Transient Receptor Potential Melastatin 7 Is Present In Human Nasopharyngeal Carcinoma Cell And Is Implicated In Its Cell Migration

Background and Objective:Although nasopharyngeal carcinoma (NPC) is rare in Western countries, it is extremely common in southern regions of China. In Guangdong, for example, NPC accounts for 18% of all cancers. Because NPC occurs close to the intracranial organs, the cancer cells invade the cranial cavity and metastasize. This leads to a poor prognosis for many NPC patients. Therefore, an improved understanding of the molecular mechanisms involved in the intracranial invasion and metastasis of NPC will lead to improved treatments and prognosis factors for NPC patients.Cell migration is required during the invasion and metastasis of tumor cells, and the ubiquitous second messenger Ca2+ is a critical regulator of cell migration. Recently, many studies have shown that Ca2+ influx and Ca2+ channels are essential for the migration of various cell types, including tumor cells such as breast cancer cells. It is possible, therefore, that Ca2+ entry pathways also exist in NPC cells. Tumor cells often lack the voltage-operated Ca2+ channels and receptor-operated Ca2+ channels that play a pivotal role in Ca2+ entry in excitable cells. However, recent studies of tumor cells have revealed two other potential pathways for Ca2+ entry:store-operated Ca2+ channels and transient receptor potential (TRP) channels. Three TRP channels, TRPM1, TRPM8 and TRPV6, have been shown to control Ca influx, and to regulate the migration of murine melanoma cells (B16-F1/10), human glioblastoma cells (DBTRG) and hepatoblastoma (HepG2) cells, respectively.TRP channels were first identified in Drosophila species. On the bases of homology and channel function, the TRP family is divided into three main subfamilies:classic (TRPC), vanilloid (TRPV), and melastatin (TRPM). TRPM7, a member of the TRPM subfamily, is a non-selective cation channel with predominant permeability for Ca2+ and Mg2+. It regulates the calcium concentration in cells, which is imperative for many processes. Recent studies have suggested more and more importance for TRPM7 in the processes of cell migration. Wei showed that calcium flickers, which arose from TRPM7 at the migrating fibroblast front, had a crucial role in guiding directional movement—after TRPM7 knockdown and inhibition of calcium signalling, all migratory, turning, and chemotactic abilities were impaired. Clark demonstrated that activating TRPM7 could promote cytoskeletal relaxation and the conversion of focal adhesions to podosomes in mouse tumor cells (N1E-115). Despite these findings, the potential function of TRPM7 channels in the migration of tumor cells is not known. We hypothesized that TRPM7 Ca2+ channels are important in the migration of human NPC cells.We used colony lines of the NPC SUNE1 cell line to investigate the presence of TRPM7 channels in 5-8F cells (highly tumorigenic with metastatic ability) and 6-10B cells (tumorigenic without metastatic ability).We found high expression of the TRPM7 gene and protein in 5-8F cells, but low expression in 6-10B cells. By activation, blockade, and knockdown of these ion channels, we showed that TRPM7 affected the migratory potential of 5-8F cells. Contrary to TRPM7 RNAi results in 5-8F cells, we found that overexpression of TRPM7 increased the migration of 6-10B cells. Moreover, we found that TRPM7 regulated the migration of 5-8F cells by controlling Ca2+ influx. Finally, pharmacological data showed that TRPM7 channels were not stores-operated channels but, instead, controlled the release of intracellular Ca2+ stores via ryanodine receptors by a calcium-induced calcium release (CICR) mechanism. Overall, our data suggest that TRPM7 might be critical for the migration of NPC cells.Methods:1. Cell and Cell cultureTwo colony lines of human NPC SUNE1 cells—5-8F cells, which are highly tumorigenic and have metastatic ability, and 6-10B cells, which are tumorigenic, but lack metastatic ability—were stored in our laboratory. Both cell lines originated from a poorly differentiated squamous cell carcinoma of the nasopharynx.2. Small interfering RNA silencingAll siRNA duplexes were synthesized by Shanghai GenePharma Co. In the wells, three TRPM7 siRNA or negative control siRNA, and Lipofect AMINE 2000 was added to Opti-MEM and mixed gently. The wells were added to plates and incubated for 48 h until ready for further assay. All western blotting and functional studies were carried out after a 48 h-72 h transfection.3. Construct and 6-10B cells transfectionThe expression construct WT human TRPM7/pCDNA4/TO were gifts from Scharenberg Lab. This recombinant hTRPM7 is tagged with the hemagglutinin epitope.6-10B cells were transfected with the hTRPM7/pCDNA4/TO construct and empty construct by using Lipofectamine 2000 and selected for stable transfectants by zeocin treatment. TRPM7 expression was induced 1 day before use by adding 1μg/ml doxycycline to the culture medium.6-10B cells transfected with the empty vector served as a control for all experiments. Western blot analysis, wound healing assay and transwell chamber migration assay were performed 16-24 h after induction.4. Calcium imagingCalcium-imaging experiments were performed as described previously. Briefly, cells were plated onto glass coverslips and loaded with Fura-2 AM. Cells were then placed in a perfusion chamber on the stage of the microscope with a 40×objective. Fluorescence images of the cells were recorded by a Ratio Vision digital fluorescence microscopy system and analysed by TILL vision 4.0 software.5. Wound healing assay5-8F cells were cultured in 12 well tissue culture plates to confluence without vacant space. A pipette tip was used to scratch a wound midline of the culture well then cells were pretreated with mitomycin C. After 16 h of culture in RPMI 1640 supplemented with 2% serum, migration of cells was evaluated by measuring the difference in width of the wounds at 0 h and at 16 h.6. Transwell chamber migration assayThe migration activities of 5-8F cells, siNC-transfected 5-8F cells, and siTRPM7-transfected 5-8F cells were assayed using transwell cell culture chambers. Cells were added to the transwell chamber. The motilities of NC and TRPM7 knockdown cells in the transwell chamber assay were stimulated by the serum alone or the serum in combination with BK. The serum in bottom wells acts as chemotatic factors and BK in both top and bottom wells acts as expected motility boosting factors. After 48 hours the cells that migrated through the membrane were counted. Cells were counted in 5 random fields and expressed as the average number of cells per field under a light microscope.7. Statistical analysisStatistical data are expressed as mean±SEM. When appropriate, Student's t-test and one-way ANOVA were applied. In all cases, p<0.05 was considered statistically significant. All experiments were repeated at least three times.Results:1. Expression of TRPM7 higher in 5-8F cells than in 6-10B cells The expression of TRPM7 in 5-8F and 6-10B cells was examined by western blot, RT-PCR and immunofluorescence staining. Levels of TRPM7—both mRNA and protein—were significantly higher in 5-8F cells than in 6-10B cells.2. Silencing of TRPM7 inhibits migration of 5-8F cells Immunoblotting analyses showed that, within 48 h after transfection of 5-8F cells with TRPM7 siRNA No.1-3, the expression of TRPM7 protein was suppressed 65%-80%. Moreover, wound-healing assays showed that serum-induced migration of 5-8F cells was inhibited 50%-70%by TRPM7 siRNA. Finally, the motility of TRPM7 knockdown cells was analyzed using the transwell chamber assay. Like the down-regulated expression of TRPM7 and the decreased influx of Ca2+, motility was also reduced in cells transfected with TRPM7 siRNA. Compared with negative control siRNA cells, TRPM7 knockdown (siRNA1) cells showed a 68%reduction in the number of cells that crossed the membrane, indicating that TRPM7 channels were required for migration.3. Overexpression of TRPM7 enhances migration of 6-10 cells Immunoblotting analyses showed that, within 48 h after transfection of 6-10B cells with hTRPM7/pCDNA4/TO, the expression of TRPM7 protein was improved significantly.Wound-healing assays and transwell chamber assays showed that BK and serum in combination or serum-induced migratory abilities increased 260%-460%in 6-10B cells transfected with WT-hTRPM7 construct when compared with control 6-10B cells.4. Ca2+ influx mediated by TRPM7 potentially critical for migration of 5-8F cells Pharmacological approaches were used to verify that influx of extracellular Ca2+ affected the migration of 5-8F cells. In the wound-healing assay, removal of extracellular Ca2+ by EGTA significantly inhibited migration of 5-8F cells, indicating that influx of extracellular Ca2+ is critical for the migration of 5-8F cells. Next, wound-healing assays were employed to investigate the effect on cell migration of pharmacological inhibitors for various ion channels that mediated Ca2+ influx. Results showed that four inhibitors of three different Ca2+ channels had no effect on migration of 5-8F cells. However, the addition of a non-specific TRPM7 channel inhibitor 2-APB reduced the migration of 5-8F cells. Likewise, another non-specific TRPM7 channel inhibitor La3+ inhibited the migration of 5-8F cells. Conversely, the addition of BK, a peptide agonist that activates and promotes expression of TRPM7 in N1E-115 cells, promoted the migration of 5-8F cells. Transwell chamber assays also revealed that both 2-APB and La3+ inhibited the migration of 5-8F cells.5. TRPM7 channel potentially a major regulator of intracellular Ca2+ response In pharmacological approaches to further confirm that TRPM7 mediated Ca2+ influx in 5-8F cells, Fura-2-based Ca2+ imaging was performed. Addition of BK triggered a rapid increase in cytosolic Ca2+ from internal stores in 5-8F cells. The initial increase in Ca2+ was transient, but was followed by a sustained phase of elevated Ca2+ that lasted for several minutes before Ca2+ returned to basal levels. Ratiometric analysis showed that compared with control (NC), silencing of TRPM7 (siRNA1) significantly decreased the transient phase of the response, but it decreased the sustained phase of the response much greater.Fura-2-based Ca2+ imaging was also used to examine the mechanism by which TRPM7 inhibitors influenced influx of extracellular Ca2+ and release of intracellular Ca2+ stores in 5-8F cells. Both of TRPM7-mediated Ca2+ influx and Ca2+ store Ca2+ release could be induced consecutively two times by BK without any desensitization in the control group. However, compared with the control group, both La3+ and 2-APB completely inhibited the amplitude of the peak and plateau phases of the second BK-induced Ca2+ response. Interestingly, similar results were observed with EGTA, which chelated extracellular Ca2+. These data indicated that Ca2+ response was not induced by BK without extracellular Ca2+.Moreover, ryanodine receptor inhibitor ryanodine and inositol-1,4,5-trisphosphate receptor inhibitor xestospongin C were used to investigate whether calcium-induced calcium release occurred via the RyR or IP3R in 5-8F cells. We compared the first response to BK in the absence of ryanodine with the corresponding second BK response after ryanodine pre-incubation. These results showed that ryanodine significantly reduced BK-induced Ca2+ peak responses while having no significant effect on BK-induced Ca2+ plateau phases. However, different results were obtained by inhibiting the IP3R with xestospongin C. The corresponding second BK response after xestospongin C pre-incubation was similar with the first BK response.Taken together, these results in 5-8F cells confirm and characterize the mediating effects of TRPM7 channels, both on influx of extracellular Ca2+ and release of intracellular Ca2+ stores via RyRs, but not IP3Rs.Conclusions:This study supports the influx of Ca2+ as a requirement for the migration of human NPC 5-8F cells and identifies TRPM7 as a novel regulator not only of Ca2+ influx but also of 5-8F cell migration, thus showing TRPM7 as a potential prognostic indicator and therapeutic target for NPC patients.Innovations of our study:1. We were the first to confirm how TRPM7 channels function to regulate the migration of tumor cells.2. Our preliminary study demonstrated TRPM7 was a key gene/protein related to migration of NPC.3. Our study identified TRPM7 as a novel potential-regulator of the Ca2+ influx that allows migration of 5-8F cells. TRPM7, therefore, might have potential as a prognostic indicator and as a therapeutic target in nasopharyngeal carcinoma.4. Stable TRPM7-transduced nasopharyngeal carcinoma cell line 6-10B was established, which will be provided a valuable tool for TRPM7 functional studies.