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Gene expression profiling of MDA231 cells depleted of MRCKalpha (CDC42BPA) and MRCKbeta (CDC42BPB)

ABSTRACT: Aim: The MRCK family of kinases (namely MRCKalpha and MRCKbeta) are downstream effectors of the Rho GTPase signaling pathways with roles in mediating cell migration via regulating the actin cytoskeleon. As such, it is thought that the MRCKs might also be involved in promoting cancer cell invasion or metastasis. However, crucial in vivo data and a general view of the signaling pathways regulated by the MRCKs are still lacking in order to substantiate the hypothesis. Therefore, the goal of this study is to perform transcriptome profiling (RNA-seq) of MDA-MB-231 cells depleted of either CDC42BPA, CDC42BPB, or both, in order to obtain insight into the differential genes expressed and whether CDC42BPA and CDC42BPB contribute as a causative factor of metastatic breast cancer. Method: We performed CRIPSR-based knockout of MRCKalpha (CDC42BPA), MRCKbeta (CDC42BPB), either alone or in combination, in the invasive breast cancer cell line MDA-MB-231. The combinatory double knockout was performed twice as two biological replicates to increase confidence in our data. The knockout cells were then prepared and sent for RNA-seq using by BGISEQ-500, and differential gene expression profile was obtained.

ORGANISM(S): Homo sapiens

PROVIDER: GSE169000 | GEO | 2021/03/17

REPOSITORIES: GEO

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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.

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Gene expression profiling of MDA231 cells depleted of MRCKalpha (CDC42BPA) and MRCKbeta (CDC42BPB)

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