Fibronectin-integrin interaction promotes fibroblast activation (nemosis) and crosstalk with tumor cells

.........................................................................................9 1. REVIEW OF LITERATURE .............................................................11 1.1 Cancer........................................................................................11 1.2 Fibroblasts ............................................................................... 12 1.2.1 Fibroblasts in tumor microenvironment ............................................... 13 1.2.2 Cells in tumor microenvironment ..........................................................14 1.2.3 Fibronectin................................................................................................16 1.2.4 Integrins as fibronectin receptors.......................................................... 17 1.3 Autophagy ................................................................................ 18 1.3.1 Induction and progression of autophagy.............................................. 19 1.3.3 Autophagy in cancer................................................................................22 1.3.3.1 Autophagy in tumor stroma and fibroblast activation...................25 1.4 The Cell cycle............................................................................26 1.4.1 Checkpoints ...............................................................................................27 1.4.3 Quiescence.................................................................................................28 1.4.4 Cellular senescence ..................................................................................28 1.5 Nemosis....................................................................................30 1.5.1 Nemosis and paracrine signaling...........................................................31 1.5.2 Nemosis and cancer.................................................................................32 2. AIMS OF THE STUDY ...................................................................34 3. MATERIALS AND METHODS .......................................................35 3.1 Cell culture (I-IV)............................................................................................35 3.1.1. Liposome-mediated siRNA transfections (I) ........................................36 3.1.2. Lentiviral mediated shRNA (II).............................................................37 3.2 Functional assays ...........................................................................................37 3.2.1 Soft-agar assay (II) .................................................................................37 3.2.2 Collagen co-culture assay (III) ..............................................................38 3.2.3 Tumor xenograft models (II)..................................................................39 3.3. Protein expression (I-IV)..............................................................................39 3.3.1 Western blot (I-IV)...................................................................................39 3.3.2 Immunohistochemistry, immunofluorescence and senescenceassociated !-galactosidase (I-IV) ........................................................39 3.3.3 Enzyme-linked immunosorbent assay (ELISA) (IV) .......................... 40 3.3.4 LDH activity assay (II, III).................................................................... 40 3.3.5 Caseinolysis assay.................................................................................. 40 3.4 mRNA expression (II-IV) ..............................................................................42 3.4.1 RT-qPCR (II, IV).......................................................................................42 3.4.2 In situ hybridization (III) .......................................................................42 3.4.3 Microarray...............................................................................................43 3.4.3.1 Microarray data analysis....................................................................43 3.5 Fluorescence-activated cell sorting (FACS) (II)...........................................44 3.6 Transmission electron microscopy (TEM) (II)............................................44 3.7 Statistical analyses .........................................................................................44 4. RESULTS AND DISCUSSION ........................................................45 4.1 Fibronectin-integrin interaction is required for fibroblast spheroid formation and activation (I).........................................................................45 4.1.1 Effect of fibronectin on spheroid formation (I).....................................45 4.1.2 FN-integrin interaction and FN matrix formation in fibroblast spheroids (I) ...........................................................................................46 4.2 Gene expression changes in fibroblast spheroids compared to adherent fibroblast cultures (II)..................................................................48 4.3 Autophagic activity increases during early time-points (II).......................49 4.3.1 ERK and Akt as possible regulators of autophagy in fibroblast spheroids (II).......................................................................................... 51 4.4 The downregulation of cytoskeleton is associated with fibroblast spheroid formation (II) ................................................................................52 4.5 Fibroblast spheroid formation relates to cell cycle arrest (II)....................53 4.6 Spheroid-activated fibroblasts acquire secretory phenotype (II-III) ........54 4.7 Fibroblast spheroids express markers of senescence (II)...........................54 4.8 Nemotic fibroblasts secrete matrix metalloproteinases to modulate their environment (III).................................................................................56 4.9 Benign keratinocytes inhibit, whereas malignant keratinocytes promote nemotic activation (IV) ................................................................. 57 4.10 Fibroblast spheroids inhibit growth of malignant keratinocytes (II)......59 4.10.1 RT3 cell growth on soft-agar is attenuated by co-culture with fibroblast spheroids (II) ........................................................................59 4.10.2 Fibroblast spheroids attenuate the growth of xenograft tumors by inducing tumor cell senescence (II) ............................................... 60 4.11 The summary of results ................................................................................ 61 CONCLUDING REMARKS ................................................................62 ACKNOWLEDGMENTS ....................................................................64 REFERENCES ...................................................................................66 ORGINAL PUBLICATIONS ...............................................................82