CANCER BIOMARKER DISCOVERY BY IN VITRO SYSTEMS BIOLOGY

This Thesis was made with the intention to mechanistically assess and further develop a multistage cell line-based (in vitro) model for oral cancer development. Efforts of establishing additional tumor cell lines for expanding the model were coupled with the application of systems biology technologies for characterization of the three entities of the start-up model, including: 1) normal, 2) immortal and non-tumorigenic, versus 3) immortal and tumorigenic stages. Omics data integration from assessment of cell lines as unique entities, and modeldriven in vitro manipulations formed the basis for construction of two bioinformatics-based pipelines for this task. Altered phenotypic and genotypic characteristics and the event of nonfunctional cell differentiation (a hallmark of cancer development) was analyzed broadly among the transformed stages of the model relative the normal counterpart, testing the overarching hypothesis that thorough analysis of cell line data might contribute clinically useful tumor biomarkers potentially hidden in existing genome-wide assessments of clinical tissue samples. The separate papers forming the Thesis, in order, generated: 1) a review of existing data from the start-up model under a selected standardized serum-free condition, 2) an omics-integrative tumor biomarker discovery pipeline based on the start-up model, 3) a model-driven tumor biomarker discovery pipeline based on assessment of influences of confluency (high cell density and cell-to-cell contact) in the seemingly most differentiation-deficient cell line in the start-up model, 4) a novel tumor cell line applicable to expand the number of serum-free entities of the model, 5) an expanded model-driven tumor biomarker discovery pipeline based on assessment of serum-induced influences of the extended model (now with four entities), and finally, 6) an analysis of the novel cell line under a further expanded omics-integrative tumor biomarker discovery pipeline. The overall results included broad description of the multiple alterations at gene, pathway and ontology levels that coupled with the transformed phenotypes and non-functional cell differentiation in the cell line models. The bioinformaticsdriven assessment using overall six different processing tools of differential expression of 44 proteins and thousands of transcripts from these analyses suggested multiple potential biomarker signatures in head and neck squamous cell carcinoma. Overall, five in vitro-based signatures could be validated for clinical significance in independent data from tumor tissue analysis, including multiple oral and non-oral patient data sets as well as body-wide transcriptomics and proteomics expression databases. The taken approaches elucidated basic mechanisms of cell transformation while simultaneously generating paradigms/protocols generally applicable to cancer biomarker discovery. Proving the hypothesis under testing, the results show that the in vitro-derived biomarkers are complementary, often with superior accuracy, to those generated from direct assessment of cancer tissue specimens. Overall, the application of technologies and methods as described possibly generated a first description of an “in vitro systems biology model of oral cancer development” with potential for wide further application in experimental and translational research. LIST OF PUBLICATIONS This thesis is based on the following publications, referred to in the text by their Roman numerals. I. Ceder, R. , Merne, M. , Staab, C.A. , Nilsson, J.A. , Hoog, J.O. , Dressler, D. , Engelhart, K. , and Grafstrom, R.C. 2007. The application of normal, SV40 T-antigen-immortalised and tumor-derived oral keratinocytes, under serumfree conditions, to the study of the probability of cancer progression as a result of environmental exposure to chemicals. Altern Lab Anim 35:621-39 II. Staab, C.A. , Ceder, R. , Jagerbrink, T. , Nilsson, J.A. , Roberg, K. , Jornvall, H. , Hoog, J.O. , and Grafstrom, R.C. 2007. Bioinformatics processing of protein and transcript profiles of normal and transformed cell lines indicates functional impairment of transcriptional regulators in buccal carcinoma. J Proteome Res 6:3705-17 III. Ceder, R. , Haig, Y. , Merne, M. , Hansson, A. , Zheng, X. , Roberg, K. , Nees, M. , Iljin, K. , Bloor, B.K. , Morgan, P.R. , Fadeel, B. , and Grafstrom, R.C. 2011. Differentiation-promoting culture of competent and non-competent keratinocytes identifies biomarkers for head and neck cancer. Am J Pathol, in press IV. Roberg, K. , Ceder, R. , Farnebo, L. , Norberg-Spaak, L. , and Grafstrom, R.C. 2008. Multiple genotypic aberrances associate to terminal differentiation-deficiency of an oral squamous cell carcinoma in serum-free culture. Differentiation. 76:868-80 V. Ceder, R. , Merne, M. , Jerhammar, F. , Nees, M. , Roberg, K. , and Grafstrom, R.C. Serum-exposure of normal and transformed keratinocytes associates novel genes to head and neck cancer, manuscript VI. Ceder, R. , Staab, C.A. , Roberg, K. , Nees, M. , Jornvall, H. , Fadeel, B. , Hoog, J-O. , and Grafstrom, R.C. Gene signatures generated from a unique serum-free medium-derived tumor line accurately classifies normal and tongue squamous cell carcinoma tissue, manuscript PUBLICATIONS NOT INCLUDED IN THE THESIS 1. Staab, C.A. , Ceder, R. , Roberg, K. , Grafstrom, R.C. , and Hoog, J.O. 2008. Serumresponsive expression of carbonyl-metabolizing enzymes in normal and transformed human buccal keratinocytes. Cell Mol Life Sci 65:3653-63 2. Ansell, A. , Jerhammar, F. , Ceder, R. , Grafstrom, R.C. , Grenman, R. , and Roberg, K. 2009. Matrix metalloproteinase-7 and -13 expression associate to cisplatin resistance in head and neck cancer cell lines. Oral Oncol 45:866-71 3. Thompson, C.M. , Ceder, R. , and Grafstrom, R.C. 2010. Formaldehyde dehydrogenase: beyond phase I metabolism. Toxicol Lett 193:1-3 4. Jerhammar, F*. , Ceder, R*. , Garvin, S. , Grenman, R. , Grafstrom, R.C. , and Roberg, K. 2011. Fibronectin 1 is a potential biomarker for radioresistance in head and neck squamous cell carcinoma. Cancer Biol Ther 10:1244-51 *These authors contributed equally to this work. 5. Kwiecinska, A. , Ottoson-Wadlund, A. , Ceder, R. , Grafstrom, R.C. , Bjorck, E. , Nordenskjold, M. , Porwit, A. , and Fadeel, B. 2011. HAX-1 expression in human B lymphoma. Leukemia 25:868-72 6. Farnebo, L. , Jerhammar, F. , Ceder, R. , Grafstrom, R.C. , Vainikka, L. , Thunell, L. , Grenman, R. , Johansson, A.C. , and Roberg, K. 2011. Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines. J Oral Pathol Med 40:739-46