goal of my project will be to begin to characterize vesiculation of these GBM cell lines, to understand whether TSAP6 is, indeed, involved in formation of Ras containing oncosomes and their biological activity. The specific objectives of my project will be as follows:
- To conduct nanoparticle tracking analysis (NTA) of GBM conditioned media using the LM500 (Nanosight instrument). During my summer project I have become familiar with this technology.
- To assess the levels of some of the key regulators of cellular vesiculation in EVs produced by GBM cells, as a function of their TSAP6 status. This will entail Western blotting of cellular and EV lysates for TSAP6, Asmase, Nsmase, Rab27a/b, Rab5a/b and Rab1a (5-8).
- To assess the level of extracellular Ras emission via the EV mediated mechanism, and as a function of TSAP6 expression. In particular, I will conduct PCR assays and Western blots for human H-ras in EVs produced by different cell lines. These assays are established in the laboratory.Significance. EVs are amongst the more intriguing and understudied entities in cancer biology today, both as mediators and also as 'messengers' of the malignant process 9. The ability to detect mutant proteins in the cargo of circulating EVs represents a very attractive biomarker platform and is being actively pursued by the MCH group. In this context it is critically important to understand the pathways that may control, modulate and alter the output of oncosomes and affect their activity and detection. My project is a part of this process.
- Al-Nedawi K, Meehan B, Micallef J et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat.Cell Biol. 2008;10:619-624.
- Yu X, Harris SL, Levine AJ. The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res. 2006;66:4795-4801.
- Lespagnol A, Duflaut D, Beekman C et al. Exosome secretion, including the DNA damage-induced p53-dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice. Cell Death.Differ. 2008;15:1723-1733.
- Wiesner SM, Decker SA, Larson JD et al. De novo induction of genetically engineered brain tumors in mice using plasmid DNA. Cancer Res. 2009;69:431-439.
- Bianco F, Perrotta C, Novellino L et al. Acid sphingomyelinase activity triggers microparticle release from glial cells. EMBO J. 2009;28:1043-1054.
- Trajkovic K, Hsu C, Chiantia S et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 2008;319:1244-1247.
- Ostrowski M, Carmo NB, Krumeich S et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat.Cell Biol. 2010;12:19-30
- Peinado H, Aleckovic M, Lavotshkin S et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat.Med. 201210.
- Al-Nedawi K, Meehan B, Rak J. Microvesicles: messengers and mediators of tumor progression. Cell Cycle. 2009;8:2014-2018.
Prerequisite: 1 term completed at McGill + CGPA of 3.0 or higher
Grading scheme (The final report must be worth at least 50% of final grade): Final grade shall be based on laboratory performance as evaluated by the research supervisor (50%) and the final written research report graded by the supervisor (50%).
Project status: This project is taken. The professor has no more '396' projects this term.
How students can apply: N/A; this project is filled.
Ethics, safety, and training: Supervisors are responsible for the ethics and safety compliance of undergraduate students. This project involves: Animal subjects; Biohazardous substances; Handling chemicals; Using lasers.