Targeted silencing of TRPM7 ion channel induces replicative senescence and produces enhanced cytotoxicity with gemcitabine in pancreatic adenocarcinoma

Abstract

The transient receptor potential TRPM7 ion channel is required for cellular proliferation in pancreatic epithelia and adenocarcinoma. To elucidate the mechanism that mediates the function of TRPM7, we examined its role in survival of pancreatic cancer cells. RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. TRPM7-deficient cells underwent replicative senescence with up-regulation of p16^CDKN2A and WRN mRNA. The combination of anti-TRPM7 siRNA and gemcitabine produced enhanced cytotoxicity as compared to gemcitabine alone. Thus, TRPM7 is required for preventing senescence, and modulation of TRPM7 expression may help improve treatment response of pancreatic cancer by combination with apoptosis-inducing agents.

1. Introduction

The goal of this study is to elucidate the mechanism that mediates the proliferative role of the transient receptor potential melastatin-subfamily member 7, TRPM7, ion channel in pancreatic cancer by examining its requirement for cell survival. Pancreatic adenocarcinoma, the most common type of primary cancer in the pancreas, is among the most lethal human diseases [1]. A number of oncogenes and tumor suppressor genes involved in the development of pancreatic neoplasia have been revealed, but their clinical utility as therapeutic targets in pancreatic adenocarcinoma remain to be demonstrated. Ion channels including the transient receptor potential (TRP) family members have been implicated in human malignancies [2], but their roles in pancreatic cancer were mostly unknown. Identification of the roles of the TRP ion channels in pancreatic adenocarcinoma and determination of the mechanisms that mediate their functions are expected to generate new insights into pancreatic carcinogenesis and provide new biomarkers and targets for therapy.

The TRP family of trans-plasma membrane channels act as cellular sensors of physical and chemical stimuli by modulating ionic homeostasis [3]. The TRP melastatin-subfamily (TRPM) ion channels mediate diverse physiological functions through regulation of cytosolic levels of Ca²⁺ and Mg²⁺ and multiple signaling pathways [4]. The TRP ion channel, TRPM7, is a Mg²⁺/Ca²⁺-permeable channel with kinase activity and it regulates various cellular processes [5,6]. These include cell proliferation [7–10], survival [5,11–14], differentiation [15], adhesion [16], volume [17], migration [18,19], and neurotransmitter release [20]. Recent discovery of Trpm7 ion channel as a developmental regulator of exocrine pancreas in zebrafish has provided a novel link of development to human pancreatic cancer [10].

The zebrafish sweetbread (swd) mutations (swd^p75fm and swd^p82mf) that affect exocrine pancreas and Trpm7 were recovered from an ethylnitrosourea-induced mutagenesis screen [21,22]. Genetic and embryological analyses of the zebrafish swd, trpm7^j124e1, and trpm7^b508 mutations have led to identification of the developmental role of Trpm7 in exocrine pancreas through controlling cell cycle progression and epithelial growth and consequently the organ size [10]. In normal adult tissues, the human orthologue TRPM7 is ubiquitously expressed [23], but in pancreatic adenocarcinoma, expression of TRPM7 is aberrantly up-regulated and required for cellular proliferation [10]. In both zebrafish larvae and human pancreatic adenocarcinoma cells, TRPM7-controlled cellular proliferation is Mg²⁺-dependent and it involves modulation of p21^CDKN1A and cyclin G1 [10]. In the developing zebrafish, supplementary Mg²⁺ or anti-sense oligos-induced repression of suppressor of cytokine signaling 3a (socs3a) is required for Trpm7-mediated epithelial proliferation in the exocrine pancreas [10]. However, the mechanisms underlying the proliferative role of TRPM7 in pancreatic cancer remain to be determined.

In this study, we examined the requirement of TRPM7 for survival of pancreatic adenocarcinoma cells. RNA interference-mediated silencing of TRPM7 induced replicative senescence, but not apoptosis, with up-regulated expression of the senescence-associated genes including the cyclin-dependent kinase inhibitor p16^CDKN2A and the Werner’s syndrome gene WRN. Combination of small interfering RNA (siRNA) directed against TRPM7 and the conventionally used apoptosis-inducing drug, gemcitabine, produced enhancement of cytotoxicity. Results of these data indicate that TRPM7 is required for preventing non-apoptotic cell death through replicative senescence and suggest that modulation of TRPM7 offers new options for therapeutic targeting in pancreatic cancer.

2. Materials and methods

2.1. Cell cultures

The human pancreatic adenocarcinoma cell lines BxPC-3 and PANC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, U.S.A.), and maintained according to the ATCC instructions. The cell culture medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone^®, Thermo Fisher Scientific Inc., Pittsburgh, Pennsylvania, U.S.A.), 100 U/ml penicillin (Gibco™, Invitrogen Corporation, Carlsbad, California, U.S.A.) and 100 µg/ml streptomycin (Gibco™). The cells were incubated in a humidified atmosphere containing 5% CO₂ at 37°C. All experiments were performed using culture medium. The cells were used within 20 passages of the stocks frozen in liquid nitrogen.

2.2. RNA interference-mediated gene silencing

BxPC-3 and PANC-1 cells were grown to 70–80% confluency, trypsinized, and resuspended at 10⁶ cells in 100 µl of Nucleofector^® Solution (Amaxa^®/ Lonza, Cologne, Germany) containing 600 nM anti-TRPM7 siRNA (sc-42662; Santa Cruz Biotechnology, Inc., Santa Cruz, California, U.S.A.) or non-targeting control siRNA (sc-37007; Santa Cruz Biotechnology). Transfection was performed using Nucleofector II (Amaxa^®/Lonza) according the manufacturer’s instructions. Forty-eight hours following transfection, total RNA was extracted and analyzed using real-time polymerase chain reaction (PCR) to verify knock down of TRPM7 [10].

2.3. Drugs and small molecules

Gemcitabine-HCl (Toronto Research Chemicals, Toronto, Canada) was dissolved in phosphate buffered saline (PBS), pH 7.4 at 10 mM. Suberoylanilide hydroxamic acid (SAHA, BioMol^®, Enzo Life Sciences International, Inc., Plymouth Meeting, Pennsylvania, U.S.A.) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich^®, St. Louis, Missouri, U.S.A.) at 50 mM. The stock solutions of gemcitabine (10 mM) and SAHA (50 mM) were divided into aliquots and stored at −20°C, and diluted with culture medium prior to addition to the cultured cells. For controls conducted in parallel, 0.01% DMSO or no drug was added to the medium.

2.4. Flow cytometric analysis of apoptosis

BxPC-3 and PANC-1 transfected with anti-TRPM7 or non-targeting control siRNA were seeded at 2×10⁵ cells / 3 ml in each well of a 6 well cell culture cluster (costar^®, Corning Incorporated, Corning, New York, U.S.A.) and incubated at 37°C for 72 h. The cells were then washed with PBS (pH 7.4), and incubated with fluorescein isothiocyanate (FITC)-conjugated Annexin V (Invitrogen™) and propidium iodide (PI, Invitrogen™), and analyzed for apoptosis by flow cytometry as described [24].

2.5. Hematoxylin and eosin staining

The cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were seeded at 10⁴ cells / 2 ml in each well of a 2 well glass slide (Lab-Tek^® Chamber Slide™, Nalge Nunc International, Rochester, New York, U.S.A.) and incubated at 37°C for 48 h. The cells were then rinsed with PBS, fixed with 2% formaldehyde / 0.2% glutaldehyde in PBS (pH 7.4), and stained with hematoxylin and eosin (Richard-Allan Scientific^®, Thermo Fisher Scientific, Pittsburgh, Pennsylvania, U.S.A.). The cell morphology was examined under a compound light microscope (Olympus BX-51, Tokyo, Japan). Images were captured using a digital camera (Olympus DP71, Tokyo, Japan) and processed using Adobe^® Photoshop^® CS3 extended.

2.6. Nuclear staining

BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were seeded at 10⁴ cells / 2 ml in each well of a 2 well glass slide (Lab-Tek^® Chamber Slide™), and incubated at 37°C for 48 h. The medium were removed, rinsed with PBS, fixed with 2% formaldehyde / 0.2% glutaldehyde in PBS (pH 7.4), and then mounted with Vectashield^® with 4’6 diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, California, U.S.A.). The DAPI-stained nuclei were examined under a compound microscope (Olympus BX-51) using fluorescence. Images were acquired using a digital camera (Olympus DP71) and processed using Adobe^® Photoshop^® CS3 extended.

2.7. Senescence assay

The transfected cells seeded at 4×10⁴ cells / 4 ml in each well of a 6 well cell culture cluster (costar^®, Corning Incorporated) and incubated at 37°C for 72 h and then analyzed for SA β-gal activity using the Senescence Detection Kit (Biovision Inc., Mountain View, California, U.S.A.) according the manufacturer’s instructions. Images were acquired under an inverted light microscope with phase contrast (Nikon TE-300, Melville, New York, U.S.A.) and processed using Adobe^® Photoshop^® CS3 extended.

2.8. Semi-quantification of p27^CDKN1B, p16^CDKN2A, and WRN mRNA

PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were seeded at 5×10⁵ cells / ml in each well of a 6 well cell culture cluster (costar^®, Corning Incorporated) and incubated at 37°C for 48 h. Total RNA was extracted using TRIzol^® (Invitrogen™) and RNeasy^® Mini Kit (Qiagen Inc., Valencia, California, U.S.A.) according to the manufacturer’s instructions. First-strand cDNA was generated using SuperScript reverse transcriptase and random primers (Invitrogen™) and amplified with SYBR^® Green (Applied Biosystems^®, Foster City, California, U.S.A.) using ABI Prism^® 7700 real-time PCR system (Applied Biosystems^®) as described [25]. The sequences of the primers used were designed based on human cDNA sequences (GenBank accession numbers: p27^CDKN1B, BC001971; p16^CDKN2A, AH005371; WRN, AY818673; GAPDH: NM_002046) as follows:

• p27^CDKN1B5’-GTCCATTTATCCACAGGAAA-3’, 5’-ATGGTTTTTCCATACACAGG-3’;

• p16^CDKN2A 5’- GGGTCGGGTAGAGGAGGT G-3’, 5’-GCGCTACCTGATTCCAATTC-3’;

• WRN 5’-GACAGCGGACTTCAACCTTC-3’, 5’-TTGGCAAACCACACAGGTAA-3’;

• GAPDH 5'-GAGTCAACGGATTTGGTCGT-3', 5'-TTGATTTTGGAGGGATCTCG-3'.

The relative mRNA levels of p27^CDKN1B, p16^CDKN2A, and WRN were determined as compared with those of GAPDH using the Comparative C_T Method (Applied Biosystems^®). Samples of PCR using primers directed against each tested gene were analyzed by agarose gel electrophoresis to validate specificity of amplification products.

2.9. Proliferation assay

PANC-1 cells were transfected with anti-TRPM7 siRNA or non-targeting control siRNA were seeded at 2×10⁴ / 100 µl in each well of a 96 well cell culture cluster (costar^®, Corning Incorporated) and incubated at 37°C. About 24 h following transfection, gemcitabine (1 µM or 5 µM) or SAHA (1 µM or 5 µM) was added and incubation was continued for a total of 72 h. Cellular proliferation was quantified using the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, Wisconsin, U.S.A.) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonylphenyl)-2H-tetrazolium (MTS) according to the manufacturer’s instructions and as described [25]. This experiment was performed with each treatment in triplicate samples.

2.10. Statistical analysis

The mean, standard deviation (s.d.), and P-values were analyzed using the Student’s t-test. Statistical significance was considered at a P-value < 0.05.

3. Results

3.1. TRPM7 is required for preventing non-apoptotic death of pancreatic adenocarcinoma cells

TRPM7 ion channel is necessary for cellular proliferation in pancreatic epithelia and adenocarcinoma [10]. In this study, we determined the role of TRPM7 in cell survival by using siRNA to silence its expression in the human pancreatic adenocarcinoma cell lines, BxPC-3 and PANC-1. The mRNA levels of TRPM7 in the cells transfected with anti-TRPM7 siRNA were reduced by up to 70% of that in the control siRNA-transfected cells, as determined by quantitative PCR. The protein levels of TRPM7 in the anti-TRPM7 siRNA-transfected cells were similarly reduced, as determined by immunoblotting. The TRPM7-deficient cells were analyzed by flow cytometry using annexin V as a marker of apoptotic cell death. As compared with controls, both BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA did not show any increase in the proportion of cells undergoing apoptosis (Fig. 1). This suggests that TRPM7 may not be required for cell survival; alternatively, the TRPM7-deficient cells might have undergone non-apoptotic cell death.

Fig. 1.

RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were incubated with FITC-conjugated annexin V and PI, and then analyzed for apoptosis by flow cytometry. The cells undergoing early apoptosis (lower right) and late apoptosis (upper right) are enclosed within the black border, with the proportion of apoptotic cells as indicated. The baseline level of apoptotic cells in the control siRNA-transfected cells may be attributed to electrical pulsation during Nucleofection^®, detachment of cells by trypsinization, and disaggregation of cells by filtration prior to flow cytometric analysis. This experiment was repeated twice with similar results.

Previously, we have shown that anti-TRPM7 siRNA induced formation of abnormally appearing nuclei and cytoplasmic vacuoles in BxPC-3 and PANC-1 as seen under phase-contrast microscopy [10]. Here, we further examined the TRPM7-deficient cells following staining of the nuclei and cytoplasm with hematoxylin and eosin, respectively. Consistent with the observations in the micrographs with phase contrast, bright field examination reveals that both BxPC-3 and PANC-1 cells with targeted knock down of TRPM7 expression exhibit morphological features indicative of a senescent phenotype (Fig. 2). The cells are enlarged and multi-nucleated with formation of cytoplasmic vacuoles. These features are suggestive of non-apoptotic cell death through replicative senescence [26].

Fig. 2.

SiRNA-mediated repression of TRPM7 induced cellular morphology indicative of a senescent phenotype. BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were stained with hematoxylin and eosin, and examined under a compound light microscope. Arrows point at the enlarged cells containing multiple nuclei in both BxPC-3 and PANC-1 and cytoplasmic vacuoles in BxPC-3. Similar results were obtained in duplicate culture dishes and the experiment was repeated twice.

To further characterize the multi-nucleated cells, BxPC-3 and PANC-1 transfected with anti-TRPM7 siRNA or non-targeting control siRNA were stained with DAPI that binds to genomic DNA. In both BxPC-3 and PANC-1, silencing of TRPM7 induced aberrant nuclear figures suggesting mitotic arrest (Fig. 3). To determine the effect of targeted inhibition of TRPM7 on cellular senescence, the TRPM7-deficient cells were assayed for SA β-gal activity, which is an established indication of cellular senescence. In the BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA but not in the controls, SA β-gal activity was detected (Fig. 4). Taken together, these data suggest that TRPM7 is required for preventing replicative senescence.

Fig. 3.

TRPM7-deficient cells contained multiple nuclei suggesting mitotic arrest. BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were stained with DAPI, and examined under a compound light microscope with fluorescence. Arrows point at multinucleated cells. This experiment was repeated twice with duplicate culture dishes in each experiment, and similar results were observed.

Fig. 4.

TRPM7-deficient cells underwent replicative senescence. BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were analyzed for SA β-gal activity. Images were acquired under an inverted light microscope with phase contrast. Arrows point at some of the cells displaying SA β-gal activity (blue color). The images shown are representative of three independent experiments with duplicate culture wells in each experiment.

3.2. RNA interference-mediated silencing of TRPM7 up-regulates expression of senescence-associated genes

To gain insights into the mechanism underlying the requirement of TRPM7 for preventing replicative senescence, we analyzed the expression of senescence-associated genes by real-time PCR. We have previously shown that, in BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA, the mRNA level of the cell cycle inhibitor p21^CDKN1A was increased whereas those of cyclin G1 and cyclin B1 were decreased [10]. In this study, we examined the mRNA levels of several markers of cellular senescence including the cyclin-dependent kinase inhibitors p27^CDKN1B and p16^CDKN2A, and the DNA helicase/exonuclease-encoding gene WRN. Up-regulated expression of p27^CDKN1B and p16^CDKN2A as well as deregulation of WRN have been associated with cell cycle arrest and replicative senescence [27,28]. In TRPM7-deficient PANC-1 cells, the mRNA levels of p16^INK4A and WRN were elevated whereas that of p27^CDKN1B remained unaltered (Fig. 5). Similarly, in TRPM7-deficient BxPC-3 cells, the mRNA levels of p16^INK4A and WRN were elevated by 1.4-fold and 26%, respectively.

Fig. 5.

Up-regulated levels of p16^CDKN2A and WRN mRNA in TRPM7-deficient cells. PANC-1 cells transfected with anti-TRPM7 or non-targeting control siRNAs were analyzed for the relative mRNA levels of p27^CDKN1B, p16^CDKN2A, and WRN by real-time PCR. Each column represents the mean mRNA level + s.d. (n = 3) relative to GAPDH. The experiment was repeated twice with similar results. *P < 0.05 indicates statistically significant difference. ^@P < 0.1 suggests a statistical trend. NS, not statistically significant.

3.3. Combination of anti-TRPM7 siRNA and gemcitabine produces enhanced cytotoxicity

The induction of non-apoptotic cell death by anti-TRPM7 siRNA might be exploited for enhancing cytotoxicity by combination with apoptosis-producing drugs, particularly in cancer cells with defects in the apoptotic pathways. The pancreatic adenocarcinoma cell line PANC-1 is relatively resistant to the conventionally used chemotherapeutic agent gemcitabine [29]. As a nucleoside analog, gemcitabine interferes with DNA metabolism and induces apoptotic cell death with modulation of the apoptosis-regulating genes, such as p53, BCL-2, BCL-xL, and BAX [30] and activation of caspases that control apoptotic pathways [31]. We hypothesize that the combination of anti-TRPM7 siRNA, which induces non-apoptotic cell death, with gemcitabine produces enhanced cytotoxicity in pancreatic cancer cells. To test this hypothesis, we assayed for proliferation in PANC-1 cells transfected with either anti-TRPM7 siRNA or non-targeting control siRNA, and incubated in the presence or absence of gemcitabine. At clinically active concentrations of 1 µM or 5 µM [32,33], gemcitabine did not produce any significant effect on cellular proliferation in PANC-1 (Fig. 6). Comparing with gemcitabine alone, the combination of gemcitabine at 1 µM or 5 µM and anti-TRPM7 siRNA significantly reduced proliferation by 43% and 32%, respectively (Fig. 6).

Fig. 6.

Combination of anti-TRPM7 siRNA with gemcitabine produced enhanced cytotoxicity. PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were incubated in the presence or absence of gemcitabine at 1 µM or 5 µM and then analyzed for proliferation using the MTS assay. Each column represents the mean (+ s.d.) proliferation as indicated by MTS absorbance relative to that of control (cells transfected with non-targeting control siRNA and incubated in the absence of gemcitabine). Statistical analysis was performed by comparing each experimental group with control except where indicated. *P < 0.01 and ^#P < 0.001 indicates statistically significant difference; ^@P < 0.1, a statistical trend; NS, not statistically significant. This experiment was repeated twice with similar results.

Besides, we examined the anti-proliferative effect of the combination of anti-TRPM7 siRNA and the clinically used inhibitor of histone deacetylases (HDACs), SAHA, which exerts anti-tumor effect on pancreatic adenocarcinoma cell lines. SAHA induces apoptosis with up-regulation of BAX expression and activation of caspases [24] as well as cellular senescence. Comparing with SAHA alone, the combination of anti-TRPM7 siRNA and SAHA at either 1 µM or 5 µM produced no enhanced cytotoxicity (Fig. 7), and this might be partly related to their overlapping mechanisms of cell death through senescence.

Fig. 7.

No enhanced cytotoxicity by anti-TRPM7 siRNA in combination with SAHA. PANC-1 cells transfected with anti-TRPM7 siRNA or non-targeting control siRNA were incubated in the presence or absence of SAHA at 1 µM or 5 µM and then analyzed for proliferation using the MTS assay. Each column represents the mean (+ s.d.) proliferation as indicated by MTS absorbance relative to that of control (cells transfected with non-targeting control siRNA and incubated in the absence of SAHA). Statistical analysis was performed by comparing each experimental group with control except where indicated. *P < 0.01 and ^#P < 0.001 indicate statistically significant differences between each treatment group and the control. NS, not statistically significant. These results are representative of three independent experiments.

4. Discussion

TRP channels have been implicated in cellular proliferation and survival, and this study suggests a novel link of an ion channel to replicative senescence. Various lines of evidence have provided strong support for the cellular effects of TRPM7 through controlling intracellular homeostasis of Mg²⁺ and Ca²⁺. We have recently shown that supplementary Mg²⁺ rescues the proliferative defect of the exocrine pancreatic epithelia of the zebrafish larvae with mutations in trpm7 and in the TRPM7-silenced human pancreatic adenocarcinoma cells [10]. These findings are in agreement with the previous study showing that TRPM7-deficient malignant B-lymphocytes or embryonic stem cells lacking TRPM7 kinase domain undergo proliferative arrest, and they can be activated to re-enter cell cycle by supplementary Mg²⁺ in the culture medium [7,34,35]. Moreover, TRPM7-mediated elevation of cytosolic Mg²⁺ or Ca²⁺ triggered by intracellular depletion of Mg²⁺ controls migration of osteoblasts and fibroblasts, respectively [18,19]. We hypothesize that metabolic sensing by TRPM7 ion channel controls cellular influx of Mg²⁺ and/or Ca²⁺ that interacts with the epidermal growth factor (EGF)-induced signaling pathways, resulting in the pro-survival and other mitogenic effects on pancreatic epithelia and cancer cells.

Mg²⁺ and Ca²⁺ are essential for normal cellular and physiological functions, and perturbed homeostasis of these ions contributes to various pathological states [36]. Chronic inadequacy of Mg²⁺ is associated with increased risk of developing aging-related diseases, possibly related to oxidative stress and inflammation [37]. In human endothelia and fibroblasts, Mg²⁺ deficiency accelerates cellular senescence as evidenced by reduced replicative lifespan and increased SA β-gal activity [38,39]. Although Ca²⁺ plays a contributory role in the regulation of apoptotic cell death by activating various pro-apoptotic factors [40], a rise in cytosolic Ca²⁺ level by stimulation with vitamin D3 and ATP can induce non-apoptotic cell death through autophagy [41]. Whether changes in cytosolic Ca²⁺ can induce other forms of non-apoptotic cell death, such as cellular senescence and mitotic catastrophe, is unclear. By using electrophysiological assay and live cell imaging analysis, we aim to determine the role of Mg²⁺ and Ca²⁺ in mediating the proliferative and pro-survival roles of TRPM7 in pancreatic epithelia and cancer cells.

Genetic silencing of TRPM7-mediated cellular senescence in pancreatic cancer cells is associated with up-regulated expression of cell cycle regulators and tumor suppressor genes. Consistent with the role of TRPM7 in Mg²⁺ transport, Mg²⁺ deficiency-induced senescence in endothelial cells is associated with up-regulated expression of p16^CDKN2A and p21^CDKN1A as well as telomeric attrition [38,39]. In agreement with these findings, we show that expression of p21^CDKN1A and p16^CDKN2A is up-regulated in TRPM7-deficient pancreatic cancer cells [10] (this study). Genetic alterations in p21^CDKN1A and p16^CDKN2A have been implicated in the multi-step pancreatic carcinogenesis [42]. Recent evidence suggests cooperative roles of p16^CDKN2A and p21^CDKN1A for mediating cellular senescence and tumor suppression [43]. Thus, the concerted up-regulation of p16^CDKN2A and p21^CDKN1A in the TRPM7-deficient cells suggest a contributory role of TRPM7 for proliferation and survival in the pathogenesis of pancreatic neoplasia through regulation of cyclin-dependent kinase inhibitors. It is notable that targeted knockdown of TRPM7 increases the mRNA level of WRN, which is a tumor suppressive gene being mutated in patients with Werner’s syndrome that is characterized by adult-onset progeria and predisposition to various malignancies including pancreatic adenocarcinoma [28]. Ongoing studies are designed to directly determine the role of the senescence-associated genes in mediating the proliferative and pro-survival functions of TRPM7.

The finding that induction of non-apoptotic cell death by inhibiting the expression of TRPM7 suggests a unique opportunity for improving therapeutic response in pancreatic adenocarcinoma. These tumor cells possess various combinations of mutations in K-RAS, TP53, p16^CDKN2A, and DPC4/SMAD4[44]. As a result of mutations in the components of the apoptotic pathways such as TP53, the pancreatic cancer cells are generally resistant to the apoptosis-inducing agents such as the clinically used chemotherapeutic drugs, gemcitabine and 5-fluorouracil. As demonstrated in this study, targeted knockdown of TRPM7 in combination with gemcitabine produces enhanced cytotoxicity in PANC-1, which contains mutations in K-RAS, TP53, and p16^CDKN2A [44]. We hypothesize that anti-TRPM7 siRNA and gemcitabine cause cell death by non-apoptotic and apoptotic pathways, respectively, such that the combination produces enhanced cytotoxicity; however, the precise mechanisms remain to be determined. In future studies, we attempt to further investigate the combined use of modulators of TRPM7 with apoptotic-inducing agents and the underlying molecular basis, with the goal of overcoming therapeutic resistance in pancreatic adenocarcinoma.

In summary, we present evidence that RNA interference-mediated silencing of TRPM7 induces replicative senescence in pancreatic adenocarcinoma. We propose that TRPM7 contributes to uncontrolled cellular proliferation at least in part by preventing non-apoptotic cell death during the multi-step pancreatic carcinogenesis. Future studies aimed to understand the signaling mechanisms that mediate the proliferative and pro-survival roles of TRPM7 are expected to help develop therapeutic interventions by targeted modulation of this ion channel and its associated signaling in pancreatic cancer and other malignancies.

Abbreviations

DAPI

4’6 diamidino-2-phenylindole

DMSO

dimethyl sulfoxide

FITC

fluorescein isothiocyanate

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonylphenyl)-2H-tetrazolium

PCR

polymerase chain reaction

PBS

phosphate buffered saline

PI

propidium iodide

siRNA

small interfering RNA

SA β-gal

senescence-associated β-galactosidase

SAHA

suberoylanilide hydroxamic acid; socs3a, suppressor of cytokine signaling 3a

swd

sweetbread

TRP

transient receptor potential

TRPM7

transient receptor potential melastatin-subfamily member 7

WRN

Werner