Non-stimulated, agonist-stimulated and store-operated ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry

Davis, Felicity M., Peters, Amelia A., Grice, Desma M., Cabot, Peter J., Parat, Marie-Odile, Roberts-Thomson, Sarah J. and Monteith, Gregory R. (2012) Non-stimulated, agonist-stimulated and store-operated ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry. PLoS One, 7 5: e36923.1-e36923.11. doi:10.1371/journal.pone.0036923


Author Davis, Felicity M.
Peters, Amelia A.
Grice, Desma M.
Cabot, Peter J.
Parat, Marie-Odile
Roberts-Thomson, Sarah J.
Monteith, Gregory R.
Title Non-stimulated, agonist-stimulated and store-operated ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry
Formatted title
Non-stimulated, agonist-stimulated and store-operated ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2012-05-01
Year available 2012
Sub-type Article (original research)
DOI 10.1371/journal.pone.0036923
Open Access Status DOI
Volume 7
Issue 5
Start page e36923.1
End page e36923.11
Total pages 11
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Language eng
Subject 1300 Biochemistry, Genetics and Molecular Biology
1100 Agricultural and Biological Sciences
Abstract Epithelial-mesenchymal transition (EMT) is an important process associated with the metastasis of breast cancer cells. Members of the Janus kinases (JAKs) and Src family kinases (SFKs) are implicated in the regulation of an invasive phenotype in various cancer cell types. Using the pharmacological inhibitors JAK Inhibitor I (a pan-JAK inhibitor) and PP2 we investigated the role of the JAKs and SFKs, respectively, in the regulation of EMT markers in the MDA-MB-468 breast cancer cell line model of epidermal growth factor (EGF)-induced EMT. We identified selective inhibition of EGF induction of the mesenchymal marker vimentin by PP2 and JAK Inhibitor I. The effect of JAK Inhibitor I on vimentin protein induction occurred at a concentration lower than that required to significantly inhibit EGF-mediated signal transducer and activator of transcription 3 (STAT3)-phosphorylation, suggesting involvement of a STAT3-independent mechanism of EGF-induced vimentin regulation by JAKs. Despite our identification of a role for the JAK family in EGF-induced vimentin protein expression, siRNA-mediated silencing of each member of the JAK family was unable to phenocopy pharmacological inhibition, indicating potential redundancy among the JAK family members in this pathway. While SFKs and JAKs do not represent global regulators of the EMT phenotype, our findings have identified a role for members of these signaling pathways in the regulation of EGF-induced vimentin expression in the MDA-MB-468 breast cancer cell line.
Formatted abstract
In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca2+ reserves, molecular components of the store-operated Ca2+ entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca2+ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca2+ influx. The potential roles for specific Ca2+ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca2+ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca2+ influx in a manner dependent on Ca2+ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca2+ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (time to peak [Ca2+]CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca2+ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca2+ influx in non-stimulated cells.
Keyword Breast cancer
Epidermal growth factor
Epithelial-mesenchymal transition
Janus kinase
Src family kinase
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 569645
Institutional Status UQ
Additional Notes Article # e36923

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2013 Collection
School of Pharmacy Publications
 
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Created: Thu, 21 Jun 2012, 22:04:49 EST by Myrtle Sahabandu on behalf of School of Pharmacy