Project description:Cyclin D1 is a well characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in ‘inhibitor of differentiation 1’ (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer. The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1 independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1 and ID1 gene expression were associated with mesenchymal-markers including SNAI1, SNAI2 and TWIST1, and with clinicopathological parameters. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like ‘claudin-low’ subtype of breast cancer than in other subtypes. Together, these results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer. MDA-MB-231 cells were transfected with cyclin D1, CDK4/6 or control siRNA.

Project description:Cyclin D1 is a well characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in ‘inhibitor of differentiation 1’ (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer. The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1 independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1 and ID1 gene expression were associated with mesenchymal-markers including SNAI1, SNAI2 and TWIST1, and with clinicopathological parameters. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like ‘claudin-low’ subtype of breast cancer than in other subtypes. Together, these results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer.

Project description:Progesterone receptor (PR) and its co-activators are direct targets of activated cyclin dependent kinases (CDKs) in response to peptide growth factors, progesterone, and deregulation of cell cycle inhibitors. Herein, using the T47D breast cancer model, we probed mechanisms of cell cycle-dependent PR action. In the absence of exogenous progestin, PR is specifically phosphorylated during the G2/M phase. Accordingly, numerous PR target genes are cell cycle regulated, including HSPB8, a heat-shock protein whose high expression is associated with tamoxifen-resistance. Progestin-induced HSPB8 expression required cyclin D1 and was insensitive to anti-estrogens, but blocked by anti-progestins or inhibition of specificity factor 1 (SP1). HSPB8 expression increased with or without ligand when cells were G2/M synchronized or contained high levels of cyclin D1. Knock-down of PR abrogated ligand-independent HSPB8 expression in synchronized cells. Notably, PR and cyclin D1 co-purified in whole cell lysates of transiently transfected COS-1 cells and in PR-positive T47D breast cancer cells expressing endogenous cyclin D1. PR, cyclin D1, and SP1 were recruited to the HSPB8 promoter in progestin-treated T47D breast cancer cells. Mutation of PR Ser345 to Ala (S345A) or inhibition of CDK2 activity using roscovitine disrupted PR/cyclin D1 interactions with DNA and blocked HSPB8 mRNA expression. Interaction of phosphorylated PRs with SP1 and cyclin D1 provides a mechanism for targeting transcriptionally active PRs to selected gene promoters relevant to breast cancer progression. Understanding the functional linkage between PR and cell cycle regulatory proteins will provide keys to targeting novel PR/cyclin D1 cross-talk in both hormone-responsive and HSPB8-high refractory disease. The study contains 4 different sample groups measured in triplicate, for a total of 12 individual samples (12 arrays). In T47D human breast cancer cell lines stably expressing PR-B, cells were synchronized (or not synchronized) before G2/M phase using nocodazole. These cell lines (synchronized or not synchronized) were treated with either (1) vehicle control (ethanol) or (2) PR ligand R5020 10e-8 M for 6 hours before total RNA harvest. Thus, the experiment contains two cell lines, and two treatments (4 sample groups) treated and analyzed in triplicate (12 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.

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Homo sapiens

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