Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

L-plastin Ser5 phosphorylation via RSKs, downstream effectors of the MAPK/ERK pathway, in (breast) cancer cells (and in vitro)

ABSTRACT: Deregulated cell migration and invasion, which are hallmarks of metastatic cancer cells, are correlated to structural and functional alterations of the actin cytoskeleton associated to a large panel of actin-binding proteins. Among these, the actin-bundling protein L-plastin, initially detected in leukocytes, is ectopically expressed in several solid tumours and is often considered as a metastatic marker. Phosphorylation of L-plastin on residue serine 5 (Ser5) has been shown to activate L-plastin and to be crucial for invasion and metastasis formation. Here, we investigate the signalling pathway leading to L-plastin Ser5 phosphorylation in four breast cancer cell lines. Whole genome microarray analysis comparing cell lines with different invasive capacities and corresponding L-plastin Ser5 phosphorylation levels revealed that genes of the MAPK/ERK pathway are differentially expressed. In line, in vitro kinase assays showed that MAPK/ERK pathway downstream kinases RSK1 and RSK2 are able to directly phosphorylate L-plastin on Ser5. In parallel to a knockdown approach, activation and inhibition studies of signalling molecules of the MAPK/ERK pathway, followed by computational modelling analysis, confirmed that RSK is an important activator of L-plastin in all four studied cell lines. Finally and rounding up our study, RSK knockdown significantly impaired migratory and invasive capacities of a selected invasive cell line, thus consolidating RSK as a promising therapeutic target in certain invasive carcinomas. Altogether, our data provide substantial evidence that the MAPK/ERK pathway is involved in L-plastin Ser5 phosphorylation in breast cancer cells with its downstream kinases RSK1 and RSK2 being able to directly phosphorylate L-plastin on Ser5.

ORGANISM(S): Homo sapiens

SUBMITTER: Muller Arnaud

PROVIDER: E-MTAB-3487 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Carcinogenesis is a multistep process in which cells accumulate multiple genetic alterations, as they progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through preneoplastic lesion/benign tumor to cancer. Histologically, progression to invasive oral squamous cell carcinoma (OSCC) follows multistep ordered series beginning with mucosal epithelial cell hyperplasia, followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore speculated that genetic alterations-mediated multistep carcinogenesis would be involved in OSCC development, but their detailed molecular mechanisms are unknown. Herein, we clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data obtained from an OSCC pathological specimen, which consists of non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion. Numerous numbers of gene expression and signal activation were altered in the OSCC development. Of them, the expression of p63 was increased and MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion as well as in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ lesions and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were merged at high frequency in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and EGFR-MEK/ERK-MAPK cooperatively induce ARL4C expression in OSCC cells. These results suggest that the stepwise activation of p63 and EGFR-MEK/ERK-MAPK contributes to OSCC tumor cell growth though regulation of ARL4C expression.

Project description:Bis-2-chloroethyl sulfide (sulfur mustard, SM) is a potent alkylating agent and vesicant. Exposure to SM results in activation of numerous signaling cascades, including mitogen-activated protein kinase (MAPK) signaling pathways. These pathways include the Erk, p38, and JNK pathways, which are involved in cell growth, inflammation, and stress signaling. However, the precise roles of these pathways in SM toxicity have not been fully elucidated. We used Western blotting and microarray analysis to examine the activation and role of each pathway following SM exposure in primary human epidermal keratinocytes. Western blotting revealed increased phosphorylation of p38 and JNK following SM exposure; however, phosphorylation of Erk was equivocal, suggesting that growth conditions may impact activation of Erk by SM. We used pharmacologic inhibitors to target each MAPK and then compared the gene expression profiles to identify SM-induced gene networks regulated by each MAPK. Cells were pretreated with 10 µM SB 203580 (p38 inhibitor), PD 98059 (Erk inhibitor), or SP 600125 (JNK inhibitor) 60 minutes before exposure to 200 µM SM. Cells were harvested at 1h, 4h, and 8h post-exposure, and RNA was extracted for synthesis of microarray probes. Probes were hybridized to Affymetrix U133 Plus 2.0 arrays for gene expression profiling. Analysis of variance was performed to identify genes significantly modulated due to pharmacologic inhibition in SM-exposed cells. Pathway mapping confirmed alterations in SM-induced Erk, JNK, and p38 MAPK signaling due to pharmacologic inhibition. SM-induced expression of IL-8, IL-6, and TNF-alpha was decreased by p38 MAPK inhibition, but not by inhibition of other MAPKs. Based on the number of significant pathways mapped to each MAPK in the presence and absence of inhibitors, the p38 MAPK pathway appeared to be the MAPK pathway most responsive to SM exposure. Interestingly, pathway mapping of the microarray data identified potential cross-talk between MAPK signaling pathways and other pathways involved in SM-induced signaling. Mining of these results will increase our understanding of the role of MAPK pathways in SM-induced signal transduction and may identify potential therapeutic targets for medical countermeasure development.

Dataset Information

L-plastin Ser5 phosphorylation via RSKs, downstream effectors of the MAPK/ERK pathway, in (breast) cancer cells (and in vitro)

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure