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A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

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Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Georgetown University

Georgetown University Medical Center

The oncogene amplified in breast cancer 1 (AIB1) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-Δ4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-Δ4 at Met(224) of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-Δ4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Δ4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-Δ4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-Δ4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-Δ4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that AIB1-Δ4 expression is correlated with metastatic capability of human cancer cell lines.

Role of the nuclear receptor coactivator AIB1-Delta4 splice variant in the control of gene transcription.

The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator that is overexpressed in various types of human cancers. However, the molecular mechanisms controlling AIB1 expression in the majority of cancers remain unclear. In this study, we identified a novel interacting protein of AIB1, forkhead-box protein G1 (FoxG1), which is an evolutionarily conserved forkhead-box transcriptional corepressor. We show that FoxG1 expression is low in breast cancer cell lines and that low levels of FoxG1 are correlated with a worse prognosis in breast cancer. We also demonstrate that transient overexpression of FoxG1 can suppress endogenous levels of AIB1 mRNA and protein in MCF-7 breast cancer cells. Exogenously expressed FoxG1 in MCF-7 cells also leads to apoptosis that can be rescued in part by AIB1 overexpression. Using chromatin immunoprecipitation, we determined that FoxG1 is recruited to a region of the AIB1 gene promoter previously characterized to be responsible for AIB1-induced, positive autoregulation of transcription through the recruitment of an activating, multiprotein complex, involving AIB1, E2F transcription factor 1, and specificity protein 1. Increased FoxG1 expression significantly reduces the recruitment of AIB1, E2F transcription factor 1 and E1A-binding protein p300 to this region of the endogenous AIB1 gene promoter. Our data imply that FoxG1 can function as a pro-apoptotic factor in part through suppression of AIB1 coactivator transcription complex formation, thereby reducing the expression of the AIB1 oncogene.

Transcriptional repression of AIB1 by FoxG1 leads to apoptosis in breast cancer cells.

Transcription of the HER2 oncogene can be repressed by estrogen (E2). We now show that, a splice isoform of the nuclear receptor coactivator AIB1, AIB1-Δ4, is able to reverse E2 repression of HER2 gene expression in breast cancer cells. The first 224 amino acids of AIB1 that are absent in AIB1-Δ4, bind a co-repressor, ANCO1. Using chromatin immunoprecipitation assay approaches in MCF7 and BT474 cell lines, we demonstrate that AIB1 and AIB1-Δ4 can bind to the E2 regulatory site in the first intron of the HER2 gene, after E2 treatment, but only full-length AIB1 recruits ANCO1. Consistent with E2-induced chromatin repression, the AIB1-ANCO1 complex recruits HDAC3 and HDAC4 to the intronic estrogen response element and the proximal promoter acquires the repressive chromatin mark H3K9me3 and loses H3K4me1. In contrast, AIB1-Δ4 does not recruit ANCO 1, HDAC3, or HDAC4 and the proximal promoter retains activation marks of H3K4me1. In cell lines with low levels of ANCO1 (T47D), E2 does not repress HER2 gene transcription but the repressive response can be restored by overexpression of ANCO1. ANCO1 can also repress other E2-responsive genes, indicating that AIB1, AIB1-Δ4 and ANCO1 are important determinants of endocrine and growth factor responsiveness in breast cancer.

Regulation of HER2 oncogene transcription by a multifunctional coactivator/corepressor complex.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the  and  loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for . These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., , , , , and ) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

The transcriptional coactivator Amplified in Breast Cancer 1 (AIB1) plays a major role in the progression of hormone and HER2-dependent breast cancers but its role in triple negative breast cancer (TNBC) is undefined. Here, we report that established TNBC cell lines, as well as cells from a TNBC patient-derived xenograft (PDX) that survive chemotherapy treatment in vitro express lower levels of AIB1 protein. The surviving cell population has an impaired tube-formation phenotype when cultured onto basement membrane, a property shared with TNBC cells that survive shRNA-mediated depletion of AIB1 (AIB1 cells). DNA analysis by exome sequencing revealed that AIB1 cells represent a distinct subpopulation. Consistent with their in vitro phenotype AIB1 cells implanted orthotopically generated slower growing tumors with less capacity for pulmonary metastases. Gene expression analysis of cultured cells and tumors revealed that AIB1 cells display a distinct expression signature of genes in pro-inflammatory pathways, cell adhesion, proteolysis and tissue remodeling. Interestingly, the presence of this AIB1 expression signature in breast cancer specimens is associated with shorter disease free survival of chemotherapy treated patients. We concluded that TNBC cell lines contain heterogeneous populations with differential dependence on AIB1 and that the gene expression pattern of AIB1 cells may represent a signature indicative of poor response to chemotherapy in TNBC patients.

Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer.

CDK4/6 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  (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 , an ovarian cancer biomarker gene, were repressed during treatment. Knockdown of  in MCFDCIS cells inhibited proliferation of invasive lesions but not progression of DCIS. After cessation of palbociclib treatment genes associated with differentiation, for example, , 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.

Short- and Long-Term Effects of CDK4/6 Inhibition on Early-Stage Breast Cancer.

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  (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.

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

Pancreatic adenocarcinoma is typically detected at a late stage and thus shows only limited sensitivity to treatment, making it one of the deadliest malignancies. In this study, we evaluate changes in microRNA (miR) patterns in peripheral blood as a potential readout of treatment responses of pancreatic cancer to inhibitors that target tumor-stroma interactions. Mice with pancreatic cancer cell (COLO357PL) xenografts were treated with inhibitors of either fibroblast growth factor receptor kinase (FGFR; PD173074) or anaplastic lymphoma kinase receptor (ALK; TAE684). While both treatments inhibited tumor angiogenesis, signal transduction, and mitogenesis to a similar extent, they resulted in distinct changes in circulating miR signatures. Comparison of the miR pattern in the tumor versus that in circulation showed that the inhibitors can be distinguished by their differential impact on tumor-derived miRs as well as host-derived circulating miRs. Distinct signatures that include circulating miR-1 and miR-22 are associated with the efficacy of ALK and FGFR inhibition, respectively. We propose that monitoring changes in circulating miR profiles can provide an early signature of treatment response or resistance to pathway-targeted drugs, and thus provide a non-invasive measurement to rapidly assess the efficacy of candidate therapies.

ArticleTotal : 3	Year
Publication title Cited by 112	2015
Publication title Cited by 1	2022
Publication title Cited by 24	2020

Article	Year
Role of the nuclear receptor coactivator AIB1-Delta4 splice variant in the control of gene transcription. The Journal of biological chemistry\| PubMed ID: 21636853	2011
Transcriptional repression of AIB1 by FoxG1 leads to apoptosis in breast cancer cells. Molecular endocrinology (Baltimore, Md.)\| PubMed ID: 23660594	2013
Regulation of HER2 oncogene transcription by a multifunctional coactivator/corepressor complex. Molecular endocrinology (Baltimore, Md.)\| PubMed ID: 24678732	2014
Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations. Genes\| PubMed ID: 30934798	2019
Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer. Neoplasia (New York, N.Y.)\| PubMed ID: 31437536	2019
Short- and Long-Term Effects of CDK4/6 Inhibition on Early-Stage Breast Cancer. Molecular cancer therapeutics\| PubMed ID: 31451564	2019
An AIB1 Isoform Alters Enhancer Access and Enables Progression of Early-Stage Triple-Negative Breast Cancer. Cancer research\| PubMed ID: 34135000	2021
Distinct Response of Circulating microRNAs to the Treatment of Pancreatic Cancer Xenografts with FGFR and ALK Kinase Inhibitors. Cancers\| PubMed ID: 35326668	2022

Anna T. Riegel

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