Cyclin D1, Id1 and EMT in breast cancer - PubMed (original) (raw)
Cyclin D1, Id1 and EMT in breast cancer
Nicholas P Tobin et al. BMC Cancer. 2011.
Abstract
Background: 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.
Methods: Protein and gene expression of ID1, CCND1 and EMT markers were determined in MDA-MB-231 and ZR75 cells by western blot and qPCR. Cell migration and promoter occupancy were monitored by transwell and ChIP assays, respectively. Gene expression was analysed from publicly available datasets.
Results: 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, CCND1low/ID1high tumours displayed increased expression of EMT markers and were associated with reduced recurrence free survival. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like 'claudin-low' subtype of breast cancer than in other subtypes.
Conclusions: 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.
Figures
Figure 1
Effect of cyclin D1 and Id1 on breast cancer cell protein expression and migration. Actively cycling MDA-MB-231 and ZR75-1 cells were monitored 20 h post-transfection with the indicated siRNA (cyclin D1/CDK4/6/Id1) or vector (Id1) for changes in gene or protein expression, and migration. Blots are representative, and plots are mean values from at least three independent experiments. Error bars represent standard deviation. MDA-MB-231 cells: (A) Microarray analysis. Left panel: CCND1 gene expression, right panel: ID1 gene expression (B) Western blot for cyclin d1, Id1 and Actin protein, (C) Cell migration as measured by Boyden chamber assay, dots indicate total number of migrated cells. (D) ChIP assay for Id1 promoter region following cyclin D1 pull down. ZR75-1: (E) Western blot for cyclin d1 and Actin protein (F) Cell migration- Boyden chamber assay. ***P ≥ 0.001, **P ≥ 0.01, *P ≥ 0.05 vs. control, two-tailed student's t-test.
Figure 2
Effect of cyclin d1 and Id1 on EMT markers. MDA-MB-231 cells were monitored 20 h post-transfection with the indicated siRNA (cyclin D1/CDK4/6/Id1) or vector (Id1) for changes in EMT-related gene expression by microarray analysis. Additionally, MDA-MB-231 and ZR75-1 gene expression was examined by qPCR assay. Plots are mean values from at least three independent experiments Error bars represent standard deviation. (A) Microarray analysis of SNAI1, SNAI2, CDH11, TWIST1 and VIM gene expression. (B-D) CCND1, ID1 and SNAI2 in MDA-MB-231 cells. (E, F) qPCR analysis of CCND1 and SNAI2 in ZR75-1 cells. ***P ≥ 0.001, **P ≥ 0.01, *P ≥ 0.05 vs. control, two-tailed student's t-test.
Figure 3
Correlation of CCND1, ID1, SNAI1 and SNAI2 expression to recurrence free survival. Expression of our genes of interest in relation to recurrence-free survival was examined in a breast cancer database containing 1,107 tumours from Sims et al. (2008). Gene expression intensity was quartiled as 1-low, 2- medium low, 3- medium high and 4- high, and assessed in all patients, ER-positive and ER-negative patients, respectively (A) CCND1 quartiles (B) ID1 quartiles (C) SNAI1 quartiles (D) SNAI2 quartiles. P-value is based on log-rank test.
Figure 4
EMT-related gene expression intensity and recurrence free survival in CCND1/ID1 high and low tumours. A breast cancer database was employed to examine (A) Mean-centered average expression of EMT-related genes of interest in CCND1/ID1 subgroups, and (B, C) Recurrence-free survival of ER-positive and negative patients in CCND1/ID1 subgroups. P-value is based on log-rank test.
Figure 5
Gene expression of Cyclin d1/Id1 and EMT markers in breast cancer cell lines and claudin-low tumours. Expression of EMT and related genes from the Neve et al cell line (A-C) and Herschkowitz et al. claudin-low (D-F) studies. (A and D) Heatmaps showing relative expression of genes of interest, Red = high, Green = low. (B and E) Scatterplots showing the relationship between breast cancer subgroups and CCND1/ID1 expression (C and F) Proportion of _CCND1-_low/_ID1_-high cell lines and tumours in breast cancer subgroups. ***P ≥ 0.001, Chi2 test.
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