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.
In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca(2+) reserves, molecular components of the store-operated Ca(2+) 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 Ca(2+) influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca(2+) influx. The potential roles for specific Ca(2+) 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 Ca(2+) influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca(2+) influx in a manner dependent on Ca(2+) influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca(2+) release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (time to peak [Ca(2+)](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 Ca(2+) 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 Ca(2+) influx in non-stimulated cells.
School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia.
Felicity M
FM
Davis
Amelia A
AA
Peters
Desma M
DM
Grice
Peter J
PJ
Cabot
Marie-Odile
MO
Parat
Sarah J
SJ
Roberts-Thomson
Gregory R
GR
Monteith
D001692:Biological Transport / Q000187:drug effects; D001943:Breast Neoplasms / Q000473:pathology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000172:deficiency / Q000235:genetics; D020013:Calcium Signaling / Q000187:drug effects*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D020868:Gene Silencing; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D000071740:ORAI1 Protein; D058913:Purinergic Agonists / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D050052:TRPC Cation Channels / Q000172:deficiency / Q000235:genetics
D015220:Calcium Channels; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D058913:Purinergic Agonists; D012333:RNA, Messenger; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D004815:Epidermal Growth Factor; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000252:Calcium-Transporting ATPases; D002118:Calcium