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Identifying human epithelial architecture genes with tumour suppressor functions

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Title: Identifying human epithelial architecture genes with tumour suppressor functions
Author(s): Myllynen, Mikko
Contributor: University of Helsinki, Faculty of Medicine, Institute of Biomedicine
Discipline: Biochemistry
Language: English
Acceptance year: 2013
Epithelial tissue is characterized by close cell-cell and cell-extracellular matrix (ECM) contacts as well as by apico-basal polarization. Integrity of these two features is important for functionality of epithelium. Additionally, proteins regulating polarity and cell junctions have been linked to cell cycle and apoptosis control. Consequently, defects in many of the polarity proteins have been linked to oncogenic events and loss of polarity is a hallmark of advanced cancers but whether it is causal to tumorigenesis is yet unknown. However, large body of knowledge on apico-basal polarity regulation and its connection on homeostasis control is derived from studies in Drosophila. This is mainly due to fact that efficient high throughput organotypic three dimensional (3D) culture methods enabling apico-basal polarization have not been available until the last decade. Large screens for epithelial polarity regulators have not been carried out in mammalian cells. Moreover, as cancer is the leading cause of death in developed countries and most of the cancers originate from epithelial tissues, knowledge of polarity regulation can be medically relevant. Oncogene MYC is overexpressed or amplified in variety of human cancers. The tumorigenic function of MYC is mainly due to its ability to drive cell cycle. We have previously shown that intact epithelial architecture is protective from cell cycle deregulating activities of MYC in 3D MCF10A mammary epithelial cell model and in vivo. This resistance can be overcome by inactivating LKB1 which is the human homologue of the polarity protein PAR4 implying a tumour suppressive role for epithelial architecture in mammalian cells. To identify regulators of epithelial architecture in mammalian cells, we have established lentiviral shRNA library (human epithelial architecture library, hEAL) encompassing 219 constructs targeting 77 genes associated with polarity regulation in Drosophila. We have previously screened the shRNA constructs for downregulation and quantified their effects on acinar morphology in the MCF10A 3D model. In this Master's Thesis I have validated the downregulation and phenotypes observed in a subset of the shRNAs during primary screening of the constructs of the library. Additionally, the possible co-operation with downregulation of the polarity regulators and conditional activation of MYC was determined. Most dramatically, downregulation of Wnt pathway gene DVL3 was shown to cause formation of enlarged multiacinar structures, which have increased proliferation. Additionally, downregulation of another Wnt pathway gene, GSK3β, resulted in acini with increased size and filled lumens. Thus these results propose a role for these genes in epithelial architecture regulation and tumour suppression in the used model even though apico-basal polarization of the acini was intact and no synergy with MYC was observed. Interestingly, no role in epithelial architecture regulation for Hippo pathway related genes FAT4 and MOBKL1A was found. Importantly, this study was able to validate primary screen showing relevance of the pipeline. Lastly, the study characterized the synthetic lethality phenotype found in the primary screen caused by downregulation of GTPase RHOA and chronic MYC activation. The shRHOA acini exhibited perturbed α6-integrin localization. When combined with MYC activation, the percentage of apoptotic acini was significantly increased. Importantly, the results suggest the observed synthetic lethality to be specific for the 3D context and to be associated with MEK/ERK and ROCK pathways. Taken together, in this study I have validated the role of novel epithelial architecture regulators and candidate tumour suppressors in MCF10A cells which may have medical relevance by helping to characterize tumorigenic processes. Furthermore, I characterized a novel 3D specific RHOA-MYC synthetic lethal interaction, which may prove to have therapeutic significance in MYC-driven cancers in future.

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