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. 2014 May 1;33(18):2307-16.
doi: 10.1038/onc.2013.187. Epub 2013 May 20.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

F M Davis  1 , I Azimi  1 , R A Faville  2 , A A Peters  1 , K Jalink  3 , J W Putney Jr  4 , G J Goodhill  5 , E W Thompson  6 , S J Roberts-Thomson  1 , G R Monteith  1
Affiliations

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

F M Davis et al. Oncogene. .

Abstract

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Stimuli known to induce EMT produce a transient increase in cytosolic calcium
A) Pseudocolor (intensity) representation of calcium wave propagation through a confluent monolayer of MDA-MB-468 cells loaded with Fluo-4 AM calcium indicator. White arrows indicate the wound edge. Scale bar, 75 µm. Representative of movies from three independent experiments. B) Scatter plots for all frames of all movies showing cell activation time (Tf) versus distance (D) from the wound edge during scratch-induced calcium wave propagation; A represents the scaling coefficient and n the power law exponent, used to estimate the mode of activation. An n of 1 (red) represents calcium wave propagation that more closely resembles intercellular communication, whilst an n of 2 (blue) signifies activation by the release of a diffusible extracellular factor. The green line indicates the line of best-fit for scratch-induced calcium wave propagation. See also Fig. S1 and Movie S1. C) MDA-MB-468 cells were incubated for 30 min with conditioned media (the supernatant of a wounded monolayer) prior to stimulation with EGF (24 h) and vimentin protein expression was assessed using immunofluorescence. Two-way ANOVA with Bonferroni’s multiple comparisons post-tests was used to assess the significance of conditioned media at each EGF concentration. D) Average relative [Ca2+]CYT transients, E) peak relative [Ca2+]CYT response and F) vimentin positivity in cells stimulated with ATP (100 µM), trypsin (30 nM) or EGF (50 ng/mL). Bar graphs show mean ± S.D. for nine individual wells from three independent experiments. Significance was assessed using one-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
Figure 2
Figure 2. Calcium regulates EGF-induced vimentin protein expression
Representative immunoblots and densitometric analysis (normalized to β-actin) of EGF-induced vimentin expression in breast cancer cells loaded with A) BAPTA-AM or B) EGTA-AM to block increases in cytosolic calcium levels. Bar graphs show mean ± S.D. for three independent experiments. The effect of calcium chelation on vimentin expression was assessed using two-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
Figure 3
Figure 3. Calcium regulates the induction of some genes implicated in EGF-induced EMT
Analysis of mRNA levels of EMT-associated genes (vimentin, Twist, N-cadherin, Snail and CD44/CD24) in MDA-MB-468 breast cancer cells with intracellular calcium chelation (BAPTA-AM). Cells were treated with EGF for 6 h (A, C, E, G and I) or 24 h (B, D, F, H and J) to induce EMT. Bar graphs show mean ± S.D. for nine individual wells from three independent experiments. The effect of BAPTA-AM on gene expression was assessed using two-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
Figure 4
Figure 4. Calcium regulates the induction of some genes implicated in hypoxia-mediated EMT
Assessment of mRNA levels of A) vimentin, B) N-cadherin, C) CD44/CD24, D) Snail and E) Twist with normoxia or hypoxia (1% O2) in MDA-MB-468 breast cancer cells with intracellular calcium chelation (BAPTA-AM). Bar graphs show mean ± S.D. for nine individual wells from three independent experiments. The effect of BAPTA-AM on gene expression was assessed using two-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
Figure 5
Figure 5. The effect of intracellular calcium chelation on EGFR phosphorylation and activation of downstream signal transduction pathways
Phosphorylation of EGFR (Tyr1173) (A–C), ERK1/2 (Thr202/Tyr204) (D–F), Akt (Ser473) (G–I) and STAT3 (Tyr705) (J–L) mediated by incubation with 50 ng/mL EGF for 20 or 60 min was assessed in cells with intracellular calcium chelation (BAPTA-AM). Bar graphs show mean ± S.D. for three independent experiments. The effect of calcium chelation on protein phosphorylation was assessed using two-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
Figure 6
Figure 6. siRNA silencing and pharmacological inhibition of TRPM7 inhibit EGF-induced vimentin protein expression
A) TRPV3, TRPC4, TRPC5, TRPC6, TRPM6, TRPM7 and ORAI1 calcium-permeable channels were silenced using Dharmacon ON-TARGETplus siRNA and cells were stimulated with EGF (10 ng/mL, 24 h). Vimentin protein expression (integrated intensity) was assessed with quantitative immunofluorescence. The scatter dot plot shows fold change relative to the non-targeting (NT) control for six individual wells from two independent experiments. Analysis of percent TRPM7 mRNA remaining following transfection with B) Dharmacon ON-TARGETplus (OTP) TRPM7 siRNA or C) Dharmacon siGENOME TRPM7 siRNA. Bar graphs show mean ± S.D. for six wells from two independent experiments. Statistical significance was assessed using a student’s t-test. D) Representative immunoblot confirming regulation by TRPM7 of EGF-induced vimentin expression and densitometric analysis of vimentin expression (normalized to β-actin) in cells transfected with E) OTP TRPM7 siRNA or F) siGENOME TRPM7 siRNA. Graphs show mean ± S.D. for three independent experiments. The effect of TRPM7 gene silencing on vimentin expression was assessed using two-way ANOVA with Bonferroni’s multiple comparisons post-tests. G) Representative immunoblot showing the effect of increasing concentrations of the TRPM7 inhibitor NS8593 on EGF-induced vimentin expression and H) densitometric analysis (normalized to β-actin) for three independent experiments. Cells were treated with NS8593 for 24 h prior to EGF treatment and NS8593 was maintained during EGF treatment. Statistical significance was assessed using one-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
Figure 7
Figure 7. TRPM7 silencing alters specific EGF signaling pathways
Phosphorylation of A) EGFR (Tyr1173), B) Akt (Ser473), C) ERK1/2 (Thr202/Tyr204), and D) STAT3 (Tyr705) following EGF stimulation (50 ng/mL, 20 min) in MDA-MB-468 breast cancer cells transfected with nontargeting (NT) siRNA or TRPM7 OTP siRNA. Graphs show mean ± S.D. for three independent experiments. The effect of TRPM7 gene silencing on protein phosphorylation was assessed using two-way ANOVA with Bonferroni’s multiple comparisons post-tests. * P < 0.05.
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