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Loss of differentiation during oncogenesisis generally thought to be a coincidental, rather than a causal, aspectoftumour progression. We tested whether master regulators of differentiationcould have more direct roles in tumour formation by acting as tumour suppressorgenes. In this work we demonstrate that the transcription factor atonal, which is necessary fordifferentiation in the Drosophilaeye, and ATOH1, which is essential forthe differentiation of Merkel cells in the skin and secretory cells in theintestine, shows all the hallmarks of a tumour suppressor gene. To investigatethe possible tumour suppressor function, we looked in tumours derived fromtissues which depend on ato-orthologuesto differentiate, namely eye tumours in Drosophila,two mouse models for colon adenocarcinoma (CAC), Merkel cell carcinoma (MCC) and CAC samples from patients and in celllines derived from MCC and CAC patients. Gain of atonalfunction leads to strong suppression of tumour formation in Drosophila.Conversely, loss of atonal (ato) in the Drosophila cancermodel enhances tumour progression and denovo tumour formation. We show that atofunctions by enhancing apoptosis and by the expression of cell cycle inhibitors.The effects of ato depend on JNKsignalling pathway. In addition, the mouse orthologue Atoh1 shows all the hallmarks of atumour suppressor gene. To this end, we conditionally ablated Atoh1 in the gut in two mouse models of CAC.The first model uses chemical carcinogenesis, the second sensitizes themice bymutations in the APC gene. In both cases, a significant increase in theincidence, the number as well as the size of the tumours is observed. Knock-outof Atoh1 in pre-neoplastic colonepithelium leads to more proliferation but no more apoptosis. In agreement with the functional data inthe Drosophila and the mouse model, humanCAC and MCC tumours show a marked decrease in the expression of ATOH1 opposed to control tissue. Moreaggressive stages of CAC have less ATOH1expression than less malignant tumours. This is due to genetic and epigeneticmutations in the form of deletions and methylation of the ATOH1 locus. Finally, gain and loss of function of ATOH1 in cell lines derived from CAC andMCC lead to a decrease and increase in growth, respectively. The decrease ingrowth upon Atoh1 activation ismediated by an increase in apoptosis and a slowing of the cell cycle. ATOH1 functions by activation ofreceptor tyrosine kinase signalling leading to phosphorylation of JNK.Similarly like in Drosophila, theeffects of ATOH1 are mediated by JNK. ABSTRACT III SAMENVATTING V ACKNOWLEDGEMENTS VII LIST OF ABBREVIATIONS IX TABLE OF CONTENTS - 1 - 1 INTRODUCTION - 5 - 1.1 ORIGIN OF CANCER - 5 - 1.1.1 Stem cells in oncogenesis - 6 - 1.1.2 Stem cell biology - 6 - 1.2 DIFFERENTIATION AND CANCER - 9 - 1.2.1 Differentiation and the cell cycle: establishing a point-of-no-return - 10 - 1.2.1.1 Transcriptional regulation - 11 - 1.2.1.2 Degrade towards differentiation - 13 - 1.2.1.3 Maintain your inhibition - 14 - 1.2.2 Differentiation factors implicated in cancer - 15 - 1.3 ATONAL, ATOH1, ATOH1: MANY NAMES, EVEN MORE FUNCTIONS. - 19 - 1.3.1 Regulation of downstream genes by atonal - 20 - 1.3.2 A progenitor-specifying function for atonal orthologues - 22 - 1.3.3 Terminal differentiation function for atonal orthologues - 24 - 1.3.4 ATOH1 implicated in tumours - 27 - 1.4 DROSOPHILA AS A MODEL FOR CANCER - 29 - 1.5 MERKEL CELL CARCINOMA - 35 - 1.6 COLON ADENOCARCINOMA - 37 - 2 AIMS - 41 - 3 METHODOLOGY - 43 - 3.1 DROSOPHILA HUSBANDRY - 43 - 3.2 IMMUNOHISTOCHEMISTRY - 43 - 3.3 GENERATION OF UAS- ATOERD TRANSGENIC FLIES - 43 - 3.4 QRT-PCR ON DROSOPHILA LARVAL EYE-ANTENNAL DISCS - 44 - 3.5 CLONING OF ATOH1ERD EXPRESSION CONSTRUCT - 44 - 3.6 ANIMALS AND TREATMENTS - 44 - 3.7 IMMUNOHISTOCHEMISTRY AND MICROSCOPIC ANALYSIS - 45 - 3.8 CELL CULTURE CONDITIONS - 46 - 3.9 DOUBLING TIME - 47 - 3.10 LENTIVIRAL VECTORS - 47 - 3.11 COLONY FORMATION IN SOFT AGAR - 48 - 3.12 CELL CYCLE DISTRIBUTION - 49 - 3.13 APOPTOSIS DETECTION - 49 - 3.14 WESTERN BLOTTING - 49 - 3.15 TYROSINE KINASE INHIBITOR, SAPK INHIBITOR II AND GAMMA-SECRETASE INHIBITOR - 50 - 3.16 BRDU STAINING - 50 - 3.17 RT-PCR AND QPCR - 51 - 3.18 90 KINASE RT-PCR ASSAY - 52 - 3.19 METHYLATION DETECTION - 52 - 3.20 ARRAY CGH - 53 - 3.21 SEQUENCING - 54 - 4 RESULTS - 55 - 4.1 ATONAL ACTS AS A TUMOUR SUPPRESSOR IN A DROSOPHILA CANCER PARADIGM - 55 - 4.1.1 Gain and loss of function of atonal, respectively repress and facilitate cancer progression - 55 - 4.1.2 ato functions through JNK-dependent inhibition of cell cycle and induction of apoptosis - 58 - 4.2 ATOH1 FUNCTIONS AS A TUMOUR SUPPRESSOR GENE IN TWO MOUSE MODELS OF CAC. - 61 - 4.3 ATOH1 IS GENETICALLY AND EPIGENETICALLY MUTATED IN HUMAN CAC AND MCC TUMOURS - 65 - 4.3.1 Frequent deletions in the ATOH1 locus - 65 - 4.3.2 ATOH1 locus is methylated during oncogenesis - 66 - 4.4 ATOH1 ACTS BY SIMILAR MECHANISMS IN HUMAN CANCER CELLS AS IN DROSOPHILA - 71 - 4.4.1 High levels of ATOH1 correlate with higher doubling times and decreased malignancy - 71 - 4.4.2 ATOH1 is not dependent on Notch signalling - 75 - 4.4.3 ATOH1 tumour suppressor function is mediated by RTK’s - 76 - 5 DISCUSSION - 81 - 5.1 ATOH1 IS A TUMOUR SUPPRESSOR: THREE PREDICTIONS - 81 - 5.2 ATONAL’S MODE OF ACTION - 82 - 5.3 THE ATOH1 LOCUS: A SPECIAL CASE - 85 - 5.4 ATOH1 STATUS MIGHT BE LINKED TO DISEASE STAGE AND OUTCOME - 88 - 5.5 LOSS OF DIFFERENTIATION AND CANCER: A “CONDITIO SINE QUA NON”? - 89 - 6 REFERENCES - 93 - 7 CURRICULUM VITAE - 105 - status: published |
