A) Quantification of the number of Zebrafish embryos with DCIS or DCIS-Î"4-hy cells that have extravasated out of the blood vessels and into the neighboring tissue. p=0.57. B) Quantification of the number of Zebrafish embryos with MCF10A, MCF10A-Î"4 or mixed cells that have extravasated out of the blood vessels and into the neighboring tissue. In the mixed cell population, only MCF10A parental line was fluorescently labeled and scored for extravasation. MCF10A and MCF10A-Î"4 are mixed at a 4:1 ratio. C) Invasion assay of an endothelial monolayer (HUVEC) using ECIS. MCFDCIS cells were treated with conditioned media (CM) from MCFDCIS-Î"4 cells for 4 hours before added to the endothelial monolayer, and vice versa. No cells added to the endothelial monolayer as a negative control. D) A schematic showing a three-chambered co-culture system. Top chamber has electrodes underneath a porous membrane to detect migrating cells. Middle chamber is only permeable to factors but not cells. Bottom chamber harbors cells that can crosstalk with migrating cells through secreted factors. E) Real-time migration rate of MCFDCIS and MCFDCIS-Î"4 in co-culture chambers. F) MCFDCIS and MCFDCIS-Î"4 cells were aggregated separately then embedded in 80% col I and 20% Matrigel together to monitor their crosstalk as separate spheres. Scale bar = 50μm.
ARTICLE ABSTRACT
AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an “enabler” phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population.
A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.
