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. 2017 Apr;19(4):288-300.
doi: 10.1016/j.neo.2017.01.004. Epub 2017 Mar 8.

The Transient Receptor Potential Melastatin 7 Channel Regulates Pancreatic Cancer Cell Invasion through the Hsp90α/uPA/MMP2 pathway

Pierre Rybarczyk  1 , Alison Vanlaeys  1 , Bertrand Brassart  2 , Isabelle Dhennin-Duthille  1 , Denis Chatelain  3 , Henri Sevestre  4 , Halima Ouadid-Ahidouch  1 , Mathieu Gautier  5
Affiliations

The Transient Receptor Potential Melastatin 7 Channel Regulates Pancreatic Cancer Cell Invasion through the Hsp90α/uPA/MMP2 pathway

Pierre Rybarczyk et al. Neoplasia. 2017 Apr.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a very poor prognosis. There is an urgent need to better understand the molecular mechanisms that regulate PDAC cell aggressiveness. The transient receptor potential melastatin 7 (TRPM7) is a nonselective cationic channel that mainly conducts Ca2+ and Mg2+. TRPM7 is overexpressed in numerous malignancies including PDAC. In the present study, we used the PANC-1 and MIA PaCa-2 cell lines to specifically assess the role of TRPM7 in cell invasion and matrix metalloproteinase secretion. We show that TRPM7 regulates Mg2+ homeostasis and constitutive cation entry in both PDAC cell lines. Moreover, cell invasion is strongly reduced by TRPM7 silencing without affecting the cell viability. Conditioned media were further studied, by gel zymography, to detect matrix metalloproteinase (MMP) secretion in PDAC cells. Our results show that MMP-2, urokinase plasminogen activator (uPA), and heat-shock protein 90α (Hsp90α) secretions are significantly decreased in TRPM7-deficient PDAC cells. Moreover, TRPM7 expression in human PDAC lymph node metastasis is correlated to the channel expression in primary tumor. Taken together, our results show that TRPM7 is involved in PDAC cell invasion through regulation of Hsp90α/uPA/MMP-2 proteolytic axis, confirming that this channel could be a promising biomarker and possibly a target for PDAC metastasis therapy.

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Figures

Figure 1
Figure 1
TRPM7 and TRPM6 channel expression in human PDAC cell lines. (A) Both TRPM7 and TRPM6 mRNAs are detected in the MIA PaCa-2 human PDAC cell line by RT-PCR in two different passages, whereas PANC-1 cells express only TRPM7 channel. Water is used as negative control. (B) Western blot indicating that TRPM7 protein is expressed in both PANC-1 and MIA PaCa-2 cell lines.
Figure 2
Figure 2
Electrophysiological properties of MIC currents in PANC-1 cells. (A) Membrane currents are recorded after 350-millisecond ramp depolarization from −100 mV to +100 mV at 0.1 Hz under whole-cell patch clamp. MIC currents are activated by dialyzing the intracellular media by an intrapipette solution without Mg2+. Both outward currents recorded at +100 mV (black circles; n=9 cells) and inward currents recorded at −100 mV (white circles; n=9 cells) increase following intracellular Mg2+ depletion. (B) Endogenous MIC currents have an inward component, a strong outward rectification, and a membrane reversal potential close to 0 mV (averaged trace of nine recordings). (C) Dialyzing of intracellular media with intrapipette solutions containing Mg2+ decreases MIC currents in a dose-dependent manner. (D) MIC currents recorded at −100 mV (white histograms) and at +100 mV (black histograms) are inhibited by increasing intracellular free Mg2+ from 0 (n=9 cells) to 210 (n=7 cells), 400 (n=7 cells), and 680 μM (n=6 cells). (E) Addition of 1 mM ATP in the intrapipette solution potentiates the inhibitory effect of Mg2+ on MIC currents (n=3 cells). (F) Interacting effects of intracellular free Mg2+ and ATP on outward MIC currents recorded at +100 mV. MIC currents are inhibited by 1 mM ATP complexed with 210 μM free Mg2+ (n=3 cells) when compared to 210 μM free Mg2+ (n=7 cells) and to 0 free Mg2+ (n=9 cells).
Figure 3
Figure 3
TRPM7 silencing inhibited MIC current in PDAC cells. (A) Effect of TRPM7 silencing by shRNA on TRPM7 mRNA expression in PANC-1 cells (N=3; P<0.001). (B) TRPM7 silencing by shRNA inhibits MIC currents in PANC-1 cells (averaged traces of six recordings for sh-Control and four recordings for sh-TRPM7 conditions). (C) Outward MIC currents recorded at +100 mV are decreased in cells treated with sh-TRPM7 (n=4 cells) when compared to control (n=6 cells; P<0.001). (D) TRPM7 silencing with siRNA decreases mRNA expression in MIA PaCa-2 (N=3; P<0.05). (E) MIC currents were decreased in MIA PaCa-2 cells transfected with siRNA (averaged trace of 6 recordings) when compared to controls (averaged trace of 12 recordings). (F) Outward MIC currents recorded at +100 mV are decreased in cells treated with si-TRPM7 (n=6 cells) when compared to control (n=12 cells; P<0.01).
Figure 4
Figure 4
TRPM7 regulated a constitutive divalent cations entry in PDAC cells. (A) TRPM7 silencing induced by shRNA decreases Mn2+ quenching of Fura-2 fluorescence in PANC-1 cells (averaged trace of 216 cells) when compared to control (averaged trace of 166 cells), with a significant reduction of quenching slope induced in sh-TRPM7 cells (histograms; P<0.001). (B) TRPM7 silencing also reduced Mn2+ quenching of Fura-2 fluorescence in MIA PaCa-2 cells (averaged trace of 112 cells) when compared to control (average trace of 171 cells), with a significant reduction of quenching slope in si-TRPM7 cells (histograms; P<0.001).
Figure 5
Figure 5
TRPM7 role in basal intracellular Ca2+ and Mg2+ levels. Ca2+ and Mg2+ homeostasis was assessed by basal Fura-2 and by MagFura-2 fluorescence ratio, respectively. (A) TRPM7 silencing by shRNA had no effect on PANC-1 basal Ca2+ levels (n=89 cells) when compared to control (n=59 cells; P>0.05). (B) Basal Ca2+ level is reduced in MIA PaCa-2 cells where TRPM7 has been silenced (n=85 cells) when compared to control (n=96 cells; P<0.001). (C) Basal Mg2+ level is reduced by TRPM7 silencing (n=132 cells) in PANC-1 when compared to control (n=110 cells; P<0.001). Basal Mg2+ level is also reduced in MIA PaCa-2 cells transfected with si-TRPM7 (n=105 cells) when compared to control (n=105 cells; P<0.01).
Figure 6
Figure 6
TRPM7 regulated PDAC cell invasiveness. (A) PANC-1 and MIA PaCa-2 cell proliferation was not affected by TRPM7 silencing (n=12 counting; N=3 for each time tested; P>0.05). (B) Both PANC-1 and MIA PaCa-2 cell invasion (40.104 cells, 24 hours) assessed in Matrigel modified Boyden chambers was decreased by TRPM7 silencing when compared to control (n=120 counting; N=3 for PANC-1; n=100 counting; N=3 for MIA PaCa-2; P<0.001 for each PDAC cell line). (C) Cell viability assessed by MTT assays (40.104 cells, 24 hours) showed no effect of TRPM7 for PANC-1 cells (n=12 counting; N=3; P>0.05) and for MIA PaCa-2 cells (n=12 counting; N=3; P>0.05).
Figure 7
Figure 7
TRPM7 regulates Hsp90α/uPA/MMP-2 pathway in PDAC cells. (A) Typical examples of reduced Pro-MMP-2 secretion following TRPM7 silencing in PANC-1 cells (left panel) and in MIA PaCa-2 cells (right panel). (B) Quantification of decreased pro-MMP-2 secretion induced by TRPM7 silencing in both PANC-1 cells (N=3) and MIA PaCa-2 cells (N=4). (C) Typical examples of reduced uPA secretion following TRPM7 silencing in PANC-1 cells (left panel) and MIA PaCa-2 cells (right panel). (D). Quantification of decreased uPA secretion induced by TRPM7 silencing in both PANC-1 cells (N=3) and MIA PaCa-2 cells (N=3). (E) Typical examples of reduced Hsp90α secretion following TRPM7 silencing in PANC-1 cells (left panel) and in MIA PaCa-2 cells (right panel). (F) Quantification of decreased Hsp90α secretion induced by TRPM7 silencing in both PANC-1 cells (N=3) and MIA PaCa-2 cells (N=4).
Figure 8
Figure 8
Extracellular Mg2+ regulates cell invasion through TRPM7 channels. (A) External application of 10 mM Mg2+ at 2 minutes induced a slight but sustained increase of intracellular Mg2+ levels in si-Control MIA PaCa-2 (black trace, n=46), whereas no effect was observed in si-TRPM7 condition (red trace, n=62). (B) Quantification of intracellular MagFura-2 fluorescence following 9-minute application of 10 mM Mg2+ in si-Control (n=46) and si-TRPM7 MIA PaCa-2 (n=62) indicating that TRPM7 mediates Mg2+ entry (P<0.001 using paired Wilcoxon signed tests). (C) External application of 10 mM Mg2+ increases MIA PaCa-2 invasion in si-Control but not in si-TRPM7 condition (n=40 counting; N=2 passages; P<0.001 using two-way analysis of variance followed by Holm-Sidak tests). (D). Pro-MMP-2 secretion is increased by adding 10 mM Mg2+ in culture media (N=4; P<0.05).
Figure 9
Figure 9
TRPM7 expression in human PDAC and LNM. (A) Examples of low (left panels) and high (right panels) apical and membranous staining in pancreatic (upper panels) and LNM (lower panels) tumor cells using TRPM7 antibody. (Inserts) Negative control obtained by omitting the primary antibody. (B) Correlation between pancreatic (n=13) and LNM (n=22) staining score for TRPM7 (P<0.001).
Figure 10
Figure 10
Graphical conclusion. TRPM7 is involved in basal Mg2+ influx in PDAC cells and secretion of Hsp90α. Hsp90α stabilizes both pro-MMP-2 and uPA/plasmin pathway that in turn enhances extracellular matrix (ECM) degradation and PDAC cell invasiveness.
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