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TRPM7 mediates breast cancer cell migration and invasion through the MAPK pathway
Cancer Letters 333 (2013) 96–102
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
TRPM7 mediates breast cancer cell migration and invasion through the MAPK pathway Xiaojing Meng a,1, Chunqing Cai a,1, Jiguo Wu a, Shaoxi Cai b, Changsheng Ye c, Haiyang Chen a, Zhengduo Yang d, Hongqiang Zeng a, Qiang Shen d,⇑, Fei Zou a,⇑ a
Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China Department of Respiratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China Breast Center, Nanfang Hospital, Southern Medical University, Guangzhou, China d Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX, USA b c
a r t i c l e
i n f o
Article history: Received 6 December 2012 Received in revised form 11 January 2013 Accepted 14 January 2013
Keywords: TRPM7 Breast cancer Invasion Src MAPK
a b s t r a c t Metastasis is an inherent feature of breast cancer and transient receptor potential (TRP) channels were found to be potentially implicated in this process. Particularly, TRPM7 may regulate cell motility. We therefore examined the expression of TRPM7 mRNA in the Oncomine database and found that TRPM7 is correlated to metastasis and invasive breast cancer. Silencing TRPM7 with RNA interference resulted in a significant decrease in migration and invasion capability of MDA-MB-435 breast cancer cells, and phosphorylation levels of Src and MAPK but not AKT. Our results suggest that TRPM7 regulates migration and invasion of metastatic breast cancer cells via MAPK pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Breast cancer is the most common tumor in women in both industrialized and developing countries [1,2]. In recent years, breast cancer incidence and mortality are clearly rising, especially in developing countries [3,4]. Currently, breast cancer ranks as the second leading cause of death in women [3,5]. Metastasis is an important biological feature and the leading cause of cancer-related death. It has been well documented that metastasis is an inherent characteristic of breast tumors [6,7]. Although metastasis is closely correlated with breast cancer survival and morbidity, its cellular and molecular mechanisms are not yet clearly understood. Recently, transient receptor potential (TRP) channels have been found to be implicated in carcinogenesis and their expression was frequently found to be altered during carcinogenesis [8–10]. TRPM7, a member of the TRP protein family, is both an ion channel and a protein kinase that is ubiquitously distributed in various normal tissues [11]. TRPM7 may phosphorylate itself and is involved in various physiologic and pathologic processes including Mg2+
⇑ Corresponding authors. Tel.: +1 713 834 6357; fax: +1 713 834 6350 (Q. Shen), tel.: +86 20 61648301; fax: +86 20 61648324 (F. Zou). E-mail addresses: [email protected] (Q. Shen), [email protected] (F. Zou). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.01.031
homeostasis, hypoxic neuronal injury, and tumor cell proliferation [11,12]. Emerging evidence has supported the notion that TRPM7 is associated with cell motility. It was found that silencing TRPM7 enhances cell adhesion and increases the number of peripheral adhesion complexes, whereas overexpression of TRPM7 in cells induces cell rounding with a concomitant loss of cell adhesion in HEK-293 cells [13] and an increase in migration in human and rat vascular smooth muscle cells [14]. Recent studies have shown that TRPM7 is overexpressed in human breast cancer and is involved in breast cancer cell proliferation [15,16], suggesting that TRPM7 plays an important role in breast carcinogenesis. However, a potential role for TRPM7 in cancer metastasis was recently reported [17]. Metastasis is a complex process that requires coordination of various signal transduction pathways that regulate cancer cell proliferation, migration, invasion and reestablishment of the tumor in a secondary organ. It is well known that one or more signaling pathways including AKT, Src and MAPK are activated in cancer progression to promote cell proliferation, inhibit apoptosis, and regulate cell adhesion, migration and invasion capabilities [18–20]. These signaling pathways are also involved in progression of breast cancer, such as control of adhesion and migration [21–23]. Although previous studies have revealed that TRPM7 regulates cell migration via regulating calcium flickers or calcium influx [24–26], whether these signaling pathways link to TRPM7 to modulate breast cancer cell migration and invasion is largely unknown.
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The present study was designed to determine whether TRPM7 regulates breast cancer cell migration and invasion through linkage with one or more signal transduction pathways, and also to determine whether its expression is increased in metastatic human cancers.
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ing. Then RPMI 1640 containing lipofectamine reagent and TRPM7-siRNA (20 nM) were added to the scratched wells. The cells were then incubated for 24 h and 48 h at 37 °C. The initial wounding and the movement of the cells in the scratched area were photographically monitored and imaged using an Olympus CKX41 inverted microscope coupled with a digital imaging system at 0 h, 24 h or 48 h. 2.6. In vitro migration assay
2. Materials and methods 2.1. Materials The rabbit polyclonal anti-TRPM7 antibody was obtained from Acris (German). The rabbit monoclonal anti-Src antibody, rabbit polyclonal anti-phospho-Src antibody, rabbit polyclonal anti-phospho-AKT (Ser473) and goat polyclonal anti-AKT antibody were from Cell Signaling (USA). The rabbit polyclonal anti-P38, anti-phospho-P38, anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK antibodies and anti-GAPDH antibody were obtained from Santa Cruz (USA). Fetal bovine serum (FBS), trypsin–EDTA, Roswell Park Memorial Institute medium (RPMI1640), OptiMEMI were obtained from GIBCO (USA). Trizol and lipofectamine reagent were obtained from Invitrogen (USA). ECL-PLUS was obtained from Thermo (USA). The RT– PCR kit was obtained from TaKaRA (China). Matrigel was obtained from BD Bioscience (USA). 2.2. Oncomine data mining Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for analysis and visualization [27,28]. The datasets from the Oncomine cancer microarray database (https://www.oncomine.com/resource/main.html) were selected to determine the alterations of TRPM7 in mRNA expression and DNA copy number change. The Oncomine is a database bank in that gene expression profile data can be queried and analyzed for selected genes across the databases available to the public. These datasets provide fold-change values of gene expression and statistical significance determined by P values, involving comparison between cancer and normal samples, cancer vs. cancer analysis, or gene expression in relation to clinical-pathological significance of patients. We extracted and compared TRPM7 expression in primary vs. metastatic human cancers, including breast cancer, ovarian cancer, colon cancer, prostate cancer and melanoma, as well as compared TRPM7 expression in breast cancer relative to normal breast.
Migration of cells was assessed in a 24-well plate Transwell system (Costar, Corning, USA) as described previously [30]. MDA-MB-435 cells were seeded at a density of 5 104 cells per well onto 8-lm Transwell inserts. Each insert was filled with 200 lL of cell suspension, and the lower chamber was filled with 500 lL RPMI1640 containing 20% FBS. The cells were incubated for 36 h at 37 °C (5% CO2). Pictures (100) of the membrane were taken in 16 random fields per chamber and the total number of cells that had migrated into plate wells was counted. The experiments were performed in triplicate. 2.7. In vitro invasion assay The invasion assay was conducted as described previously [5]. Briefly, the Transwell inserts were coated with 50 lL Matrigel solution (concentration in culture media: 8.5 lg/L1) for 15 min at 37 °C and then 30 min at room temperature. 200 lL cell suspensions (5 104 cells mL1) were added into the Transwell insert. The plate wells were filled with RPMI1640 (supplemented with 20% FBS). After incubation for 48 h at 37 °C, photographs were taken and cells were counted as described above. 2.8. Western blotting Western blotting was conducted routinely. At the indicated time points (24– 48 h), cells were rinsed with ice-cold PBS and lysed in protein lysis buffer solution containing Complete Protease Inhibitor (Roche, USA) for 30 min at 4 °C, followed by 15 min of centrifugation at 14,000 rpm/min. Protein concentrations of the supernatants were determined using the BCA protein assay (Pierce, USA). Lysates were applied to sodium dodecyl sulfate–polyacrylamide electrophoresis gels, and then transferred to polyvinylidene difluoride membranes for Western blot detection by specific antibodies against TRPM7, phospho-AKT, AKT, phospho-Src, Src, phosphoERK, ERK, phospho-JNK, JNK, phospho-P38 or P38 antibody. Antibody to GAPDH was used as a loading control.
2.3. Cell culture The human breast cancer cell line MDA-MB-435 was obtained from American Type Culture Collection (ATCC). MDA-MB-435 cells were grown in RPMI1640 supplemented with penicillin (100 U ml1), streptomycin (100 mg ml1), and 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified atmosphere containing 5% (v/v) CO2 and 95% air.
2.9. Statistical analysis Data are expressed as means ± SD. Statistical significance was evaluated using the SPSS 13.0 software. When the data meet the requirements of variance, data were analyzed by one-way ANOVA and LSD, otherwise by Welch approximate analysis of variance and Dunnett T3 multiple comparison method. Differences were considered statistically significant when p < 0.05.
2.4. Design of TRPM7-siRNA and transfection Two targeted sequences specific to the TRPM7 gene were chosen as potential small interference RNA (siRNA) target sites and a non-targeted sequence (NC) was chosen for control (Table 1) following the instructions from Shanghai GenePharma Co., Ltd. TRPM7 specific siRNA was transfected in MDA-MB-435 breast cancer cells to suppress TRPM7 expression according to the manufacturer’s instructions. Briefly, MDA-MB-435 cells were routinely cultured for 24 h in a 6-well plate prior to transfection. MDA-MB-435 cells at 40% confluence were rinsed twice with PBS and transiently transfected using transfection mixtures. Transfection was allowed to continue for 8–12 h before removal of the transfection mixtures. The transfection efficiency was assessed by quantitative real-time PCR and western blot analysis. After transfection for the desired time, wound scratch assays, transwell migration and invasion assays were performed. 2.5. Wound scratch assay For wound scratch assays, cell monolayers were wounded by scratching the surface on the 6-well plate as uniformly as possible with a pipette tip. The scratched wells were washed several times with PBS to remove detached debris after scratchTable 1 The siRNA sequences for the TRPM7 gene and a negative control. Name
Sequence
TRPM7-siRNA1
Sense 50 -GUCUUGCCAUGAAAUACUCTT-30 Anti-sense 50 -GAGUAUUUCAUGGCAAGACTT-30 Sense 50 -AGGAGAAGAUGCAAUUAAATT-30 Anti-sense 50 -UUUAAUUGCAUCUUCUCCUAG-30 Sense 50 -UUCUCCGAACGUGUCACGUTT-30 Anti-sense 50 -ACGUGACACGUUCGGAGAATT-30
TRPM7-siRNA2 NC
3. Results 3.1. Overexpression of TRPM7 is correlated to cancer metastasis and invasive breast cancers We examined the Oncomine™ (Compendia Bioscience, Ann Arbor, MI) for analysis and visualization of TRPM7 in breast and other cancer types for its expression levels in human cancer tissues and cell lines with the methods described [27,28]. The datasets from the Oncomine cancer microarray database (https:// www.oncomine.com/resource/main.html) were selected to determine the alterations of TRPM7 in DNA copy numbers and mRNA expression levels. TRPM7 mRNA levels were significantly higher in metastatic cancers of breast, [31,32], ovary [33,34], prostate (4 out of 5 databases) [35–39], stomach [40], melanoma [41]and sarcoma [42], and marginal in colon [42,43], compared with TRPM7 expression in the primary tumor site. However, TRPM7 gene copy number remained the same in primary or metastatic breast tumors [44]. Representative datasets are shown for each indicated cancer types in mRNA levels and DNA copy numbers (Fig. 1A–H). Invasive ductal breast carcinoma, invasive lobular breast carcinoma, invasive ductal and lobular breast carcinoma, and mixed lobular and ductal breast carcinoma expressed significantly higher TRPM7 mRNA (Fig. 2A–D), compared to the breast tissue control in TCGA breast datasets. In addition,
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Fig. 1. TRPM7 expression level is related to cancer metastasis. Selected datasets from the Oncomine cancer microarray database (https://www.oncomine.com/resource/ main.html) were mined to determine the alterations of TRPM7 in mRNA expression levels and DNA copy numbers. Representative datasets from breast, ovary, prostate, gastric, melanoma, sarcoma, and colon cancer types were compared with TRPM7 expression in the primary tumors vs. metastatic tumors. (A) Breast cancer; (B) ovarian cancer; (C) prostate cancer; (D) gastric cancer; (E) melanoma; (F) sarcoma; (G) colon cancer; (H) copy number variations for breast cancer. T-tests were performed for statistical significance for the entire series of analysis. Median values are indicated.
lobular breast carcinoma expressed significantly more TRPM7 mRNA among seven major pathological subtypes of breast carcinoma (Fig. 2E), while ductal carcinoma and mixed lobular and ductal breast carcinoma demonstrated marginal significance in TRPM7 transcription. There is no significant alteration in DNA copy numbers among the tested subtypes (data not shown). 3.2. Knockdown of TRPM7 mRNA reduces TRPM7 protein levels Since these data suggest that TRPM7 expression is correlated with invasive breast cancer, we used highly metastatic MDA-MB435 breast cancer cells to explore the potential role of the TRPM7 channel in breast cancer migration and invasion. Firstly, TRPM7 siRNA was used to downregulate TRPM7 expression. To assess the interference efficiency of TRPM7 specific small RNA, the expression of TRPM7 protein was analyzed by Western blot. The result showed that TRPM7 expression in terms of protein was significantly (p < 0.01) silenced with TRPM7-siRNA in MDA-MB-435 breast cancer cells (Fig. 3). 3.3. Silencing TRPM7 suppresses cancer cell migration and invasion To evaluate the effect of TRPM7 on cancer cell migration, wound-scratch assays were used to determine the migration capability of MDA-MB-435 breast cancer cells after silencing of TRPM7. The results showed that the cells migrated more slowly to close the scratched wounds after transfection with TRPM7 specific siRNA for 48 h and the number of migrating cells was significantly (p < 0.01) decreased, compared with control (Fig. 4A and B). Furthermore, we conducted Transwell invasion assays to evaluate the effect of
TRPM7 on cancer cell invasion. The results showed that MDAMB-435 breast cancer cells significantly (p < 0.01) lost their ability to invade through the Matrigel matrix and the number of cells that invaded through the Matrigel matrix was significantly decreased after silencing of TRPM7 with specific siRNA as compared with control (Fig. 4C and D). In line, we measured the effect of TRPM7 knockdown on proliferation of MDA-MB-435 cells using MTT assays. We found that cell proliferation was not significantly affected by siRNA interference (Fig. S1). These data suggest that TRPM7 expression is correlated to the invasion capability of MDA-MB435 breast cancer cells. Taken together, the results suggest that TRPM7 plays an important role in mediating migration and invasion of invasive breast cancer cells.
3.4. TRPM7 mediates migration and invasion of breast cancer cell through the MAPK pathway To explore the mechanism by which TRPM7 mediated migration and invasion of breast cancer cells, the effect of silencing TRPM7 on the activity of various signaling molecules, including AKT, Src and mitogen-activated protein kinases (MAPKs), was investigated. We first determined whether AKT, a major transducer of the phosphoinositide 3-kinase pathway, [45], is involved in TRPM7 mediated cancer cell migration and invasion. The present results show that no significant (p > 0.05) difference was observed in the expression and phosphorylation of AKT after silencing TRPM7, compared with control (Fig. 5A and B), suggesting that AKT was not involved in breast cancer cell migration and invasion mediated by TRPM7.
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Fig. 2. TRPM7 expression is upregulated in invasive breast cancers. Comparisons of normal breast tissue vs. invasive ductal or lobular breast carcinoma were made using TCGA datasets. (A) Invasive ductal breast carcinoma vs. normal control; (B) invasive lobular breast carcinoma vs. normal control; (C) invasive ductal and lobular breast carcinoma vs. normal control; (D) mixed lobular and ductal breast carcinoma vs. normal control; and (E) TRPM7 mRNA in lobular breast carcinoma vs. six other pathological subtypes of breast carcinoma. T-tests were performed for statistical significance for this series of analysis.
MDA-MB-435 breast cancer cells resulted in a significant decrease in Src activity. We next measured the expression and activation of the MAPK signaling pathway. The phosphorylation level of signal molecules (P38, ERK and JNK) was significantly reduced after silencing TRPM7 with TRPM7 specific small interfering RNA as compared with control (Fig. 5A, D, E, F), indicating that silencing of TRPM7 in MDAMB-435 breast cancer cells resulted in a significant inactivation of MAPKs. Taken together, silencing TRPM7 led to a significant reduction in migration and invasion potentials of MDA-MB-435 breast cancer cells, as well as inactivation of Src and MAPKs, suggesting that the Src and MAPKs signaling pathways are involved in TRPM7-mediated migration and invasion of MDA-MB-435 breast cancer cells. 4. Discussion Fig. 3. Downregulation of TRPM7 in MDA-MB-435 breast cancer cells by 48 h of siRNA interference. (A) ECL image of western blot of TRPM7, GAPDH was used as a loading control. (B) Quantification analysis of TRPM7 protein band densities. Indicates significant difference at p < 0.05, and indicates significant difference at p < 0.01. Error bars represent one standard deviation (n = 3).
Next, we determined whether Src, a regulatory protein that plays a key role in cell differentiation, motility, proliferation and survival [46], is involved in TRPM7 mediated cancer cell migration and invasion. We found that the phosphorylation level of Src significantly (p < 0.01) decreased after silencing TRPM7 compared with control (Fig. 5C), indicating that silencing of TRPM7 in
Metastasis is considered as an inherent feature and a main lethal factor of breast cancer [6,7]. Despite the importance of metastasis to breast cancer survival and morbidity, our knowledge is still far from understanding the cellular and molecular mechanisms of breast cancer metastasis. During carcinogenesis, cancer cell migration and invasion are the most subtle and critical steps for cancer progression and metastasis. Recently, transient receptor potential (TRP) channels have been implicated in carcinogenesis [8–10]; as well as in cancer metastasis. TRPM7, a member of TRP protein family, is involved in adhesion in HEK-293 cells [13], migration in vascular smooth muscle cells [14], and proliferation of breast cancer cells [15,47], suggesting that TRPM7 plays a crucial role in carcinogenesis including breast cancer. Our
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Fig. 4. TRPM7 knockdown suppresses MDA-MB-435 cell migration and invasion. Before the wound-scratch assay, MDA-MB-435 cells were transfected with TRPM7-siRNA for 48 h to silence TRPM7. (A) The cell images of wound-scratch assay at 0, 24, 48 h; (B) the number of cells migrating to the scratched wounds. Indicates significant difference at p < 0.05, and indicates significant difference at p < 0.01. Error bars represent one standard deviation (n = 3). Before the transwell assay, cells were transfected with TRPM7-siRNA for 48 h to silence TRPM7. (C) The images of cells that migrated into plate wells (lower chamber) in transwell invasion assay. (D) The number of cells that invaded through the Matrigel matrix into plate wells in Transwell invasion assay. Pictures (100) of the membrane were taken in 16 random fields per chamber. Indicates significant difference at p < 0.05.
Fig. 5. TRPM7 downregulation suppresses activation of Src, P38, ERK and JNK. (A) ECL image of western blot of total and phosph-AKT, Src, P38, ERK and JNK, GAPDH was used as a loading control. B, C, D, E, F. Quantification analysis of AKT, Src, P38, ERK and JNK protein band densities. Error bars represent one standard deviation (n = 3).
data from wound scratch assays and Transwell assays clearly demonstrate that the migration and invasion capability of MDAMB-435 breast cancer cells is significantly reduced after silencing TRPM7 (Figs. 3–5), while cell proliferation is not affected (Fig. S1),
suggesting that TRPM7 is required for the invasive phenotype of MDA-MB-435 breast cancer cells. These results are consistent with recent studies that depressing the function of TRPM7 with RNA interference or non-specific pharmacological agents inhibits
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the migration of A549 lung cancer cells, while up-regulation of TRPM7 enhances the migration of A549 cells [48] and vascular smooth muscle cells [14]. Furthermore, our results are also supported indirectly by previous studies showing that silencing TRPM7 enhances cell adhesion and increases the number of peripheral adhesion complexes, whereas overexpression of TRPM7 in cells induces cell rounding with a concomitant loss of cell adhesion in HEK-293 cells [13] because low adhesion capability of cell is beneficial for cell migration. Although numerous evidence supports the notion that TRPM7 is involved in regulating cell migration and invasion, the precise signaling pathway that control the regulation process are largely unknown. In the present study, we measured expression and activation of multiple signal molecules to explore the signaling pathways involved in breast cancer migration and invasion mediated by TRPM7. AKT is a primary signaling transducer of the phosphoinositide 3-kinase (PI3K) pathway and mainly contributes to cancer progression by inhibiting apoptosis, promoting metabolism changes and cell proliferation as well as regulating migration and invasion [45]. Previous studies have shown that the PI3K/AKT pathway is frequently activated in malignant breast cancer cells [49] and AKT is activated as frequently as 24% in breast cancer patients [50]. Recently, Chin and Toker [51] suggested that AKT mediates breast cancer cell migration in an isoform-specific manner and that AKT1 decreases migration, while AKT2 enhances migration. However, our data indicated that the activity of AKT was not altered when the migration and invasion capability of breast cancer cell was reduced by silencing TRPM7 (Fig. 4). We thus speculate that TRPM7-mediated migration and invasion of breast cancer cells is not associated with the AKT signaling pathway. Since cancer metastasis frequently involves the coordination of various signal-transduction pathways that regulate cancer cell migration, invasion and reestablishment of the tumor, we also investigated Src and Mitogen-activated protein kinase (MAPK), two important signaling pathways that control the essential events for metastasis including cancer cell migration and invasion [18,19]. Previous studies have indicated that Src is intimately involved in the control of matrix adhesion and cell migration in breast cancer cells [21], and MAPK is frequently activated in tumorigenesis [19] and breast cancer [23]. MAPKs (including P38, ERK1/2 and JNK1/2 members) signaling pathway play a crucial role in tumor cell proliferation, adhension and migration [22,52–54]. As upstream of the MAPK signaling pathway, Src is suggested to be essential for MAPK activation during breast cancer cell proliferation and invasion [47]. Our data showed that silencing TRPM7 with specific small interfering RNA led to a significant reduction in migration and invasion capability of MDA-MB-435 breast cancer cells and a concomitant inactivation of Src and MAPK (Fig. 5), suggesting that TRPM7 modulation of migration and invasion of breast cancer cells involves the Src-MAPK signaling pathway. Previous results show that TRPM7 modulates cell migration by means of regulating calcium flickers [25,26]. In addition, elevation of intracellular calcium or calcium influx frequently involves activation of MAPK pathway in normal and cancer cells [55–58], suggesting that intracellular calcium level is associated with MAPK activity. In the present study, it would be expected that silencing TRPM7 would lead to a low intracellular calcium level since TRPM7 is a Ca2+-permeable channel protein and a low activity of MAPK, which would result in a reduction in migration and invasion capability of MDA-MB-435 breast cancer cells. We are in the process of testing this interesting hypothesis in breast cancer cells. Our findings that TRPM7 modulates migration and invasion of MDA-MB435 breast cancer cells through the Src–MAPK signaling pathway suggest that depressing this signaling pathway or/and the TRPM7 channel protein may be beneficial in treating breast cancer patients with or without metastasis lesions.
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Acknowledgments This work was supported by grants from National Basic Research Program of China, No. 2012CB518200 (FZ), No.2012CB525004 (XM); National Natural Science Foundation of China No. 30971193(FZ), No. 81071611 (CC).
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2013.01. 031.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
TRPM7 mediates breast cancer cell migration and invasion through the MAPK pathway Xiaojing Meng a,1, Chunqing Cai a,1, Jiguo Wu a, Shaoxi Cai b, Changsheng Ye c, Haiyang Chen a, Zhengduo Yang d, Hongqiang Zeng a, Qiang Shen d,⇑, Fei Zou a,⇑ a
Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China Department of Respiratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China Breast Center, Nanfang Hospital, Southern Medical University, Guangzhou, China d Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX, USA b c
a r t i c l e
i n f o
Article history: Received 6 December 2012 Received in revised form 11 January 2013 Accepted 14 January 2013
Keywords: TRPM7 Breast cancer Invasion Src MAPK
a b s t r a c t Metastasis is an inherent feature of breast cancer and transient receptor potential (TRP) channels were found to be potentially implicated in this process. Particularly, TRPM7 may regulate cell motility. We therefore examined the expression of TRPM7 mRNA in the Oncomine database and found that TRPM7 is correlated to metastasis and invasive breast cancer. Silencing TRPM7 with RNA interference resulted in a significant decrease in migration and invasion capability of MDA-MB-435 breast cancer cells, and phosphorylation levels of Src and MAPK but not AKT. Our results suggest that TRPM7 regulates migration and invasion of metastatic breast cancer cells via MAPK pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Breast cancer is the most common tumor in women in both industrialized and developing countries [1,2]. In recent years, breast cancer incidence and mortality are clearly rising, especially in developing countries [3,4]. Currently, breast cancer ranks as the second leading cause of death in women [3,5]. Metastasis is an important biological feature and the leading cause of cancer-related death. It has been well documented that metastasis is an inherent characteristic of breast tumors [6,7]. Although metastasis is closely correlated with breast cancer survival and morbidity, its cellular and molecular mechanisms are not yet clearly understood. Recently, transient receptor potential (TRP) channels have been found to be implicated in carcinogenesis and their expression was frequently found to be altered during carcinogenesis [8–10]. TRPM7, a member of the TRP protein family, is both an ion channel and a protein kinase that is ubiquitously distributed in various normal tissues [11]. TRPM7 may phosphorylate itself and is involved in various physiologic and pathologic processes including Mg2+
⇑ Corresponding authors. Tel.: +1 713 834 6357; fax: +1 713 834 6350 (Q. Shen), tel.: +86 20 61648301; fax: +86 20 61648324 (F. Zou). E-mail addresses: [email protected] (Q. Shen), [email protected] (F. Zou). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.01.031
homeostasis, hypoxic neuronal injury, and tumor cell proliferation [11,12]. Emerging evidence has supported the notion that TRPM7 is associated with cell motility. It was found that silencing TRPM7 enhances cell adhesion and increases the number of peripheral adhesion complexes, whereas overexpression of TRPM7 in cells induces cell rounding with a concomitant loss of cell adhesion in HEK-293 cells [13] and an increase in migration in human and rat vascular smooth muscle cells [14]. Recent studies have shown that TRPM7 is overexpressed in human breast cancer and is involved in breast cancer cell proliferation [15,16], suggesting that TRPM7 plays an important role in breast carcinogenesis. However, a potential role for TRPM7 in cancer metastasis was recently reported [17]. Metastasis is a complex process that requires coordination of various signal transduction pathways that regulate cancer cell proliferation, migration, invasion and reestablishment of the tumor in a secondary organ. It is well known that one or more signaling pathways including AKT, Src and MAPK are activated in cancer progression to promote cell proliferation, inhibit apoptosis, and regulate cell adhesion, migration and invasion capabilities [18–20]. These signaling pathways are also involved in progression of breast cancer, such as control of adhesion and migration [21–23]. Although previous studies have revealed that TRPM7 regulates cell migration via regulating calcium flickers or calcium influx [24–26], whether these signaling pathways link to TRPM7 to modulate breast cancer cell migration and invasion is largely unknown.
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The present study was designed to determine whether TRPM7 regulates breast cancer cell migration and invasion through linkage with one or more signal transduction pathways, and also to determine whether its expression is increased in metastatic human cancers.
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ing. Then RPMI 1640 containing lipofectamine reagent and TRPM7-siRNA (20 nM) were added to the scratched wells. The cells were then incubated for 24 h and 48 h at 37 °C. The initial wounding and the movement of the cells in the scratched area were photographically monitored and imaged using an Olympus CKX41 inverted microscope coupled with a digital imaging system at 0 h, 24 h or 48 h. 2.6. In vitro migration assay
2. Materials and methods 2.1. Materials The rabbit polyclonal anti-TRPM7 antibody was obtained from Acris (German). The rabbit monoclonal anti-Src antibody, rabbit polyclonal anti-phospho-Src antibody, rabbit polyclonal anti-phospho-AKT (Ser473) and goat polyclonal anti-AKT antibody were from Cell Signaling (USA). The rabbit polyclonal anti-P38, anti-phospho-P38, anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK antibodies and anti-GAPDH antibody were obtained from Santa Cruz (USA). Fetal bovine serum (FBS), trypsin–EDTA, Roswell Park Memorial Institute medium (RPMI1640), OptiMEMI were obtained from GIBCO (USA). Trizol and lipofectamine reagent were obtained from Invitrogen (USA). ECL-PLUS was obtained from Thermo (USA). The RT– PCR kit was obtained from TaKaRA (China). Matrigel was obtained from BD Bioscience (USA). 2.2. Oncomine data mining Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for analysis and visualization [27,28]. The datasets from the Oncomine cancer microarray database (https://www.oncomine.com/resource/main.html) were selected to determine the alterations of TRPM7 in mRNA expression and DNA copy number change. The Oncomine is a database bank in that gene expression profile data can be queried and analyzed for selected genes across the databases available to the public. These datasets provide fold-change values of gene expression and statistical significance determined by P values, involving comparison between cancer and normal samples, cancer vs. cancer analysis, or gene expression in relation to clinical-pathological significance of patients. We extracted and compared TRPM7 expression in primary vs. metastatic human cancers, including breast cancer, ovarian cancer, colon cancer, prostate cancer and melanoma, as well as compared TRPM7 expression in breast cancer relative to normal breast.
Migration of cells was assessed in a 24-well plate Transwell system (Costar, Corning, USA) as described previously [30]. MDA-MB-435 cells were seeded at a density of 5 104 cells per well onto 8-lm Transwell inserts. Each insert was filled with 200 lL of cell suspension, and the lower chamber was filled with 500 lL RPMI1640 containing 20% FBS. The cells were incubated for 36 h at 37 °C (5% CO2). Pictures (100) of the membrane were taken in 16 random fields per chamber and the total number of cells that had migrated into plate wells was counted. The experiments were performed in triplicate. 2.7. In vitro invasion assay The invasion assay was conducted as described previously [5]. Briefly, the Transwell inserts were coated with 50 lL Matrigel solution (concentration in culture media: 8.5 lg/L1) for 15 min at 37 °C and then 30 min at room temperature. 200 lL cell suspensions (5 104 cells mL1) were added into the Transwell insert. The plate wells were filled with RPMI1640 (supplemented with 20% FBS). After incubation for 48 h at 37 °C, photographs were taken and cells were counted as described above. 2.8. Western blotting Western blotting was conducted routinely. At the indicated time points (24– 48 h), cells were rinsed with ice-cold PBS and lysed in protein lysis buffer solution containing Complete Protease Inhibitor (Roche, USA) for 30 min at 4 °C, followed by 15 min of centrifugation at 14,000 rpm/min. Protein concentrations of the supernatants were determined using the BCA protein assay (Pierce, USA). Lysates were applied to sodium dodecyl sulfate–polyacrylamide electrophoresis gels, and then transferred to polyvinylidene difluoride membranes for Western blot detection by specific antibodies against TRPM7, phospho-AKT, AKT, phospho-Src, Src, phosphoERK, ERK, phospho-JNK, JNK, phospho-P38 or P38 antibody. Antibody to GAPDH was used as a loading control.
2.3. Cell culture The human breast cancer cell line MDA-MB-435 was obtained from American Type Culture Collection (ATCC). MDA-MB-435 cells were grown in RPMI1640 supplemented with penicillin (100 U ml1), streptomycin (100 mg ml1), and 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified atmosphere containing 5% (v/v) CO2 and 95% air.
2.9. Statistical analysis Data are expressed as means ± SD. Statistical significance was evaluated using the SPSS 13.0 software. When the data meet the requirements of variance, data were analyzed by one-way ANOVA and LSD, otherwise by Welch approximate analysis of variance and Dunnett T3 multiple comparison method. Differences were considered statistically significant when p < 0.05.
2.4. Design of TRPM7-siRNA and transfection Two targeted sequences specific to the TRPM7 gene were chosen as potential small interference RNA (siRNA) target sites and a non-targeted sequence (NC) was chosen for control (Table 1) following the instructions from Shanghai GenePharma Co., Ltd. TRPM7 specific siRNA was transfected in MDA-MB-435 breast cancer cells to suppress TRPM7 expression according to the manufacturer’s instructions. Briefly, MDA-MB-435 cells were routinely cultured for 24 h in a 6-well plate prior to transfection. MDA-MB-435 cells at 40% confluence were rinsed twice with PBS and transiently transfected using transfection mixtures. Transfection was allowed to continue for 8–12 h before removal of the transfection mixtures. The transfection efficiency was assessed by quantitative real-time PCR and western blot analysis. After transfection for the desired time, wound scratch assays, transwell migration and invasion assays were performed. 2.5. Wound scratch assay For wound scratch assays, cell monolayers were wounded by scratching the surface on the 6-well plate as uniformly as possible with a pipette tip. The scratched wells were washed several times with PBS to remove detached debris after scratchTable 1 The siRNA sequences for the TRPM7 gene and a negative control. Name
Sequence
TRPM7-siRNA1
Sense 50 -GUCUUGCCAUGAAAUACUCTT-30 Anti-sense 50 -GAGUAUUUCAUGGCAAGACTT-30 Sense 50 -AGGAGAAGAUGCAAUUAAATT-30 Anti-sense 50 -UUUAAUUGCAUCUUCUCCUAG-30 Sense 50 -UUCUCCGAACGUGUCACGUTT-30 Anti-sense 50 -ACGUGACACGUUCGGAGAATT-30
TRPM7-siRNA2 NC
3. Results 3.1. Overexpression of TRPM7 is correlated to cancer metastasis and invasive breast cancers We examined the Oncomine™ (Compendia Bioscience, Ann Arbor, MI) for analysis and visualization of TRPM7 in breast and other cancer types for its expression levels in human cancer tissues and cell lines with the methods described [27,28]. The datasets from the Oncomine cancer microarray database (https:// www.oncomine.com/resource/main.html) were selected to determine the alterations of TRPM7 in DNA copy numbers and mRNA expression levels. TRPM7 mRNA levels were significantly higher in metastatic cancers of breast, [31,32], ovary [33,34], prostate (4 out of 5 databases) [35–39], stomach [40], melanoma [41]and sarcoma [42], and marginal in colon [42,43], compared with TRPM7 expression in the primary tumor site. However, TRPM7 gene copy number remained the same in primary or metastatic breast tumors [44]. Representative datasets are shown for each indicated cancer types in mRNA levels and DNA copy numbers (Fig. 1A–H). Invasive ductal breast carcinoma, invasive lobular breast carcinoma, invasive ductal and lobular breast carcinoma, and mixed lobular and ductal breast carcinoma expressed significantly higher TRPM7 mRNA (Fig. 2A–D), compared to the breast tissue control in TCGA breast datasets. In addition,
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Fig. 1. TRPM7 expression level is related to cancer metastasis. Selected datasets from the Oncomine cancer microarray database (https://www.oncomine.com/resource/ main.html) were mined to determine the alterations of TRPM7 in mRNA expression levels and DNA copy numbers. Representative datasets from breast, ovary, prostate, gastric, melanoma, sarcoma, and colon cancer types were compared with TRPM7 expression in the primary tumors vs. metastatic tumors. (A) Breast cancer; (B) ovarian cancer; (C) prostate cancer; (D) gastric cancer; (E) melanoma; (F) sarcoma; (G) colon cancer; (H) copy number variations for breast cancer. T-tests were performed for statistical significance for the entire series of analysis. Median values are indicated.
lobular breast carcinoma expressed significantly more TRPM7 mRNA among seven major pathological subtypes of breast carcinoma (Fig. 2E), while ductal carcinoma and mixed lobular and ductal breast carcinoma demonstrated marginal significance in TRPM7 transcription. There is no significant alteration in DNA copy numbers among the tested subtypes (data not shown). 3.2. Knockdown of TRPM7 mRNA reduces TRPM7 protein levels Since these data suggest that TRPM7 expression is correlated with invasive breast cancer, we used highly metastatic MDA-MB435 breast cancer cells to explore the potential role of the TRPM7 channel in breast cancer migration and invasion. Firstly, TRPM7 siRNA was used to downregulate TRPM7 expression. To assess the interference efficiency of TRPM7 specific small RNA, the expression of TRPM7 protein was analyzed by Western blot. The result showed that TRPM7 expression in terms of protein was significantly (p < 0.01) silenced with TRPM7-siRNA in MDA-MB-435 breast cancer cells (Fig. 3). 3.3. Silencing TRPM7 suppresses cancer cell migration and invasion To evaluate the effect of TRPM7 on cancer cell migration, wound-scratch assays were used to determine the migration capability of MDA-MB-435 breast cancer cells after silencing of TRPM7. The results showed that the cells migrated more slowly to close the scratched wounds after transfection with TRPM7 specific siRNA for 48 h and the number of migrating cells was significantly (p < 0.01) decreased, compared with control (Fig. 4A and B). Furthermore, we conducted Transwell invasion assays to evaluate the effect of
TRPM7 on cancer cell invasion. The results showed that MDAMB-435 breast cancer cells significantly (p < 0.01) lost their ability to invade through the Matrigel matrix and the number of cells that invaded through the Matrigel matrix was significantly decreased after silencing of TRPM7 with specific siRNA as compared with control (Fig. 4C and D). In line, we measured the effect of TRPM7 knockdown on proliferation of MDA-MB-435 cells using MTT assays. We found that cell proliferation was not significantly affected by siRNA interference (Fig. S1). These data suggest that TRPM7 expression is correlated to the invasion capability of MDA-MB435 breast cancer cells. Taken together, the results suggest that TRPM7 plays an important role in mediating migration and invasion of invasive breast cancer cells.
3.4. TRPM7 mediates migration and invasion of breast cancer cell through the MAPK pathway To explore the mechanism by which TRPM7 mediated migration and invasion of breast cancer cells, the effect of silencing TRPM7 on the activity of various signaling molecules, including AKT, Src and mitogen-activated protein kinases (MAPKs), was investigated. We first determined whether AKT, a major transducer of the phosphoinositide 3-kinase pathway, [45], is involved in TRPM7 mediated cancer cell migration and invasion. The present results show that no significant (p > 0.05) difference was observed in the expression and phosphorylation of AKT after silencing TRPM7, compared with control (Fig. 5A and B), suggesting that AKT was not involved in breast cancer cell migration and invasion mediated by TRPM7.
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Fig. 2. TRPM7 expression is upregulated in invasive breast cancers. Comparisons of normal breast tissue vs. invasive ductal or lobular breast carcinoma were made using TCGA datasets. (A) Invasive ductal breast carcinoma vs. normal control; (B) invasive lobular breast carcinoma vs. normal control; (C) invasive ductal and lobular breast carcinoma vs. normal control; (D) mixed lobular and ductal breast carcinoma vs. normal control; and (E) TRPM7 mRNA in lobular breast carcinoma vs. six other pathological subtypes of breast carcinoma. T-tests were performed for statistical significance for this series of analysis.
MDA-MB-435 breast cancer cells resulted in a significant decrease in Src activity. We next measured the expression and activation of the MAPK signaling pathway. The phosphorylation level of signal molecules (P38, ERK and JNK) was significantly reduced after silencing TRPM7 with TRPM7 specific small interfering RNA as compared with control (Fig. 5A, D, E, F), indicating that silencing of TRPM7 in MDAMB-435 breast cancer cells resulted in a significant inactivation of MAPKs. Taken together, silencing TRPM7 led to a significant reduction in migration and invasion potentials of MDA-MB-435 breast cancer cells, as well as inactivation of Src and MAPKs, suggesting that the Src and MAPKs signaling pathways are involved in TRPM7-mediated migration and invasion of MDA-MB-435 breast cancer cells. 4. Discussion Fig. 3. Downregulation of TRPM7 in MDA-MB-435 breast cancer cells by 48 h of siRNA interference. (A) ECL image of western blot of TRPM7, GAPDH was used as a loading control. (B) Quantification analysis of TRPM7 protein band densities. Indicates significant difference at p < 0.05, and indicates significant difference at p < 0.01. Error bars represent one standard deviation (n = 3).
Next, we determined whether Src, a regulatory protein that plays a key role in cell differentiation, motility, proliferation and survival [46], is involved in TRPM7 mediated cancer cell migration and invasion. We found that the phosphorylation level of Src significantly (p < 0.01) decreased after silencing TRPM7 compared with control (Fig. 5C), indicating that silencing of TRPM7 in
Metastasis is considered as an inherent feature and a main lethal factor of breast cancer [6,7]. Despite the importance of metastasis to breast cancer survival and morbidity, our knowledge is still far from understanding the cellular and molecular mechanisms of breast cancer metastasis. During carcinogenesis, cancer cell migration and invasion are the most subtle and critical steps for cancer progression and metastasis. Recently, transient receptor potential (TRP) channels have been implicated in carcinogenesis [8–10]; as well as in cancer metastasis. TRPM7, a member of TRP protein family, is involved in adhesion in HEK-293 cells [13], migration in vascular smooth muscle cells [14], and proliferation of breast cancer cells [15,47], suggesting that TRPM7 plays a crucial role in carcinogenesis including breast cancer. Our
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Fig. 4. TRPM7 knockdown suppresses MDA-MB-435 cell migration and invasion. Before the wound-scratch assay, MDA-MB-435 cells were transfected with TRPM7-siRNA for 48 h to silence TRPM7. (A) The cell images of wound-scratch assay at 0, 24, 48 h; (B) the number of cells migrating to the scratched wounds. Indicates significant difference at p < 0.05, and indicates significant difference at p < 0.01. Error bars represent one standard deviation (n = 3). Before the transwell assay, cells were transfected with TRPM7-siRNA for 48 h to silence TRPM7. (C) The images of cells that migrated into plate wells (lower chamber) in transwell invasion assay. (D) The number of cells that invaded through the Matrigel matrix into plate wells in Transwell invasion assay. Pictures (100) of the membrane were taken in 16 random fields per chamber. Indicates significant difference at p < 0.05.
Fig. 5. TRPM7 downregulation suppresses activation of Src, P38, ERK and JNK. (A) ECL image of western blot of total and phosph-AKT, Src, P38, ERK and JNK, GAPDH was used as a loading control. B, C, D, E, F. Quantification analysis of AKT, Src, P38, ERK and JNK protein band densities. Error bars represent one standard deviation (n = 3).
data from wound scratch assays and Transwell assays clearly demonstrate that the migration and invasion capability of MDAMB-435 breast cancer cells is significantly reduced after silencing TRPM7 (Figs. 3–5), while cell proliferation is not affected (Fig. S1),
suggesting that TRPM7 is required for the invasive phenotype of MDA-MB-435 breast cancer cells. These results are consistent with recent studies that depressing the function of TRPM7 with RNA interference or non-specific pharmacological agents inhibits
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the migration of A549 lung cancer cells, while up-regulation of TRPM7 enhances the migration of A549 cells [48] and vascular smooth muscle cells [14]. Furthermore, our results are also supported indirectly by previous studies showing that silencing TRPM7 enhances cell adhesion and increases the number of peripheral adhesion complexes, whereas overexpression of TRPM7 in cells induces cell rounding with a concomitant loss of cell adhesion in HEK-293 cells [13] because low adhesion capability of cell is beneficial for cell migration. Although numerous evidence supports the notion that TRPM7 is involved in regulating cell migration and invasion, the precise signaling pathway that control the regulation process are largely unknown. In the present study, we measured expression and activation of multiple signal molecules to explore the signaling pathways involved in breast cancer migration and invasion mediated by TRPM7. AKT is a primary signaling transducer of the phosphoinositide 3-kinase (PI3K) pathway and mainly contributes to cancer progression by inhibiting apoptosis, promoting metabolism changes and cell proliferation as well as regulating migration and invasion [45]. Previous studies have shown that the PI3K/AKT pathway is frequently activated in malignant breast cancer cells [49] and AKT is activated as frequently as 24% in breast cancer patients [50]. Recently, Chin and Toker [51] suggested that AKT mediates breast cancer cell migration in an isoform-specific manner and that AKT1 decreases migration, while AKT2 enhances migration. However, our data indicated that the activity of AKT was not altered when the migration and invasion capability of breast cancer cell was reduced by silencing TRPM7 (Fig. 4). We thus speculate that TRPM7-mediated migration and invasion of breast cancer cells is not associated with the AKT signaling pathway. Since cancer metastasis frequently involves the coordination of various signal-transduction pathways that regulate cancer cell migration, invasion and reestablishment of the tumor, we also investigated Src and Mitogen-activated protein kinase (MAPK), two important signaling pathways that control the essential events for metastasis including cancer cell migration and invasion [18,19]. Previous studies have indicated that Src is intimately involved in the control of matrix adhesion and cell migration in breast cancer cells [21], and MAPK is frequently activated in tumorigenesis [19] and breast cancer [23]. MAPKs (including P38, ERK1/2 and JNK1/2 members) signaling pathway play a crucial role in tumor cell proliferation, adhension and migration [22,52–54]. As upstream of the MAPK signaling pathway, Src is suggested to be essential for MAPK activation during breast cancer cell proliferation and invasion [47]. Our data showed that silencing TRPM7 with specific small interfering RNA led to a significant reduction in migration and invasion capability of MDA-MB-435 breast cancer cells and a concomitant inactivation of Src and MAPK (Fig. 5), suggesting that TRPM7 modulation of migration and invasion of breast cancer cells involves the Src-MAPK signaling pathway. Previous results show that TRPM7 modulates cell migration by means of regulating calcium flickers [25,26]. In addition, elevation of intracellular calcium or calcium influx frequently involves activation of MAPK pathway in normal and cancer cells [55–58], suggesting that intracellular calcium level is associated with MAPK activity. In the present study, it would be expected that silencing TRPM7 would lead to a low intracellular calcium level since TRPM7 is a Ca2+-permeable channel protein and a low activity of MAPK, which would result in a reduction in migration and invasion capability of MDA-MB-435 breast cancer cells. We are in the process of testing this interesting hypothesis in breast cancer cells. Our findings that TRPM7 modulates migration and invasion of MDA-MB435 breast cancer cells through the Src–MAPK signaling pathway suggest that depressing this signaling pathway or/and the TRPM7 channel protein may be beneficial in treating breast cancer patients with or without metastasis lesions.
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Acknowledgments This work was supported by grants from National Basic Research Program of China, No. 2012CB518200 (FZ), No.2012CB525004 (XM); National Natural Science Foundation of China No. 30971193(FZ), No. 81071611 (CC).
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2013.01. 031.
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