A) FDR-corrected shared and unique ChIP peaks of AIB1 and AIB1Î"4 in MCF10A parental cell line (10A) versus AIB1Î"4-expressing isogenic line (10A-Î"4). Shared peaks have at least 1bp overlap. B) AIB1 ChIP peaks in MCF7 cells treated for 3 hours with estradiol (E2) or vehicle (V) overlapped with MCF10A and MCFDCIS (parental and AIB1Î"4 derivative lines). MCF7 data set obtained from Zwart et al(32). C) Motifs within 200 bp of the peak summit for significant ChIP-Seq peaks were analyzed for motif enrichment using HOMER. In red are the motifs unique to AIB1Î"4 in MCF10A cells. D) FDR-corrected histone modification peaks in DCIS and DCIS-Î"4 overlapping with enhancer sites from human mammary epithelial cells (hMEC enh.) (42). E) The distribution of AIB1, H3K27ac or H3K4me1 ChIP peaks 500 kb up- and downstream of gene transcription start site (TSS). Genomic distance was divided into 5kb bins.

A list of differentially expressed genes in tumors and cells.

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.

Palbociclib treated 3D time course

A list of enriched pathways using Gene Set Enrichment Analysis (GSEA).

A list of AIB1Î"4 signature genes that were used to generate KM-plots in Figure S5.

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.

A) Sequence alignment of PCR amplicons of genomic DNA spanning exon 4 of AIB1 (UCSC genome browser). Black filled boxes are the sequences that align to the genome and dotted lines represent the missing/deleted sequences. AIB1 in the DCIS-Î"4 cell line is missing part of intron 3 and exon 4. Two deleted regions around exon 4 of AIB1 were identified in the 10A-Î"4 cell line; both are missing exon 4 acceptor site. B) Sanger sequencing of PCR amplicons spanning exons 2, 3, 4, 5 splice junctions. C) AIB1 mRNA between exon 2 and exon 5 in the MCFDCIS-Î"4 cell clones and the parental MCFDCIS cells. D) Western blot shows AIB1 isoforms' protein expression in MCFDCIS-Î"4 cell clones and the parental MCFDCIS cells. E) Schematic of the migration, trans-endothelial invasion and proliferation assays using real time electric cell impedance sensing (ECIS) to detect migrating cells, invasion across an endothelial monolayer or proliferation rate. F) Invasion of DCIS-Î"4-hy across an endothelial monolayer using ECIS compared to parental control DCIS cells. G) Cell proliferation of MCFDCIS (DCIS), DCIS-Î"4 and DCIS-Î"4-hy measured by electric cell impedance sensing (ECIS). A single well coated with electrodes was used to measure real time change in cell impedance as they proliferate over 80 hours (See diagram in S1E). H) Cell proliferation of MCFDCIS and MCF10A parental lines and their AIB1Î"4 expressing counterpart. ***p<0.001.

Supplementary Methods: Supplementary Table 1 and 2

A) Representative images of hematoxylin and eosin (H&E) staining of MCFDCIS, MCFDCIS-Î"4 and mixed (4:1 respectively) tumors that were injected in the mammary fat pad of SCID/Beige mice after two or five weeks. MCFDCIS-Î"4 tumors were not collected at two weeks due to small tumor size. Scale bar=50μm. B) Growth of subcutaneous tumors of DCIS or DCIS-Î"4-hy in athymic nude mice. n=5. **p<0.01. H&E tumor histology at 3 weeks. Scale bar = 50μm. C) Quantification of P63 and Luciferase (unique to DCIS) co-localization in mixed tumors at 2 and 5 weeks. ROI= 2007x1500um. **p<0.01. D) Representative images and quantification of p63 and CD44 IHC on tumors from S3B. **p<0.01, ***p<0.001. Scale bar = 50μm. E) Bioluminescence images showing lung metastases at 2-3 months. Signal is detected from parental DCIS that harbor the luciferase construct. DCIS-Î"4 cells are luciferase negative. F) qPCR of genomic DNA from the lungs of mice harboring mix tumors. Primers are specific to either the unique region on AIB1 gene that was created by CRISPR editing (CRISPR) or human intronic DNA as a control (Intronic). Mouse intronic DNA was used for normalization. G) Bioluminescence images and graph showing tumor recurrence rate after DCIS or Mix primary tumor removal when they reached an area of ~80mm2. Two million cells were injected in the MFP of SCID mice. H) qPCR of human genomic DNA for either the unique region on AIB1 gene that was created by CRISPR editing (CRISPR) or human intronic DNA as a control (Intronic). MCFDCIS and MCFDCIS-Î"4 cell lines were used as reference points for MCFDCIS and Mix tumors (MCFDCIS and MCFDCIS-Î"4).

AbstractAIB1Δ4 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.Significance: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.

AbstractCDK4/6 inhibitors are used in the treatment of advanced estrogen receptor (ER)(+) breast cancer. Their efficacy in ER(−) and early-stage breast cancer is currently under investigation. Here, we show that palbociclib, a CDK4/6 inhibitor, can inhibit both progression of ductal carcinoma in situ (DCIS) and growth of invasive disease in both an ER(−) basal breast cancer model (MCFDCIS) and an ER(+) luminal model (MCF7 intraductal injection). In MCFDCIS cells, palbociclib repressed cell-cycle gene expression, inhibited proliferation, induced senescence, and normalized tumorspheres formed in Matrigel while the formation of acini by normal mammary epithelial cells (MCF10A) was not affected. Palbociclib treatment of mice with MCFDCIS tumors inhibited their malignant progression and reduced proliferation of invasive lesions. Transcriptomic analysis of the tumor and stromal cell compartments showed that cell cycle and senescence genes, and MUC16, an ovarian cancer biomarker gene, were repressed during treatment. Knockdown of MUC16 in MCFDCIS cells inhibited proliferation of invasive lesions but not progression of DCIS. After cessation of palbociclib treatment genes associated with differentiation, for example, P63, inflammation, IFNγ response, and antigen processing and presentation remained suppressed in the tumor and surrounding stroma. We conclude that palbociclib can prevent progression of DCIS and is antiproliferative in ER(−) invasive disease mediated in part via MUC16. Lasting effects of CDK4/6 inhibition after drug withdrawal on differentiation and the immune response could impact the approach to treatment of early-stage ER(−) breast cancer.

Transcription factors critical for the transition of normal breast epithelium to ductal carcinoma in situ (DCIS) and invasive breast cancer are not clearly defined. Here, we report that the expression of a subset of YAP-activated and YAP-repressed genes in normal mammary and early-stage breast cancer cells is dependent on the nuclear co-activator AIB1. Gene expression, sequential ChIP, and ChIP-seq analyses show that AIB1 and YAP converge upon TEAD for transcriptional activation and repression. We find that AIB1-YAP repression of genes at the 1q21.3 locus is mediated by AIB1-dependent recruitment of ANCO1, a tumor suppressor whose expression is progressively lost during breast cancer progression. Reducing ANCO1 reverts AIB1-YAP-dependent repression, increases cell size, and enhances YAP-driven aberrant 3D growth. Loss of endogenous ANCO1 occurs during DCIS xenograft progression, a pattern associated with poor prognosis in human breast cancer. We conclude that increased expression of AIB1-YAP co-activated targets coupled with a loss of normal ANCO1 repression is critical to patterns of gene expression that mediate malignant progression of early-stage breast cancer.

Ductal carcinoma in situ (DCIS) is a pre-invasive lesion of the breast considered a precursor of invasive ductal carcinoma. This study aimed to determine whether activated PPARγ acts as a tumor suppressor in human DCIS progression.

Breast cancer can be defined as a group of diseases with heterogeneous origins, molecular profiles and behaviors characterized by uncontrolled proliferation of cells within the mammary tissue. Around one in eight women in the US will develop breast cancer in their lifetime, making it the second most frequently diagnosed cancer behind skin cancer [1]. In 2015, an estimated 231,840 cases of invasive carcinoma were diagnosed, and over 40,000 deaths were caused by breast cancer which accounts for almost 7% of all cancer mortality each year [1, 2]. In 2015, 60,290 cases of in situ breast cancer were diagnosed, representing over 14% of all new cancer cases among women and men [1]. The steep increase in diagnosis of early‐stage breast cancer over the past 10 years is believed to be a result of more frequent mammography. However, since over half of these in situ lesions will not progress to invasive breast cancer, controversies have arisen about approaches to treatment and prevention of progression of early‐stage in situ breast cancer. Understanding the mechanisms of transition of normal breast to in situ pre‐neoplastic lesions and invasive breast cancer is currently a major focus of breast cancer research with implications for preventive and clinical management of breast cancer. In this review, we give an overview of current knowledge on the molecular and pathological changes that occur during early‐stage progression of breast cancer and describe some of the current models that are used to study this process.

Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2 −/− mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2 −/− model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure.

Breast cancer can be defined as a group of diseases with heterogeneous origins, molecu - lar profiles and behaviors characterized by uncontrolled proliferation of cells within the mammary tissue. Around one in eight women in the US will develop breast cancer in their lifetime, making it the second most frequently diagnosed cancer behind skin cancer [1 ]. In 2015, an estimated 231,840 cases of invasive carcinoma were diagnosed, and over 40,000 deaths were caused by breast cancer which accounts for almost 7% of all cancer mortality each year [ 1 , 2]. In 2015, 60,290 cases of in situ breast cancer were diagnosed, rep - resenting over 14% of all new cancer cases among women and men [ 1 ]. The steep increase in diagnosis of early‐stage breast cancer over the past 10 years is believed to be a result of more frequent mammography. However, since over half of these in situ lesions will not progress to invasive breast cancer, controversies have arisen about approaches to treat - ment and prevention of progression of early‐stage in situ breast cancer. Understanding the mechanisms of transition of normal breast to in situ pre‐neoplastic lesions and inva - sive breast cancer is currently a major focus of breast cancer research with implications for preventive and clinical management of breast cancer. In this review, we give an over - view of current knowledge on the molecular and pathological changes that occur during early‐stage progression of breast cancer and describe some of the current models that are used to study this process.

Palbociclib (PD), a CDK4/6 inhibitor, has recently been approved for use in advanced ER positive breast cancer, demonstrating significant benefit in combination with endocrine therapies. Ductal carcinoma in situ (DCIS) is an inobligate precursor to invasive ductal carcinoma with anywhere from 14-53% of cases progressing. Progression is unpredictable and driven by poorly understood mechanisms. In this study, we assess the efficacy of PD in the context of this early stage disease using a triple negative DCIS model(MCF10DCIS), not only in inhibiting cell cycle but also in hindering the progression from in situ to invasive lesion. We demonstrate similar efficacy of PD in 2D on MCF10DCIS and immortal mammary epithelial cells (MCF10As) in preventing proliferation, inhibiting phosphorylation of RB and stopping cell cycle progression. Yet in 3D, MCF10DCIS cells form smaller, more organized spheres in the presence of drug while MCF10As appear unaffected, highlighting potential disease selectivity in vivo. Interestingly, when a cDNA array is performed on MCF10DCIS cells grown in 3D +/- PD, the most regulated genes are not well characterized cell cycle genes as one might predict based on previously published data and 2D response to treatment, suggesting alternative CDK4/6 targets may be responsible for the phenotype observed in 3D. We also demonstrate in a nude mouse xenograft model of MCF10DCIS that PD is able to significantly reduce overall tumor burden while simultaneously delaying the progression of lesions to invasive disease. Though the growth of PD treated MCF10DCIS lesions is delayed we demonstrate that proliferation is unaffected in vivo by the presence of the drug, based on equivalent Ki67 signal within the lesions. This reiterates the possibility that in 3D and in vivo contexts PD targets mechanisms outside of cell cycle regulation in order to slow tumor growth and delay invasive transition. We believe that investigation into alternative targets of CDK4/6 altered by PD in 3D and in vivo may provide insights on important mechanisms of DCIS progression in DCIS models and human patients. Citation Format: William B. Kietzman, Virginie Ory, Fransisco Saenz, Ghada Sharif, Anton Wellstein, Anna T. Riegel. CDK4/6 inhibition in early stage triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2357. doi:10.1158/1538-7445.AM2017-2357