Project description: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.

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: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:The hormonal luminal-A is the most pre-dominant sub type of breast cancer (BC), and it is associated with a high level of cyclin D1 in Saudi patients. Tamoxifen is the golden therapy for hormonal BC, but resistance of cancer cells to tamoxifen contributes to the recurrence of BC due to many reasons, including high levels of AIB1 and cyclin D1. Overcoming drug resistance could be achieved by exploring alternative targetable therapeutic pathways and new drugs or combinations. The objective of this study was to determine the differentially enriched pathways in 12 samples of Saudi women diagnosed with luminal-A using the PamChip peptide microarray-based kinase activity profiling, and to compare the activity of HAA2020 and dinaciclib with tamoxifen in singles and combinations in the MCF7 luminal-A cell line. Our results of network and pathway analysis of the 12 samples highlighted the importance of VEGFR and CDKs in promoting luminal-A breast cancer. The activation of VEGF signaling via VEGFR-2 leads to activation of PI3K/AKT kinases and an increase of cell survival, and leads to activation of Hsp90, which induces the phosphorylation of FAK1, resulting in cytoskeleton remodeling. PLC-gamma 1 is also activated, leading to FAK-2 and PKC activation. Notably, the G1/S cell cycle phases and phosphorylation processes contribute to the top seven tumorigenesis processes in the 12 samples. Further, the MTT combination of HAA2020 and dinaciclib showed the best combination index (CI), was more clonogenic against MCF7 cells compared to the other combinations, and it also showed the best selectivity index (SI) in normal MRC5 cells. Interestingly, HAA2020 and dinaciclib showed a synergistic apoptotic and G1 cell cycle effect in MCF7 cells, which was supported by their synergistic CDK2, cyclin D1, and PCNA inhibition activities. Additionally, the combination showed VEGFR-2 and Hsp90 inhibition activities in MCF7 cells. The results show the significance of targeting VEGFR-2 and cyclin D1 in Saudi luminal-A breast cancer patients, and the effect of combining HAA2020 and dinaciclib on those targets in the MCF7 model. It also warrants further preclinical and in vivo investigations for the combination of HAA2020 and dinaciclib as a possible future second-line treatment for luminal-A breast cancers.

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Cyclin D1, Id1 and EMT in breast cancer

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