Transient receptor potential melastatin-related 7 channel is overexpressed in human pancreatic ductal adenocarcinomas and regulates human pancreatic cancer cell migration

Authors


Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer with a tendency to invade surrounding healthy tissues, leading to a largely incurable disease. Despite many advances in modern medicine, there is still a lack of early biomarkers as well as efficient therapeutical strategies. The melastatin-related transient receptor potential 7 channel (TRPM7) is a nonselective cation channel that is involved in maintaining Ca2+ and Mg2+ homeostasis. It has been recently reported to regulate cell differentiation, proliferation and migration. However, the role of TRPM7 in PDAC progression is far to be understood. In our study, we show that TRPM7 is 13-fold overexpressed in cancer tissues compared to the healthy ones. Furthermore, TRPM7 staining is stronger in tumors with high grade, suggesting a correlation between TRPM7 expression and PDAC progression. Importantly, TRPM7 expression is inversely related to patient survival. In BxPC-3 cell line, dialyzing the cytoplasm during the patch-clamp whole-cell recording with a 0-Mg2+ solution activated a nonselective current with a strong outward rectification. This cation current is inhibited by intracellular Mg2+ and by TRPM7 silencing. The downregulation of TRPM7 by small interference RNA dramatically inhibited intracellular Mg2+ fluorescence and cell migration without affecting cell proliferation, suggesting that TRPM7 contributes to Mg2+ entry and cell migration. Moreover, external Mg2+ following TRPM7 silencing fully restored the cell migration. In summary, our results indicate that TRPM7 is involved in the BxPC-3 cell migration via a Mg2+-dependent mechanism and may be a potential biomarker of poor prognosis of PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the fifth most frequent cause of cancer-related mortality in the European Union1 and the the fourth most frequent cause in the United States.2 PDAC has a poor long-term prognosis with 5-year survival rates of only 1–4%. Current therapeutic strategies generally result in only a few months of extended life because PDAC develops insidiously and metastasizes quickly and widely. Thus, a greater understanding of the molecular processes involved in PDAC progression and metastasis is urgently needed.

Ion channels are transmembrane proteins that are involved in a wide panel of physiological cell mechanisms, including excitability, secretion, proliferation, apoptosis and motility. Nevertheless, a growing amount of studies show that ion channels expression is altered in several cancers including breast and prostate.3 Among these ion channels, members of the transient receptor potential (TRP) family have been proposed as prognosis markers in breast and prostate cancers.4, 5

TRP melastatin-related 7 (TRPM7) channels are a member of “chanzymes,” which are unique feature of cation channels fused with a kinase function.6, 7 They allow magnesium and calcium entries; however, they are mainly involved in magnesium homeostasis.8 In the digestive system, TRPM7 channels are expressed and involved in magnesium (Mg2+) absorption9 and in intestinal motor activity.10 Recently, TRPM7 have been shown to be involved in cell proliferation in head and neck,11 breast cancers,12 as well as in cell survival in gastric cancer13, 14 and in rat hepatoma cells.15 Moreover, TRPM7 regulates cell migration in nasopharyngeal carcinoma cells.16 The ability of TRPM7 channel blockade to inhibit the growth and survival of breast cancer and gastric cells suggests TRPM7 as a potential marker of these cancers.12, 14

The cation current through TRPM7 channel is inhibited by free intracellular Mg2+ as well as Mg2+ complexed with nucleotides, like MgATP. Indeed, this current has been described as magnesium-inhibited cation channel (MIC) current or magnesium nucleotide-regulated metal ion (MAGNUM) current.17, 18 In a recent study, TRPM7 has been identified as a key regulator of pancreatic development in zebrafish and as a regulator of cell proliferation in human pancreatic cancer cell lines.19 The goal of our study was to characterize the activity and the possible role of TRPM7 in PDAC cell migration, to determine the expression of TRPM7 in human PDAC tissue compared to exocrine pancreatic tissue from healthy donors and finally to investigate whether TRPM7 expression could be related to patient characteristics including tumor grade and survival.

Material and Methods

Human pancreatic tissues

Human tissue samples from PDAC were used with the agreement of patients treated by surgery in the University Hospital of Amiens (Picardie, France). Experiments on human tissues were approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Picardie (CCPPRBP, Amiens, France). Pancreata (n = 6), recovered from adult deceased donors in accordance with the French Regulations and with the local Institutional Ethical Committee (University Hospital of Lille, France), were cold preserved (<8 hr). A routine sample of pancreatic tissue was fixed for histology in paraformaldehyde (4%).

Histological examination was performed by an experimented pathologist (D.C.) on paraffin-embedded tissues using hematoxylin-phloxin-saffron staining. The human PDAC samples (n = 18) were classified according to the Scarff, Bloom and Richardson histoprognosis grading system based on cell differentiation, nuclear polymorphism and mitotic activity. In summary, human pancreatic tissues included six healthy tissues, seven Grade 1 adenocarcinomas, seven Grade 2 adenocarcinomas and four Grade 3 adenocarcinomas.

Immunohistochemistry of TRPM7

Immunohistochemical studies were performed using the indirect immunoperoxidase staining technique on the paraffin-embedded material with a Ventana XT instrument (Ventana Medical Systems, Roche Diagnostics, Basel, Switzeland) and a hematoxylin counterstain as previously described.20 The sections were incubated with anti-TRPM7 antibody (1/100; Chemicon, Billerica, MA), and negative controls were performed by omitting the primary antibodies. The specificity of the antibody was already tested using blocking peptide on breast adenocarcinoma tissues.12 Analysis of tissue section was done by light microscopy by a pathologist (D.C.) at 400× magnification. Because PDAC tissues showed a nonhomogenous staining, TRPM7 staining in human PDAC was determined by the previously published method.21 Briefly, the percentages of tumors cells (in well differentiated and undifferentiated areas) that were stained at each of the intensity score 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining) were added, and the sum of these scores gave a final score for each tissue ranging from 0 to 0.99 (weak TRPM7 staining), 1 to 1.99 (moderate TRPM7 staining) or 2 to 2.99 (strong TRPM7 staining). The p-value was calculated using Kruskal-Wallis one-way ANOVA on ranks comparing the scores of each of the PDAC grades with the healthy pancreatic tissue sample followed by post hoc tests between each grade (Dunn's Method). The relationship between TRPM7 staining and patient survival was also assessed using Student's t-test comparing the average patient survival for each of the PDAC staining score range (weak versus moderate TRPM7 staining).

Cell culture

BxPC-3 cell line was from ATCC (London, UK) (CRL-1687™). Cells were grown in RPM1 1640 (Sigma-Aldrich, St Louis, MO) supplemented with 1% L-glutamine (Sigma-Aldrich, St Louis, MO) and 10% fetal calf serum (FCS; Lonza, Bâle, Switzerland). Cells were trypsinized once a week using trypsin-EDTA (Sigma-Aldrich, St Louis, MO) and incubated at 37°C in a humidified atmosphere with 5% CO2. For all experiments, cells were used from passages 12 to 22 after decongelation from ATCC.

Quantitative and semiquantitative RT-PCR

RNA extraction was performed using the standard Trizol-phenol-chloroform protocol or the RNAeasy Micro Kit (Qiagen, Venlo, Netherlands) for tissues (20 mg) after homogenization with a Polytron homogenizer (PRO-200; Fisher Bioblock Scientific, Illkirsh, France). Total RNA (1 μg) was reverse transcribed into cDNA with random hexamers and MultiScribe™ Reverse Transcriptase (Applied Biosystems, Carlsbad, CA) as already described.12 PCRs were carried out using a iCycler thermal cycler (Biorad, Hercules, CA) and Taq DNA polymerase (Invitrogen, Carlsbad, CA) with 30 and 40 cycles for β-actin (forward 5′-CAGAGCAAGAGAGGCATCCT-3′ and reverse 5′-ACGTACATGGCTGGGGTG-3′), TRPM6 (forward 5′-GAGGAGATGGATGGGGGC-3′ and reverse 5′-GG TCCAGTGAGAGAAAGCCAA-3′) and TRPM7 (forward 5′-GTCACTTGGAAACTGGAACC-3′ and reverse 5′-CGGTAG ATGGCCTTCTACTG-3′) primers, respectively. For semiquantitative experiments, 30 cycles were performed for TRPM7, 40 for TRPM6 and 23 for β-actin. PCR products were visualized in agarose gels and quantified using Quantity One Software (Biorad, Hercules, CA). Results were expressed as TRPM7 expression normalized to β-actin expression for semiquantitative RT-PCR experiments. Quantitative RT-PCR was performed on a LightCycler system (Roche, Basel, Switzerland) using a mix containing SYBR green (Applied Biosystem, Carlsbad, CA). TRPM7 mRNA quantities were normalized to total mRNA and to 18S rRNA (forward 5′-CAGCCACCCGAGATTGAGCA-3′ and reverse 5′-TAGTAGCGACGGGCGGTGTG-3′) and β-actin as housekeeping genes.

TRPM7 silencing

TRPM7 silencing was made using small interference RNA (siRNA) strategy as already described.12 Briefly, siRNA were included in pancreatic cancer cells by nucleofection using a Nucleofector™ II device (Lonza, Bâle, Switzerland). BxPC-3 cells (106 cells) were transfected with 2 μg siRNA (corresponding to 1.5 μM) according to the optimized protocol recommended by Lonza. BxPC-3 cells were nucleofected with a scrambled siRNA (SiCTL) or with a siRNA targeting TRPM7 (SiTRPM7; targeting nucleotide sequence: 5′-GTCTTGCCATGAAATACTC-3′). All siRNAs were purchased from Dharmacon Research, USA, Lafayette, CO. All the experiments were made 48 hr following the nucleofection.

Electrophysiological recordings

TRPM7 currents were recorded using the conventional technique of patch-clamp in the whole-cell configuration as previously described.12 Briefly, holding membrane potential was held to −40 mV, and currents were elicited by a ramp depolarization from −100 to +100 mV for 350 msec. Interval between each ramp depolarization was 10 sec. The patch pipettes (3–5 MΩ) were made from hematocrit glass using a vertical puller (P30 vertical micropipette puller; Sutter Instrument). The following extracellular solution was used (in mM): Na-gluconate, 140; K-gluconate, 5; Mg-gluconate, 2; Ca-gluconate, 2; HEPES, 10; glucose, 5 and TEA-Cl, 5 (pH adjusted to 7.4 with NaOH). The following intrapipette solution was used (in mM): Cs-gluconate, 145; Na-gluconate, 8; EGTA, 10; Mg-gluconate or MgATP (as indicated) and HEPES, 10 (pH adjusted to 7.2 with CsOH). For external divalent-free (DVF) solution, Ca-gluconate and Mg-gluconate were removed, and EDTA (4 mM) was added. Free [Mg2+]i was estimated using the program WEBMAXC STANDARD (http://www.stanford.edu/∼cpatton/webmaxcS.htm). Signals were filtered at 1 kHz and digitized at 5 kHz using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) combined with a 1322A digidata (Molecular Devices, Sunnyvale, CA). The MIC current was recorded after the dialysis of intracellular media by 0 Mg intrapipette solution. The MIC current was calculated as the difference between the steady-state current activated by the depletion of [Mg2+]i and the basal current recorded few minutes after patch rupture. MIC currents were expressed as current–densities (in pA/pF) by dividing the current intensity (in pA) by the cell capacitance (in pF). Electrophysiological protocols and analyses were made using pClamp 10, Clampfit (both by Molecular Devices) and Origin 6.0 (Microcal Software, Northampton, MA). All experiments were performed at room temperature.

Cell imaging

BxPC-3 cells were placed on glass cover slips in 35-mm-diameter dishes at a density of 8 × 104 cells after nucleofection with SiCTL or siTRPM7. After 48 hr, cells were loaded in cell growth medium at 37°C for 1 hr with 3 μM of Fura 2-AM (Sigma-Aldrich, St Louis, MO) or 3 μM of Mag-Fura 2-AM (Invitrogen, Carlsbad, CA) and subsequently washed three times with the extracellular solution. The cover slip was then transferred onto a perfusion chamber on a Zeiss microscope equipped for fluorescence.

To estimate the divalent cation influx, we used Mn2+ quenching. Cells were perfused for 1 min with extracellular solution containing (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 0.4 MgCl2, 10 HEPES and 5 glucose (pH adjusted to 7.4 with NaOH). After this period, Ca2+ was replaced by Mn2+ (2 mM). To measure Mn2+ influx, cells were excited at 360 nm with a monochromator (TILL® Photonics, Munich, Germany), and fluorescence emission was monitored at 510 nm by a CCD camera coupled to a Zeiss inverted microscope (Carl Zeiss MicroImaging, LLC, Oberkochen, Germany). After Mn2+ perfusion, the decrease of Fura-2 fluorescence described a linear decay, and the slope is correlated with the rate of the Mn2+ influx. The calculated slope is obtained by subtracting the slope of basal decreasing Fura-2 fluorescence obtained in basal conditions (culture conditions) and after Mn2+ application.

To measure the Ca2+ basal fluorescence ratio, BxPC-3 were incubated with Fura-2AM as previously indicated. Cells were then perfused with the extracellular solution containing 2 mM of Ca2+. Cells were excited alternatively at 350 and 380 nm. The fluorescence emission was monitored at 510 nm. The ratio of Fura-2 fluorescence intensities measured with excitation at 350 and 380 nm (RF350/F380) was used as a [Ca2+]i-related signal.

For Mg2+ basal ratio recording, the same protocol as previously described for calcium fluorescence was used. Cells were perfused by extracellular solution which contain only 0.4 mM Mg2+ (as cultured conditions) and MAG-Fura-2AM was used as a fluoroprobe. The ratio of MAG-Fura-2 fluorescence intensities measured with excitation at 330 and 370 nm (RF330/F370) was used as a [Mg2+]i-related signal.

In vitro proliferation assays

Cell viability and proliferation were assessed using Trypan blue and methylthiazol tetrazolium (MTT) colorimetric tests. For Trypan blue assays, BxPC-3 cells were grown in 35-mm Petri dishes for 48 hr at the density of 4 × 104 cells. Cell growth was assessed with the standard Malassez cell method. Briefly, cells were removed by trypsinization and diluted in Trypan blue. Cell counts were performed six times (in a blinded manner), and the results were expressed as the percentage of viable cells measured compared to those measured under control conditions.

For MTT assays, cells were seeded in 35-mm Petri dishes at a density of 4 × 104 cells after nucleofection and were incubated at 37°C for further 48 hr. Cell proliferation was assessed by the MTT salt assay as previously described.22 The results are expressed as the percentage of formazan absorbance at 570 nm relative to the siCTL condition. In vitro viability and proliferation assays were performed in three different passages.

In vitro migration assays

Migration tests were performed in 8-μm pore size polyethylene terephthalate membrane cell culture inserts (BD FALCON™ Cell Culture Inserts, BD Biosciences, Franklin Lakes, NJ). The upper compartment was seeded with 4 × 104 cells of 24 hr-transfected BxPC-3 cells (SiCTL or SiTRPM7) in growth medium supplemented with 10% FCS. The lower compartment was filled with growth medium supplemented with 20% FCS as chemoattractant. After 24 hr of further incubation at 37°C, the remaining cells were removed from the upper side of the membrane by scrubbing. Migrated cells on the lower side were washed by phosphate buffered saline, fixed by methanol and stained by hematoxylin. Migrated cells were counted under an inverted microscope (Zeiss, Oberkochen, Germany) in duplicate (20 contiguous areas at 400× magnification for each insert). For each experiment, the number of migrating cells per area for each condition (SiCTL or SiTRPM7) was normalized by the mean of siCTL migrating cells. Experiments were repeated in three different passages. For each passage, a MTT test was carried out in the same condition than the migration assays (24 hr after cell nucleofection).

Statistical analysis

Data are presented as mean ± SEM, n refers to the number of experiments and N refers to the number of passages. Statistical analyses were made using Student's t-tests or Mann-Whitney rank sum test depending on sample normality using Sigma-Stat 3.0 (Systat Software). When more than two conditions were compared, a Kruskal-Wallis one-way ANOVA was used followed by post hoc Dunn's Method tests using Sigma-Stat 3.0.

Results

A MIC current is recorded in the human PDAC BxPC-3 cell line

The average membrane capacitance of BxPC-3 cells was 16.7 ± 0.7 pF (n = 68). The dialysis of intracellular media with patch pipette solution without Mg2+ induced the generation of a nonselective cation current in almost 15 min (Fig. 1a, left panel). This current displayed the shape of the MIC current with a reversal potential (11.7 ± 3.4 mV; n = 11) and a strong outward rectification (Fig. 1a, right panel). The current–density of the dialyzed-activated current was −3.3 ± 1.0 pA/pF at −100 mV and 32.2 ± 4.4 pA/pF at +100 mV (n = 11). The outward component of the MIC current was significantly inhibited by dialyzing with a patch pipette solution containing 680 μM free-[Mg2+]i (−2.8 ± 1.8 pA/pF at −100 mV and 10.6 ± 0.8 pA/pF at +100 mV; n = 5; p < 0.01) and it was fully inhibited by 850 μM free-[Mg2+]i (−0.6 ± 0.5 pA/pF at −100 mV and 0.5 ± 0.6 pA/pF at +100 mV; n = 5; p < 0.01; Fig. 1b, left panel). The addition of ATP in the patch pipette sensitized the MIC current to free-[Mg2+]i (Fig. 1b, right panel). Indeed, addition of 7 mM [MgATP] in the patch pipette (calculated free-[ATP]i = 900 μM and free-[Mg2+]i = 680 μM) fully abolished the MIC current in BxPC-3 cells (0.1 ± 0.4 pA/pF at −100 mV; −0.1 ± 0.1 pA/pF at +100 mV; n = 4; p < 0.001). Finally, cell perfusion with a DVF solution induced a large and a reversible linear current with inward (−42.8 ± 2.7 pA/pF at −100 mV; n = 5; p < 0.001) and outward (53.9 ± 6.8 pA/pF at +100 mV; n = 5; p < 0.05) components (Fig. 1c).

Figure 1.

Electrophysiological recording of TRPM7-like current in BxPC-3 human pancreatic ductal adenocarcinoma cells. (a) Left panel: Membrane cationic currents were recorded using the conventional technique of patch-clamp in the whole-cell configuration. Membrane was depolarized from −100 to +100 mV during 350 ms from a holding potential of −40 mV every 10 sec. Recording at +100 mV indicated that outward current was generated in almost 15 min during the dialysis of intracellular media by an intrapipette solution containing 0 Mg2+. Right panel: Average current–voltage (IV) trace of TRPM7-like currents recorded in BxPC-3 cells (n = 11) exhibiting an inward component (−3.3 ± 1.0 pA/pF at −100 mV), a strong outward rectification (32.2 ± 4.4 pA/pF at +100 mV) and a reversal potential (11.7 ± 3.4 mV). (b) The TRPM7-like current is sensitive to free [Mg2+]i and [MgATP]i. Left panel: The presence of 680 μM free-[Mg2+]i in the patch-pipette significantly decreased the TRPM7-like current (−2.8 ± 1.8 pA/pF at −100 mV and 10.6 ± 0.8 pA/pF at +100 mV; n = 5; p < 0.01), whereas it is fully abolished by 850 μM free-[Mg2+]i (−0.6 ± 0.5 pA/pF at −100 mV and 0.5 ± 0.6 pA/pF at +100 mV; n = 5; p < 0.01). Right panel: The addition of Mg2+ complexed to ATP in the intrapipette solution to obtain 680 μM free-[Mg2+]i fully abolished the TRPM7-like current (0.1 ± 0.4 pA/pF at −100 mV; −0.1 ± 0.1 pA/pF at +100 mV; n = 4; p < 0.001). (c) Left panel: At the steady state of TRPM7-like current recording (1), a divalent-free (DVF) solution was perfused to the cell. This induced a reversible increase of both inward and outward components recorded at −100 and +100 mV, respectively (2). Right panel: DVF perfusion induced a modification of the IV relationship shape which became linear. The inward component was −42.8 ± 2.7 pA/pF at −100 mV (n = 5; p < 0.001), and the outward component was 53.9 ± 6.8 pA/pF at +100 mV (n = 5; p < 0.05).

TRPM7 is functional in BxPC-3 cells, allows cation entry and regulates intracellular [Mg2+]

We investigated the expression of TRPM7 and TRPM6 in BxPC-3 cells. Human breast cancer MCF-7 cells are used as positive control, as they express both TRPM7 and TRPM6.12 As shown in Figure 2a, only TRPM7 was detected in BxPC-3 cells, but not TRPM6.

Figure 2.

Effect of SiTRPM7 on the MIC current in BxPC-3 cells. (a) TRPM7 mRNA are detected in BxPC-3 human pancreatic ductal adenocarcinoma cell line by RT-PCR in three different passages, whereas TRPM6 mRNA are not detected. MCF-7 human breast cancer cell line is used as positive control. (b) The 48-hr transfection of BxPC-3 cells with a siRNA targeting TRPM7 induced the decrease of TRPM7 expression normalized to β-actin by RT-PCR. The results are a typical example from three separate experiments in three different passages. TRPM7 expression measured by quantitative RT-PCR indicates that TRPM7 silencing induced a decrease of TRPM7/β-actin mRNA quantity of 66.4% ± 6.7% compared to a scrambled siRNA (N = 3; p < 0.001). (c) Typical traces of IV relationships recorded in BxPC-3 cells treated with a SiTRPM7 and with a SiCTL. IV curves were recorded using the same protocol than for (a). (d) TRPM7 silencing significantly decreased the TRPM7 current–density in BxPC-3 cells (8.2 ± 2.7 pA/pF at +100 mV; n = 7) compared to the cells treated with a SiCTL (22.7 ± 5.3 pA/pF at +100 mV; n = 6: p < 0.05).

To demonstrate the involvement of TRPM7 in the MIC current in BxPC-3 cells, we conducted a series of siRNA-mediated knockdown experiments. As shown in Figure 2b, a 48-hr treatment with siRNA against TRPM7 (SiTRPM7) significantly reduced TRPM7 mRNA of 66.4% ± 6.7% (N = 3; p < 0.001). This was related to a significant decrease in the MIC current–density from 22.7 ± 5.3 pA/pF (n = 6) to 8.2 ± 2.7 pA/pF (n = 7; p < 0.05; Figs. 2c and 2d).

As TRPM7 channel is involved in Ca2+ and Mg2+ homeostasis, we assessed the effect of TRPM7 silencing on divalent entries using the Mn2+-quenching method. Compared to control BxPC-3 cells, transfected cells exhibited a marked decrease in cation entry (Fura-2 fluorescence intensity: 26% ± 2.6%; n = 192, p < 0.05; Figs. 3a and 3b), confirming the transmembrane flux of cations into the cell through TRPM7 channels in our culture conditions.

Figure 3.

TRPM7 is involved in the control of the basal intracellular Mg2+ homeostasis. (a) Typical traces of external application of Mn2+ that induced the quenching of the Fura-2 fluorescent probe in BxPC-3 cells treated with a SiTRPM7 or with a scrambled siRNA. (b) SiTRPM7 induced a significant reduction of the slope of the Mn2+ quenching (26% ± 2.6%; n = 192; N = 3; p < 0.05) compared to the SiCTL condition. (c) TRPM7 silencing failed to affect Fura-2 F350/F380 fluorescence ratio in BxPC-3 cells (0.97 ± 0.04 for SiCTL; n = 67; N = 3 vs. 1.04 ± 0.04 for SiTRPM7; n = 74; N = 3; p > 0.05). (d) Transfection of BxPC-3 cells by SiTRPM7 induced a decrease of MAG-Fura-2 F330/F370 fluorescence ratio (0.69 ± 0.01 for SiCTL; n = 111; N = 2 vs. 0.55 ± 0.01 for SiTRPM7; n = 100; N = 2; p < 0.001) in culture conditions (in the presence of 0.4 mM of Mg2+). The addition of 1 mM of MgCl2 in the medium culture (total concentration of Mg2+ = 1.4 mM) normalized the effect of SiTRPM7 on the MAG-Fura-2 F330/F370 fluorescence ratio.

To determine whether TRPM7 regulates basal [Mg2+]i or [Ca2+]i, we used MAG-Fura and Fura-2 probes (see Material and Methods section). No variation in [Ca2+]i was observed in BxPC-3 cells transfected with SiTRPM7 compared to SiCTL (n = 74; p > 0.05; Fig. 3c), whereas the basal [Mg2+]i was significantly decreased in cells transfected by SiTRPM7 (n = 100; p < 0.001; Fig. 3d). To investigate whether the increase of extracellular Mg2+ could restore the [Mg2+]i inhibited by SiTRPM7, we added 1 mM of MgCl2 in the culture medium and incubated cells transfected with SiCTL or SiTRPM7 during 24 hr. The addition of 1 mM of MgCl2 fully normalize [Mg2+]i in cells transfected by SiTRPM7 (26.4% ± 3.3%, N = 2; Fig. 3d). Moreover, the omission of extracellular Mg2+ decreased [Mg2+]i by −36.6% ± 5% (N = 2, data not shown).

Downregulation of TRPM7 reduces the BxPC-3 cell migration

We used Trypan blue, MTT and cell culture insert techniques to measure viability, proliferation and migration rates of BxPC-3 cells, respectively (Fig. 4). Cells were transfected with either SiTRPM7 or SiCTL for 48hr. TRPM7 silencing neither affected the cell viability nor the cell proliferation (for each condition, the number of dead cells was <10%; Figs. 4a and 4b). However, TRPM7 silencing decreased the cell migration (Fig. 4c). The cell migration was significantly decreased in BxPC-3 cells transfected with SiTRPM7 (48% ± 4%; N = 6) compared to the SiCTL (100% ± 5%; N = 6; p < 0.001). There is some evidence that external Mg2+ regulates cell migration in PDAC.23 To assess whether the addition of Mg2+ could restore the migration inhibited by SiTRPM7, we added MgCl2 in the upper compartment of the culture insert. The addition of 1 mM of MgCl2 fully restored the migration in BxPC-3 treated with SiTRPM7 (103% ± 7%; N = 3; p > 0.05 compared to SiCTL; Fig. 4c). For these experiments, the different conditions did not affect the cell proliferation (data not shown).

Figure 4.

Role of TRPM7 in BxPC-3 cell proliferation and migration. (a) BxPC-3 cell viability measured by Trypan blue assays after 48-hr transfection was not different in the SiTRPM7 condition (120% ± 9%; N = 3) compared to the SiCTL condition (100% ± 10%; N = 3; p > 0.05). For each of these experiments, the number of dead cells was always <10%. (b) BxPC-3 proliferation was measured by MTT assays after 48-hr transfection, and no difference was found between SiTRPM7 condition (128% ± 12%; N = 3) and SiCTL condition (100% ± 2%; N = 3; p > 0.05). (c) BxPC-3 migration was measured in Boyden chambers. The migration duration was the last 24 hr within the 48-hr transfection. TRPM7 silencing significantly decreased the BxPC-3 cell migration (48% ± 4%; N = 6) compared to SiCTL (100% ± 5%; N = 6; p < 0.001). The addition of 1 mM of MgCl2 fully restored the migration in SiTRPM7 cells (103% ± 7%; N = 3; p > 0.05) compared to SiCTL. SiTRPM7 and SiTRPM7 + MgCl2 conditions were compared to the SiCTL condition (100% ± 6%; N = 6) by Kruskal-Wallis one-way ANOVA and post hoc Dunn's Method.

TRPM7 is overexpressed in human PDAC tissues compared to healthy tissues

To strengthen the importance of this channel as a potential biomarker of PDAC, TRPM7 expression was first assessed in six samples of healthy human exocrine pancreatic tissues. No TRPM7 staining was observed in healthy exocrine ducts, whereas a brown TRPM7 staining was observed in the duct areas from PDAC tissues (n = 18; Fig. 5a). TRPM7 mRNA, normalized to 18S rRNA, was assessed in PDAC and healthy tissues using quantitative RT-PCR. The relative quantity of TRPM7 mRNA was 12.5-fold ±1.0 higher in PDAC tissues (n = 8) compared to the healthy ones used as a control and assigned to the value of 1 (n = 6; p < 0.05; Fig. 5b). Normalization to total mRNA and β-actin as control gene produced similar results in separate experiments (Supporting Information S1). We also investigate the TRPM6 expression in PDAC tissues compared to the healthy ones using 18S rRNA as a housekeeping gene. We observed a very low level of TRPM6 transcript compared to TRPM7 in PDAC tissues (see Supporting Information S2). Moreover, the averaged relative amount of TRPM6 mRNA in PDAC tissues (n = 8) was not different compared to the healthy ones (n = 6; p > 0.05; see Supporting Information S2).

Figure 5.

TRPM7 is overexpressed in PDAC; its expression depends on tumor grade and survival. (a) Left panel: no staining was observed in healthy tissue (magnification, ×200). Inset: Negative control obtained by omitting the primary antibody. Right panel: TRPM7 staining with a marked reactivity in poorly differentiated pancreatic ductal adenocarcinoma (magnification, ×200). Inset: Negative control. (b) TRPM7 mRNA levels were measured in eight adenocarcinoma and six healthy samples by quantitative RT-PCR and normalized to 18S rRNA. Adenocarcinoma samples have a higher expression of TRPM7 compared to the average expression in the healthy tissue (SEM p < 0.05, Kruskal-Wallis one-way ANOVA). (c) Typical examples of TRPM7 immunoreactivity in no-tumoral adjacent tissue (weak staining), in Grade 1 PDAC tissue (weak staining) and in Grade 2 + 3 PDAC tissues (moderate staining) at a magnification of ×200. Insets: Negative control obtained by omitting the primary antibody. (d) TRPM7 immunoreactivity was related to patient survival. We used the Pearson product moment correlation to analyze this linear regression. The correlation coefficient R was −0.64 and the p-value was 0.004, which indicated that patient survival tend to decrease while TRPM7 expression increases.

The overexpression of TRPM7 in PDAC suggests an involvement of the channel in the pathophysiology of this disease, which might be linked to patient survival. To test this hypothesis, statistical quantifications were performed. TRPM7 staining was assessed in Grade 1 (n = 7, well differentiated), Grade 2 (n = 7, moderately differentiated) and Grade 3 (n = 4, poorly differentiated) PDACs compared to the healthy tissue (n = 6). The intensity of TRPM7 staining was stronger in high-grade PDACs (2 and 3), whereas no staining was found in healthy tissue and only a weak staining was found in Grade 1 PDACs (Fig. 5c and Table 1 for numerical values).

Table 1. Analysis of TRPM7 expression in healthy human and PDAC samples
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Patient survival was also investigated, and overall survival (OS) was compared between tumors with weak and moderate staining for TRPM7. We found that TRPM7 staining intensity is inversely associated with mean survival patient (Fig. 5d). Indeed, the OS was strongly reduced in patient with moderate TRPM7 staining (mean OS, 9.1 ± 2.2 weeks; n = 7 vs. 19.3 ± 2.4 weeks; n = 11; p < 0.05). Moreover, no correlation was found between TRPM7 immunoreactivity and lymph node metastasis as all patients were positive for metastasis.

Discussion

In spite of significant advances in surgery and the use of new, more effective chemotherapeutic agents, the overall 5-year survival of patients with PDAC is 1–4%. Identification of new marker factors might be of value for PDAC detection and/or in directing therapy. The involvement of TRPM7 in pancreatic cancer had not been systematically studied. In our study, we report four major findings: (i) TRPM7 is functional in human pancreatic cancer cells and regulates [Mg2+]i but not [Ca2+]i; (ii) TRPM7 regulates cell migration through a Mg2+-dependent process; (iii) TRPM7 is overexpressed in human PDAC and (iv) importantly, TRPM7 expression is positively correlated to the poorly differentiated status and is associated with a reduction of patient survival.

TRPM7 channels are involved in Ca2+ and Mg2+ uptake in the digestive system and are expressed physiologically in the brush-border membrane of the colon.9 In a recent study, Yee et al.19 showed that the Trpm7 gene regulates exocrine pancreatic development via the Mg2+-sensitive Socs3a pathway in Zebrafish. Moreover, they found that TRPM7 is overexpressed at the mRNA level in five of seven human pancreatic cancer cell lines, including BxPC-3, when compared to the human pancreatic ductal epithelial cell line H6c7.19 Finally, immunohistochemistry of TRPM7 in human pancreatic adenocarcinomas and matched non-tumoral tissues indicates that TRPM7 staining is stronger in pancreatic adenocarcinomas.19 Nevertheless, no quantification of TRPM7 expression was made to confirm the immunohistological data or correlation with patient characteristics (tumor grade, lymph node metastasis, survival). In the present study, the immunohistochemistry of TRPM7 was done in eighteen human PDAC tissues and compared to six human exocrine pancreatic tissues from healthy donors in addition to the comparison with the matched non-tumoral tissues. Our results confirm that TRPM7 is overexpressed in human PDAC tissues compared to the non-tumoral adjacent one. Moreover, we reported, for the first time, an association between the TRPM7 expression and both tumor differentiation and patient survival. Indeed, 11 patients with high-grade tumor (moderately and poorly differentiated tissues) show a stronger TRPM7 immunostaining than in Grade 1 tumors. Thus, we compared the intensity of TRPM7 staining with the OS of the patients, and we found a reduction of the patient survival in TRPM7-positive patients. TRPM7 mRNA level was also studied in eight PDAC tissues compared to the six healthy ones and our data show that TRPM7 is 12.5-fold increased in PDAC tissues. TRPM7 mRNA levels were normalized to 18S rRNA. 18S rRNA has been previously shown to demonstrate variations of less than 10% and the lowest variability in expression between cancerous and normal pancreas tissue.24 Moreover, TRPM7 mRNA normalization to total mRNA and β-actin as housekeeping gene produced similar results in separate experiments (Supporting Information). The high level of TRPM7 mRNA (12.5-fold) we report in PDAC tissue compared to the healthy ones is consistent with levels reported for other biomarker of PDAC progression like high-mobility group A1 protein.25

Taken together, our data strongly suggest that TRPM7 could be used as a promising marker of human PDAC progression and prognosis. However, further studies with a larger group of patients will be necessary to substantiate these data, particularly for Grade 3 adenocarcinomas.

TRPM7 and its close homolog TRPM6 are dual-activity enzymes comprising both an ion channel as well as a kinase domain. These cation channels are inhibited by intracellular Mg2+ and MgATP, and the resultant nonselective cation current has been identified as MIC or MAGNUM current.17, 18 MIC current has been recorded in cancer cell lines including human head and neck,11 breast12 and gastric13 adenocarcinomas and rat hepatoma.15 Our electrophysiological data show a similar MIC current in human BxPC-3 cell line with sensitivity to both intracellular Mg2+ and MgATP. TRPM7 regulates both Ca2+ and Mg2+ influx. Indeed, in breast, head and neck and nasopharyngeal cancers, it regulates Ca2+ cell influx,6, 7, 12 whereas it regulates Mg2+ influx in polarized cells,26 vascular smooth muscle cells27 and osteoblasts.28 In BxPC-3 human PDAC cell line, our data show that TRPM7 silencing decreased [Mg2+]i, suggesting that this channel is required for magnesium uptake in pancreatic cancer cells. Recently, Yee et al.19 indicated that TRPM7 regulates both BxPC-3 and Panc-1 cells proliferation via a Mg2+-sensitive mechanism. We neither observed any effect of TRPM7 silencing in BxPC-3 proliferation by MTT assays nor by viable cell counting in Trypan blue after 48 and 72 hr of total treatment. These apparent contradictory results could be explained by the different experimental procedures. However, here, we show that TRPM7 strongly regulates BxPC-3 cell migration. Cell motility is involved in the processes regulating cancer cell dissemination and metastasis. TRPM7 has been reported to be involved in migration by Ca2+- or Mg2+-dependent mechanisms depending on cell types. Indeed, in human embryonic lung fibroblasts and nasopharyngeal carcinoma, TRPM7 channel promotes migration by Ca2+-dependent mechanisms16, 29 and it promotes vascular smooth muscle cells27 and osteoblast28 migration by regulating Mg2+ uptake. More recently, it has been shown that TRPM7 knockdown alters cell morphology, the cytoskeleton, and the ability of cells to form lamellipodia and to execute polarized cell movements. The expression of the Mg2+ transporter SLC41A2 is able to restore these phenotypic changes,26 whereas the SLC41A1 transporter is post-transcriptionally regulated by extracellular Mg2+ in TRPM7-deficient cells.30, 31 Another Mg2+ entry pathway, the Mg2+ transporter MagT1, has also been reported to rescue cell growth and Mg2+ uptake in TRPM7-deficient cells.32 Here, in PDAC cells, we show a significant decrease in migration in cells deficient for TRPM7 associated with a decrease of [Mg2+]i. Moreover, supplementation of culture media with Mg2+ was able to restore the migration in cells lacking TRPM7. Therefore, our data strongly suggest that TRPM7 regulates BxPC-3 cell migration by Mg2+-dependent mechanisms. However, the determination of molecular mechanisms involved in TRPM7-mediated pancreatic cell migration are far to be understood and need further investigations.

In conclusion, this study provides evidence that TRPM7 is a promising biomarker of PDAC progression and prognosis. Moreover, in human BxPC-3 cell line, TRPM7 regulates cell migration by a Mg2+-dependent mechanism.

Acknowledgements

The authors thank Marie-Pierre Mabille from the Anatomy and Pathology Department of Amiens Hospital for her technical assistance. P.R. is a recipient of a PhD stipend from the Ministère de la Recherche et de l'Enseignement Supérieur.

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