Tumor Biol. DOI 10.1007/s13277-014-2077-8
RESEARCH ARTICLE
TRPM8 promotes aggressiveness of breast cancer cells by regulating EMT via activating AKT/GSK-3β pathway Jinxin Liu & Yizhi Chen & Shuai Shuai & Dapeng Ding & Rong Li & Rongcheng Luo
Received: 18 March 2014 / Accepted: 7 May 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Breast cancer already taken the first place of incidence in Chinese female cancer patients. TRPM8 is found to be over-expressed in breast cancer, but whether it promotes breast cancer aggressiveness remains unknown. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB231, BT549, SKBR3 and ZR-75-30, while it just could be detected in MCF-10A, the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore expression status of cancer-related proteins. The Liu and Chen contributed equally to this work J. Liu : S. Shuai : R. Luo (*) Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, Guangdong, China e-mail: [email protected] Y. Chen Department of Health Records, Longgang District Central Hospital of Shenzhen, Shenzhen, China D. Ding Institute of Genetic Engineering, Southern Medical University, Guangzhou, Guangdong, China R. Li (*) Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China e-mail: [email protected] J. Liu Department of Oncology, Longgang District Central Hospital of Shenzhen, Shenzhen, China J. Liu : S. Shuai : R. Luo Cancer Center, Southern Medical University, Guangzhou, Guangdong, China
Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3β pathway, and the hypothesis had been supported by cell function tests. All the results demonstrated that TRPM8 significantly upexpressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/GSK-3β pathway, indicating TRPM8 gets the prospects of to be developed as medication or diagnostic indicator to be applied in clinical work. Keywords TRPM8 . EMT . Breast cancer . AKTGSK-3β
Introduction Breast cancer is the most common form of cancer in women in Western countries and already taking the first place of incidence in Chinese female cancer patients. Many studies have revealed that female steroid hormones play an essential role in the development of breast cancer [1, 2], and they regulate the expression of many genes including transient receptor potential (TRP) channels that have emerged as new channels implicated in carcinogenesis [3–7]. TRPM8 channel is a Ca2+-permeable cation channel and belongs to the TRPM (melastatin) subfamily of TRP proteins. It is better known as a temperature-sensitive ion channel activated by mild cooling, such as by environmental temperatures below 28 °C and by the cooling agents Menthol and Icilin, which elicit the sensation of coolness [8, 9]. Apart from its role in thermosensation, acute activation or inhibition of TRPM8 can have analgesic effects either to diminish neuropathic and visceral pain [10–12] or to attenuate cold hypersensitivity in inflammatory and nerve-injury pain models [13], suggesting that neuronal TRPM8 also play a neurogenic anti-inflammatory role in certain settings. But in recent years, many studies demonstrated that TRPM8 is found to be over-expressing in
Tumor Biol.
Fig. 1 TRPM8 expression is upregulated in breast cancer cell lines. Expressions of TRPM8 were detected in breast epithelial cell MCF10A and breast metastatic cancer cell lines MCF-7, T47D, MDA-MB231, BT549, SKBR3 and ZR-75-30. Protein expressions were shown by
Western blotting bands with α-Tubulin as loading control (left panel). TRPM8 expressions in transcriptional level were tested by qRT-PCR (right panel)
several types of primary tumors including colon, lung, skin, prostate and breast cancers [14–18]. But whether TRPM8 could regulate the occurrence and metastasis of breast cancer remains unknown, it’s a prospective study point of breast cancer research. Epithelial–mesenchymal transition (EMT) is a certain event describing the key step of tumor cell metastasis which includes consecutive processes of cell-detaching, migrating, invading, dispersing and final residing [19]. It has been abundantly identified as a hallmark in metastasis of multiple tumors such as breast cancer, connecting to plenty of transcriptional factors as well as PI3K/Akt/ GSK-3β pathway [20–23]. Activation of the PI3K/Akt/GSK-3β pathway is emerging as a central feature of EMT [24–28], followed by the degradation of E-cadherin which is a critical step in EMT [29, 30]. To clarify the relations between TRPM8, metastasis and EMT is meaningful for researches about diagnosis and treatment of breast cancer. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR75-30, while it just could be detected in MCF-10A the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore the expression status of cancer-related proteins. The Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3β pathway, and the hypothesis had been supported by cell function tests. Hence, we concluded that TRPM8 significantly upexpressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/ GSK-3β pathway.
Materials and methods Tissue samples Primary cancer tissues and adjacent nontumor tissues were obtained from surgical treatments of breast cancer. All samples were formalin-fixed and paraffin-embedded (FFPE) with standard procedures. The histological diagnosis was made by a pathologist and has been reconfirmed by a second pathologist. The study was approved by the Institutional Review Board (IRB) in the Southern Medical University and consented by patients involved. Cell culture Breast epithelial cell line MCF-10A and metastatic breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR-75-30 were purchased from American Type Culture Collection (ATCC) and preserved in Central Laboratory in Southern Medical University. Each cell line is maintained in culture medium and atmosphere instructed by each manuscripts. Generation of stable cell lines The sequence of TRPM8 and small hairpin RNA (shRNA)TRPM8 were cloned into pMSCV-puromycin and pSuperpuromycin retroviral vectors, respectively, and the resulting Table 1 mRNA expressions of TRPM8 in breast cell lines
Cell line
Folds change
MCF-10A MCF-7 T47D MDA-MB231 BT549 SKBR3 ZR-75-30
1 7.713671 12.21725 30.52901 22.22653 19.80036 5.609732
Tumor Biol.
Fig. 2 TRPM8 expression is upregulated in breast cancer tissues. Expressions of TRPM8 were detected in primary breast cancer samples and surrounding adjacent nontumor tissues. TRPM8 protein expressions were
shown by Western blotting bands with α-Tubulin as loading control (left panel). Expression of TRPM8 mRNA in each specimen was tested by qRT-PCR (right panel)
plasmid was transfected into 293FT cells to generate virus as described previously [31]. After incubation at 37 °C for 6 h, the transfected cells were cultured in fresh media overnight. In the following days, media were collected three times a day to gather the produced virus until the 293FT cells reached total confluency. Media containing pMSCV/TRPM8 or pSuper/ shRNA-TRPM8 were used to infect MCF-7 or MDA-MB231, respectively, for 24 h. After removal of the inoculum and replacement with fresh media, infected cells were selected by adding puromycin. Stable cell lines were verified by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR).
Briefly, cells were trypsinized and suspended in serum-free medium. Then 1.5×105 cells were added to the upper chamber, whereas lower chamber was filled with medium with 10 % FBS. After incubated for 48 h, cells were invaded through the coated membrane to the lower surface, in which cells were fixed with 4 % paraformaldehyde and stained with hematoxylin. The cell count was done under the microscope (100×).
Quantitative real-time PCR (qRT-PCR) analysis Total RNA from different cell lines and human tissues was extracted using Trizol reagent (Gibco). cDNAwas synthesized from 2.5 μg of total RNA using random hexamers. Real-time PCR was carried out using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Reactions were run in triplicate in three independent experiments. The geometric mean of the housekeeping gene β-actin was used as internal control to normalize the variability in expression levels. Wound-healing assay One day before scratch, MCF-7/TRPM8 and MDA-MB-231/ RNAi cells were trypsinized and seeded equally into six-well tissue culture plates and grew to reach almost total confluence in 24 h. When nonserum starvation kept for 24 h after cell monolayer formed, an artificial homogenous wound was created onto the monolayer with a sterile 100-ml tip. After scratching, the cells were washed with serum-free medium. Images of cells migrating into the wound were captured at time points of 0, 12 and 24 h by inverted microscope (40×).
Western blotting For the expression analysis of EMT-related proteins, immunoblotting assay was carried out. MCF-7/TRPM8 and MDA-MB-231/RNAi cells were seeded in 100 mm tissue culture dishes. After 24 h, cells were washed with prechilled phosphate buffered saline (PBS) when the confluence reached to 60–70 %, followed by being harvested in sample buffer [62.5 mmol/l Tris–HCl (pH 6.8), 2 % SDS, 10 % glycerol, and 5 % 2-bmercaptoethanol]. Equal amounts of protein from the supernatant were loaded per lane and resolved by SDS-polyacrylamide electrophoresis. In sequence, protein was transferred onto PVDF membrane (Millipore), blocked by 5 % nonfat milk for 1 h at room temperature and probed with primary antibodies (1:1000) for 3 h. Primary antibodies for E-Cadherin, Fibronectin and Vimentin were purchased from BD Biosciences. Those for AKT, p-AKT, GSK-3β, p-GSK-3β, Snail
Table 2 mRNA expressions of TRPM8 in breast tissues
In vitro invasion assay The invasion assay was done by using Transwell chamber consisting of 8-mm membrane filter inserts (Corning) coated with Matrigel (BD Biosciences) as previously described [32].
ANT adjacent nontumor tissue, T tumor
Sample tissues
Folds change
ANT 1 T1
1 4.029055
ANT 2 T2 ANT 3 T3 ANT 4 T4
1.022101 4.845752 2.577143 6.335187 1.393722 4.554236
Tumor Biol.
Tumor Biol.
Fig. 3
TRPM8 activated Akt/GSK-3β pathway and regulated EMT phenotypical proteins. a Expression of TRPM8 was detected in MDAMB-231 cells which was transfected with scrambled sequence plasmids or with silencing plasmids, respectively, and b for MCF-7 cells with control vector or TRPM8-expressing plasmid. TRPM8 protein expressions were shown by Western blotting bands with α-Tubulin as loading control (left panels) and mRNA of TRPM8 in each cell line was tested by qRT-PCR (right panels). c Expression of epithelial phenotypical protein E-Cadherin and mesenchymal phenotypical proteins Fibronectin and Vimentin were detected by Western blotting in MDA-MB-231 and MCF-7 cell lines, both of which got altered TRPM8 expressions. Followingly, activity of Akt/GSK-3β pathway and its phosphorylation were also determined as well as Snail expression. α-Tubulin was used as a loading control. d MDA-MB-231/scramble, MDA-MB-231/RNAi, MCF-7/vector and MCF-7/TRPM8 cells were stained for E-Cadherin, Vimentin and DAPI and analyzed by confocal microscopy. The green signal represents staining for the corresponding protein, while the blue signal signifies nuclear DNA staining with DAPI
and α-Tubulin were purchased from Abcam. Membranes were washed thrice (10 min each) in TBS-T buffer and incubated for 40 min at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibodies. Blots were washed thrice (10 min each) in TBST and developed using the ECL system. Protein loading was normalized by reprobing the blots with α-Tubulin antibody (Abcam). Immunofluorescence analysis Cells were stained for immunofluorescence on coverslips as described previously [33]. Briefly, the cells were incubated with primary antibodies against E-cadherin and Vimentin and then incubated with rhodamineconjugated or FITC-conjugated goat antibodies against rabbit or mouse IgG (Jackson ImmunoResearch Laboratories). The coverslips were counterstained with DAPI and imaged with a confocal laser-scanning microscope (Olympus FV1000). 3D morphogenesis assay As previously described [34], 24-well dishes were coated with growth factor-reduced matrigel (BD Biosciences) and covered with growth medium supplemented with 2 % Matrigel. Cells were trypsinized and seeded at a density of 104 cells/well, and medium was replaced with 2 % Matrigel for 3 to 4 days. Microscopic images were captured at 2-day intervals for 2–3 weeks. Statistical analysis Statistics were assessed using SPSS 17.0 (SPSS, Chicago, IL, USA). When counting cells in invasion assay
experiments, data were compared by Student’s t test. p values of >0.05 were considered significant.
Results TRPM8 expression is upregulated in breast cancer cell lines Expressions of TRPM8 were examined in one breast epithelial cell line and six breast cancer cell lines. The analyses were conducted at translational level using Western blotting assays (Fig. 1, left panel) and at transcriptional levels using qRT-PCR (Fig. 1, right panel). A very strong to moderate expression of TRPM8 was observed in all cell lines except for MCF-10A cell that showed such low expression which was normalized as baseline in qRT-PCR. Among the six breast cancer cell lines, MDA-MB-231, BT549 and SKBR3 showed highest expression of TRPM8, while MCF-7, T47D and ZR-75-30 demonstrated relatively lower TRPM8 expressions (Table 1). TRPM8 expression is upregulated in breast cancer tissues Six pairs of surgical resected breast tissues were included to our study. As shown in Fig. 2, TRPM8 protein levels were obviously higher in tumor tissues than in the pairing adjacent nontumor tissues (left panel). To consolidate this result, we detected mRNA expressions of TRPM8 in each samples, and outcomes were in consistent with findings above (right panel and Table 2). Upregulation of TRPM8 promoted EMT of breast cancer cells via activating PI3K/Akt pathway To investigate whether TRPM8 regulates metastatic features of breast cancer cells via promoting EMT, we interfered endogenous expression of TRPM8 in MDA-MB-231 by transfecting silencing plasmids (Fig. 3a) while increased ectopic TRPM8 expression in MCF-7 by introducing TRPM8expressing constructs (Fig. 3b). As shown in Fig. 3c, overexpression of TRPM8 repressed E-cadherin, one of epithelial phenotypical markers and supposed to be downregulated during EMT. Nevertheless, E-cadherin was increased in breast cancer cells transfected with TRPM8 silencing plasmids. Moreover, Fibronectin and Vimentin, which are mesenchymal pheotypical markers and should be upregulated during EMT, were inhibited in TRPM8-silenced cells. However, they were significantly increased in cells transfected with TRPM8expressing plasmids. In order to observe visually, we probed the cells by immunofluorescent antibodies. As shown in Fig. 3c, cells with higher cells exhibited obviously lower
Tumor Biol.
expression of E-cadherin while Vimentin upregulated concordantly with TRPM8 and vice versa. These findings suggested that TRPM8 had positive effects on inducing EMT in breast cancer and might mediate epithelial cells to transform toward mesenchymal cells. Because activation of the PI3K/Akt pathway is emerging as a central feature of EMT [24–28], we speculated TRPM8 regulate Akt activity in breast cancer cells. As shown in Fig. 3b, changed TRPM8 expression did not obviously influence Akt level, but phosphorylation of Akt was significantly inhibited in cells transfected with small interfering (siRNA) against TRPM8. Followingly, the increase in Akt phosphorylation was accompanied by a change in phosphorylation of GSK-3β, a downstream target protein of Akt, suggesting that upregulation of TRPM8 activates the Akt/GSK-3β pathway in breast cancer cells. Previously, GSK-3β activity was demonstrated to be necessary for the maintenance of epithelial architecture by dual regulation of Snail [35, 36]. As shown in Fig. 3b, Western blotting revealed that compared with vector cells, expression of Snail significantly increased in breast cancer cells transfected with TRPM8 plasmids but decreased in those with silencing constructs. Upregulation of TRPM8 increased aggressiveness of breast cancer cells in vitro To investigate the role of TRPM8 in the development and progression of breast cancer, TRPM8 over-expressing cells were established by plasmid transfection to perform migration and invasion assays in vitro. As we expect, alterations of cellmigrating ability observed by wound-healing assay demonstrated that cells transfected with TRPM8 plasmids significantly ran faster than those with control vectors. On the other hand, cells transfected with siTRPM8 moved more slowly than those with scrambled sequence vectors (Fig. 4a). The invasive property of PC-3 cells was examined by TranswellMatrigel penetration assay, which depicted cells transfected with TRPM8 plasmids penetrated through the gel-membrane section much more than cells with control vectors. Meanwhile, siTRPM8-transfected breast cancer cells got obviously repressed invasive ability. Morphologically, high expression of TRPM8 induced major shape changes of cells, with the formation of thorns or legs which suggest more motile and higher invasiveness (Fig. 4c).
Discussion For the first time, our study demonstrated that TRPM8 facilitates breast cancer metastasis via promoting EMT process, and especially by activating Akt/GSK-3β pathway. This finding is a great supplement for the ongoing researches about
TRPM8 regulating breast cancer, which always focus on molecular structure of TRPM8 and its ability of sustaining Ca2+ influx [37]. TRPM8 was initially cloned as a novel prostate-specific gene by screening a prostate cDNA library [14], and has been found in a number of other cancers, including melanoma, breast adenocarcinoma, colorectal cancer, lung cancer and bladder cancer [14, 37, 38]. Prostate cells normally express low amounts of TRPM8 while its expression is much higher in cancerous cells [14, 39], and out data showed similar result. When the mild expression of TRPM8 in MCF-10A was normalized to onefold, higher expressions of TRPM8 in other cancer cell lines ranged from fivefolds to 30-folds. So we selected the MDA-MB231 due to its highest expression and the MCF-7 which did not get lowest TRPM8 but was involved more common in research than ZR-75-30. Function of TRPM8 is well established in the majority of cell-signaling pathways involved in carcinogenesis [40], in which EMT is a key step for cell transformation, migration, adhesion and invasion. Actually, lots of studies had identified the significance of EMT during breast cancer development. Cells undergoing EMT typically show both an increase in protein abundances of Vimentin, N-cadherin, Fibronectin, etc. and a decrease in E-cadherin, Cytokeratins and Occludin, etc. [41]. Our results are in complete agreement with that theory. Since TRPM8 was significantly knocked down in MDA-MB-231 cell line, expression of E-cadherin, a mark of epithelial cell phenotype, was obviously upregulated in those cells. Meanwhile, Vimentin and Fibronectin both of which are marks of mesenchymal cell phenotype correspondently decreased in the transfected cell line. Converse results showed in MCF-7 cells when expression of TRPM8 was increased by plasmids. These findings strongly suggest that TRPM8 could regulate the EMT phenotype of breast cancer cells. Besides regulating EMT marker proteins of breast cancer cells, TRPM8 was firstly identified to be involved in the activation of AKT/GSK-3β pathway. We can see in each transfected cell line that expressions of AKT and GSK-3β seemed extremely unchanged, because TRPM8 might not influence transcriptions of AKT and GSK-3β. But upregulated TRPM8 significantly increased the phosphorylation of AKT in breast cancer cells; this result suggests TRPM8 activating AKT via phosphorylation. Importantly, Ser 9 phosphorylation of GSK-3β by activated AKT results in the downregulation of GSK-3β, and GSK-3β binds to and phosphorylates Snail transcriptional repressor to induce its cytoplasmic translocation and degradation [36, 42]. Consequently, increasing of TRPM8 promoted expression of Snail that represses the transcription of E-cadherin, of which the degradation is key step of the initiation of EMT [29, 43, 44]. Our data demonstrated that activation AKT/GSK-3β-signaling pathway directly leads to the activation of Snail-EMT-signaling cascade. Since EMT is critical for metastasis of cancer cells
Tumor Biol.
[45], our results elucidate that TRPM8 activates EMT through inducing AKT/GSK-3β pathway and strongly supplements researches about breast cancer metastasis. Studies have underlined the involvement of ion channels such as potassium channels in cell cycle progression and migration [46–48], as well as sodium channels in invasion processes [49, 50]. For example, Yang et al.
Fig. 4 TRPM8 altered breast cancer cells invasiveness. a Shapes of MDA-MB-231 and MCF-7 cell lines of altered TRPM8 expressions were photographed under microscope. b The invasive properties of cells were analyzed by invasion assay using a Matrigel-coated Boyden chamber. Migrated cells were plotted as the average number of cells per field of view from three different experiments, as described in “Materials and methods” section. Original magnification, ×200. Error bars represent SEM. **P
RESEARCH ARTICLE
TRPM8 promotes aggressiveness of breast cancer cells by regulating EMT via activating AKT/GSK-3β pathway Jinxin Liu & Yizhi Chen & Shuai Shuai & Dapeng Ding & Rong Li & Rongcheng Luo
Received: 18 March 2014 / Accepted: 7 May 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Breast cancer already taken the first place of incidence in Chinese female cancer patients. TRPM8 is found to be over-expressed in breast cancer, but whether it promotes breast cancer aggressiveness remains unknown. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB231, BT549, SKBR3 and ZR-75-30, while it just could be detected in MCF-10A, the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore expression status of cancer-related proteins. The Liu and Chen contributed equally to this work J. Liu : S. Shuai : R. Luo (*) Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, Guangdong, China e-mail: [email protected] Y. Chen Department of Health Records, Longgang District Central Hospital of Shenzhen, Shenzhen, China D. Ding Institute of Genetic Engineering, Southern Medical University, Guangzhou, Guangdong, China R. Li (*) Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China e-mail: [email protected] J. Liu Department of Oncology, Longgang District Central Hospital of Shenzhen, Shenzhen, China J. Liu : S. Shuai : R. Luo Cancer Center, Southern Medical University, Guangzhou, Guangdong, China
Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3β pathway, and the hypothesis had been supported by cell function tests. All the results demonstrated that TRPM8 significantly upexpressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/GSK-3β pathway, indicating TRPM8 gets the prospects of to be developed as medication or diagnostic indicator to be applied in clinical work. Keywords TRPM8 . EMT . Breast cancer . AKTGSK-3β
Introduction Breast cancer is the most common form of cancer in women in Western countries and already taking the first place of incidence in Chinese female cancer patients. Many studies have revealed that female steroid hormones play an essential role in the development of breast cancer [1, 2], and they regulate the expression of many genes including transient receptor potential (TRP) channels that have emerged as new channels implicated in carcinogenesis [3–7]. TRPM8 channel is a Ca2+-permeable cation channel and belongs to the TRPM (melastatin) subfamily of TRP proteins. It is better known as a temperature-sensitive ion channel activated by mild cooling, such as by environmental temperatures below 28 °C and by the cooling agents Menthol and Icilin, which elicit the sensation of coolness [8, 9]. Apart from its role in thermosensation, acute activation or inhibition of TRPM8 can have analgesic effects either to diminish neuropathic and visceral pain [10–12] or to attenuate cold hypersensitivity in inflammatory and nerve-injury pain models [13], suggesting that neuronal TRPM8 also play a neurogenic anti-inflammatory role in certain settings. But in recent years, many studies demonstrated that TRPM8 is found to be over-expressing in
Tumor Biol.
Fig. 1 TRPM8 expression is upregulated in breast cancer cell lines. Expressions of TRPM8 were detected in breast epithelial cell MCF10A and breast metastatic cancer cell lines MCF-7, T47D, MDA-MB231, BT549, SKBR3 and ZR-75-30. Protein expressions were shown by
Western blotting bands with α-Tubulin as loading control (left panel). TRPM8 expressions in transcriptional level were tested by qRT-PCR (right panel)
several types of primary tumors including colon, lung, skin, prostate and breast cancers [14–18]. But whether TRPM8 could regulate the occurrence and metastasis of breast cancer remains unknown, it’s a prospective study point of breast cancer research. Epithelial–mesenchymal transition (EMT) is a certain event describing the key step of tumor cell metastasis which includes consecutive processes of cell-detaching, migrating, invading, dispersing and final residing [19]. It has been abundantly identified as a hallmark in metastasis of multiple tumors such as breast cancer, connecting to plenty of transcriptional factors as well as PI3K/Akt/ GSK-3β pathway [20–23]. Activation of the PI3K/Akt/GSK-3β pathway is emerging as a central feature of EMT [24–28], followed by the degradation of E-cadherin which is a critical step in EMT [29, 30]. To clarify the relations between TRPM8, metastasis and EMT is meaningful for researches about diagnosis and treatment of breast cancer. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR75-30, while it just could be detected in MCF-10A the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore the expression status of cancer-related proteins. The Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3β pathway, and the hypothesis had been supported by cell function tests. Hence, we concluded that TRPM8 significantly upexpressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/ GSK-3β pathway.
Materials and methods Tissue samples Primary cancer tissues and adjacent nontumor tissues were obtained from surgical treatments of breast cancer. All samples were formalin-fixed and paraffin-embedded (FFPE) with standard procedures. The histological diagnosis was made by a pathologist and has been reconfirmed by a second pathologist. The study was approved by the Institutional Review Board (IRB) in the Southern Medical University and consented by patients involved. Cell culture Breast epithelial cell line MCF-10A and metastatic breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR-75-30 were purchased from American Type Culture Collection (ATCC) and preserved in Central Laboratory in Southern Medical University. Each cell line is maintained in culture medium and atmosphere instructed by each manuscripts. Generation of stable cell lines The sequence of TRPM8 and small hairpin RNA (shRNA)TRPM8 were cloned into pMSCV-puromycin and pSuperpuromycin retroviral vectors, respectively, and the resulting Table 1 mRNA expressions of TRPM8 in breast cell lines
Cell line
Folds change
MCF-10A MCF-7 T47D MDA-MB231 BT549 SKBR3 ZR-75-30
1 7.713671 12.21725 30.52901 22.22653 19.80036 5.609732
Tumor Biol.
Fig. 2 TRPM8 expression is upregulated in breast cancer tissues. Expressions of TRPM8 were detected in primary breast cancer samples and surrounding adjacent nontumor tissues. TRPM8 protein expressions were
shown by Western blotting bands with α-Tubulin as loading control (left panel). Expression of TRPM8 mRNA in each specimen was tested by qRT-PCR (right panel)
plasmid was transfected into 293FT cells to generate virus as described previously [31]. After incubation at 37 °C for 6 h, the transfected cells were cultured in fresh media overnight. In the following days, media were collected three times a day to gather the produced virus until the 293FT cells reached total confluency. Media containing pMSCV/TRPM8 or pSuper/ shRNA-TRPM8 were used to infect MCF-7 or MDA-MB231, respectively, for 24 h. After removal of the inoculum and replacement with fresh media, infected cells were selected by adding puromycin. Stable cell lines were verified by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR).
Briefly, cells were trypsinized and suspended in serum-free medium. Then 1.5×105 cells were added to the upper chamber, whereas lower chamber was filled with medium with 10 % FBS. After incubated for 48 h, cells were invaded through the coated membrane to the lower surface, in which cells were fixed with 4 % paraformaldehyde and stained with hematoxylin. The cell count was done under the microscope (100×).
Quantitative real-time PCR (qRT-PCR) analysis Total RNA from different cell lines and human tissues was extracted using Trizol reagent (Gibco). cDNAwas synthesized from 2.5 μg of total RNA using random hexamers. Real-time PCR was carried out using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Reactions were run in triplicate in three independent experiments. The geometric mean of the housekeeping gene β-actin was used as internal control to normalize the variability in expression levels. Wound-healing assay One day before scratch, MCF-7/TRPM8 and MDA-MB-231/ RNAi cells were trypsinized and seeded equally into six-well tissue culture plates and grew to reach almost total confluence in 24 h. When nonserum starvation kept for 24 h after cell monolayer formed, an artificial homogenous wound was created onto the monolayer with a sterile 100-ml tip. After scratching, the cells were washed with serum-free medium. Images of cells migrating into the wound were captured at time points of 0, 12 and 24 h by inverted microscope (40×).
Western blotting For the expression analysis of EMT-related proteins, immunoblotting assay was carried out. MCF-7/TRPM8 and MDA-MB-231/RNAi cells were seeded in 100 mm tissue culture dishes. After 24 h, cells were washed with prechilled phosphate buffered saline (PBS) when the confluence reached to 60–70 %, followed by being harvested in sample buffer [62.5 mmol/l Tris–HCl (pH 6.8), 2 % SDS, 10 % glycerol, and 5 % 2-bmercaptoethanol]. Equal amounts of protein from the supernatant were loaded per lane and resolved by SDS-polyacrylamide electrophoresis. In sequence, protein was transferred onto PVDF membrane (Millipore), blocked by 5 % nonfat milk for 1 h at room temperature and probed with primary antibodies (1:1000) for 3 h. Primary antibodies for E-Cadherin, Fibronectin and Vimentin were purchased from BD Biosciences. Those for AKT, p-AKT, GSK-3β, p-GSK-3β, Snail
Table 2 mRNA expressions of TRPM8 in breast tissues
In vitro invasion assay The invasion assay was done by using Transwell chamber consisting of 8-mm membrane filter inserts (Corning) coated with Matrigel (BD Biosciences) as previously described [32].
ANT adjacent nontumor tissue, T tumor
Sample tissues
Folds change
ANT 1 T1
1 4.029055
ANT 2 T2 ANT 3 T3 ANT 4 T4
1.022101 4.845752 2.577143 6.335187 1.393722 4.554236
Tumor Biol.
Tumor Biol.
Fig. 3
TRPM8 activated Akt/GSK-3β pathway and regulated EMT phenotypical proteins. a Expression of TRPM8 was detected in MDAMB-231 cells which was transfected with scrambled sequence plasmids or with silencing plasmids, respectively, and b for MCF-7 cells with control vector or TRPM8-expressing plasmid. TRPM8 protein expressions were shown by Western blotting bands with α-Tubulin as loading control (left panels) and mRNA of TRPM8 in each cell line was tested by qRT-PCR (right panels). c Expression of epithelial phenotypical protein E-Cadherin and mesenchymal phenotypical proteins Fibronectin and Vimentin were detected by Western blotting in MDA-MB-231 and MCF-7 cell lines, both of which got altered TRPM8 expressions. Followingly, activity of Akt/GSK-3β pathway and its phosphorylation were also determined as well as Snail expression. α-Tubulin was used as a loading control. d MDA-MB-231/scramble, MDA-MB-231/RNAi, MCF-7/vector and MCF-7/TRPM8 cells were stained for E-Cadherin, Vimentin and DAPI and analyzed by confocal microscopy. The green signal represents staining for the corresponding protein, while the blue signal signifies nuclear DNA staining with DAPI
and α-Tubulin were purchased from Abcam. Membranes were washed thrice (10 min each) in TBS-T buffer and incubated for 40 min at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibodies. Blots were washed thrice (10 min each) in TBST and developed using the ECL system. Protein loading was normalized by reprobing the blots with α-Tubulin antibody (Abcam). Immunofluorescence analysis Cells were stained for immunofluorescence on coverslips as described previously [33]. Briefly, the cells were incubated with primary antibodies against E-cadherin and Vimentin and then incubated with rhodamineconjugated or FITC-conjugated goat antibodies against rabbit or mouse IgG (Jackson ImmunoResearch Laboratories). The coverslips were counterstained with DAPI and imaged with a confocal laser-scanning microscope (Olympus FV1000). 3D morphogenesis assay As previously described [34], 24-well dishes were coated with growth factor-reduced matrigel (BD Biosciences) and covered with growth medium supplemented with 2 % Matrigel. Cells were trypsinized and seeded at a density of 104 cells/well, and medium was replaced with 2 % Matrigel for 3 to 4 days. Microscopic images were captured at 2-day intervals for 2–3 weeks. Statistical analysis Statistics were assessed using SPSS 17.0 (SPSS, Chicago, IL, USA). When counting cells in invasion assay
experiments, data were compared by Student’s t test. p values of >0.05 were considered significant.
Results TRPM8 expression is upregulated in breast cancer cell lines Expressions of TRPM8 were examined in one breast epithelial cell line and six breast cancer cell lines. The analyses were conducted at translational level using Western blotting assays (Fig. 1, left panel) and at transcriptional levels using qRT-PCR (Fig. 1, right panel). A very strong to moderate expression of TRPM8 was observed in all cell lines except for MCF-10A cell that showed such low expression which was normalized as baseline in qRT-PCR. Among the six breast cancer cell lines, MDA-MB-231, BT549 and SKBR3 showed highest expression of TRPM8, while MCF-7, T47D and ZR-75-30 demonstrated relatively lower TRPM8 expressions (Table 1). TRPM8 expression is upregulated in breast cancer tissues Six pairs of surgical resected breast tissues were included to our study. As shown in Fig. 2, TRPM8 protein levels were obviously higher in tumor tissues than in the pairing adjacent nontumor tissues (left panel). To consolidate this result, we detected mRNA expressions of TRPM8 in each samples, and outcomes were in consistent with findings above (right panel and Table 2). Upregulation of TRPM8 promoted EMT of breast cancer cells via activating PI3K/Akt pathway To investigate whether TRPM8 regulates metastatic features of breast cancer cells via promoting EMT, we interfered endogenous expression of TRPM8 in MDA-MB-231 by transfecting silencing plasmids (Fig. 3a) while increased ectopic TRPM8 expression in MCF-7 by introducing TRPM8expressing constructs (Fig. 3b). As shown in Fig. 3c, overexpression of TRPM8 repressed E-cadherin, one of epithelial phenotypical markers and supposed to be downregulated during EMT. Nevertheless, E-cadherin was increased in breast cancer cells transfected with TRPM8 silencing plasmids. Moreover, Fibronectin and Vimentin, which are mesenchymal pheotypical markers and should be upregulated during EMT, were inhibited in TRPM8-silenced cells. However, they were significantly increased in cells transfected with TRPM8expressing plasmids. In order to observe visually, we probed the cells by immunofluorescent antibodies. As shown in Fig. 3c, cells with higher cells exhibited obviously lower
Tumor Biol.
expression of E-cadherin while Vimentin upregulated concordantly with TRPM8 and vice versa. These findings suggested that TRPM8 had positive effects on inducing EMT in breast cancer and might mediate epithelial cells to transform toward mesenchymal cells. Because activation of the PI3K/Akt pathway is emerging as a central feature of EMT [24–28], we speculated TRPM8 regulate Akt activity in breast cancer cells. As shown in Fig. 3b, changed TRPM8 expression did not obviously influence Akt level, but phosphorylation of Akt was significantly inhibited in cells transfected with small interfering (siRNA) against TRPM8. Followingly, the increase in Akt phosphorylation was accompanied by a change in phosphorylation of GSK-3β, a downstream target protein of Akt, suggesting that upregulation of TRPM8 activates the Akt/GSK-3β pathway in breast cancer cells. Previously, GSK-3β activity was demonstrated to be necessary for the maintenance of epithelial architecture by dual regulation of Snail [35, 36]. As shown in Fig. 3b, Western blotting revealed that compared with vector cells, expression of Snail significantly increased in breast cancer cells transfected with TRPM8 plasmids but decreased in those with silencing constructs. Upregulation of TRPM8 increased aggressiveness of breast cancer cells in vitro To investigate the role of TRPM8 in the development and progression of breast cancer, TRPM8 over-expressing cells were established by plasmid transfection to perform migration and invasion assays in vitro. As we expect, alterations of cellmigrating ability observed by wound-healing assay demonstrated that cells transfected with TRPM8 plasmids significantly ran faster than those with control vectors. On the other hand, cells transfected with siTRPM8 moved more slowly than those with scrambled sequence vectors (Fig. 4a). The invasive property of PC-3 cells was examined by TranswellMatrigel penetration assay, which depicted cells transfected with TRPM8 plasmids penetrated through the gel-membrane section much more than cells with control vectors. Meanwhile, siTRPM8-transfected breast cancer cells got obviously repressed invasive ability. Morphologically, high expression of TRPM8 induced major shape changes of cells, with the formation of thorns or legs which suggest more motile and higher invasiveness (Fig. 4c).
Discussion For the first time, our study demonstrated that TRPM8 facilitates breast cancer metastasis via promoting EMT process, and especially by activating Akt/GSK-3β pathway. This finding is a great supplement for the ongoing researches about
TRPM8 regulating breast cancer, which always focus on molecular structure of TRPM8 and its ability of sustaining Ca2+ influx [37]. TRPM8 was initially cloned as a novel prostate-specific gene by screening a prostate cDNA library [14], and has been found in a number of other cancers, including melanoma, breast adenocarcinoma, colorectal cancer, lung cancer and bladder cancer [14, 37, 38]. Prostate cells normally express low amounts of TRPM8 while its expression is much higher in cancerous cells [14, 39], and out data showed similar result. When the mild expression of TRPM8 in MCF-10A was normalized to onefold, higher expressions of TRPM8 in other cancer cell lines ranged from fivefolds to 30-folds. So we selected the MDA-MB231 due to its highest expression and the MCF-7 which did not get lowest TRPM8 but was involved more common in research than ZR-75-30. Function of TRPM8 is well established in the majority of cell-signaling pathways involved in carcinogenesis [40], in which EMT is a key step for cell transformation, migration, adhesion and invasion. Actually, lots of studies had identified the significance of EMT during breast cancer development. Cells undergoing EMT typically show both an increase in protein abundances of Vimentin, N-cadherin, Fibronectin, etc. and a decrease in E-cadherin, Cytokeratins and Occludin, etc. [41]. Our results are in complete agreement with that theory. Since TRPM8 was significantly knocked down in MDA-MB-231 cell line, expression of E-cadherin, a mark of epithelial cell phenotype, was obviously upregulated in those cells. Meanwhile, Vimentin and Fibronectin both of which are marks of mesenchymal cell phenotype correspondently decreased in the transfected cell line. Converse results showed in MCF-7 cells when expression of TRPM8 was increased by plasmids. These findings strongly suggest that TRPM8 could regulate the EMT phenotype of breast cancer cells. Besides regulating EMT marker proteins of breast cancer cells, TRPM8 was firstly identified to be involved in the activation of AKT/GSK-3β pathway. We can see in each transfected cell line that expressions of AKT and GSK-3β seemed extremely unchanged, because TRPM8 might not influence transcriptions of AKT and GSK-3β. But upregulated TRPM8 significantly increased the phosphorylation of AKT in breast cancer cells; this result suggests TRPM8 activating AKT via phosphorylation. Importantly, Ser 9 phosphorylation of GSK-3β by activated AKT results in the downregulation of GSK-3β, and GSK-3β binds to and phosphorylates Snail transcriptional repressor to induce its cytoplasmic translocation and degradation [36, 42]. Consequently, increasing of TRPM8 promoted expression of Snail that represses the transcription of E-cadherin, of which the degradation is key step of the initiation of EMT [29, 43, 44]. Our data demonstrated that activation AKT/GSK-3β-signaling pathway directly leads to the activation of Snail-EMT-signaling cascade. Since EMT is critical for metastasis of cancer cells
Tumor Biol.
[45], our results elucidate that TRPM8 activates EMT through inducing AKT/GSK-3β pathway and strongly supplements researches about breast cancer metastasis. Studies have underlined the involvement of ion channels such as potassium channels in cell cycle progression and migration [46–48], as well as sodium channels in invasion processes [49, 50]. For example, Yang et al.
Fig. 4 TRPM8 altered breast cancer cells invasiveness. a Shapes of MDA-MB-231 and MCF-7 cell lines of altered TRPM8 expressions were photographed under microscope. b The invasive properties of cells were analyzed by invasion assay using a Matrigel-coated Boyden chamber. Migrated cells were plotted as the average number of cells per field of view from three different experiments, as described in “Materials and methods” section. Original magnification, ×200. Error bars represent SEM. **P
