Deciphering the effect of HSP90 inhibition on the expression of heart shock proteins in human mantle cell lymphoma cells, by means of computive proteomics (Master thesis)

Τσετσάκος, Σαράντης/ Tsetsakos, Sarantis

Mantle Cell Lymphoma (MCL) is a rare type of B-cell non-Hodgkin lymphoma (NHL), characterized by various deregulated mechanisms, including, cell cycle, apoptosis, B-cell receptor signaling and p53 signaling pathway. In this context, Heat Shock Proteins (HSPs) bare a significant role for both cellular homeostasis and lymphomatogenesis. HSPs serve as molecular chaperones, stabilizing a plethora of “client-proteins”, some of which are regarded as indispensable for cellular physiology and others as oncogenic. Subsequently, their inhibition; more prominently HSP90 inhibition, is currently being explored as a potential therapeutic intervention. However, MCL remains among the most ominous malignancies. Research advancements, within the systems biology framework, can enhance the development of novel, combinatory therapeutic objectives. In this study, we used comparative techniques, namely western blotting (WB) and mass-spectrometry–based proteomics (MS), in order to elucidate the differential expression of HSPs in JMP-1 cells, an in-vitro MCL model, treated with 1μΜ - 2μΜ of the HSP90 inhibitor 17-AAG for 24 - 48h. In parallel, genomic (DNA) and transcriptomic (RNA) material was also isolated, allowing for a holistic assessment of HSP90 inhibition’s effect on the lymphoma cells. Moreover, cells were treated either with 1μΜ - 2μΜ of 17-AAGfor 24-48h, or with a combination of 17- AAG and Nutlin-3a (N3a), a potent proapoptotic p53 activator, across a spectrum of concentrations, for 24h. The two agents’ effect was evaluated by determining cellular viability and cell growth, using light microscopy. We ascertain that HSP90 inhibition not only reduces both cellular viability and growth in a dose-dependent manner, but also reinforces the anti-lymphoma activity of N3a, especially in low concentrations of both agents, implying a synergic interaction. Quantitation of HSP90, HSP70, Grp94 and of the transcriptional factor HSF-1 by means of WB indicates that HSP90 inhibition indeed affects the expression levels of the aforementioned proteins. We observe an upregulation trend for HSP70 but also a downregulation trend for HSP90 and its ER homologue GRP94, in the presence of 17-AAG. These observations coincide, partially, with those derived from the MS analysis of one biological replicate. In that assessment we also noticed an upregulation trend for HSP70 and for the organelle HSPs, while HSP90 itself displayed a different pattern. Some of the differentially expressed HSPs exhibit a similar motif of differential expression, implying shared regulation, as indicated by Principal Component Analysis (PCA). As a final note, mapping the differentially expressed HSPs in a network of protein-protein interactions a high degree of functional connectivity. The aforementioned findings may be regarded as a stepping-stone towards understanding the HSPs’ role and regulation mechanisms in MCL, especially in relation with other therapeutic interventions.
Institution and School/Department of submitter: Δημοκρίτειο Πανεπιστήμιο Θράκης. Σχολή Επιστημών Υγείας. Τμήμα Μοριακής Βιολογίας και Γενετικής
Subject classification: Heat shock proteins
Keywords: Heat shock proteins,Mandle cell lymphoma,Proteomics,Πρωτεΐνες θερμικού σοκ,Λέμφωμα κυττάρων του μανδύα,Πρωτεϊνωματική ανάλυση

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