Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca 2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

Laboratory Investigation

Marcial

et al. 2020

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The Ca 2+ signal is essential in both hypoxia-and epidermal growth factor (EGF)-mediated epithelial to mesenchymal transition (EMT) in MDA-MB-468 breast cancer cells. This finding suggests that Ca 2+-permeable ion channels participate in the induction of expression of some mesenchymal markers such as vimentin. However, the ion channels involved in vimentin expression induction have not been fully characterized. This work sought to define how differential modulation of the calcium signal effects the induction of vimentin and the Ca 2+ influx pathways involved. We identified that the intracellular Ca 2+ chelator EGTA-AM, cytochalasin D (a modulator of cytoskeletal dynamics and cell morphology) and the sarco/endoplasmic reticulum ATPase inhibitor thapsigargin are all inducers of vimentin in MDA-MB-468 breast cancer cells. EGTA-AM-and thapsigargin-mediated induction of vimentin expression in MDA-MB-468 cells involves store-operated Ca 2+ entry, as evidenced by sensitivity to silencing of the molecular components of this pathway, STIM1 and ORAI1. In stark contrast, cytochalasin D-mediated vimentin induction was insensitive to silencing of ORAI1, despite sensitivity to silencing of its canonical activator the endoplasmic reticulum Ca 2+ sensor STIM1. Cytochalasin D-mediated vimentin induction was, however, sensitive to silencing of another reported STIM1 target, TRPC1. Subsequent studies identified that EGTA-AM-induced vimentin expression also partially involved a TRPC1-dependent pathway. These studies define a complex interplay between vimentin expression in this model and the specific Ca 2+-permeable ion channels involved. The complexity in the engagement of different Ca 2+ influx pathways that regulate vimentin induction are opportunities but also potential challenges in targeting Ca 2+ signaling to block EMT in cancer cells. Our findings further highlight the need to identify potential indispensable ion channels that can regulate induction of specific mesenchymal markers via different stimuli.

Section: Resultsmentioning

confidence: 99%

Differential engagement of ORAI1 and TRPC1 in the induction of vimentin expression by different stimuli

Stewart

Laboratory Investigation

Marcial

et al. 2020

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“…Cell culture and induction of hypoxia MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were obtained from The Brisbane Breast Bank, UQCCR, Australia and American Type Culture Collection (ATCC) respectively, and cultured and maintained as previously described (Azimi et al, 2015(Azimi et al, , 2017. HCC1569 cells were obtained from the ATCC and maintained in RPMI 1640 medium (SigmaAldrich) supplemented with 10% FBS at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

TRPC1 is a differential regulator of hypoxia-mediated events and Akt signalling in PTEN-deficient breast cancer cells

Journal of Cell Science

Milevskiy

Kaemmerer

et al. 2017

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Hypoxia is a feature of the tumour microenvironment that promotes invasiveness, resistance to chemotherapeutics and cell survival. Our studies identify the transient receptor potential canonical-1 (TRPC1) ion channel as a key component of responses to hypoxia in breast cancer cells. This regulation includes control of specific epithelial to mesenchymal transition (EMT) events and hypoxia-mediated activation of signalling pathways such as activation of the EGFR, STAT3 and the autophagy marker LC3B, through hypoxia-inducible factor-1α (HIF1α)-dependent and -independent mechanisms. TRPC1 regulated HIF1α levels in PTEN-deficient MDA-MB-468 and HCC1569 breast cancer cell lines. This regulation arises from effects on the constitutive translation of HIF1α under normoxic conditions via an Akt-dependent pathway. In further support of the role of TRPC1 in EMT, its expression is closely associated with EMT-and metastasisrelated genes in breast tumours, and is enhanced in basal B breast cancer cell lines. TRPC1 expression is also significantly prognostic for basal breast cancers, particularly those classified as lymph node positive. The defined roles of TRPC1 identified here could be therapeutically exploited for the control of oncogenic pathways in breast cancer cells.

“…Cells were allowed to incubate for a further 15 min in the dark at ambient temperature in physiological salt solution (PSS [nominal]; 10 mM HEPES, 5.9 mM KCl, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 140 mM NaCl, 11.5 mM glucose, 1.8 mM CaCl 2 , pH 7.3). Loading-dye solution was removed and cells were washed twice with PSS Ca 2+ (PSS supplemented with 1.8 mM CaCl 2 ) and then twice with PSS no added extracellular (nominal) to ensure less than 100 µM Ca 2+ was remaining in the test wells [56]. Fresh PSS containing 1.8 mM CaCl 2 was added for intracellular calcium measurements.…”

Section: Intracellular Calcium Imaging and Quantification In Mcf-7 Cementioning

confidence: 99%

Ca²⁺ mediates extracellular vesicle biogenesis through alternate pathways in malignancy

Taylor

J of Extracellular Vesicle

Monteith

et al. 2020

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Extracellular vesicles (EVs) are small membrane vesicles that serve as important intercellular signalling intermediaries in both malignant and non-malignant cells. For EVs formed by the plasma membrane, their biogenesis is characterized by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. EV-production is significantly higher in malignant cells relative to non-malignant cells and previous work suggests this is dependent on increased calcium mobilization and activity of calpain. However, calcium-signalling pathways involved in malignant and non-malignant EV biogenesis remain unexplored. Here we demonstrate; malignant cells have high basal production of plasma membrane EVs compared to non-malignant cells and this is driven by a calcium-calpain dependent pathway. Resting vesiculation in malignant cells occurs via mobilization of calcium from endoplasmic reticulum (ER) stores rather than from the activity of plasma membrane calcium channels. In the event of ER store depletion however, the store-operated calcium entry (SOCE) pathway is activated to restore ER calcium stores. Depleting both ER calcium stores and blocking SOCE, inhibits EV biogenesis. In contrast, calcium signalling pathways are not activated in resting non-malignant cells. Consequently, these cells are relatively low vesiculators in the resting state. Following cellular activation however, an increase in cytosolic calcium and activation of calpain increase in EV biogenesis. These findings contribute to furthering our understanding of extracellular vesicle biogenesis. As EVs are key mediators in the intercellular transfer of deleterious cancer traits such as cancer multidrug resistance (MDR), understanding the molecular mechanisms governing their biogenesis in cancer is the crucial first step in finding novel therapeutic targets that circumvent EV-mediated MDR. ARTICLE HISTORY