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Figure S5 from An AIB1 Isoform Alters Enhancer Access and Enables Progression of Early-Stage Triple-Negative Breast Cancer

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posted on 2023-03-31, 04:28 authored by Ghada M. Sharif, Moray J. Campbell, Apsra Nasir, Surojeet Sengupta, Garrett T. Graham, Max H. Kushner, William B. Kietzman, Marcel O. Schmidt, Gray W. Pearson, Olivier Loudig, Susan Fineberg, Anton Wellstein, Anna T. Riegel

A) Principal component analyses of RNA-Seq from MCFDCIS and MCF10A cell lines and the AIB1Î"4 corresponding clones. B) Relapse free survival and overall survival KM-plots in patients with ER-negative breast tumors separated by AIB1Î"4 top regulated genes in MCFDCIS cells. AIB1Î"4 signature gene list used to generate the KM-plots is in Supplementary table S3. C) Volcano plots illustrating differentially expressed genes (DEGs) regulated by AIB1Î"4 in MCF10A cell clones (|log2(FC)|>1.5 and -log(adj p-val)>1.3). Full list of DEGs in Supplementary table S1. D) Significantly enriched hallmark pathways from gene set enrichment analyses (GSEA) associated with AIB1Î"4 expression in MCF10A cells. Full list of enriched pathways in Supplementary table S2. E) Top canonical pathways altered in MCF10A-Î"4 compared to control MCF10A cells identified by Ingenuity Pathway Analysis (IPA). |z-score|>1, p-val <0.05. F) Relapse free survival KM-plots in patients with ER-negative breast tumors separated by AIB1Î"4 top regulated genes in MCF10A cells. AIB1Î"4 signature gene list used to generate the KM-plots in Supplementary table S3. G) Western blot of EMT markers of DCIS cells induced by DCIS-Î"4 for the indicated times. Actin is used as a loading control. Band quantification is indicated in red. H) A volcano plot illustrating differentially expressed genes (DEGs) in MCFDCIS-Î"4 when co-cultured with MCFDCIS compared to MCFDCIS-Î"4 alone. Mixed cells were then separated by flow cytometry prior to RNA-seq analysis. (|log2(FC)|>1.5 and -log(adj p-val)>1.3). Full list of DEGs in Supplementary table S1.

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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.

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