Breast cancer is one of the leading heterogeneous diseases in women worldwide which can be divided into several subtypes [1
]. According to the statistics from the National Cancer Institute (SEER 18, 2008–2014), the 5-year relative survival rate of female patients with localized breast cancer is 98.7%, whereas the rate for the female patients with metastatic breast cancer is only about 27.0%. Surgery in combination with endocrine therapy can provide better treatments for the patients with estrogen receptor (ER) positive, progesterone receptor (PR) positive and human epidermal growth factor receptor 2 (HER2) positive breast cancer [3
]. The treatment of triple-negative breast cancer (TNBC), a highly metastatic subtype, still remains challenging due to the lack of targeted therapy.
Apoptosis is a key regulator of tissue homeostasis [4
]. An imbalance between cell proliferation and apoptosis promotes tumorigenesis. Chemotherapy, radiation therapy and immunotherapy, through inducing DNA damage and triggering apoptosis of cancer cells, are major treatment strategies for TNBC [5
]. However, the side effects of these conventional treatments are severe. Antibody-drug conjugates (ADCs), which can allow exact targeting to tumour cell-surface proteins, are a new class of therapeutic agents for targeted cancer therapy [7
]. Therefore, identification of differentially expressed cell-surface proteins in TNBC is deemed necessary for an effective and specific treatment.
Transient receptor potential (TRP) channels, a group of non-selective cation channels, modulates a diversity of cellular physiological traits. Differential expression as well as dysregulation of specific TRP channels have presented positive correlations with different breast cancer subtypes. Upregulated TRP channels worsen breast cancer progression through increasing cell proliferation, migration and invasion. Thus, TRP channels have been proposed as potential breast cancer diagnostic markers and therapeutic targets [8
Canonical TRP isoform 3 (TRPC3) channel was reported to be upregulated in breast cancer biopsy tissues when compared to normal breast tissues [11
]. However, the biological role of TRPC3 in breast cancer still remains to be elucidated. In the present study, we aimed to investigate if TRPC3 is responsible for the proliferation and apoptosis resistance of the TNBC cells, and, if yes, the underlying mechanisms involved.
The major novel findings of this study are as follows: (1) TNBC cell line MDA-MB-231 over-expresses TRPC3 channel on the plasma membrane; (2) functional presence of TRPC3 regulates extracellular calcium entry across plasma membrane into cytosol; (3) blockade of TRPC3 decreases MDA-MB-231 proliferation but does not affect cell cycle distribution; (4) blockade of TRPC3 induces apoptosis via the activation of ERK1/2 in MDA-MB-231; (5) RASA4, a Ca2+
-promoted Ras-MAPK pathway suppressor, is located on the plasma membrane of MDA-MB-231; blockade of TRPC3 causes the translocation of RASA4 from the plasma membrane to the cytosol. Taken all these findings together, we highlight a key functional role of the TRPC3-RASA4-MAPK signaling cascade in maintaining the proliferation and apoptosis resistance of TNBC cells. A schematic illustration is shown in Figure 6
Over-expressed TRPC6 was found to promote breast cancer cell growth and metastasis [22
]. TRPC1 was reported to play an important role in basal-like breast cancer cell migrations with regulation of the epithelial to mesenchymal transition (EMT) procedure [23
]. TRPC5 was reported to be essential for the survival of adriamycin-resistant MCF-7 cells through induction of the expression of a key efflux transporter P-glycoprotein [24
]. In our present study, we aimed to identify a potential molecular therapeutic target of TNBC cells distinguished from hormone receptor positive breast cancer cells. A previous study has reported the abnormal upregulation of TRPC3 and TRPC6 in breast cancer tissues from patients [11
]; the differential expression of TRPC3 in MCF-7 and MDA-MB-231 has attracted our attention. In our current study, by Western blot and immunocytochemistry, TRPC3 was found to be over-expressed on the plasma membrane of MDA-MB-231 when compared to MCF-7, consistent with this previous study [11
]. In yet other studies, TRPC3 was reported to contribute to the proliferation of ovarian cancer cells and lung cancer cells [25
]; our current findings that the upregulated TRPC3 in MDA-MB-231 plays a positive role in cancer progression are in line with those previous studies.
Expression of DNA repair genes are downregulated in TNBC; and this has been suggested to increase the effectiveness of DNA damage response inhibitors for the treatment of TNBC [30
]. Patients with basal-like TNBC are suggested to be preferentially treated with agents that engage DNA damage signaling response pathways (e.g., PARP inhibitors) [1
]. We found that blocking TRPC3 induced apoptosis of MDA-MB-231 which was characterized by morphological and biochemical changes including cell shrinkage, membrane blebbing, DNA fragmentation, cleavage of caspase-3/7 and PARP [31
]. It has been known for long that caspases-3/7 cleaves PARP and inactivates its DNA-repairing abilities during apoptosis [32
]. In our study, TRPC3 blockade was found to increase the amount of cleaved caspase-3/7, suggesting that blocking TRPC3 induces caspase-dependent apoptosis in MDA-MB-231.
Our study revealed that TRPC3 was oncogenic in MDA-MB-231 with suppression of ERK1/2 phosphorylation. Dysregulation of Ras-MAPK pathway is commonly observed in cancer [33
]. Multiple anti-cancer drugs targeting Ras-MAPK pathway are currently under clinical trials [34
]. While MDA-MB-231 is a KRas mutant (G13D) cell line [35
], we found that there was no significant change of cell proliferation in MEK-ERK inhibitor PD98059-treated MDA-MB-231 cells. In contrast, decrease of cell proliferation caused by TRPC3 blockade was attenuated in PD98059 pre-treated cells. Therefore, our results suggested that mutated KRas may lead to constitutive activation of other MAPK pathway targets but not ERK1/2. In fact, reversible phosphorylation and dephosphorylation of MAPK subfamilies at serine and threonine residues play dual roles in cell death [36
]. On one hand, activation of MEK-ERK pathway was commonly regarded to serve an anti-apoptotic function by protecting caspase-9 from cleavage [37
]. On the other hand, constitutively activated ERK was reported to sensitize cancer cells to different chemotheraputic agents [38
]. Our results indicated that blocking TRPC3 caused apoptosis through activation of ERK1/2.
A loss of intracellular Ca2+
homeostasis triggers cell apoptosis [40
]. Endoplasmic reticulum (ER)-Ca2+
store depletion and mitochondrial Ca2+
uptake are key pro-apoptotic players [8
]. In addition, Ca2+
influx through TRP channels was reported to act as anti-apoptotic regulators through activation of Ca2+
-sensitive downstream pathways [43
]. Our present study firstly demonstrated that Ca2+
-dependent mechanism for activation of MAPK pathway caused by TRPC3 blockade is mediated through the TRPC3-RASA4-MAPK signaling cascade. In RAS mutated tumor cells, the signaling transduction upstream of MAPK pathway is complex. Both RASAL2 and RASA4 are members of RAS GTPase-activating proteins (RAS-GAPs) catalyzing GTP into GDP and therefore inactivating RAS. RASAL2 has recently been found to activate RAC1 and contribute to TNBC tumorigenesis [46
]. On the other hand, numerous previous studies showed that plasma membrane RASA4 switched off the MAPK pathway in response to the elevating intracellular Ca2+
]. In our study, RASA4 was found to be over-expressed on the plasma membrane of MDA-MB-231 when compared to MCF-7 where it inhibited MAPK pathway. RASA4 on MDA-MB-231 plasma membrane was decreased in response to a decreased free Ca2+
concentration induced by Pyr3, with concomitant activation of MAPK pathway.
In summary, our study found that in TNBC MDA-MB-231 cells, Ca2+ influx through TRPC3 channel sustains the presence of RASA4 on the plasma membrane where it inhibits Ras-MAPK pathway, leading to proliferation and apoptosis resistance. Our study reveals this novel TRPC3-RASA4-MAPK signaling cascade in TNBC cells and suggests that TRPC3 may be exploited as a potential therapeutic target for TNBC.
4. Materials and Methods
4.1. Cell Culture
This study was approved by the Ethics Committee (the Clinical Research Ethics Committee (CREC) code is 2014.236), the Chinese University of Hong Kong and followed the tenant of the Declaration of Helsinki. Two human breast cancer cell lines, MDA-MB-231 (ER–, PR– and HER2–) and MCF-7 (ER+, PR+/− and HER2–) (American Type Culture Collection, Manassas, VA, USA), were selected as our in vitro research model. They were cultured at 37 °C under an atmosphere of 5% CO2/95% air. MCF-7 cells were cultured in the phenol red-free RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), whereas MDA-MB-231 cells were cultured in the same RPMI 1640 medium supplemented with 5% heat-inactivated FBS.
4.2. Treatment Regimen
The effects of TRPC3 blocker Pyr3 (Tocris, Bristol, UK) on MDA-MB-231 were investigated. Cells were seeded at the density of 6.67 × 104 MDA-MB-231 cells cm−2 on 0.1% gelatin (Sigma-Aldrich, Missouri, MO, USA)-coated dish (96-well dish for MTT and Trypan blue exclusion assay, 24-well dish for immunocytochemistry and 6-well dish for protein harvesting) and were allowed for adhesion overnight. MDA-MB-231 cells were then treated with TRPC3 blocker Pyr3 or DMSO (solvent control) for three to five days. SP600125 (JNK inhibitor, 1 μmol/L, Tocris), PD98059 (MEK-ERK inhibitor, 5 μmol/L, Tocris), and SB202190 (p38 MAPK inhibitor, 1 μmol/L, Tocris) were used to treat cells for 24 h prior to Pyr3 exposure. Trypan blue exclusion, MTT, cell cycle, Western blot and immunocytochemistry analyses were then performed.
4.3. Western Blot
MCF-7 and MDA-MB-231 cell lysates were prepared and Western blot was performed as previously described [47
]. To assay for the presence of TRPC3, 1:1000 rabbit anti-TRPC3 (Alomone) and 1:1000 mouse anti-TRPC3 (Santa Cruz) were used. To validate the specificity of the anti-TRPC3 antibody, the anti-TRPC3 was pre-incubated with its blocking peptide according to the manufacturer’s instructions for 2 h at 37 °C prior to the membrane incubation. To assay for apoptotic cell death, primary antibodies 1:1000 rabbit anti-caspase-7, 1:200 rabbit anti-caspase-3, 1:1000 rabbit anti-PARP (Cell Signaling, Danvers, MA, USA) were used. To assay for MAPK pathway involvement, 1:1000 rabbit anti-phosphorylated or total p38 MAPK, ERK1/2 and JNK (Cell Signaling, Danvers, MA, USA) were used. In all cases, the membranes were stripped and probed with 1:1000 rabbit anti-β-tubulin (Cell Signaling) as an internal control. After primary antibody probing, membranes were washed in TBST, and incubated with HRP-conjugated secondary antibody (Dako, Glostrup, Denmark) in the dilution of 1:3000 for 1 h at room temperature. Protein expression was detected by enhanced chemiluminescent substrate (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and protein bands were visualized by film exposure. The density of the bands was quantified using Image J software (version 1.48v, National Institutes of Health, Bethesda, MD, USA).
MCF-7 and MDA-MB-231 cells were seeded on 0.1% gelatin-coated glass coverslips in 24-well culture plates (Thermo Fisher Scientific) for 24 h and were allowed to proliferate for 48 h. Cells were then fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min at 37 °C, then rinsed in PBS twice for 5 min, and subsequently incubated in 0.1% Triton X-100 (Sigma-Aldrich) for 15 min. Coverslips were then washed with PBS twice, and incubated in a blocking solution containing 2% BSA and 5% normal goat serum (NGS) (Invitrogen) for 1 h followed by an overnight incubation in the blocking solution containing antibodies at 4 °C in the dark. To assay for the presence of TRPC3, the coverslips were incubated with 1:100 rabbit anti-TRPC3 (Abcam) and 1:100 mouse anti-TRPC3 (Abnova), respectively. To assay for the presence of RASA4, 1:100 rabbit anti-RASA4 (Abcam) was used. After three times being washed with PBS supplemented with 0.1% Tween (Sigma-Aldrich), secondary antibodies, 1:100 Alexa Fluor 488/594 goat anti-mouse/rabbit (Invitrogen), were diluted in 1% NGS/PBS and applied to incubate the cover slides for 1 h at room temperature. Then 1:5000 DAPI (Roche, Basel, Switzerland) in PBS was used to stain nuclei for 10 min at room temperature. Slides were affixed with mounting medium (Dako, Carpinteria, CA, USA) and viewed using an Olympus FluoView FV1000 confocal laser scanning microscope with a 60 × objective. Images were analyzed using the FV1000 software (Olympus, Tokyo, Japan).
4.5. Subcellular Fractionation Followed by Western Blot
Whole cell pellets of MDA-MB-231 were fractionated into cytosol and membrane fractions. Cells were lysed by hypotonic fractionation buffer (0.32 M sucrose, 5 mM Tris at pH 7.4) freshly supplemented with protease inhibitor cocktail (Roche). After vortex and passing through a syringe with a 27 gauge needle for ten times, the supernatant (membrane and cytosol) and pellet (nuclear fraction) were separated by centrifugation at 500× g for 10 min at 4 °C. The supernatant was further centrifuged at 100,000× g for 1 h at 4 °C to separate the cytosol and the membrane fraction. The pellet was resuspended with membrane resuspension buffer (20 mM HEPES, 1 mM EDTA, 10% glycerol, 120 mM KCl and 2% Triton X-100) freshly supplemented with protease inhibitor cocktail. Protein concentration of each fraction was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). α1 sodium/potassium-ATPase (Na/K-ATPase α1) and β-tubulin were used as the protein makers of the membrane fraction and cytosolic fraction, respectively. Mouse anti-Na/K-ATPase α1 (1:1000, Abcam) and rabbit anti-β-tubulin (1:1000, Cell Signaling) were used in primary antibody incubation step and all the subsequent processes for Western blot were conducted as described above under ‘4.3 Western Blot’.
4.6. Confocal Ca2+ Imaging
imaging using Fluo-4 AM (Thermo Fisher Scientific) was performed as previously described [17
]. Drugs including adenosine-triphosphate disodium salt hydrate (ATP) (Sigma), CaCl2
(Sigma) and Pyr3 were added at their appropriate concentrations at a given time. Equal volumes of dimethyl sulfoxide (DMSO) (Sigma) were also added in the solvent control group. Raw traces reflected the changes in cytosolic Ca2+
level were expressed as F/F0 which was defined by the fluorescence intensity at a given time normalized to its baseline. Data was analyzed using with FV1000 software (Olympus).
4.7. Proliferation Assay
MDA-MB-231 cells were treated with TRPC3 blocker Pyr3 or DMSO for 3–5 days. Previous studies have shown that expression of the N-terminal fragment of TRPC3 (N-terminal domain consisted of amino acids 1–302 of human TRPC3) would lead to a dominant negative (DN) effect on TRPC3 channel function [17
]. Recombinant adenoviruses transferring green fluorescent protein (GFP) and DN of TRPC3 were constructed previously by our group [17
] and were used to infect MDA-MB-231 cells. Cell viability and cell proliferation were measured by MTT assay. Viable cell numbers were measured by Trypan blue exclusion assay as previously described [47
4.8. Propidium Iodide (PI) Staining Followed by Flow Cytometry for Cell Cycle Analysis
Cells were seeded at the density of 3.33 × 104 MDA-MB-231 cells cm−2 on the 100-mm cell culture dishes (Cellstar, Greiner bio-one, Kre Austria). In addition, 1 × 106 cells per treatment group were harvested with 0.05% trypsin-EDTA (Invitrogen), then fixed with 70% ethanol (Sigma) on ice for 30 min. Cells were then centrifuged at 200× g and the cell pellet was resuspended with staining solution containing 2 μg/mL PI (Sigma) and 10 mg/mL RNase A (Thermo Fisher Scientific) in PBS for 30 min in dark at 37 °C and analyzed using a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). The percentages of viable cells residing in G0/G1, S, and G2/M phases and apoptotic cells residing in sub-G1 phase were calculated using the ModFit LT software (Verity Software House, Topsham, ME, USA).
4.9. Fluorescence Imaging
Living cell morphology of MDA-MB-231 cells and green fluorescence of adenovirus-infected MDA-MB-231 cells were captured using Nikon TE300 eclipse microscope with a 10× objective. For observation of programmed cell death, living cells were stained with 100 nM Mitotracker Red CMXRos (Invitrogen) at 37 °C under an atmosphere of 5% CO2
/95% air for 30 min. Cells were then fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min at 37 °C, rinsed in PBS twice for 5 min, and subsequently incubated in 0.1% Triton X-100 (Sigma-Aldrich) for 15 min. 1:5000 DAPI (Roche) in PBS was used to stain nuclei for 10 min at room temperature. Cell morphology was observed using the Olympus FluoView FV1000 confocal laser scanning microscope with a 60× objective and further analyzed using the FV1000 software [49
4.10. Statistical Analysis
Each experiment was repeated at least three times. The results were expressed as mean ± SEM. Statistical significance between two groups of means was determined by student’s t-test. Statistical significance between three or more groups of means was determined by analysis of variance (ANOVA). p < 0.05 was considered to be statistically significant.