Università degli Studi di Milano PhD program in Experimental medicine and medical biotechnologies XXIX cycle The role of NOTCH pathway in multiple myeloma progression: control of IL-6 expression in malignant plasma cells and bone marrow niche PhD thesis: Serena GALLETTI R10586 Tutor: Professor Antonino NERI Academic Year: 2015/2016 Summary ABSTRACT ...................................................................................................................................................... 4 INTRODUCTION ............................................................................................................................................. 5 1. Multiple Myeloma ..................................................................................................................................... 6 1.1 Multiple Myeloma pathogenesis and progression ............................................................................. 6 1.2 Genetic of Multiple Myeloma ........................................................................................................... 9 1.3 Diagnosis and clinical features ........................................................................................................ 12 1.4 The role of bone marrow niche in MM ........................................................................................... 12 1.5 Pharmacological treatments of MM ................................................................................................ 16 2 NOTCH pathway ..................................................................................................................................... 18 2.1 NOTCH components structure ........................................................................................................ 18 2.1.1 NOTCH receptors structure ............................................................................................................ 18 2.1.2 NOTCH ligands structure ............................................................................................................... 20 2.2 Activation and intracellular signaling of NOTCH ........................................................................... 20 2.3 NOTCH pathway regulation ............................................................................................................ 22 2.3.1 Receptors regulation ....................................................................................................................... 22 2.3.2 Ligands regulation .......................................................................................................................... 24 2.4 NOTCH interaction with other signaling pathway .......................................................................... 26 2.4.1 Nuclear factor-κB ........................................................................................................................... 26 2.4.2 mTOR ............................................................................................................................................. 28 2.4.3 Phosphatidylinositol 3-kinase/AKT................................................................................................ 30 2.4.4 Epidermal growth factor receptor ................................................................................................... 31 2.4.5 Wnt pathway ................................................................................................................................... 32 2.4.6 Hedgehog pathway ......................................................................................................................... 33 2.5 NOTCH involvement in cancer disease .......................................................................................... 34 2.5.1 NOTCH in Multiple Myeloma ....................................................................................................... 38 3 Interleukin-6 pathway .............................................................................................................................. 40 3.1 IL-6 signaling ........................................................................................................................................ 41 3.2 IL-6 and cancers .................................................................................................................................... 42 3.2.1 IL-6 in Multiple Myeloma .............................................................................................................. 44 3.3. NOTCH pathway and IL-6 ................................................................................................................... 45 MATERIALS and METHODS ....................................................................................................................... 46 1. Cell cultures ............................................................................................................................................. 47 2. Treatments ............................................................................................................................................... 48 2.1 NOTCH pathway inhibition ............................................................................................................ 48 2.2 NOTCH pathway activation ............................................................................................................ 49 3. Gene expression analysis ......................................................................................................................... 49 3.1 RNA extraction ................................................................................................................................ 49 3.2 RNA quantification ......................................................................................................................... 50 3.3 Reverse transcription ....................................................................................................................... 50 3.4 Quantitative RT-PCR ...................................................................................................................... 51 3.5 Gene Expression Profiling (GEP) analysis ...................................................................................... 52 4. Western Blot ............................................................................................................................................ 53 5. RNA interference ..................................................................................................................................... 53 6. Luciferase reporter assay ......................................................................................................................... 54 7. Flow cytometry analysis .......................................................................................................................... 55 7.1 Apoptosis assay ............................................................................................................................... 55 7.2 Cell cycle assay ............................................................................................................................... 55 7.3 Intracellular IL-6 detection .............................................................................................................. 55 8. Immunohistochemistry (IHC).................................................................................................................. 56 9. Statistical analysis ................................................................................................................................... 57 RESULTS ........................................................................................................................................................ 58 1. Gene expression profiling (GEP) analysis on a cohort of MM patients .................................................. 59 2. IL-6 independence is a consequence of NOTCH pathway activity ......................................................... 61 3. IL-6 production is induced by NOTCH signaling in MM cells ............................................................... 66 4. BMSCs are able to support MM tumor growth through IL-6 production induced by NOTCH signaling pathway ........................................................................................................................................................... 70 5. NOTCH pathway activation in BMSCs mediated by JAGGED ligands presented on MM cells promotes IL-6 production by BMSCs and their ability to support tumor growth ........................................................... 74 6. NOTCH signaling withdrawal reduces the release of IL-6 in co-cultures of primary MM cells and BMSCs ............................................................................................................................................................ 78 CONCLUSIONS and DISCUSSION .............................................................................................................. 82 BIBLIOGRAPHY ........................................................................................................................................... 90 ABSTRACT Multiple myeloma (MM) is an incurable hematological tumor characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow (BM). MM cells create a very strictly interaction with the surrounding BM niche that directly supports tumor growth through adhesion