Abnormal expression, localization and interaction of canonical transient receptor potential ion channels in human breast cancer cell lines and tissues: a potential target for breast cancer diagnosis and therapy
© Aydar et al; licensee BioMed Central Ltd. 2009
Received: 27 October 2008
Accepted: 18 August 2009
Published: 18 August 2009
Ca2+ is known to be involved in a number of metastatic processes including motility and proliferation which can result in store-depletion of Ca2+. Up regulation of genes which contribute to store operated channel (SOC) activity may plausibly be necessary for these processes to take place efficiently. TRPC proteins constitute a family of conserved Ca2+-permeable channels that have been shown to contribute to SOC activity.
In breast cancer biopsy tissues, TRPC3 and TRPC6 were the predominant TRPC genes expressed with TRPC3 and TRPC6 being significantly up regulated compared to normal breast tissue. In the lowly metastatic breast cancer cell line MCF-7, TRPC6 was the chief TRPC gene expressed while in the highly metastatic breast cancer cell line MDA-MB-231 both TRPC3 and TRPC6 were the predominant TRPC genes expressed. Western blotting, immunoconfocal analysis and immunoprecipitation experiments confirmed that the MDA-MB-231 cell line expressed both TRPC3 and TRPC6 protein with the majority of protein being intracellular. TRPC3 and TRPC6 were found to be in an immunoprecipitatble complex and co-localize within the cell. To demonstrate the potential of targeting TRP channels in breast cancer, hyperforin reportably a specific activator of TRPC6 significantly reduced the growth and viability of the breast cancer cell lines but had no effect on the non-cancerous breast cell line. Silencing of TRPC6 in MDA-MB-231 cells resulted in a significant reduction in cell growth but not viability.
TRPC channels may be potential future targets for breast cancer diagnosis and therapy and deserve further investigation to evaluate their role in cancer cell physiology.
Breast cancer is a leading cause of cancer-associated death in women . It most commonly metastasizes to the bone, with 70% of patients who develop bone metastases dying . Finding early markers of metastasis and developing effective therapies against their development is a priority. Investigation of functional expression of membrane ion channels is an exciting development in cancer research. Increased expression of voltage-sensitive ion channels is directly associated with malignancy, as evidenced by their role in cell proliferation, migration and survival . As such, some of these channels have begun to be developed as targets for cancer drug design . Whilst considerable work has so far been done on K+ and Na+ channels, surprisingly little is known about the role of mechanosensitive channels and Ca2+ signalling in cancer. Both are likely to have a significant role in the cancer process and Ca2+ is a regulator of proliferation and apoptosis . In this regard, a key group of ion channels of increasing prominence are the Transient Receptor Potential (TRP) family . There is evidence that expression of TRPV6, TRPM8, TRPM1 and TRPV1 are significantly altered in human cancer cells . TRPC proteins in humans (TRPC1, 3, 4, 5, 6 and 7) constitute a family of conserved Ca2+-permeable cation channels which are activated in response to agonist-stimulated PIP(2) hydrolysis and subsequent studies have provided substantial evidence that some TRPCs contribute to SOC activity . TRPC proteins have also been shown to form agonist-stimulated calcium entry channels that are not store-operated but are likely regulated by PIP(2) or possibly diacylglycerol . TRPC1 and 6 are also sensors of mechanically and osmotically induced membrane stretch . In human PCa LNCaP cells, agonist-mediated stimulation of alpha1-adrenergic receptors required coupled activation of TRPC6 channels and nuclear factor of activated T-cells to promote proliferation . Additionally, in the same cells, store-operated Ca2+ channels were found to be important determinants of the transition to androgen-independent state . Consistent expression of TRPC1, 3, 5, 6 in glioma cell lines and acute patient-derived tissues has been described. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the non-selective TRPC inhibitors GdCl3, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest . Finally in liver tumor samples TRPC6 was expressed more strongly than in isolated hepatocytes from healthy patients and experiments suggested that TRPC6 may play a role in control of human hepatoma cell proliferation . Little is known about canonical TRP (TRPC) ion channels in breast cancer apart from a few emerging papers to date [13, 14], such channels could play a major role in cellular activities involved in the cancer process since TRPC channels are predominantly Ca2+ permeable. The primary aim of this paper is to determine which TRPC channels are expressed in breast cancer cell lines and tissues and the roles these channels may play in cell proliferation.
A mouse anti-TRPC3 polyclonal antibody was obtained from the Abnova Corporation and used at a 1:200 dilution for immunoblotting and 1:20 for immunochemistry. A rabbit anti-TRPC6 polyclonal antibody was obtained from Sigma-Aldrich and used at a 1:200 dilution for immunoblotting and 1:20 for immunochemistry. TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). PBS buffer (40 mM Na2HPO4, 10 mM KH2PO4, and 120 mM NaCl with a resulting pH of 7.2).
Culture of cell lines
Human breast cancer cell lines (MCF-7 and MDA-MB-231) were cultured in DMEM (Gibco) containing 10% FBS and 4 mM glutamine with penicillin-streptomycin. The 'normal' human breast epithelial cell line (MCF-10A) was cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 medium (Gibco, DMEM-F12) supplemented with 5% FBS, hydrocortisone (0.5 μg/ml), insulin (10 μg/ml), epidermal growth factor (20 ng/ml), and penicillin-streptomycin. All cell lines were maintained in a 37°C CO2 incubator in 100 mm culture dishes.
RNA extraction from biopsy samples and breast cell lines
An absolutely RNA miniprep kit (Stratagene) was used to extract total RNA from breast cell lines or homogenized breast biopsy tissue. cDNA synthesis was conducted using 1 μg of RNA, 1 μg of Oligo (dT)18 and Superscript II reverse transcriptase (Gibco) according to the manufacturers instructions. All samples were tested for absence of genomic DNA contamination before use.
Primers utilized in this investigation
Expected product size (bp)
5'-GTA AGT GGA TTT GCT CTC AT-3'
298 for TRPC1B splice variant
5'-TGG TTA ATT TCT TGG ATA AA-3'
5'-TAC TCA ACA TGC TAA TTG CTA TGA T-3'
5'-CAC AGT TGC TTG GCT CTT GTC TTC C-3'
5'-CTC TGG TTG TTC TAC TCA ACA TG-3'
5'-CCT GTT GAC GAG CAA CTT CTT CT-3'
5'-GCT CGC AGC CAC CCC AAA GGG AGG A-3'
5'-CCA ATG TCC CTA CCC TGT TCT CCC AGC TCT C-3'
5'-GAA CTT AGC AAT GAA CTG GCA GT-3'
625 or 277
5'-CAT ATC ATG CCT ATT ACC CAG GA-3'
5'-GTC CGA ATG CAA GGA AAT CT-3'
5'-TGG GTT GTA TTT GGC ACC TC-3'
5'-AAA CCA GTT CTG CCT CCT CCA C-3'
5'-GTC CAT CGG AAG TCT TAT CTT TCT TTC-3'
5'-GAT TTT CAC CAA TGA CCG CCG-3'
5'-GAG CAG TCC CTG CTG GAA CT-3'
5'-GGT ACT TCG AGA CAC TGA GG-3'
5'-GAA GGA CAC GCT GCG CGA GT-3'
5'-GCA TCC TCC CGT GCC TCC TT-3'
5'-ATG GAT GAT GAT ATC GCC GC-3'
5'-ATC TTC TCG CGG TTG GCC TT-3'
TRPC1 F nested
TRPC1 R nested
TRPC3 F nested
TRPC3 R nested
TRPC4 F nested
TRPC4 R nested
TRPC5 F nested
TRPC5 R nested
TRPC6 F nested
5'-CAA CCA GAA ACA GAA GCA TG-3'
TRPC6 R nested
5'-CTC GCA ATG AAT GAT GCT GC-3'
TRPC7 F nested
TRPC7 R nested
Real time PCR
Real time PCR was performed on a DNA Engine Opticon 2 machine with Opticon Monitor™ software. PCR with SYBR® Green PCR Master Mix (Qiagen) was performed under the following conditions: 95°C for 30 s, 55°C for 15 s, and 72°C for 1 min for 40 cycles. Heating for 5 min at 95°C preceded the cycles. Melting curves and agarose gel electrophoresis were performed to verify the specificity of the products. As variations in cDNA quality within different samples could occur, β-actin was included as an internal control. By subtracting the β-actin threshold (Ct) value from the TRPC Ct value of each sample, the cDNA quality of each sample is taken into account. The relative quantification (R) of the mRNA levels in each individual sample was calculated according to following equation: R = EΔCt (control-sample) . The real time PCR efficiency of each TRPC amplicon (E) was calculated according to the method described previously .
Cellular protein extracts at a concentration of 1 mg/mL were mixed with SDS sample buffer (Sigma-Aldrich) and 5 μg of protein were loaded per lane and separated on acrylamide 4% – 20% gradient Tris-glycine minigels (Cambrex). The proteins were transferred to nitrocellulose membrane for 3 h at 4°C; transfer was verified using Ponceau red (Sigma) and the blots were blocked with a solution containing 2.5% skimmed milk and 2.5% bovine serum albumin (BSA) in TBST overnight at 4°C before probing with specific antibodies. The antibodies were diluted in TBST with 0.5% skimmed milk and 0.5% BSA and incubated with the blot for 4 h at room temperature. Following four 10-min washes with TBST (with constant agitation) at room temperature, the blots were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DAKO- at the manufacturers' suggested concentrations) as appropriate, and diluted in TBST with 0.5% skimmed milk and 0.5% BSA for 2 h at room temperature. The blots were again washed as before and then developed with an enhanced chemiluminescence Western blot kit (Amersham). Immunoprecipitations were carried out with a Classic Seize kit from Pierce. As controls, the immunoprecipitation was performed in the absence of immunoprecipitating antibody or with a isotype control antibody.
Sterile 13 mm diameter glass coverslips were placed in 24 well costar plates. To each well 1 ml of cell suspension were seeded at a density of 2 × 104 cells/ml and then allowed to grow for 48 h. The cells were washed twice with PBS and then incubated with concanavalin-A (FITC conjugated) for 10 mins at room temperature in PBS with 1% BSA. Cells were washed twice in PBS for 1 min and then fixed in 2% paraformaldehyde for 7 mins at room temperature. The cells were washed four times with PBS and subsequently incubated in saponin (0.1%) in PBS for 5 mins at room temperature. The permeabilization solution was aspirated, and the cells were washed four times in PBS before being blocked with 8% BSA in PBS for 30 mins. Cells were again washed twice with PBS for 5 min and excess liquid was removed before addition of TRPC antibodies (1/20 dilution) in 1% BSA in PBS for 1 h. A further four PBS washes were performed before the cells were incubated with either an alexifluor 568 conjugated anti-rabbit IgG (1:100) or a FITC conjugated anti-mouse IgG (1:100) in PBS with 1% BSA for 30 mins at room temperature in the dark. Four 5 min PBS washes were performed in the dark and coverslips were mounted in Vectashield (Vector Laboratories Inc.) and allowed to set overnight at 4°C. Controls to assess auto-fluorescence or non-specific labeling were also performed and consisted of treatment with 1% BSA without either primary antibody, secondary antibody or both. Confocal microscopy was performed on a Leica 650T instrument using 568 and 488 nm filters and analysis performed with Leica confocal software using a cross section tool. Images were analyzed with excitation at both 568 and 488 nm and each singly to rule out 'cross' bleed from either wavelength.
Silencing of TRPC6
Transfection was accomplished using a double transfection procedure using the following target sequences for TRPC6 (SiRNA1: AACGAGAGCCAGGACTATCTG; SiRNA2: AAGACGGCTGCCCGCAAGCCC; SiRNACon: AM4635-Ambion) Briefly, 24-well plates were seeded at 2.5 × 104 cells/well 24 h prior to transfection. Transfection mixes were set-up with 0.2 μg of siRNA in 50 μl of Optimem and 1 μl of lipofectamine in 50 μl of Optimem (per well). Transfection mixes were applied to the cells for 6 hours, subsequently removed and replaced with 1 ml of growth media. After 48 hours the cells were removed with trypsin and reseeded into 24-well plates and the transfection repeated as above. Seventy two hours after this second transfection the levels of TRPC6 protein was ascertained by SDS-PAGE electrophoresis and western blotting as described in the section above.
Cell viability was assayed using a trypan blue exclusion assay. Cells were removed with trypsin-EDTA and mixed with 0.4% trypan blue in PBS for 5 mins at room temperature. Subsequently the number of cells which were able to exclude the dye was counted.
To six well dishes 600 μl of fresh medium and 150 μl of 5 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were added to the wells and incubated at 37°C for 2 h. The solution was removed and replaced by 890 μl of DMSO and 110 μl of Glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) for 10 mins. The numbers of viable cells were then quantified by measuring the absorbance at 570 nm with a spectrophotometer.
Data were analyzed by one-way ANOVA. Differences were taken as statistically significant when P < 0.05.
Expression of TRPC mRNA in normal and breast cancer biopsy tissue
Expression of TRPC mRNA in breast cancer epithelial cell lines
Relative expressions of TRPC channel sub-types in non-cancerous and cancerous breast cell lines
2.3 × 10-6 +/- 1.6 × 10-6
1.5 × 10-7 +/- 3.9 × 10-8
5.0 × 10-4 +/- 5.0 × 10-4
0.36 +/- 0.19
0.0012 +/- 7.3 × 10-4
0.46 +/- 0.08
Protein expression and interaction of TRPC6 and TRPC3 in breast cancer epithelial cell lines
Localization and co-localization of TRPC6 and TRPC3 in breast cancer epithelial cell lines
Effect of a TRPC6 activator and gene silencing of TRPC6 on growth of breast epithelial cell lines
Our experiments suggested that normal breast tissues expressed the TRPC1 and TRPC3 genes. Comparison of normal and breast cancer biopsy tissues indicated a significant alteration in TRPC channel expression both in terms of channel type expressed and levels of channel expression. In breast cancer biopsy tissues TRPC6 was expressed and appeared to be the predominant TRPC channel expressed. Additionally TRPC3 appeared to be significantly up regulated in breast cancer tissues. Of course our experiments could not distinguish which cell types these TRPC channels were expressed in, since the breast tissue could contain a variety of cell types.
In the epithelial non-cancerous cell line MCF-10A only relatively very low amounts of TRPC1 expression was observed. In lowly metastatic breast cancer cell line MCF-7 the predominant channel expressed was a short splice variant of TRPC6. In the highly metastatic breast cancer cell line MDA-MB-231 the predominant TRPC channels expressed were TRPC3 and TRPC6 (of longer splice form variants). The amounts of TRPC3 and TRPC6 expressed in the MDA-MB-231 cell line were comparable in level, which leads us to speculate that they may form heteromultimers. The sizes of PCR products for TRPC3 and C6 found in the cancer tissues appeared to be consistent to that found in the MDA-MB-231 cell line.
Protein expression of TRPC3 and TRPC6 in breast cancer cell lines appeared to be consistent with the PCR results. TRPC6 and TRPC3 expression was observed in the MDA-MB-231 cell line with the majority of protein being expressed in an intracellular location.
Our data provides strong evidence that TRPC3 and TRPC6 form heteromutimeric channels in breast cancer epithelial cell line MDA-MB-231, which is consistent with previous reports of TRPC3/6 expression .
Hyperforin a specific activator of TRPC6 produced a significant reduction in cell growth for the breast cancer cell lines, while the non-cancerous breast cell line was unaffected. Viability was significantly reduced in the breast cancer cell lines. TRPC6 activation is known to promote cell proliferation. This is an opposite result which is obtained here when using hyperforin however it may be expected that overactivation of TRPC6 by hyperforin could cause a disruption in Ca2+ signalling hence affecting cell proliferation. In an earlier study, hyperforin induced apoptosis and inhibited the growth of various human and rat tumour cell lines in vivo, . Our results provide preliminary functional data that a rationale to target TRPC channels in breast cancer cells may have some promise.
Complete silencing of TRPC6 in MDA-MB-231 cells using transfected SiRNA resulted in a significant reduction in cell growth but not viability.
On the whole our results with the MCF-7 cell line confer with the recent report by Bolanz et al  who found that TRPC6 and TRPV6 are the predominant TRP channels expressed in the lowly metastatic cell line T47D. Interestingly our data suggests that in the highly metastatic cell line MDA-MB-231 TRPV6 was down regulated and TRPC3 was up regulated. Our results are also consistent with the previous report by El Hiani et al  who suggested that, in MCF-7 cells, TRPC1 and TRPC6 are the most likely candidates for the highly selective Ca2+ current described in these cells. We propose that TRPC6 is the major TRPC channel expressed in MCF-7 cells. However correlation of the permeation and pharmacological profiles of the cationic current in MCF-7 cells as described by El Hiani et al,  with those described for TRPC6 shows a number of notable differences. The authors of that paper suggest that the cationic channels in MCF-7 cells are probably heteromultimers that include both TRPC6 and TRPC1 together with some other TRP members. Given the presence of TRPV6 and TRPC6 in MCF-7 cells we support this view. More recently Guilbert et al  demonstrated that TRPC6 was expressed and functional in breast cancer epithelial cells and that this channel was overexpressed in tumour tissues without any correlation with tumour grade, ER expression and lymph node metastasis. TRPC proteins constitute a family of conserved Ca2+-permeable cation channels which are activated in response to agonist-stimulated PIP(2) hydrolysis and subsequent studies have provided substantial evidence that some TRPCs contribute to SOC activity . Since Ca2+ is known to be involved in a number of metastatic processes including motility and proliferation  these processes may result in store-depletion thus up regulation of genes which contribute to SOC activity may plausibly be necessary for these processes to take place efficiently.
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