Project description:Advanced prostate cancer is a highly heterogenous disease with few in vitro models. We report generation of seven novel 3D organoid lines of patient-derived prostate cancer. We determine the copy number alterations of these lines using array CGH. They contain may classic alterations of prostate cancer such as lost of the short arm of chromosome 8 and gain of the long arm of chromosome 8. In addition, we found homozygous deleteion of tumor suppressor PTEN and CHD1 as well as the TMPRSS2-ERG interstitial deletion. Prostate cancer organoid lines, ~2 months after in vitro propagation, were used for profiling on Agilent 1M aCGH arrays per manufacturer's instructions. A pooled reference normal DNA was used as the reference.
Project description:Organotypic in vitro culture is useful to model mammalian disease in numerous tissues. Normal epithelial differentiation and carcinogenesis both undergo in vivo regulation by stroma, but current culture methods exclude stroma. To mimic this in vivo environment, we developed and characterized a human 3D prostate organoid co-culture model that incorporates prostate stroma. Primary prostate stromal cells supported increased organoid formation and expressed growth factors and WNT-related genes involved in epithelial differentiation. Organoid branching occurred distal to physical contact with stromal cells, demonstrating non-random branching. Tumoroids derived from primary prostate cancer maintained differential expression of the prostate cancer marker AMACR only in the presence of stroma. Stroma-induced phenotypes were similar in all patients examined, yet maintained inter-patient heterogeneity in the degree of response. Addition of stroma to in vitro organoid culture recapitulated the in vivo microenvironment by inducing organization of benign organoids into branching structures and preserving prostate cancer phenotypes.
Project description:Mutations in the FOXA1 transcription factor define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown. By annotating the FOXA1 mutation landscape from 3086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (~50% of all mutations) and R219 (~5%), a highly conserved DNA contact residue. Clinically, Wing2 mutations are seen in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumors with neuroendocrine histology. Interrogation of the biologic properties of FOXA1WT and 14 FOXA1 mutants revealed gain-of-function in mouse prostate organoid proliferation assays. ATAC-seq of FOXA1WT and representative Wing2 and R219 mutants revealed dramatic, mutant-specific changes in open chromatin at thousands of genomic loci, together with novel sites of FOXA1 binding and associated increases in gene expression. Of note, peaks in R219 mutant expressing cells lack the canonical core FOXA1 binding motifs (GTAAAC/T) but are enriched for a related motif (GTAAAG/A), which is preferentially activated by R219 mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its normal pioneering function and promote oncogenesis through perturbation of normal luminal epithelial differentiation programs, providing further support to the role of lineage plasticity in cancer progression.
Project description:The goal of this study was to determine how modulation of mitochondrial pyruvate metabolism regulates gene expression in a three-dimensional organoid model of mouse prostate epithelium. Organoids were treated with the small molecule UK5099, which impairs mitochondrial pyruvate uptake, and subjected to single-cell RNA-sequencing. Our analysis revealed considerable heterogeneity within prostate organoids characterized by distinct cell clusters expressing markers associated with basal, luminal and mesenchymal-like cells. UK5099 treatment led to a reduction in epithelial populations and an enrichment in mesenchymal-like cells. These findings indicate that pyruvate metabolism can modulate features of cell fate in prostate epithelium.
