To My Family “We must have perseverance and above all confidence in ourselves. We must believe that we are gifted for something and that this thing must be attained.” ― Maria Skłodowska-Curie List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Zieba A, Pardali K, Söderberg O, Lindbom L, Nyström E, Moustakas A, Heldin C-H and Landegren U. Intercellular varia- tion in signaling through the TGF-β pathway and its relation to cell density and cell cycle phase. Mol Cell Proteomics. 2012 Mar 22. [Epub ahead of print] II Sundqvist A, Zieba A, Vasilaki E, Herrera Hidalgo C, Söder- berg O, Heldin C-H, Landegren U, ten Dijke P and van Dam H. Specific interactions between Smad proteins and AP1 compo- nents determine TGFβ–induced breast cancer cell invasion. Submitted. III Pinidiyaarachchi A, Zieba A, Allalou A, Pardali K, Wählby C. A detailed analysis of 3D subcellular signal localization. Cy- tometry A. 2009 Apr;75(4):319-28. IV Zieba A, Wählby C, Hjelm F, Jordan L, Berg J, Landegren U, Pardali K . Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with perox- idase detection. Clin Chem. 2010 Jan;56(1):99-110. Reprints were made with permission from the respective publishers. Other publications by the author V Raja E, Zieba A, Morén A, Voytyuk I, Söderberg O, Heldin C-H and Moustakas A. The tumour suppressor kinase LKB1 negatively regu- lates bone morphogenetic protein signalling. Submitted. VI Dahl M, Lönn P, Vanlandewijck, M, Zieba A, Hottiger M, Söder- berg O, Heldin C-H and Moustakas A. PARP-2 activation and asso- ciation with Smads during regulation of TGF TGF-β signaling. Man- uscript. VII Dieterich L, Mellberg S, Langenkamp E, Zhang L, Zieba A, Sa- lomäki H, Teichert M, Huang H, Edqvist P-H, Kraus T, Augustin H, Olofsson T, Larsson E, Söderberg O, Molema G, Pontén P, Georgii- Hemming P, Alafuzoff I, Dimberg A. Transcriptional profiling of tumor vessels reveals a gene expression signature indicative of a key role of VEGF-A and TGF-β2 in vascular abnormalization in human glioblastoma. Submitted. VIII Zieba A, Grannas K, Söderberg O, Nilsson M and Landegren L. Molecular tools for companion diagnostics. Submitted review. IX Leja J, Yu D, Nilsson B, Gedda L, Zieba A, Hakkarainen T, Åker- ström G, Öberg K, Giandomenico V, Essand M. Oncolytic adenovi- rus modified with somatostatin motifs for selective infection of neu- roendocrine tumor cells. Gene Ther. 2011 Nov;18(11):1052-62. X Florczyk U, Golda S, Zieba A, Cisowski J, Jozkowicz A, Dulak J. Overexpression of biliverdin reductase enhances resistance to chemotherapeutics. Cancer Lett. 2011 Jan 1;300(1):40-7. XI Wang X, Hu B, Zieba A, Neumann NG, Kasper-Sonnenberg M, Honsbein A, Hultqvist G, Conze T, Witt W, Limbach C, Geitmann M, Danielson H, Kolarow R, Niemann G, Lessmann V, Kilimann MW. A protein interaction node at the neurotransmitter release site: domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-1. Neurosci. 2009 Oct 7;29(40):12584-96. XII Looman C, Sun T, Yu Y, Zieba A, Ahgren A, Feinstein R, Forsberg H, Hellberg C, Heldin CH, Zhang XQ, Forsberg-Nilsson K, Khoo N, Fundele R, Heuchel R. An activating mutation in the PDGF receptor- beta causes abnormal morphology in the mouse placenta. Int J Dev Biol. 2007;51(5):361-70. Contents Introduction ................................................................................................... 11 TGF-β signaling- the classical frame ............................................................ 12 Down to the nucleus ................................................................................. 12 They are not alone .................................................................................... 14 Phospho-“diversion” – when one is not enough ........................................... 15 TGF-β in cancer ............................................................................................ 18 TGF-β in colon carcinogenesis ................................................................ 19 TGF-β in biomarker discovery ................................................................. 20 Paper I and II - With the tool in my hand ..................................................... 22 Paper I: Intercellular variation in signaling through the TGF-β pathway and its relation to cell density and cell cycle phase .................................. 23 Paper II: Specific interactions between Smad proteins and AP1 components determine TGFβ–induced breast cancer cell invasion. ........ 24 Paper III and IV - Necessity is the mother of invention ............................... 26 Paper III: A detailed analysis of 3D subcellular signal localization......... 26 Paper IV: Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection. ........ 27 Current investigations and future plans ........................................................ 28 The context-dependent signaling: Perspectives on the single cell analysis of the TGF-β pathway ................................................................ 28 Smad phosphorylation ......................................................................... 28 Hippo pathway- The window on the world ......................................... 29 PLA in cancer diagnostics ........................................................................ 32 Identification of a gene expression signature for colorectal cancer outcome ............................................................................................... 32 TGF-β and tumor vasculature .............................................................. 34 Acknowledgments......................................................................................... 36 References ..................................................................................................... 39 Abbreviations ActR-II activin receptor type II ALK1-7 activin receptor-like kinases 1-7 AMHR-II anti-Müllerian hormone receptor type II AP-1 activating protein-1 ATF activating transcription factor BMP bone morphogenetic protein BMPR-II bone morphogenetic protein type II bZIP basic leucine zipper CDK cyclin-dependent kinase cFos FBJ murine osteosarcoma viral oncogene homolog chIP chromatin-immunoprecipitation Co-Smad common mediator Smad CRC colorectal cancer EGF epidermal growth factor EMT epithelial to mesenchymal transition EPSC EMT promoting Smad complexes ERK extracellular signal-regulated kinases FGF fibroblast growth factor Fra1/2 Fos-related antigen 1/2 HRP horseradish peroxidase I-Smad inhibitory Smad isPLA in situ proximity ligation assay JNK c-Jun N-terminal kinase K-ras Kirsten rat sarcoma viral oncogene homolog LNA locked nucleic acid sequences Maf musculoaponeurotic fibrosarcoma oncogene homolog MSI microsatellite instability Noch neurogenic locus notch homolog protein PI3 kinase phosphatidylinositol 3-kinase PLA proximity ligation assay PTM post-translational modification Ras rat sarcoma RCA rolling circle amplification R-Smad receptor regulated Smad RCP rolling circle product Smad small mothers against decapentaplegic Ski Sloan-Kettering avian retrovirus transforming protein SnoN Ski-related novel protein N Taz transcriptional coactivator with PDZ-binding motif TGF-β transforming growth factor β TGF-β RI TGF-β Receptor Type I TGF-β RII TGF-β Receptor Type II TMA tissue microarrays TS thymidylate synthase Wnt wingless-type MMTV integration site family Yap Yes-associated protein Introduction A number of mechanisms can modulate Smad signaling on different levels. To understand all aspects of that signaling it is important to build a “map” of interactions and modifications that are involved. Smads never work alone and there is not only one path of signaling through Smads. To project a com- plete picture of TGF-β signal transduction one needs to change the percep- tion of Smad signaling pathway and see it as a sophisticated network instead of a linear signaling pipeline. The core components of Smad signaling func- tion as a “backbone” of TGF-β signaling, while the most important regulato- ry elements for the outcome of the cascade activity come from the outside of the canonical pathway. These are the ones we now need to focus on. Analyses of cellular responses are generally done at the population level, ignoring sources of heterogeneity and thereby complicating interpretation of how cells coordinate responses to changes in the external and internal envi- ronments. Outcomes of analysis of in vitro cultured cell lines are often ex- trapolated to biology of cells in their natural environment, the body. Taking into account the variety of cellular responses in tissue of organs or tumors it is unlikely that this is the right path to follow. Therefore analysis of signal- ing events needs to be performed at the single cell level within the cells normal environment, i.e. within tissue sections. 11 TGF-β signaling- the classical frame The TGF-β pathway is one of the most well studied regulatory systems of the cell that is involved in most of major cell activities under physiological and pathological conditions. Deregulation in signaling downstream of TGF-β is associated with many diseases such as cancer and fibrosis [1-4]. TGF-β has a very complex role in tumorigenesis, exhibiting both oncogenic and suppressive properties, depending on tumor stage. At early stages TGF-β induces apoptosis and growth arrest, it also has the ability to suppress