Project description:Epithelial-mesenchymal transition (EMT) is a diverse and dynamic biological process which is involved in cancer progression. It is important for carcinoma cells during invasion and metastasis. EMT has been in the spotlight for cancer cells to disseminate to distant organs by gaining partial EMT phenotype. Cancer cells with partial EMT are believed to be more cancerogenic/invasive than cells that have undergone complete EMT. The proteomic changes that occur following EMT in breast epithelial cells and how these relate to changes in cellular metabolism are incompletely understood. To study metabolic reprogramming in different mesenchymal states, we analyzed proteomic changes following EMT in the breast epithelial cell model D492, with single-shot LFQ supported by SILAC proteomic approach. The D492 EMT cell model contains three isogenic cell lines: epithelial D492 cells, mesenchymal D492M cells, and partial mesenchymal, HER2 overexpressing, tumorigenic D492HER2 cells. Proteomic analysis positioned the D492 and D492M cells as basal-like while D492HER2 as claudin-low. Further comparison of the non-tumorigenic D492 and D492M cells to tumorigenic D492HER2 differentiated the metabolic EMT markers of migration from those of invasion. Among these were markers of glycan metabolism. We identified glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2) as the top dysregulated enzyme in glycan metabolism and found increased GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA knockdown of GFPT2 influenced both cell growth and invasion in vitro and was accompanied by lowered flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway which regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 expression was regulated by the level of reduced glutathione (GSH) and suppressed by the oxidative stress regulator, GSK3-β. Our results demonstrate that GFPT2 is a marker for oxidative stress. It is upregulated and controls growth and invasion in the D492 EMT model and is associated with claudin-low and poor prognosis in 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: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.

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Claudin 1 mediates TNFα-induced gene expression and cell migration in human lung carcinoma cells

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