Role of ARID1A in endometrial cancer organoid model

Endometrium is the inner lining of the uterus. Endometrial cancer is the most prevalent malignancy of the female reproductive tract in developed countries and the incidence rates are steadily increasing. One of the most frequently mutated genes in endometrial tumours is ARID1A, a component of the SWI/SNF chromatin remodelling complex, however the mechanism by which its inactivation promotes tumorigenesis is unclear, not least due to the lack of a suitable experimental model. Recently, we and others have established 3D endometrial organoid cultures that faithfully recapitulate the architecture, expression patterns, hormonal responsiveness and secretory abilities of the in vivo endometrial epithelium. We propose to employ this system to elucidate the function of ARID1A in human endometrium and its role in initiation and progression of endometrial cancer. Using loss of function experiments, expression analyses and chromatin immunoprecipitation, we seek to decipher the set of genes directly regulated by ARID1A in human endometrial epithelium. To gain novel insights into the ARID1A mode of action we aim to determine ARID1A protein interactors using immunoprecipitation followed by mass spectrometry. We expect to identify transcription factors that recruit ARID1A (as part of the SWI/SNF chromatin remodelling complex) to specific genomic loci as well as endometrium-specific subunits of this complex. Next, we aim to generate an organoid model of endometrial cancer by introduction of mutation in ARID1A, KRAS and/or PIKCA3 genes using the CRISPR/Cas9 technology. We will follow effects of these mutations on niche factor dependency, organoid growth and tumorigenicity upon transplantation into mice and in the context of high estrogen signalling. Overall, here we propose to use a unique, comprehensive approach by combining mechanistic advances with functional assays in a novel cancer model system to understand the role of ARID1A in endometrial biology and malignancy.

Despite advances in reproductive medicine, pregnancy-related disorders associated with impaired placental and uterine development and function still pose a large risk to the mother and the embryo. The human placenta is a vital organ to sustain mammalian development in utero. It consists of three main lineages: the extravillous trophoblast (EVT) that invades the maternal uterus and remodels blood vessels, the multinucleated syncytiotrophoblast (STB) that is the actual site of exchange between the maternal and foetal bloodstreams, and the progenitor population cytotrophoblast (CTB), that gives rise to both STB and EVT. Failures in the differentiation of these lineages during development lead to placental pathologies. This funding enabled us to uncover the MSX2 protein as a vital regulator of the CTB progenitor population. Using human trophoblast stem cells (hTSCs), we demonstrated that depletion of MSX2 results in spontaneous STB differentiation, while MSX2 forced expression blocks it. We found that MSX2 interacts and co-binds many target genes with components of the SWI/SNF chromatin remodelling complex. In addition, we specifically investigated the function of the SWI/SNF complex during STB differentiation of hTSCs. Enhanced SWI/SNF binding at the regions with increased chromatin accessibility, as measured by ChIP-seq and ATAC-seq, indicated a role of SWI/SNF in actively reshaping the chromatin landscape during this process. Chemical and genetic perturbations of SWI/SNF function resulted in delayed STB differentiation of hTSCs, trophoblast organoids, and primary culture models. The observed phenotype manifested in deregulating differentiation and self-renewal genes, reduced chromatin accessibility, and reduced H3K27ac deposition at important regulatory regions. Together, these findings expand our understanding and provide important novel insights into the molecular mechanisms regulating human placental development and disease.