Abstract 421: Deciphering the molecular mechanisms behind Oct4(hi) tumor-initiating breast cancer cells with global gene expression

LIN28 is an evolutionarily conserved RNA-binding protein with critical functions in developmental timing and cancer. However molecular mechanisms underlying LIN28's oncogenic properties are yet to be described. RIP-Seq analysis revealed significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28 bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28 interacting partner. Subsequently, we use a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 siRNA-treated cells. Results reveal these proteins regulate alternative splicing and steady state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of ENAH exon 11a isoform. Expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available TCGA array data reveals LIN28 expression is significantly different in HER2 compared to other breast cancer subtypes. Collectively, our data suggests that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes.

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Comparison of the global gene expression profiles produced by methylparaben,n-butylparaben and 17β-oestradiol in MCF7 human breast cancer cells

Journal of Applied Toxicology ◽

10.1002/jat.1200 ◽

2006 ◽

Vol 27 (1) ◽

pp. 67-77 ◽

Cited By ~ 34

Author(s):

D. Pugazhendhi ◽

A. J. Sadler ◽

P. D. Darbre

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cancer Cells ◽

Human Breast Cancer ◽

Breast Cancer Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Global Gene Expression ◽

Human Breast Cancer Cells ◽

Human Breast

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Inhibition of Cytokine Receptor gp130 Signaling in Breast Cancer Cells Markedly Suppresses Secretion of Tissue Factor-Expressing Tumor Microparticles.

Blood ◽

10.1182/blood.v106.11.1935.1935 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1935-1935

Author(s):

Diego A. de Idiaquez ◽

Dannielle E. Branam ◽

Larry Lamb ◽

Dongquan Chen ◽

Lihong Teng ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Flow Cytometry ◽

Cancer Cells ◽

Tissue Factor ◽

Tumor Invasion ◽

Breast Cancer Cells ◽

Molecular Mechanisms ◽

Extracellular Proteins ◽

Dominant Negative

Abstract gp130 is the common signaling receptor subunit for the IL-6 family of cytokines. We have previously shown that expression of a dominant negative (DN) gp130 inhibitory protein in MDA-MB-231 breast cancer cells decreased tumor invasion and angiogenesis in an orthotopic animal model (Cancer Research64:6924, 2004). In order to better understand the molecular mechanisms of this decreased malignancy, we determined global MDA-MB-231 gene expression in the presence of DN gp130. RNA was obtained from multiple independent single cell clones of MDA-MB-231 cells expressing either DN gp130 (n=5) or vector-only (n=5). Each clone was studied using Affymetrix U133A chips containing >22,000 genes. We identified 237 genes that were up- or down-regulated in the presence of DN gp130 at least 2-fold with p<0.05. Genes of interest were further studied by real-time PCR, western blot, and flow cytometry. We focused on extracellular proteins involved in proteolytic pathways. Six coagulation regulatory proteins were found to be differentially regulated by DN gp130: tissue factor (decreased), PAI-1 (decreased), alpha-2 antiplasmin (decreased), TFPI (increased), thrombomodulin (increased), and MMP-14 (increased). Thus, inhibition of gp130 signaling results in an anticoagulant phenotype in these cells. Tissue factor regulation was further studied. Cell surface tissue factor protein was down-regulated 10-fold in MDA-MB-231 DN gp130 cells compared to MDA-MB-231 control cells. Tumor microparticles (MP) were isolated from conditioned media by differential centrifugation. MDA-MB-231 MP contained uPA and MMP-9, as previously reported for MP from other tumors. Tissue factor was strongly expressed in control MP and nearly absent in MP from DN gp130 cells. Flow cytometry of tumor MP was used to quantitate tissue factor-expressing MP. There was a 50-fold decrease in double-labeled annexin V positive/tissue factor positive MP obtained from DN gp130 cells compared to control cells. In addition, the absolute number of tumor MP was decreased by 50% in the presence of DN gp130. In order to determine the mechanism of this regulation of breast cancer MP by gp130, the above list of 237 differentially expressed genes was re-examined. Proteins known to be enriched in MP- CD55, CD59, and three members of the tetraspanin family (including CD9) were significantly decreased in the presence of DN gp130. The expression of three proteins that are involved in the cellular production of MP were also significantly affected by DN gp130: Ca2+-dependent activator of protein for secretion 2, Rab11 interacting protein, and syntaxin 3. Our data supports a hypothesis in which signaling via IL-6 family cytokines induces a procoagulant gene expression program in breast cancer cells. As part of this program, the production of tumor MP are increased via gene regulation of cellular proteins that control MP processing. Additionally, the procoagulant properties of the tumor MP are increased due to an increase in tissue factor content. It has been reported that both activation of coagulation and production of tumor MP are involved in solid tumor invasion and metastasis. Inflammatory signaling by IL-6 family members in the tumor milieu could be important in this process. This hypothesis has clinical significance since potential pharmacologic approaches to specific gp130 inhibition are available.

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Endoxifen, a Secondary Metabolite of Tamoxifen, and 4-OH-Tamoxifen Induce Similar Changes in Global Gene Expression Patterns in MCF-7 Breast Cancer Cells

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.105.100511 ◽

2006 ◽

Vol 318 (2) ◽

pp. 503-512 ◽

Cited By ~ 96

Author(s):

Young Chai Lim ◽

Lang Li ◽

Zeruesenay Desta ◽

Qianqian Zhao ◽

James M. Rae ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cancer Cells ◽

Secondary Metabolite ◽

Breast Cancer Cells ◽

Expression Patterns ◽

Global Gene Expression ◽

Gene Expression Patterns ◽

Mcf 7

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Nuclear and Extranuclear Pathway Inputs in the Regulation of Global Gene Expression by Estrogen Receptors

Molecular Endocrinology ◽

10.1210/me.2008-0059 ◽

2008 ◽

Vol 22 (9) ◽

pp. 2116-2127 ◽

Cited By ~ 123

Author(s):

Zeynep Madak-Erdogan ◽

Karen J. Kieser ◽

Sung Hoon Kim ◽

Barry Komm ◽

John A. Katzenellenbogen ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Estrogen Receptors ◽

Cancer Cells ◽

Gene Transcription ◽

Breast Cancer Cells ◽

Global Gene Expression ◽

Selective Ligand ◽

Extranuclear Signaling ◽

The Impact

Abstract Whereas estrogens exert their effects by binding to nuclear estrogen receptors (ERs) and directly altering target gene transcription, they can also initiate extranuclear signaling through activation of kinase cascades. We have investigated the impact of estrogen-mediated extranuclear-initiated pathways on global gene expression by using estrogen-dendrimer conjugates (EDCs), which because of their charge and size remain outside the nucleus and can only initiate extranuclear signaling. Genome-wide cDNA microarray analysis of MCF-7 breast cancer cells identified a subset of 17β-estradiol (E2)-regulated genes (∼25%) as EDC responsive. The EDC and E2-elicited increases in gene expression were due to increases in gene transcription, as observed in nuclear run-on assays and RNA polymerase II recruitment and phosphorylation. Treatment with antiestrogen or ERα knockdown using small interfering RNA abolished EDC-mediated gene stimulation, whereas GPR30 knockdown or treatment with a GPR30-selective ligand was without effect, indicating ER as the mediator of these gene regulations. Inhibitors of MAPK kinase and c-Src suppressed both E2 and EDC stimulated gene expression. Of note, in chromatin immunoprecipitation assays, EDC was unable to recruit ERα to estrogen-responsive regions of regulated genes, whereas ERα recruitment by E2 was very effective. These findings suggest that other transcription factors or kinases that are downstream effectors of EDC-initiated extranuclear signaling cascades are recruited to regulatory regions of EDC-responsive genes in order to elicit gene stimulation. This study thus highlights the importance of inputs from both nuclear and extranuclear ER signaling pathways in regulating patterns of gene expression in breast cancer cells.

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The histone variant macroH2A1.1 regulates gene expression by direct association with their transcription start site

10.1101/2020.01.29.924704 ◽

2020 ◽

Author(s):

Ludmila Recoules ◽

Alexandre Heurteau ◽

Flavien Raynal ◽

Fatima Moutahir ◽

Fabienne Bejjani ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cancer Cells ◽

Transcription Start Site ◽

Breast Cancer Cells ◽

Molecular Mechanisms ◽

Histone Variants ◽

Histone Variant ◽

Start Site ◽

Transcription Start

AbstractThe histone variant macroH2A1 (mH2A1) is involved in cellular growth, differentiation and reprogramming, but the underlying molecular mechanisms are a matter of debate. Different roles of mH2A1 in gene expression may relate to functional differences of its two splicing isoforms, mH2A1.1 and mH2A1.2. Here, we map for the first time genome-wide localization of endogenous mH2A1.1 and link the distribution of mH2A1.1 to control of gene expression in human breast cancer cells. In addition to localization shared with mH2A1.2 to facultative heterochromatin, mH2A1.1 specifically associates with regulatory elements required for gene activation, super-enhancers and promoters of highly expressed genes. Depending on the recruitment profile of mH2A1.1 to these elements, selective depletion of mH2A1.1 up- or downregulates its target genes. mH2A1.1 represses transcription when its binding is spread over the entire gene and promoter, and activates transcription when its binding is strictly confined to the transcription start site (TSS). Notably, RNA Polymerase II was frequently in pause at mH2A1.1-activated genes. Functionally, mH2A1.1-dependent regulation of a subset of paused genes impedes mammary tumor cell migration. Molecular mechanisms of mH2A1.1 function at the TSS uncovered by our study define an intriguing new mode of transcription regulation in cancer cells.Author SummaryControl of gene expression driving cellular functions from differentiation to epistasis and causing, when dysfunctional, uncountable diseases, relies on modifications of chromatin structure. One key element enabling chromatin plasticity is the replacement of canonical histones by histone variants. Among histone variants macroH2A1 (mH2A) is an extraordinary H2A variant possessing a large non-histone domain placed outside of the nucleosome. Two splicing isoforms, mH2A1.1 and mH2A1.2, are produced, but these are rarely studied separately because they only differ in a 30 amino acid region and are difficult to distinguish experimentally, which likely explains contradictory functions reported in the literature. Here, we take advantage of a mH2A1.1 specific antibody to generate the first genome-wide chromatin-associated map of this histone variant in the invasive breast cancer cells line MDA-MB231. We confirm that mH2A1.1, like mH2A1.2, is enriched at facultative heterochromatin in agreement with its reported role as a repressor. However, we discovered that unlike its splicing isoform, mH2A1.1 specifically binds to super-enhancers and the transcription start site of highly transcribed genes. mH2A1.1 is necessary for regulating transcription of these genes. At the cellular level, we demonstrate that mH2A1.1 inhibits migration capacity of highly metastatic breast cancer cells. Our study characterizes for the first time binding profiles of mH2A1.1 that are linked to regulation of gene expression, thereby providing a new molecular mechanisms which govern the plasticity of human tumor cells.

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