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:Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. (1), which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells. The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.
